NIST SPECIAL PUBLICATION 260-155

U. S. DEPARTMENT OF COMMERCE/Technology Administration 

National Institute of Standards and Technology 

Standard Reference Materials®

Human Mitochondrial DNA—Amplification and 

Sequencing Standard Reference Materials— 

SRM 2392 and SRM 2392-I 

B. C. Levin, D. K. Hancock, K. A. Holland, H. Cheng and K. L. Richie 
NIST Special Publication 260-155 

Standard Reference Materials®

Human Mitochondrial DNA—Amplification and 

Sequencing - Standard Reference Materials— 

SRM 2392 and SRM 2392-I 

B. C. Levin, D. K. Hancock, K. A. Holland, H. Cheng and K. L. Richie 

Biotechnology Division 

Chemical Science and Technology Laboratory 

National Institute of Standards and Technology 

Gaithersburg, MD 20899-8311 

U.S. DEPARTMENT OF COMMERCE, Donald L. Evans, Secretary

TECHNOLOGY ADMINISTRATION, Phillip J. Bond, Under Secretary of Commerce for Technology

NATIONAL INSTITUTE OF STANDARDS AND TECHNOLOGY, Arden L. Bement, Jr., Director 

Issued September 2003
Certain commercial equipment, instruments, or materials are identified in this paper in 

order to specify the experimental procedure adequately. Such identification is not intended 

to imply recommendation or endorsement by the National Institute of Standards and 

Technology, nor is it intended to imply that the materials or equipment identified are 

necessarily the best available for the purpose. 

National Institute of Standards and Technology Special Publication 260-155

 Natl. Inst. Stand. Technol. Spec. Publ. 260-155, 89 pages (September 2003) 

 CODEN: NSPUE2

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 For sale by the Superintendent of Documents, U.S. Government Printing Office 

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iii

Contents 

Abstract......................................................................................................................................... vi 

1. Introduction................................................................................................................................1 

2. Materials and Methods..............................................................................................................1 

2.1. Origin of Extracted DNA...................................................................................................1 

2.2. Isolation and Cloning of mtDNA Containing the C-Stretch in SRM 2392 ...................2 

2.3 Bacteriophage DNA Isolation and Sequencing................................................................3 

2.4. mtDNA Primers used in Both SRM 2392 and 2392-I .....................................................4

2.5. Polymerase Chain Reaction (PCR) SRM 2392 Completed in 1999...............................4 

2.6. Polymerase Chain Reaction (PCR) SRM 2392-I Completed in 2002............................4 

2.7. Sequencing SRM 2392 Completed in 1999.......................................................................4 

2.8. Sequencing SRM 2392-I Completed in 2002 ...................................................................5 

2.9. Interlaboratory Evaluation for SRM 2392 Completed in 1999 ....................................5 

2.10. Differences in Methodology used by Laboratories in Interlaboratory Evaluation of 

SRM 2392, 1999 ................................................................................................................6

2.11. Interlaboratory Evaluation for SRM 2392-I, 2002 .......................................................6 

2.12. Differences in Methodology used by the Laboratories in the Interlaboratory 

Evaluation for SRM 2392-I, 2002 ...................................................................................7 

2.12.1. Armed Forces DNA Identification Laboratory (AFDIL), SRM 2392-I .........7 

2.12.1.1. AFDIL PCR Amplification, SRM 2391-I ...........................................7 

2.12.1.2. AFDIL Sequencing, SRM 2392-I ........................................................7 

2.12.2. Georgia Bureau of Investigation (GBI), Interlaboratory Evaluation, SRM 

2392-1 ...................................................................................................................8 

2.12.3. Federal Bureau of Investigation (FBI), Interlaboratory Evaluation, SRM 

2392-I ...................................................................................................................9
iv

2.12.3.1. FBI Polymerase Chain Reaction (PCR), Interlaboratory 

Evaluation, SRM 2392-I .......................................................................9

2.12.3.2. FBI Sequencing, Interlaboratory Evaluation, SRM 2391-I .............9 

2.13. Permissions .....................................................................................................................10

2.14. Final Preparation of DNA for SRM 2392 and SRM 2392-I ......................................10

2.14.1. CHR DNA Preparation and Confirmation, SRM 2392 .................................10

2.14.2. CHR Cloned DNA Production, SRM 2392 .....................................................10 

2.14.3. 9947A DNA Production, SRM 2392 ................................................................10 

2.14.4. HL-60 Production, SRM 2392-I .......................................................................11 

3. Results and Discussion ...........................................................................................................12

3.1. SRM Templates ............................................................................................................12

3.2. Primers ..........................................................................................................................12 

3.3. Differences between the SRM DNAs and the Cambridge Reference Sequence ......13

3.4. Meaning of the Differences from the Cambridge Reference Sequence ..................13 

3.5. The Interlaboratory Evaluation of the CHR and HL-60 Templates ......................14

4. Conclusions .............................................................................................................................15

5. Acknowledgments ..................................................................................................................16 

6. References ...............................................................................................................................16

Table 1. Sequences for Primer Sets Used for PCR Amplification of Human DNA ..............19 

Table 2. Certified Human mtDNA Sequence Differences from the Cambridge Reference 

Sequence (CRS) found in the Two Templates (CHR and 9947A) in NIST SRM 2392, 

One Template (HL-60) in NIST SRM 2392-I and in GM03798 and GM10742A.........22 

Table 3. Universal Genetic Code and (Human mtDNA Differences) ....................................29 
v

Figure 1. Schematic of Human mtDNA Showing all the Differences from the Cambridge 

Reference Sequence in CHR, 9947A, HL-60, GM03798 and GM10742A ....................30 

Appendixes....................................................................................................................................33

Appendix A. Certificate of Analysis, SRM 2392 

Appendix B. Certificate of Analysis, SRM 2392-I 

Appendix C. “A Human Mitochondrial DNA Standard Reference Material for Quality 

Control in Forensic Identification, Medical Diagnosis, and Mutation Detection,” 

B. C. Levin, H. Cheng, and D. J. Reeder [Genomics 55, 135-146 (1999)]. 

Appendix D. “Comparison of the Complete mtDNA Genome Sequences of Human Cell 

Lines - HL-60 and GM10742A - From Individuals with Pro-Myelocytic Leukemia and 

Leber Hereditary Optic Neuropathy, Respectively, and the Inclusion of HL-60 in the 

NIST Human Mitochondrial DNA Standard Reference Material - SRM 2392-I,” 

 B. C. Levin, K. A. Holland, D. K. Hancock, M. Coble, T. J. Parsons, L. J. Kienker, D. 

W. Williams, MP. Jones, and K. L. Richie [Mitochondrion 2, 387-400 (2003)]. 
vi

NIST Special Publication 260-155 

Standard Reference Materials: 

Human Mitochondrial DNA – Amplification and Sequencing – SRM 2392 and SRM 2392-I 

Barbara C. Levin, Diane K. Hancock, Koren A. Holland, Haiyan Cheng and Kristy L. Richie 

Biotechnology Division 

Chemical Science and Technology Laboratory 

National Institute of Standards and Technology 

Gaithersburg, MD 20899-8311 

Abstract 

Standard Reference Materials SRM 2392 and 2392-I are intended to provide quality control when 

performing the polymerase chain reaction (PCR) and sequencing of human mitochondrial DNA 

(mtDNA) for forensic identification, medical diagnosis, or mutation detection. They may also serve 

as controls when amplifying (PCR) and sequencing any DNA. These SRMs can also be used for 

quality assurance when determining the sequence of in-house DNA controls. SRM 2392 is certified 

for the sequences of the entire human mtDNA (16,569 base pairs) from two lymphoblastoid cell 

culture lines (CHR and 9947A) from apparently normal individuals and the cloned HV1 region of 

CHR containing a C-stretch through which it is difficult to sequence. SRM 2392-I is certified for the 

mtDNA sequence from HL-60, a promyelocytic cell line prepared from the peripheral blood 

leukocytes from an individual with acute promyelocytic leukemia. The mtDNA sequence 

information (but not the DNA) of two additional DNA templates (GM03798 and GM10742A) that 

were amplified and sequenced in their entirety multiple times at NIST are provided in this Special 

Publication for comparison purposes. The sequences of fifty-eight unique primer sets that allow any 

area or the entire mtDNA (16,569 base pairs) to be amplified and sequenced are also provided. We 

found during the sequencing of these five mtDNA templates that some of the single nucleotide 

polymorphisms (SNPs) did result in amino acid changes when compared with the Cambridge 

Reference Sequence (Anderson et al.,1981; Andrews et al.,1999). Two interlaboratory evaluations, 

one for the amplification, sequencing, and data analysis of the CHR template and one for the HL-60 

template were each conducted by three different laboratories and NIST. Corroboration of the results 

in SRMs 2392 and 2392-I will provide quality assurance that any unknown mtDNA is also being 

amplified and sequenced correctly. 

Keywords: CHR; 9947A; HL-60; GM03798; GM10742A; Forensic Identification; Medical 

Diagnosis; Mitochondrial DNA sequence; Mutation Detection; Single Nucleotide Polymorphism 

(SNP); Standard Reference Material (SRM); 
1

1. Introduction 

Each human cell can have a few dozen to several thousand molecules of mtDNA (Bogenhagen and 

Clayton, 1974; King and Attardi, 1989). Sequence analysis of mtDNA is used by the forensic 

community for human identification especially in those cases where the genomic DNA is highly 

degraded or non-existent (Holland et al., 1993; Holland et al., 1995). Forensic analysis to 

distinguish between non-maternally related individuals is primarily based on the considerable 

sequence variation found in the two hypervariable regions (HV1, HV2) located in the non-coding 

displacement loop (D-loop). The medical community is also using sequence analysis of mtDNA for 

diagnoses of diseases associated with specific mutations and deletions in the coding regions 

(Wallace et al., 1997). A third area of research which is largely unexplored and which needs 

sequence analysis is the examination of the mutagenic effects of chemical and physical agents on 

mtDNA (Grossman, 1995; Ballinger et al., 1996). 

Human mitochondrial DNA (mtDNA) has been completely sequenced and found to be circular 

double-stranded molecules containing 16,569 base pairs (Fig. 1) (Anderson et al., 1981). The 

sequence determined by Anderson et al. (1981) is referred to as the Cambridge Reference Sequence 

and was a consensus sequence based primarily on the results from a human placenta, partly on the 

sequence from HeLa cells, and in five areas where the results were ambiguous, the authors inserted 

the results from the bovine mtDNA that they had also sequenced. In 1999, Andrews et al. 

resequenced the original placenta and found that the 1981 sequence contained a number of errors 

and rare polymorphisms that were specific for that placenta. In SRM 2392, which became available 

in 1999, we compared the mtDNA sequence of three apparently normal individuals with the 1981 

Cambridge Reference Sequence. We also found a number of places that appeared to be errors and 

which were later confirmed by Andrews et al. (1999). In this NIST Special Publication on the 

development of the mtDNA amplification and sequencing SRM 2392 (Levin et al., 1999) and SRM 

2392-I (Levin et al., 2003, in press), we compare the sequencing results from three apparently 

normal individuals and two individuals with diseases with both the 1981 and 1999 Cambridge 

Reference Sequences. Human mtDNA SRMs 2392 and 2392-I can be used for quality control in 

amplification, sequencing, forensic identifications, medical diagnostics, and mutation detection. 

2. Materials and Methods

2.1. Origin of Extracted DNA

In SRM 2392, the DNA template designated CHR came from human white blood cells that were 

transformed with the Epstein-Barr virus and immortalized as a cell culture line (CHR cells) by the 

American Type Culture Collection (ATCC, Manassas, VA). After transformation, the cells were 

grown in Iscove’s Modified Dulbecco’s Media or RPMI 1640 media with L-glutamine, sodium 

bicarbonate, penicillin, streptomycin and 20% fetal calf serum (Life Technologies, Inc., Grand 

Island, NY). The cell cultures were grown at 37 o

C in an humidified atmosphere containing 5% CO2

and 95% air. The DNA was extracted from 2 x 108

 CHR cells by the Qiagen Plasmid/Cosmid 

Purification Protocol (Qiagen, Inc., Chatsworth, CA). This procedure enhanced the concentration of 

mtDNA and reduced, but did not eliminate, nuclear DNA. 
2

The CHR data presented in this paper were obtained primarily with the above mentioned 

immortalized CHR cell culture line. However, before starting production of the final SRM 2392, it 

was necessary to obtain fresh blood from CHR and to reestablish the cell line. This second CHR cell 

line was established by ATCC as above. The sequence of the mtDNA of this second CHR cell line 

was examined and found to be identical to that of the first CHR cell line, with the single exception 

that no heterplasmy was noted at nucleotide position (np) 68491

; the mtDNA of the second CHR cell 

line agreed with the Cambridge Reference Sequence at np 6849. It is the DNA from the second 

CHR cell line that is included in SRM 2392. 

Also in SRM 2392, the DNA template 9947A was obtained from Life Technologies, Inc. (Grand 

Island, NY) who prepared it from a Epstein-Barr virus immortalized human lymphoid cell line. 

DNA from 9947A is also used in the PCR-based DNA profiling standard (SRM 23912

) designed for 

forensic and paternity testing, law enforcement training, and research. 

SRM 2392-I contains the extracted DNA from HL-60; this DNA was prepared by the Professional 

Services Department of ATCC (Manassas, VA). 

The entire mtDNA of two additional cell lines (GM03798 and GM10742A) were amplified and 

sequenced multiple times at NIST and these sequences are provided in this NIST Special Publication 

 for information and comparison purposes. Although the extracted DNA from GM03798 and 

GM10742A are not part of SRM 2392 or 2392-I, the cell cultures can be obtained from the National 

Institute of General Medical Sciences (NIGMS) Human Genetic Mutant Cell Repository, Coriell 

Institute for Medical Research, Camden, NJ. GM03798, an apparently normal human 

lymphoblastoid cell culture, was grown in the same manner as the CHR cells and the DNA was 

extracted using DNA NOWTM, a phenol-free DNA isolation reagent (BIOGENTEX, Seabrook, TX). 

 The lymphoblast cell line GM10742A came from a patient with Leber Hereditary Optic Neuropathy 

(LHON), a disease that causes blindness in young adults and that has been associated with a number 

of mtDNA primary, intermediate and secondary mutations (Wallace et al., 1997). This cell line 

(GM10742A) was purchased from NIGMS. The cells were grown at NIST in RPMI 1640 plus Lglutamine and sodium bicarbonate growth media (Sigma, St. Louis, MO), fetal calf serum (20%) 

(Sigma), and the antibiotics streptomycin and penicillin (final concentration: 100 U/mL) (Sigma). 

DNA was extracted from GM10742A using the QIAGEN Plasmid Kit following the Plasmid Mini 

Purification Protocol. 

 

2.2. Isolation and Cloning of mtDNA Containing the C-Stretch in SRM 2392 

Confluent CHR cells were harvested by centrifugation at 1500 rpm for 5 min. The mtDNA was 

isolated using the Qiagen Plasmid/Cosmid Purification Protocol (Qiagen Inc., Chatsworth, CA). 

Following isolation, the mtDNA was digested with restriction enzymes SacI and KpnI (New 

England Biolabs, Inc., Beverly, MA) into five fragments which were separated on a 0.7% low 

 

1

All nucleotide numbers referred to in this paper are based on the numbering system of the 

Cambridge Reference Sequence (Anderson et al., 1981 and Andrews et al., 1999). 

2

SRM 2391 may be obtained from the Standard Reference Material Program, NIST, Gaithersburg, 

MD 20899.
3

melting agarose gel. Bands of the size of the fragment containing the HV1 region were cut from the 

gel and melted at 65 oC. DNA was extracted with phenol twice and precipitated by adding sodium 

acetate (150 mmol/L) and two volumes of 100% ethanol. The final product was resuspended in 10 

mmol/LTris-1mmol/L EDTA (TE) buffer. The cloning vector, M13mp18, was also digested with 

SacI and KpnI, treated with Calf Intestinal Alkaline Phosphatase (New England Biolabs, Inc., 

Beverly, MA) extracted with phenol and precipitated with sodium acetate and ethanol as described 

above. The vector was incubated with the mtDNA product and T4 DNA ligase (Life Technologies, 

Inc., Grand Island, NY) at 4 o

C overnight. An overnight culture of E. coli host TG-1 cells was 

diluted and grown at 37 oC in LB media (Sambrook et al., 1989) until the OD650 reached 0.4 - 0.5. 

The cells were harvested by centrifugation at 1500 rpm for 5 min. The cell pellet was resuspended in 

10 mL calcium chloride (50 mmol/L) and incubated for one hour on ice, centrifuged, and 

resuspended in 1 mL calcium chloride (50 mmol/L) and incubated for 30 min on ice. The treated 

TG-1 cell suspension (0.3 mL) was incubated for 30 min on ice with 20 μL of a ligation mixture 

containing the isolated mtDNA fragment, the cloning vector that had been treated overnight, T4

Ligase and ligation buffer (Life Technologies, Inc., Gaithersburg, MD). This mixture was heat 

shocked at 42 oC for 2 min, mixed with 0.2 mL of untreated TG-1 cells (from the overnight culture), 

4 μL of a 1mol/L isopropylthio-β-D-galactoside (IPTG), 40 μL of a 20 mg/mL 5-bromo-4-chloro-3-

indolyl-β-D-galactopyranoside (X-gal) and 3 mL of melted (55 o

C) top agar and spread on the 

surface of freshly prepared LB agarose plates (Sambrook et al., 1989). The plates were incubated at 

37 o

C overnight. Both colorless and blue plaques were visible in the morning. The colorless plaques 

indicate that insertion of the mtDNA fragment into the vector has occurred; whereas, the blue 

plaques have no insertion. 

2.3. Bacteriophage DNA Isolation and Sequencing

Single, well isolated colorless plaques from the above LB plates were each placed in a sterile tube 

with 1.5 mL of a TG-1/ LB cell suspension which contained TG-1 cells that were grown overnight 

and diluted 1/100 in LB media and grown for 1 hour at 37 o

C. The plaques and the TG-1/ LB cell 

suspension were grown at 37 o

C for 5 hours. Cells were removed by centrifugation at 15000 rpm for 

5 min. The supernatant containing the bacteriophage was incubated with 0.2 mL polyethylene 

glycol (20% PEG in 2.5 mol/L NaCl) overnight at 4 o

C and the resultant precipitate containing the 

DNA phage particles was pelleted by centrifugation at 15000 rpm for 15 min. The bacteriophage 

DNA was isolated by phenol extraction and sodium acetate/ethanol precipitation as described above 

and then dissolved in 25 μL of TE buffer. The bacteriophage DNA was cycle sequenced with 

AmpliTaq DNA polymerase, FS and the -21M13 primer: 5'-TGTAAAACGACGGCCAGT-3' 

according to the protocol in the ABI PRISMTM Dye Terminator Cycle Sequencing Ready Reaction 

Kit (PerkinElmer, Foster city, CA). The cycle sequencing was conducted in a PerkinElmer Model 

9600 thermocycler by first heating the DNA reaction mixture at 96 o

C for 1 min and then subjecting 

the mixture to 25 cycles of 96 o

C for 10 sec, 50 oC for 5 sec and 60 oC for 2 min. The cycle 

sequencing product was purified using a Centri-Sep spin column (Princeton Separations, Inc., 

Adelphia, NJ). The DNA pellet was rinsed with 70% ethanol, vacuum dried, resuspended in loading 

buffer prepared by combining deionized formamide and 25 mmol/L EDTA (pH 8.0) in a ratio of 5:1, 

loaded onto a 4.75% acrylamide gel and electrophoresed on an ABI 373A DNA sequencer. One of 

the clones containing the C-stretch sequence was used as the source of the cloned DNA for the 

SRM. 
4

2.4. mtDNA Primers used in both SRM 2392 and 2392-I

Fifty-eight sets of unique primers (19-28 bp) for sequencing the entire human mtDNA (16,569 bp) 

were computer-designed using GENE RUNNER FOR WINDOWS (Hastings Software, Inc., 

Hastings, NY). The -21M13 primer was used to sequence the cloned HV1 region of the DNA from 

the CHR template. The sequences of all the primers are shown in Table 1 and are the same as those 

used for SRM 2392 (Levin et al., 1999; Appendix C) with the exception of the reverse primer of set 

51. During the course of the development of SRM 2392-I, this primer was changed since it 

contained a C at np 14368. Since this is the reverse primer, it would bind to a G at np 14368. 

Andrews et al. (1999) in their reevaluation of the placenta originally used to sequence human 

mtDNA in 1981 (Anderson et al., 1981) found that np 14368 should have a C at that position. 

Therefore, the primer should have a G at np 14368. The new reverse primer 51 is 

5’-TTAGCGATGGAGGTAGGATTGG-3’ (np 14368 is in bold and underlined) which binds to the 

C at position 14368. The 5’ end is np 14388 and the 3’ end is 14367. The 58 sets of primers were 

custom-made by Bio-Synthesis, Inc. (Lewisville, TX); the new reverse primer 51 was obtained from 

Invitrogen (Carlsbad, CA). 

2.5. Polymerase Chain Reaction (PCR) SRM 2392 completed in 1999 

Extracted DNA was resuspended in TE buffer (pH 7.5) containing 10 mmol/L Tris and 1 mmol/L 

EDTA. The PCR reaction mixture contained: DNA (1μL), Taq DNA polymerase (0.5 μL or 2.5 

units)((Boehringer Mannheim, Indianapolis IN) and 10x buffer (5 μL) (Boehringer Mannheim), 

dNTP’s (0.2 mmol/L each) (Life Technologies, Inc., Gaithersburg, MD), forward and reverse 

primers (0.4 μmol/L each) plus H2O to a final volume of 50 μL. The 10x buffer (pH 8.3) contained 

Tris-HCl (100 mmol/L), MgCl2 (15 mmol/L), and KCl (500 mmol/L). Thermal cycling was 

conducted in a PerkinElmer Model 9600 thermocycler and consisted of 1 min at 96 o

C, followed by 

32 cycles of 15 sec at 94 o

C (denaturation), 30 sec at 56 oC (annealing), and 15 sec at 72 oC 

(extension), and ending with a final extension of 7 min at 72 o

C. A sample of the amplified DNA 

was electrophoresed in 0.7% agarose and stained with ethidium bromide to assess the purity and size 

of the PCR product. Before sequencing, extraneous materials were removed from the PCR product 

with a QIAquick PCR Purification Kit (QIAGEN, Inc., Chatsworth, CA). 

2.6. Polymerase Chain Reaction (PCR) SRM 2392-I completed in 2002 

The PCR mixture contained: DNA (1μL; 1.4 ng), AmpliTaq Gold® DNA polymerase, (0.5 μL; 2.5 

units) (Applied Biosystems), 10x buffer (5 μL) containing 100 mmol/L Tris-HCl, pH 8.3, 500 

mmol/L KCl, 15 mmol/L MgCl2 and 0.01% (w/v) gelatin (Applied Biosystems), 10 mmol/L dNTP 

mix (1 μL) (Invitrogen), 10 μmol/L forward and reverse primers (1 or 2 μL), plus water to make a 

final volume of 50 μL. Thermal cycling was conducted in a PerkinElmer thermocycler Model 9700 

and started with 10 minutes at 95 oC, followed by 35 cycles of 20 sec at 94 oC (denaturation), 10 sec 

at 56 oC (annealing), 30 sec at 72 oC (extension) and ended with a final extension of 7 min at 72 oC. 

Amplified DNA was purified with a QIAquick PCR Purification Kit (Qiagen) and the purity and size 

of the PCR product was determined by electrophoresis in 2% agarose gels in 1x TBE buffer 

containing 0.5 μg/mL ethidium bromide (Sigma). 

2.7. Sequencing SRM 2392 completed in 1999 
5

Cycle sequencing using fluorescent dye-labeled terminators was performed with an ABI PRISMTM

Dye Terminator Cycle Sequencing Kit with AmpliTaq® DNA Polymerase, FS (PerkinElmer). 

Thermal cycling was conducted in a PerkinElmer Model 9600 thermocycler and started with one 

minute at 96 o

C. The reaction then underwent 25 cycles of 96 oC for 15 sec (denaturation), 50 oC 

for 5 sec (annealing), and 60 o

C for 2 min (extension). The DNA products were purified by passage 

through a Centri-Sep spin column (Princeton Separations, Inc.). Electrophoresis and sequencing of 

the fluorescently-labeled purified DNA were performed with a 373A ABI Sequencer (PerkinElmer) 

using a 4.75% acrylamide gel. Data analysis was executed with the Sequence Navigator software 

package (PerkinElmer). 

2.8. Sequencing SRM 2392-I completed in 2002 

Cycle sequencing using fluorescent dye-labeled terminators was performed with an ABI PRISM® 

BigDye® Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq® DNA Polymerase, FS 

(Applied Biosystems). Cycle sequencing reactions in both the forward and reverse modes were 

conducted with a 9700 PerkinElmer thermal cycler and started with one minute at 96 oC. The 

reaction then underwent 25 cycles of 96 oC for 15 sec (denaturation), 50 oC for 5 sec (annealing), 

and 60 oC for 2 min (extension). The DNA products were purified using Edge Gel Filtration 

Cartridges (Edge BioSystems, Gaithersburg, MD). Sequencing and data analysis of the purified 

DNA were performed using an Applied Biosystems PRISM® Model 310 Genetic Analyzer with 

POP-6TM polymer system and 47 cm x 50 μm capillaries (Applied Biosystems). Sequence data 

were analyzed with Sequencing Analysis Software 3.3, and comparisons to the Cambridge 

Reference Sequence (Anderson et al.,1981) were performed with Sequence Navigator Software 

1.01. 

2.9. Interlaboratory Evaluation for SRM 2392 completed in 1999 

Three laboratories in addition to NIST participated in an interlaboratory evaluation of the CHR 

template. These laboratories were The Bode Technology Group, Inc., 21515 Ridgetop Circle, Suite 

140, Sterling, VA 20166; IIT Research Institute, Virginia Technology Center, 8510 Cinderbed Road, 

Suite 300, P.O. Box 899, Newington, VA 22122; and Lark Technologies, Inc., 9545 Katy Fwy, Suite 

465, Houston, TX 77024. 

Each laboratory was sent: 

1. Two tubes of DNA from the first CHR cell culture line. One tube contained extracted DNA 

ready for PCR amplification of the entire mtDNA. The other contained the cloned DNA ready for 

cycle sequencing of the HV1 region (this DNA did not need to be PCR amplified). 

2. Fifty-eight sets of primers labeled with either F# (forward primer) or R# (reverse primer). 

Forward and reverse primers with the same number were paired and numbered from the 5' end. 

Primers were diluted to 10 μL and ready for use. Also enclosed was the -21M13 primer to do the 

sequencing of the cloned HV1 region of the CHR DNA, which covered base pairs 16133 through 

16569 and 1 through 40. 

3. The protocol used at NIST to amplify and sequence the DNA. The laboratories, however, were 
6

free to use any protocol with which they were familiar and felt comfortable. 

4. A form table to record the results. This table provided the number of the Primer Set, the region 

that each Primer Set amplified, and the length of the amplified region. We requested that the 

laboratory fill in the differences found when they compared the sequence that they determined for 

the mtDNA from CHR with that of the Cambridge Reference Sequence (Anderson et al., 1981). 

5. All the laboratories received the following cautionary note: 

 WARNING: The DNA and cells were derived from a cell culture line from an apparently 

healthy human subject. The cell culture line has been tested and found to be nonreactive for 

hepatitis B surface antigen and HIV. However, no test method can ensure that a product derived 

from human blood does not contain HIV, hepatitis or other infectious agents. HANDLE AS IF 

CAPABLE OF TRANSMITTING DISEASE. (The second CHR cell culture line generated from the 

same individual was not tested again for hepatitis or HIV. Normal precautions should be used.) 

2.10. Differences in Methodology used by Laboratories in Interlaboratory Evaluation of SRM 2392, 

1999.

The Bode Technology Group, Inc. essentially followed the NIST protocol except used a 6% 

acrylamide/8.3 mol/L urea gel for the sequencing electrophoresis instead of a 4.75% acrylamide. 

IIT Research Institute also followed the NIST protocol except used Taq Gold (PerkinElmer) for the 

amplification reaction which was modified to include a hot start of 95 o

C for 11 minutes. Microcon 

100 microconcentrators (Amicon, Inc., Beverly, MA) were used to purify the PCR products. The 

quantities of DNA were determined by capillary electrophoresis (CE) with a Beckman P/ACE 5010 

System (Beckman Instruments, Inc., Fullerton, CA) as follows: 1 μL of the amplified product was 

mixed with 25 μL of sterile deionized H2O containing 0.52 ng/μL of a 200 bp internal standard 

(GenSura Laboratories, Inc., Del Mar, CA) and run on the CE. One determines the quantity of the 

amplified product from the ratio of the PCR product peak area to the internal standard peak area 

multiplied by a migration standard. A 6% acrylamide gel was used for the sequencing 

electrophoresis instead of a 4.75% acrylamide. 

Lark Technologies, Inc., followed the NIST protocol with the following differences: Ampli-Taq 

DNA polymerase (PerkinElmer) was used to amplify the DNA; the dNTP’s were purchased from 

Pharmacia Biotech, Inc. (Piscataway,NJ); the products were purified with Qiaquick PCR purification 

kit (Qiagen); in the sequencing reactions, the amount of PCR product used varied from 1 to 3 μL 

based on the concentration estimated from agarose gels; cycling conditions were 95 o

C for one 

minute, followed by 25 cycles of 96 o

C for 15 seconds, 50 oC for 15 seconds, and 60 oC for 4 

minutes; the sequence reactions were cleaned up by ethanol precipitation; a 4.25% polyacrylamide 

gel was used for the sequencing electrophoresis instead of a 4.75% acrylamide; and a ABI 377 

automated sequencer was used instead of the ABI 373. Electropherograms were printed for each 

reaction and the sequences were manually edited based on the electropherogram patterns. Printed 

electropherograms and a floppy disc with the sequence data were sent to NIST where the data were 

compared to the Cambridge Reference Sequence (Anderson et al.,1981). 

2.11. Interlaboratory Evaluation for SRM 2392-I, 2002 
7

Three laboratories, in addition to NIST, participated in an interlaboratory evaluation of the mtDNA 

sequence of HL-60. These laboratories included the FBI Laboratory, FBI Academy, Quantico, VA 

22135; Armed Forces DNA Identification Laboratory (AFDIL), Armed Forces Institute of 

Pathology, Rockville, MD 20850; and the Georgia Bureau of Investigation (GBI), Decatur, GA 

30034. Each laboratory was asked to amplify and sequence the entire mtDNA of HL-60. NIST 

provided: 1. A tube of DNA containing the extracted DNA ready for PCR amplification, 2. The 58 

sets of primers labeled with either F# (forward primer) or R# (reverse primer), 3. A table to record 

the results, and 4. Any other supplies that were needed and requested by the participants. They were 

allowed to use any protocol for amplification or sequencing that they wished, but were requested to 

provide a copy of that protocol to NIST. NIST also requested copies of the electropherograms to 

enable us to resolve any discrepancies; although, as it turned out, there were no discrepancies. 

2.12. Differences in Methodology Used by the Laboratories in the Interlaboratory Evaluation for 

SRM 2392-I 

2.12.1. Armed Forces DNA Identification Laboratory (AFDIL), SRM 2392-I

AFDIL has developed a high-throughput, automated sequencing procedure using 12 primer sets that 

produce overlapping PCR products ranging from 825 to 1886 bp. The primers used to amplify the 

first 11 products are based on those published in Levin et al. (1999). They are designated: Amp01 – 

F361/R2216; Amp02 – F1993/R3557; Amp03 – F3441/R4983; Amp04 – F4797/R6526; Amp05 – 

F6426/R8311; Amp06 – F8164/R9848; Amp07 – F9754/R11600; Amp08 – F11403/R13123; 

Amp09 – F12793/R14388; Amp10 – F14189/R15396; Amp11 – F15260/R16084. The primers used 

to amplify the control region were developed at AFDIL and are Amp12 – F15878/R649 (F15878 is 

TTAACTCCACCATTAGCACC and R649 is TTTGTTTATGGGGTGATGTGA). 

2.12.1.1. AFDIL PCR Amplification, SRM 2392-I: 

The PCR mixture contained HL-60 DNA (1 μL), AmpliTaqGold® DNA polymerase (1 μL) 

(Applied Biosystems), 10x PCR buffer (5 μL) (Applied Biosystems), dNTP’s (0.2 mmol/L) 

(Invitrogen), 2 μL of forward and reverse primers (10 μmol/L) (MWG Biotech, High Point, NC) 

plus dH2O to a final volume of 50 μL. The 10x PCR buffer was the same as that used by NIST. 

Thermal cycling was conducted in a PerkinElmer 9700 thermocycler with the following conditions: 

10 min at 96 oC (activation of AmpliTaq Gold®), plus 40 cycles of 94 o

C for 15 sec, 56 oCfor 30 sec, 

and 72 o

C for 1 min. The purity and size of the PCR products were assessed by electrophoresis in a 

0.7% agarose gel containing 0.3 μg/mL of ethidium bromide. The PCR products were purified with 

Shrimp Alkaline Phosphatase/Exonuclease I (Amersham Pharmacia, Piscataway, NJ). 5 μL of 

exonuclease I (10 U/μL) and 10 μL of Shrimp Alkaline Phosphatase (1 U/μL) was added to each 

tube containing PCR product. The tubes were heated at 37o

C for 15 min followed by 94oC for 15 

min in a PerkinElmer 9700 thermocycler. 

2.12.1.2. AFDIL Sequencing, SRM 2392-I : 

Cycle sequencing was performed with the ABI PRISM® BigDye® terminators (original version) 

cycle sequencing kit (Applied Biosystems). The sequencing mixture contained 9 μL dH2O, 6 μL 
8

BigDye® dilution buffer (400 mmol/L TRIS, 10 mmol/L MgCl2, pH 9.0), 2 μL BigDye® 

terminator reaction mixture, 1 μL of forward or reverse primer (10 μmol/L each) and 2 μL of HL-60 

PCR product for a total volume of 20 μL. A few of the sequencing primers (e.g., R649) required the 

use of the ABI PRISM® dGTP BigDye® terminator kit (Applied Biosystems). Thermal cycling was 

conducted in a PerkinElmer 9700 thermocycler at the following conditions: an initial 1 min 

denaturation at 96 o

C, followed by 25 cycles of 94 oC for 15 sec, 50 oC for 5 sec, and 60 oC for 2 min. 

The DNA product was purified by filtration through a spin column matrix (Edge BioSystems, 

Gaithersburg, MD). Electrophoresis and sequencing were performed with an ABI PRISM® 3100 

Genetic Analyzer (Applied Biosystems) using POP-6TM polymer (Applied Biosystems) with a 50 cm 

capillary. Data analysis was executed using Sequencher Plus 4.0.5b11 (Gene Codes, Ann Arbor, 

MI). The HL-60 sequence differences were identified by comparison to the Cambridge Reference 

Sequence as revised by Andrews et al. (1999). In most cases, sequence information was acquired for 

both the forward and reverse directions. In some regions, two separate reactions using the same 

primer were conducted (indicated by 2X in the following list of primers). A total of 95 sequencing 

reactions plus 1 pGEM reaction were conducted in a 96 well format. If the primer failed the first 

trial, the reaction was repeated. The finding of a heteroplasmy at np 12071 was also confirmed by an

additional PCR and sequencing reaction. The following primers from Levin et al. (1999) were used 

to sequence the 12 PCR amplicons: 

Amp01 (F361/R2216): F361, R921, F1234, R1425, F873, R2216, F1657, R1769 

Amp02 (F1993/R3557): F1993, R2660, R2834, R3557, F2417, R3006, F3234 

Amp03 (F3441/R4983): F3441, R3940, F3931 (2X), R4982, F4392, R4162 

Amp04 (F4797/R6526): F4797 (2X), R6526, F5700 (2X), F5318, R5882, F6242 

Amp05 (F6426/R8311): F6426 (2X), R7255, F7645 (2X), R8311, F7075, R7792 

Amp06 (F8164/R9848): F8164 (2X), R9059, F8903, R9848, F8539, R9403, F9309 

Amp07 (F9754/R11600): F9754 (2X), R10556, F11001 (2X), R11600, F10386, R11267 

Amp08 (F11403/R13123): F11403 (2X), F12357, R13123, F11901 (2X), F12601, R12876 Amp09 

(F12793/R14388): F12793, R13611, F13518 (2X), R14388, F13188, R13343, 

F13899, R13935 

Amp10 (F14189/R15396): F14189 (2X), R15396, F14909, R14996, F14470 

Amp11 (F15260/R16084): F15260, R16084, F15574, R15774 

Amp12 (F15878/R649): F15971, R16175 (2X), F16450 (2X), R274, F314 (2X), R649 (2X), 

 F16190, R16400 

In Amp12, F15971 came from Levin et al. (1999). The other primers were designed by AFDIL and 

were as follows: 

R16175: TGGATTGGGTTTTTATGTA 

F16450: GCTCCGGGCCCATAACACTTG 

R274: TGTGTGGAAAGTGGCTGTGC 

F314: CCGCTTCTGGCCACAGCACT 

R649: TTTGTTTATGGGGTGATGTGA 

F16190: CCCCATGCTTACAAGCAAGT 

R16400: GTCAAGGGACCCCTATCTGA 

2.12.2. Georgia Bureau of Investigation (GBI), Interlaboratory Evaluation, SRM 2392-I

The Georgia Bureau of Investigation used the same protocol as that used by NIST with the 
9

following exceptions: 1. A PerkinElmer model 9600 was used for thermal cycling; 2. Amplified 

DNA was electrophoresed in 2.75% agarose gels; 3. The cycle sequencing was performed with a 

PerkinElmer model 9600 using the program provided with the BigDye® kit (25 cycles of 96 o

C for 

10 sec, 50 oC for 5 sec, and 60 oC for 4 min); 4. Some samples that needed to be cycle sequenced 

again using more amplicon were not purified before precipitation; 5. The samples were precipitated 

using isopropanol precipitation as per the BigDye® instructions; and 6. Comparison of the sequence 

data was performed with Sequencer 3.1.1 software (Gene Codes, Ann Arbor, MI). 

2.12.3. Federal Bureau of Investigation (FBI), Interlaboratory Evaluation, SRM 2392-I 

2.12.3.1. FBI Polymerase Chain Reaction (PCR): 

The PCR mixture contained from 0.1 to 1.4 ng HL-60, AmpliTaq Gold® Polymerase (2.5 units) 

(Applied Biosystems), 10x PCR buffer (5 μL) (Applied Biosystems), GeneAmp® dNTPs (0.2 

mmol/L each) (Applied Biosystems), forward and reverse primers (0.4 μmol/L each), plus dH2O to a 

final volume of 50 μL. The 10x buffer (pH 8.3) was the same as used by NIST. Thermal cycling was 

conducted in a GeneAmp® PCR System 9700 (PerkinElmer) and consisted of 10 min at 95 o

C 

followed by 35 cycles of 94 oC for 20 sec, annealing temperatures of 50 o

C (primer set 49), 51 oC 

(primer sets 1, 7, 44, 57), 52 o

C (primer sets 6, 8, 30, 45), 53 oC (primer set 53) and 56 oC (all other 

primer sets) for 10 sec, and 72 oC for 30 sec and ending with a final extension of 7 min at 72 o

C. 

Amplified products were purified by treatment with Exo-SAP-IT (5 μL for every 25 μL of PCR 

product) (USB Corp., Cleveland, OH). Samples of the PCR products were electrophoresed in 1.2% 

agarose gels containing ethidium bromide to assess the purity, size, and quantity of the PCR 

products. 

2.12.3.2. FBI Sequencing: 

Cycle sequencing using fluorescent dye–labeled terminators was performed with an ABI PRISM® 

dRhodamine Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq® DNA Polymerase, 

FS (Applied Biosystems). Thermal cycling was conducted in a GeneAmp® PCR System 9700 

(Applied Biosystems) and started with 1 min at 96 o

C followed by 25 cycles of 96 oC for 15 sec, 50 o

C for 1 sec and 60 oC for 1 min and ended with a final incubation at 15 oC for 10 min. These 

products were purified with Centri-SepTM spin columns (Princeton Separation, Inc., Adelphia, NJ) 

and electrophoresed on an ABI PRISM® 310 Genetic Analyzer (Applied Biosystems) using POP6TM and either a 47 or 61 cm capillary. Comparison of the sequence data was performed with 

Sequencer 4.1 software (Gene Codes). 

The FBI also discovered the problem with original primer set 51 (see Materials and Methods). The 

reverse primer incorporated a G at position 14368 [an error in the original Cambridge Reference 

Sequence (Anderson et al., 1981) that was found and corrected by Andrews et al. (1999)]. However, 

sequencing with primer set 52 showed a C at position 14368. They prepared a new set of primers 

(primer set 51.5) to amplify approximately a 400 bp fragment that encompassed np 14368. The new 

primers were: 

F14217: 5’-CTAATCAACGCCCATAATCATAC-3’ and 

R14620: 5’-GTTTTCTTCTAAGCCTTCTCC-3’. 

The new primer set confirmed that the correct base at position 14368 was a C. 
10

2.13. Permissions 

The development of SRM 2392-I was deemed exempt from the policy of Part 27 of Title 15 of the 

Code of Federal Regulations by the NIST Institutional Review Board and the Director of the 

Chemical Science and Technology Laboratory. This work fit into the exemption category described 

in 15 CFR 27.101(b)(4) which states: “Research, involving the collection or study of existing data, 

documents, pathological specimens, or diagnostic specimens, if, these sources are publicly available 

or if the information is recorded by the investigator in such a manner that subjects cannot be 

identified, directly or through identifiers linked to the subjects.” 

ATCC also waived condition 3(c) in their Material Transfer Agreement which states that the 

“purchaser shall not sell, lend, distribute or otherwise transfer the material or replicates to any 

others” for the use of HL-60 in the NIST mtDNA SRM. They stated that, in their view, “as a 

government agency, NIST will not be providing this material as a commercial product despite the 

collection of fees for the SRM.” 

2.14. Final Preparation of DNA for SRM 2392 and SRM 2392-I 

2.14.1. CHR DNA Preparation and Confirmation SRM 2392:

CHR cells were grown in RPMI-1640 media supplemented with L-glutamine, sodium bicarbonate, 

penicillin, streptomycin and 10% fetal calf serum (Sigma Chemical Co.), centrifuged and 

resuspended in 10 mL of phosphate buffered saline (PBS) to a final cell count of 1.2 x 106

 cells/mL. 

DNA was extracted using the Qiagen Maxi Kit according to the Qiagen protocol (Qiagen Inc.). The 

concentration of the DNA in the extract was determined with an UV spectrophotometer to be 82.3 

ng/μL. 

PCR amplification was performed on the extract using primer set 57 (HV1 region) and primer set 1 

(HV2 region) in order to verify the sequence. The extract was diluted 1/10 (8.23 ng/μL), and 1 μL 

was used in PCR reaction. The PCR reaction mixture was the same as that described in the 

polymerase chain reaction section of Materials and Methods, with the exception that the forward and 

reverse primers were used at a concentration of 0.2 μM. 

Thermal cycling was conducted in a PerkinElmer 9600 (HV1) or 9700 (HV2) and consisted of 30 

sec at 95 o

C, followed by 32 cycles of 20 sec at 94 oC (denaturation), 10 sec at 56 oC (annealing), and 

30 sec at 72 o

C (extension), and ending with a final extension of 7 minutes at 72 oC. A sample of the 

amplified DNA was electrophoresed and stained with ethidium bromide and purified as described in 

the polymerase chain reaction section of Materials and Methods except that a 2% agarose gel was 

used. Cycle sequencing, electrophoresis of the products, and the analysis of the sequence data were 

performed as described in the sequencing section of Materials and Methods. The polymorphisms of 

the HV1 and HV2 regions and the C-stretch (HV1 region) seen in the pre-production mode samples 

were confirmed in the production mode samples. 

The DNA extract was diluted to 1 ng/μL with TE buffer and PCR amplified using Primer Set 1 

(HV2) to verify that this concentration was appropriate to use for PCR. Sixty μL of the CHR DNA 

extract at a concentration of 1 ng/μL was aliquotted into 260 tubes. 
11

2.14.2. CHR Cloned DNA Production, SRM 2392:

DNA from one of the C-stretch clones (containing 12 C’s) prepared as described earlier was used to 

transform TG-1 cells. Following the transformation and growth of the clones, the bacteriophage 

DNA was isolated and sequenced to verify the presence of the 12 C region. Following this 

verification, a large scale transformation was performed for SRM production. The cloned DNA that 

was obtained following this transformation was quantitated by UV spectrophotometry and diluted to 

100 ng/μL with TE buffer. The cloned DNA was sequenced and analyzed as described in section 

entitled Bacteriophage DNA Isolation and Sequencing in Materials and Methods to verify that it 

contained the C-stretch of 12 C’s plus all the additional polymorphisms previously found in the HV1 

region of the CHR cells. For the final SRM, 260 tubes containing 10 μL at a concentration of 100 

ng/μL were prepared. 

2.14.3. 9947A DNA Production, SRM 2392: 

Cell culture line 9947A was grown by Life Technologies, Inc. (Gaithersburg, MD) who extracted the 

DNA and submitted a sample (at a concentration of 1 ng/μL) to NIST for verification of the 

sequence. The HV1 (primer set 57) and HV2 (primer set 1) regions as well as the region amplified 

by primer set 30 were checked by PCR and sequencing to verify that the DNA was the same as 

sequenced earlier. One μL of DNA (1 ng) was used as the template in the PCR reaction mixture, 

which was performed as described in the polymerase chain reaction section of the Materials and 

Methods section, with the exception that the forward and reverse primers were used at a 

concentration of 0.2 μmol/L. Thermal cycling was conducted in a PerkinElmer 9700 and consisted 

of 30 sec at 95o

C, followed by 32 cycles of 20 sec at 94 oC (denaturation), 10 sec at 56 oC 

(annealing), and 30 sec at 72 o

C (extension), and ending with a final extension of 7 minutes at 72 oC. 

A sample of the amplified DNA was electrophoresed and stained with ethidium bromide and 

purified as described in the section entitled polymerase chain reaction except that a 1% agarose gel 

was used. Cycle sequencing, electrophoresis of products, and analysis of sequence data were 

performed as described earlier. Examination of the results from primer sets 57 (HV1), 1 (HV2), and 

30 indicated that the DNA supplied by Life Technologies was the same as that examined earlier in 

the pre-production mode. 

After NIST verified that the sequence was okay, Life Technologies, Inc. prepared 250 tubes 

containing 60 μL of 9947A DNA at a concentration of 1 ng/μL. 

2.14.4.HL-60 Production, SRM 2392-I: 

DNA from the HL-60 cell culture was extracted, isolated, and quantified by the Professional 

Services Department of the American Type Culture Collection (ATCC, Manassas, VA). DNA was 

isolated from the HL-60 cell culture using the QIAamp DNA Blood Mini Kit (Qiagen, Inc.). 

Quantification was determined by the Quantiblot Human DNA Quantification Kit (Applied 

Biosystems). The final concentration, 1.4 ng/μL, is based on 12 replicate tests. The DNA purity 

(A260:A280 = 1.9) was determined spectrophotometrically. The integrity of DNA was determined 

electrophoretically using 0.4% agarose gels. 
12

Sixty-five μL of HL-60 DNA were dispensed into 260 tubes. Three randomly chosen tubes were 

used to send to the laboratories that agreed to participate in the Interlaboratory Evaluation. Each of 

the three laboratories plus NIST used all 58 primer sets to sequence the entire 16,569 base pairs of 

the HL-60 mtDNA. All four laboratories found the identical sequence, thereby confirming the 

accuracy of the sequence determined at NIST. 

3. Results And Discussion

3.1. SRM Templates: 

Two DNA templates, CHR and 9947A, are included in the NIST human mtDNA amplification and 

sequencing SRM 2392. The CHR and 9947A DNA samples came from human cell culture lines that 

were developed from apparently normal individuals. The DNA template in SRM 2392-I, HL-60, 

came from a promyelocytic cell line prepared from the peripheral blood leukocytes from an 

individual with acute promyelocytic leukemia. The DNA extracted from 9947A and HL-60 is the 

total DNA including nuclear DNA. The DNA from CHR was isolated in a manner which enhanced 

the concentration of the mtDNA, but did not totally eliminate the nuclear DNA. However, both types 

of extracted DNA amplified and sequenced accurately. SRM 2392 also provides cloned DNA from 

the HV1 region of the CHR template which contains a C-stretch. In most people, the HV1 region 

has a string of cytosine (C) residues interrupted by a thymine (T) at np 16189. In some individuals, 

however, a transition occurs which changes the T to a C producing a long string of C’s called the Cstretch. When this happens, sequencing beyond the C-stretch becomes very difficult, if not 

impossible. Clones of the HV1 region containing the C-stretch indicated that the number of C’s 

differed among the different clones and the difficulty in sequencing was due to frameshifts that 

resulted from the simultaneous sequencing of templates with differing numbers of C’s (Bendall and 

Sykes, 1995; Levin et al., 1995; Levin et al., 1997). We found, however, that one could sequence 

through the entire HV1 region including the C-stretch without problems if one used the clone of the 

area. Therefore, we included the cloned HV1 region of the CHR DNA template in SRM 2392. 

In addition to templates CHR and 9947A in SRM 2392 and HL-60 in SRM 2392-I, we have included 

all the mtDNA sequence information from two additional templates in Table 2: 1. GM03798, a 

normal lymphoblastoid cell line and 2. GM10742A from an individual with the mtDNA disease, 

Leber Hereditary Optic Neuropathy (LHON), which causes blindness in early adulthood. Both of 

these cell lines were obtained from the NIGMS Human Genetic Mutant Cell Repository. The DNA 

from these cell lines are not part of SRMs 2392 or 2392-I and the data are included for information 

and comparison purposes only. 

3.2. Primers: 

The 58 sets of unique primers were designed to allow the amplification and sequencing of any 

region or the entire 16,569 base pairs that comprise human mtDNA. The sequence of both the 

forward and reverse primers that are in each set are shown in Table 1. The numbers indicate the 5' 

end of the primer. They are all between 19 and 28 base pairs long and the criteria that were used to 

choose these primers were primer melting temperatures (Tm) between 50 and 65 o

C, primer length 

between 15 and 30 base pairs, and PCR product length between 220 and 1000 base pairs. In 

actuality, these primers gave PCR product lengths between 224 and 976 base pairs. The primers 
13

were designed to produce sequences that overlapped with both the previous and following regions to 

allow those areas in the beginning and end of electropherograms which are difficult to sequence to 

become readable and to prevent any gaps in the sequence. The readable region is always smaller 

than that of the amplified region. In the process of developing SRM 2392-I, we found that reverse 

primer of primer set 51 needed to be changed (see explanation in section entitled “mtDNA primers” 

in the Materials and Methods section). The new reverse primer 51 is shown in Table 1. The change 

is in bold and underlined. 

In addition to the designed primers, we also used the -21M13 primer (Table 1) to sequence the 

cloned DNA from the HV1 region of the CHR template which contained the C-stretch. The PCR 

products were produced under the same conditions for all 58 primer sets and generated single, 

distinct bands in all cases (Levin et al., 1999). 

3.3. Differences between the SRM DNAs and the Cambridge Reference Sequence: 

Human mtDNA was completely sequenced in 1981 and is referred to as the Cambridge Reference 

Sequence (Anderson et al., 1981). This sequence is a consensus sequence based primarily on a 

placenta, a few areas sequenced from HeLa cells, and five ambiguous areas where the sequence from 

bovine mtDNA was used. All investigators, who subsequently examined human mtDNA, have used 

the numbering system of the Cambridge Reference Sequence and have compared their sequence 

findings to those found in 1981. In 1999, Andrews et al. resequenced the original placenta used in 

1981. They found that the 1981 sequence had a number of errors and that the placenta had a number 

of rare polymorphisms. At the present time, investigators compare their sequences to the 1999 

revised sequence. However, the DNA from this placenta is not available for use as a positive control 

during actual experiments; whereas, NIST SRMs 2392 and 2392-I are available. Table 2 shows the 

mtDNA differences compared to the Cambridge Reference Sequence that were found at NIST with 

the three templates - CHR and 9947A (in SRM 2392) and HL-60 (in SRM 2392-I) and the two 

additional templates - GM03798, and GM10742A - that are included for information and 

comparison purposes. In all five templates, all 58 areas comprising the entire mtDNA were 

completely amplified and sequenced at least twice. There are 13, 9, 11, 4, and 12 differences in the 

non-coding regions of templates CHR, 9947A, HL-60, GM03798, and GM10742A, respectively and 

33, 23, 33, 19, and 31 differences in the coding regions of templates CHR, 9947A, HL-60, 

GM03798, and GM10742A, respectively. All of the differences from the Cambridge Reference 

Sequence found in CHR, 9947A, HL-60, GM03798, and GM10742A are shown in Figure 1 along 

with many of the mutations associated with Leber Hereditary Optic Neuropathy (LHON) that have 

been noted in the literature (Wallace et al., 1997). With one exception, none of the base pair changes 

found in the coding regions of the apparently normal templates (CHR, 9947A, GM03798) sequenced 

at NIST correlate with any of the changes found associated with the published disease states found 

in the MitoMap web site: http://www.mitomap.org The one exception was a secondary LHON 

mutation found in CHR. HL-60 and GM10742A, however, did have some mutations that have been 

associated with LHON. 

3.4. Meaning of the Differences from Cambridge Reference Sequence: 

Since three templates came from apparently normal individuals, one from an individual with acute 

promyelocytic leukemia, and one from an individual with LHON, it was of interest to determine if 
14

the differences in the coding regions would actually cause amino acid changes in the resultant 

protein structures. The genetic code for human mtDNA is slightly different from the universal 

genetic code. Table 3 shows the differences in the universal genetic code that one needs to consider 

when determining the amino acid sequence designated by the three base pair codons in mtDNA. 

Many of the differences from the Cambridge Reference Sequence were in the third position wobble 

and did not affect the amino acid sequence. However, CHR, 9947A, HL-60, GM03798, and 

GM10742A had 11, 12, 15, 8, and 14 different base pairs, respectively, that would result in a 

different amino acid from that designated by the Cambridge Reference Sequence of 1981. However, 

comparison with the revised Cambridge Reference Sequence of 1999, indicate that only 3, 4, 7 plus 

2 in tRNAs, 0, and 7 would result in amino acid changes in CHR, 9947A, HL-60, GM03798, and 

GM10742A, respectively. These structural changes, however, do not necessarily mean a functional 

change has occurred in the protein. To determine if a functional change has occurred, one still needs 

to decipher whether the amino acid change is in an active site on the protein. We have developed an 

interactive web site that will easily and quickly determine if the DNA change causes an amino acid 

change (Lee and Levin, 2002). The changes that result in amino acid changes are shown in Table 2. 

3.5. The Interlaboratory Evaluation of the CHR and HL-60 Templates: 

The interlaboratory evaluation of CHR was conducted by four laboratories including NIST. The 

interlaboratory evaluation of HL-60 was conducted by four laboratories including NIST. Any 

changes made by the various laboratories to the NIST protocol are listed in the section on Materials 

and Methods. Each laboratory was instructed to amplify and sequence the 58 areas designated by 

the 58 primer sets and those laboratories that sequenced CHR were also to sequence the cloned DNA 

from the HV1 region. Those sequencing CHR were designated Labs 1, 2, 3, and 4. Labs 1, 2 and 3 

found essentially the same polymorphisms. Laboratory 4 had less experience with sequencing 

mtDNA and did find differences that the other laboratories did not observe. Interlaboratory data was 

considered ambiguous and excluded if the following problems were observed: 1. If the computer 

results were ambiguous as indicated by calling a peak “N” rather than A, C, G or T. 2. If the 

differences from the Cambridge Reference Sequence were not consistently seen within a laboratory, 

i.e., if the laboratory sequenced in both the forward and reverse directions and one direction agreed 

with the Cambridge Reference Sequence and the other direction did not agree, we assumed the 

results that agreed with the Cambridge Reference Sequence were correct. 3. If, within any one 

laboratory, the difference from Cambridge Reference Sequence was seen with one primer set, but 

not in the overlapping sequences seen in the previous or subsequent primer sets, the results agreeing 

with the Cambridge Reference Sequence were considered correct. Even with these exclusions, 

Laboratory 4 had many differences that were not seen by the other labs. One problem was that they 

did not provide data from primer sets 29, 39, and 41 and we were unable to check the overlapping 

sequences. Laboratory 2 was unable to sequence the clone, which was not a problem for the other 

laboratories. Laboratory 3 was missing data from primer sets 36 and 48. Laboratories 1 and 3 noted 

a heteroplasmy3

 at base number 6849 (the Cambridge Reference Sequence found an A at this site). 

 

3

In final preparation of SRM 2392, a new blood sample was obtained from CHR and a new cell 

culture line was established. The sequence analysis of the new CHR was identical to the first cell 

line except no heteroplasmy was found at bp 6849. Therefore, the cell line supplied with SRM 

2392 does not have this heteroplasmy. The data on the first cell line are included in the text to 

indicate the agreement in the interlaboratory evaluation which was done with the first cell line. 
15

Laboratory 1 found a G at this site, but closer examination of the electropherogram showed that an A 

peak existed under the G peak. Laboratory 3 also noted the A/G heteroplasmy at this site. 

Laboratory 4 did not note the heteroplasmy, but when their electropherograms were examined at 

NIST, the A/G heteroplasmy was noted. NIST did not have the electropherograms of Laboratory 2, 

but on questioning them, they agreed that the heteroplasmy was there, but that they had missed it. 

One of the problems with finding heteroplasmic sites is that if the computer call is the same as the 

Cambridge Reference Sequence, one would not necessarily examine that site more closely. If the 

computer call is different from the Cambridge Reference Sequence, one would look more closely at 

the electropherogram and then note the presence of a smaller peak under the main peak. The cell 

line CHR was reestablished with fresh blood and the entire 16,569 base pairs were amplified and 

sequenced again. The heteroplasmy at 6849 was not present in the new cell line. The new cell line 

agreed with the Cambridge Reference Sequence at this site. It is the DNA from the new cell line that 

is in SRM 2392. 

With the exceptions of the differences noted here, the interlaboratory evaluation of CHR was 

successful in that most of the laboratories found the same results. The many differences noted by 

Laboratory 4, who was less experienced at sequencing mtDNA, confirms and emphasizes the need 

for Standard Reference Materials for sequencing mtDNA. If Laboratory 4 had the NIST mtDNA 

SRM 2392 or SRM 2392-I and had run it along side of their unknown sample, they would have 

realized that they were finding an undue number of differences and could have reexamined their 

procedures to try to determine the reason for these excessive differences. 

The Interlaboratory Evaluation of the mtDNA sequence of HL-60 was conducted by four 

laboratories (see Materials and Methods section) including NIST. All the laboratories produced 

identical results ). 

4. Conclusions

Two NIST Standard Reference Materials (SRM 2392 and 2392-I) have been prepared. These SRMs 

allow one to amplify and sequence any region or the entire 16,569 base pairs which comprise human 

mtDNA. Fifty-eight pairs of unique primers have been designed, tested and shown to work well in 

the amplification and sequencing procedures (note: the reverse primer for primer set 51 has been 

changed from that originally listed in SRM 2392, in Levin et al., 1999, and in Appendix C). The 

three DNA templates (CHR and 9947A in SRM 2392, and HL-60 in SRM 2392-I) have 

characteristic polymorphisms throughout the non-coding and coding regions of the DNA and 

therefore, can serve as positive controls during PCR amplification and sequencing. With one 

exception, none of the polymorphisms found in CHR and 9947A correspond to any of the published 

base pair changes that have been correlated with specific diseases. The one exception is that CHR 

has one mutation that has been associated with a secondary mutation associated with LHON. HL60, however, has 4 mutations that have been associated with the mitochondrial disease LHON. 

Compared to the 1981 Cambridge Reference Sequence, CHR mtDNA had 13 differences in the noncoding regions and 33 differences in the coding regions, 9947A mtDNA had 9 differences in the 

non-coding regions and 23 differences in the coding regions, HL-60 had 11 differences in the noncoding regions and 33 differences in the coding regions. GM03798 and GM 10742A, whose data 

are included for comparison and information, had 4 and 12 differences in the non-coding regions, 
16

respectively and 19 and 14 differences in the coding regions, respectively. All of these differences 

are shown in Fig. 1 and Table 2. These differences in the coding regions do result in some amino 

acid changes in the proteins coded for by mtDNA. Four laboratories participated in an 

interlaboratory evaluation of the CHR template and four laboratories participated in the 

interlaboratory evaluation of HL-60; some differences between laboratories were noted in the CHR 

evaluation, but, in general, agreement was good. No differences were noted in the HL-60 

interlaboratory evaluation. The use of NIST SRM 2392 and SRM 2392-I will provide quality control 

to the scientific and medical communities when they amplify and sequence human mtDNA. 

5. Acknowledgments

The authors are grateful to The Bode Technology Group, Inc., Sterling, VA; IIT Research Institute, 

Newington, VA; and Lark Technologies, Inc., Houston, TX for their participation in the 

Interlaboratory Evaluation of the CHR template and to the FBI Laboratory, FBI Academy, Quantico, 

VA; the Armed Forces DNA Identification Laboratory (AFDIL), Armed Forces Institute of 

Pathology, Rockville, MD; and the Georgia Bureau of Investigation (GBI), Decatur, GA for their 

participation in the Interlaboratory Evaluation of HL-60. The research to prepare SRM 2392-I 

containing HL-60 was supported by Interagency Agreement number 1999-IJ-R-094 from the 

National Institute of Justice. The authors also acknowledge the funding received from the National 

Institute of Justice through the NIST Office of Law Enforcement Standards and the support from the 

Advanced Technology Program at NIST to develop SRM 2392. Points of view are those of the 

authors and do not necessarily represent the position of the U.S. Department of Justice. The authors 

thank Prasad Reddy for his help and advice on cloning, Lois A. Tully (when serving as a postdoctoral research associate at NIST) and Julie T. Chen for checking selected sequence areas of DNA 

samples from CHR, the CHR clone, and 9947A before the final assembly of SRM 2392 that became 

available in 1999. We also thank MaryPat Jones for confirming the sequences of some areas of the 

second generation of the CHR cell line. The authors are grateful to Gettysburg College for the partial 

funding of Koren A. Holland during her sabbatical year at NIST. We also acknowledge and thank 

Mary Pat Jones and Kathryn Kapfer for help in sequencing GM10742A at NIST. 

6. References

Anderson, S., Bankier, A. T., Barrell, B. G., deBrujin, M. H. L., Coulson, A. R., Drouin, J., Eperon, 

I. C., Nierlich, D. P., Roe, B. A., Sanger, F., Schreier, P. H., Smith, A. J. H., Staden, R., and Young, 

I. G. (1981). Sequence and organization of the human mitochondrial genome. Nature 290: 457-465. 

Andrews, R.M., Kubacka, I., Chinnery, P.F., Lightowlers, R.N., Turnbull, D.M., Howell, N. (1999). 

Reanalysis and revision of the Cambridge Reference Sequence for human mitochondrial DNA. 

Nature Genetics 23:147. 

Ballinger, S. W., Bouder, T. G., Davis, G. S., Judice, S. A., Nicklas, J. A., and Albertini, R. J. 

(1996). Mitochondrial genome damage associated with cigarette smoking. Cancer Research 56: 

5692-5697. 

Bendall, K. E., and Sykes, B. C. (1995). Length heteroplasmy in the first hypervariable segment of 

the human mtDNA control region. Am. J. Hum. Genet. 57: 248-256. 
17

Bogenhagen, D., and Clayton, D. A. (1974). The number of mitochondrial deoxyribonucleic acid 

genomes in mouse L and human HeLa cells. J. Biol. Chem. 249: 7991-7995. 

Grossman, L. I. (1995). Mitochondrial mutations and human disease. Env. & Mol. Mutag. 25: 

(supplement 26) 30-37. 

Holland, M. M., Fisher, D. L., Mitchell, L. G., Rodriquez, W. C., Canik, J. J., Merril, C. R., and 

Weedn, V. W. (1993) Mitochondrial DNA sequence analysis of human skeletal remains: 

Identification of remains from the Vietnam War. J. Forensic Sciences 38: 542-553. 

Holland, M. M., Fisher, D. L., Roby, R. K., Ruderman, J., Bryson, C., and Weedn, V. W. (1995) 

Mitochondrial DNA sequence analysis of human remains. Crime Laboratory Digest 22: 109-115. 

Howell, N., McCullough, D. A., Kubacka, I., Halvorson, S., and Mackey, D. (1992). The sequence 

of human mtDNA: the question of errors versus polymorphisms. Am. J. Hum. Genet. 50: 1333-1337. 

King, M. P., and Attardi, G. (1989). Human cells lacking mtDNA: Repopulation with exogenous 

mitochondria by complementation. Science 246: 500-503. 

Lee, M.S. and Levin, B.C. (2002). MitoAnalyzer, a computer program and interactive web site to 

determine the effects of single nucleotide polymorphisms and mutations in human mitochondrial 

DNA. Mitochondrion 1: 321-326. 

Levin, B. C., Cheng, H., O'Connell, C., Reeder, D. J., and Holland, M. (1995). Evaluation of 

alternative enzymes and additives to improve sequencing of mitochondrial DNA. Proceedings of the 

Fifth International Symposium on Human Identification, Scottsdale, AZ, October 8-12, 1994, p. 

170. 

Levin, B. C., Cheng, H., Holland, M., and Reeder, D. J. (1997). Heteroplasmy responsible for 

difficulty experienced in sequencing the human mitochondrial DNA HV1 region containing the Cstretch. Proceedings of the Seventh International Symposium on Human Identification, Scottsdale, 

AZ, September 19-21, 1996, p. 166. 

Levin, B. C., Cheng, H., and Reeder, D. J. (1999). A human mitochondrial DNA Standard Reference 

Material for quality control in forensic identification, medical diagnosis, and mutation detection. 

Genomics 55: 135-146. 

Levin, B.C., Holland, K.A., Hancock, D.K., Coble, M., Parsons, T.J., Kienker, L.J., Williams, D.W., 

Jones, MP., and Richie, K.L., (2003). Comparison of the complete mtDNA genome sequences of 

human cell lines - HL-60 and GM10742A - from individuals with promyelocytic leukemia and leber 

hereditary optic neuropathy, respectively, and the inclusion of HL-60 in the NIST human 

mitochondrial DNA Standard Reference Material - SRM 2392-I. Mitochondrion (in press, 2003).

Marzuki, S., Lertrit, P., Noer, A. S., Kapsa, R. M. I., Sudoyo, H., Byrne, E., and Thyagarajan, D. 

(1992). Reply to Howell et al.: the need for a joint effort in the construction of a reference data base 
18

for normal sequence variants of human mtDNA. Am. J. Hum. Genet. 50: 1337-1340. 

Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning, A Laboratory Manual, 

Second Edition, Cold Spring Harbor Laboratory Press, NY. 

Wallace, D. C. (1992). Mitochondrial genetics: a paradigm for aging and degenerative diseases.

Science 256: 628-632. 

Wallace, D. C., Brown, M. D., and Lott, M. T. (1997). Mitochondrial genetics. In “Emery and 

Rimoin’s Principles and Practice of Medical Genetics” (D. L. Rimoin, J. M. Connor, and R. E. 

Pyeritz RE, Eds.), Vol. 1, Third Edition, pp. 277-332, Churchill Livingstone, NY. 
19

Table 1. Sequences for Primer Sets Used for PCR Amplification of Human mtDNA 

Primer 

Set 

Number 

 Primer Sequence 

1(HV2) F15 

R484 

CACCCTATTAACCACTCACG 

TGAGATTAGTAGTATGGGAG 

2 F361 

R921 

ACAAAGAACCCTAACACCAGC 

ACTTGGGTTAATCGTGTGACC 

3 F756 

R1425 

CATCAAGCACGCAGCAATG 

AATCCACCTTCGACCCTTAAG 

4 F873 

R1425 

GGTTGGTCAATTTCGTGCCAG 

AATCCACCTTCGACCCTTAAG 

5 F1234 

R1769 

CTCACCACCTCTTGCTCAGC 

GCCAGGTTTCAATTTCTATCG 

6 F1587 

R2216 

TGCACTTGGACGAACCAGAG 

TGTTGAGCTTGAACGCTTTC 

7 F1657 

R2216 

CTTGACCGCTCTGAGCTAAAC 

TGTTGAGCTTGAACGCTTTC 

8 F1993 

R2216 

AAACCTACCGAGCCTGGTG 

TGTTGAGCTTGAACGCTTTC 

9 F2105 

R2660 

GAGGAACAGCTCTTTGGACAC 

AGAGACAGCTGAACCCTCGTG 

10 F2417 

R3006 

CACTGTCAACCCAACACAGG 

ATGTCCTGATCCAACATCGAG 

11 F2834 

R3557 

CCCAACCTCCGAGCAGTACATG 

AGAAGAGCGATGGTGAGAGC 

12 F2972 

R3557 

ATAGGGTTTACGACCTCGATG 

AGAAGAGCGATGGTGAGAGC 

13 F3234 

R3557 

AGATGGCAGAGCCCGGTAATC 

AGAAGAGCGATGGTGAGAGC 

14 F3441 

R3940 

ACTACAACCCTTCGCTGACG 

TGAAGCCTGAGACTAGTTCGG 

15 F3635 

R4162 

GCCTAGCCGTTTACTCAATCC 

TGAGTTGGTCGTAGCGGAATC 

16 F3931 

R4728 

TCAGGCTTCAACATCGAATACG 

TTATGGTTCATTGTCCGGAGAG 

17 F4183 

R4728 

TTTCTACCACTCACCCTAGCATTAC 

TTATGGTTCATTGTCCGGAGAG 

18 F4392 

R4983 

CCCATCCTAAAGTAAGGTCAGC 

GGTTTAATCCACCTCAACTGCC 

19 F4447 

R4982 

TTGGTTATACCCTTCCCGTAC 

GTTTAATCCACCTCAACTGCC 

20 F4797 

R5553 

CCCTTTCACTTCTGAGTCCCAG 

AGGGCTTTGAAGGCTCTTG 

21 F4976 

R5553 

ATTAAACCAGACCCAGCTACG 

AGGGCTTTGAAGGCTCTTG 
20

22 F5318 

R5882 

CACCATCACCCTCCTTAACC 

GCTGAGTGAAGCATTGGACTG 

23 F5700 

R6262 

TAAGCACCCTAATCAACTGGC 

GCCTCCACTATAGCAGATGCG 

24 F5999 

R6526 

TCTAAGCCTCCTTATTCGAGC 

ATAGTGATGCCAGCAGCTAGG 

25 F6242 

R6526 

CGCATCTGCTATAGTGGAGG 

ATAGTGATGCCAGCAGCTAGG 

26 F6426 

R7030 

GCCATAACCCAATACCAAACG 

TGGGCTACAACGTAGTACGTG 

27 F6744 

R7255 

GGCTTCCTAGGGTTTATCGTG 

TTTCATGTGGTGTATGCATCG 

28 F7075 

R7792 

GAGGCTTCATTCACTGATTTCC 

GGGCAGGATAGTTCAGACGG 

29 F7215 

R7792 

CGACGTTACTCGGACTACCC 

GGGCAGGATAGTTCAGACGG 

30 F7645 

R8215 

TATCACCTTTCATGATCACGC 

GACGATGGGCATGAAACTG 

31 F7901 

R8311 

TGAACCTACGAGTACACCGACTAC 

AAGTTAGCTTTACAGTGGGCTCTAG 

32 F8164 

R8669 

CGGTCAATGCTCTGAAATCTGTG 

CATTGTTGGGTGGTGATTAGTCG 

33 F8539 

R9059 

CTGTTCGCTTCATTCATTGCC 

GTGGCGCTTCCAATTAGGTG 

34 F8903 

R9403 

CCCACTTCTTACCACAAGGC 

GTGCTTTCTCGTGTTACATCG 

35 F9309 

R9848 

TTTCACTTCCACTCCATAACGC 

GAAAGTTGAGCCAATAATGACG 

36 F9449 

R9995 

CGGGATAATCCTATTTATTACCTCAG 

AGAGTAAGACCCTCATCAATAGATGG 

37 F9754 

R10275 

AGTCTCCCTTCACCATTTCCG 

AAAGGAGGGCAATTTCTAGATC 

38 F10127 

R10556 

ACTACCACAACTCAACGGCTAC 

GGAGGATATGAGGTGTGAGCG 

39 F10386 

R11166 

GGATTAGACTGAACCGAATTGG 

CATCGGGTGATGATAGCCAAG 

40 F10704 

R11267 

GTCTCAATCTCCAACACATATGG 

TGTTGTGAGTGTAAATTAGTGCG 

41 F11001 

R11600 

AACGCCACTTATCCAGTGAACC 

CTGTTTGTCGTAGGCAGATGG 

42 F11403 

R11927 

GACTCCCTAAAGCCCATGTCG 

TTGATCAGGAGAACGTGGTTAC 

43 F11760 

R12189 

ACGAACGCACTCACAGTCG 

AAGCCTCTGTTGTCAGATTCAC 

44 F11901 

R12876 

TGCTAGTAACCACGTTCTGGTG 

GATATCGCCGATACGGTTG 

45 F12357 AACCACCCTAACCCTGACTTCC 
21

R12876 GATATCGCCGATACGGTTG 

46 F12601 

R13123 

TTCATCCCTGTAGCATTGTTCG 

AGCGGATGAGTAAGAAGATTCC 

47 F12793 

R13343 

TTGCTCATCAGTTGATGATACG 

TTGAAGAAGGCGTGGGTACAG 

48 F13188 

R13611 

CACTCTGTTCGCAGCAGTATG 

TCGAGTGCTATAGGCGCTTGTC 

49 F13518 

R13935 

CATCATCGAAACCGCAAAC 

TGTGATGCTAGGGTAGAATCCG 

50 F13715 

R14118 

GAAGCCTATTCGCAGGATTTC 

TGGGAAGAAGAAAGAGAGGAAG 

51 

F13899 

R14388 

R14388 

TTTCTCCAACATACTCGGATTC 

TTAGCGATGGAGGTAGGATTGG (New Primer) 

TTAGCGATGGAGGTAGGATTCG (Old Primer) 

52 F14189 

R14926 

ACAAACAATGGTCAACCAGTAAC 

TGAGGCGTCTGGTGAGTAGTGC 

53 F14470 

R14996 

TCCAAAGACAACCATCATTCC 

CGTGAAGGTAGCGGATGATTC 

54 F14909 

R15396 

TACTCACCAGACGCCTCAACCG 

TTATCGGAATGGGAGGTGATTC 

55 F15260 

R15774 

AGTCCCACCCTCACACGATTC 

ACTGGTTGTCCTCCGATTCAGG 

56 F15574 

R16084 

CGCCTACACAATTCTCCGATC 

CGGTTGTTGATGGGTGAGTC 

57 (HV1) F15971 

R16451 

TTAACTCCACCATTAGCACC 

GCGAGGAGAGTAGCACTCTTG 

58 F16097 

R336 

TACATTACTGCCAGCCACCATG 

TTAAGTGCTGTGGCCAGAAG 

-21M13 F TGTAAAACGACGGCCAGT 

HV2: Hypervariable region 2 

HV1: Hypervariable region 1 

F: forward primer 

R: reverse primer 

These are the same primers used for SRM 2392, Levin et al., 1999, and Appendix C except the 

reverse primer of set 51 has been changed to: TTAGCGATGGAGGTAGGATTGG. The change (C 

to G) occurs at np 14368 and is in bold and underlined. Those using SRM 2392 or SRM 2392-I 

should use the new reverse primer 51. 
22

Table 2. Certified Human mtDNA Sequence Differences from the Cambridge Reference Sequence (CRS) Templates (CHR and 9947A) in NIST SRM 2392, One Template (HL-60) in NIST SRM 2392-I and in GM

Comparison with the Cambridge Reference Sequence (CRS) 

CRS 

# a 

Baseb

1981/1999 

Template

CHRd 

Template

9947Ad Template HL-60 Template GM03798 Template GM10742A Amin

cha

73 93 

150 152 185 195 204 207 214 228 

263*R 

295 

303-309 

311-315*R 

462 

A A 

C 

T 

G 

T T 

G A G A 

C 

- - 

C 

G 

- - - - 

C C 

A 

- - 

G 

- 

C (ins) C (ins) 

- 

- 

G 

- - - 

C 

- - 

G 

- 

G 

- 

CC (ins) 

C (ins) 

- 

G 

- 

T 

C 

- - - - - - 

G 

T 

- 

C (ins) 

- 

- - - - - - - - - - 

G 

- - 

C (ins) 

- 

G 

- - - 

A 

- - - - 

A G 

T 

- 

C (ins) 

T 
23

482 489 709 

750 *R 

T T 

G A 

- - 

A G 

- - - 

G 

- 

C 

- 

G 

- - 

A G 

C C 

- 

G 

1438*R 

1719 

A G 

G A 

G 

- 

G 

- 

G 

- 

G 

- 

2706 2841 3010 

A 

T 

G 

G 

- - 

- - - 

G 

- - 

- - 

A 

G A A 

3106-3107*E 

3394 

C/del 

T 

del C 

- 

del C 

- 

del C 

- 

del C 

- 

del C 

C Tyr → 

3423*E G/T T T T T T Sil

4135 4216 

T T 

- - 

C 

- 

- 

C 

- - 

- 

C 

Tyr → 

Tyr → 

4769*R A G G G G G Sil

4985*E 

5186 5228 5633 

G/A 

A 

C C 

A G 

- - 

A 

- - - 

A 

- 

G 

T 

A 

- - - 

A 

- - - 

SilSilSil

6221 6371 

T 

C 

C 

T 

- - 

- - 

- - 

- - 

SilSil
24

6791 

6849h 

A A 

G 

G(0.3A)h

- - 

- - 

- - 

- - 

Sil

Thr → 

7028 7476 

C C 

T 

- 

- - 

T T 

- - 

T 

- 

Sil

7645 T - C - - - Sil7861 8020 

T 

G 

- - 

C 

- 

- - 

- - 

- 

A 

SilSil

8448 8503 

T T 

- 

C 

C 

- 

- - 

- - 

- - 

Met → 

Sil

8860*R A G G G G nd Thr → 

9315 T - C - - - Phe → 

9559*E 

10172 10398 11251 11287 

G/C 

G A A 

T 

C 

- - - - 

C 

- - - - 

C 

A G G 

- 

C 

- - - - 

C 

- 

G G 

C 

Arg → 

Sil

Thr → 

SilSil

11335*E T/C C C C C C Sil

11719 11778 

G G 

A 

- 

- - 

A 

- 

- - 

A A 

Sil

Arg → 

11878 

12071het 

T T 

C 

- 

- - 

- 

C/Thet

- - 

- - 

Sil

Phe→

12612 12705 

A 

C 

G 

T 

- - 

G 

- 

- - 

G 

- 

SilSil

13572 T - C - - - Sil
25

13702*E 

13708 13759 

G/C 

G G 

C 

A 

- 

C 

- 

A 

C 

A 

- 

C 

- - 

C 

A 

- 

Gly → 

Ala → 

Ala → 

13966 A G - - - - Thr → 

14199*E 14272*E 14365*E 

G/T 

G/C G/C 

T 

C C 

T 

C C 

T 

C C 

T 

C C 

T 

C C 

Pro → 

Phe → 

Sil

14368*E 

14470 14569 

14766*E 

14798 15257 

G/C 

T 

G 

T/C 

T 

G 

C C 

- 

T 

- - 

C 

- - 

C 

- - 

C 

- 

A 

T 

- 

A 

C 

- - 

C 

- - 

C 

- - 

T 

C 

- 

Phe → 

SilSil

Ile → 

Phe → 

Asp→ 

15326*R 

15452 

A 

C 

G 

- 

G 

- 

G A 

G 

- 

G A 

Thr → 

Leu → 

15646 C - - - T - Sil15812 16069 16126 

G 

C 

T 

- - - 

- - - 

A 

T 

- 

- - - 

- 

T 

C 

Val → 
26

16183 

16184-93 

16189 16193 16223 16278 16292 16311 16357 

A 

- 

T 

C C C C 

T T 

C 

C (ins) 

C 

- 

T T 

- - - 

- - - - - - - 

C 

- 

- - - 

T 

- 

T 

- - - 

- - - - - - - - 

C 

- - - - - - 

T 

- - 

16362 T - - C - - 16519 T C C - C nd 

a : Numbers correspond to Cambridge Reference Sequence (Anderson et al., 1981). b: Base found in 1981 (Anderson et al., 1981)/ Base found in 1999 (Andrews et al., 1999). 

d: SRM 2392 (Levin et al., 1999). e: SRM 2392-I (Levin et al., 2003 in press). 

- : Base pair same as in 1981 Cambridge Reference Sequence (Anderson et al., 1981). 

h : Possible heteroplasmic site. This heteroplasmy seen in the mtDNA from the first CHR cell culture line is n

from the second CHR cell culture line. The second CHR cell culture line agrees with the CRS at np 6849. second CHR cell culture line that is supplied in NIST SRM 2392. 

*R: Rare polymorphisms in Cambridge Reference Sequence discovered by reanalysis of original placenta (And

*E: Error in Cambridge Reference Sequence discovered by reanalysis of original placenta (Andrews et al., 199del: Deletion ins: Insertion het: Heteroplasmy found in HL-60 at np 12071. HV1: Non-coding region found from 16024 and 16569. HV2: Non-coding region found from 1 and 576. CHR DNA: Sequence based on two amplifications and cycle sequencing procedures with DNA from the first c

least one amplification and cycle sequencing procedure with DNA from the second cell culture line. 

9947A DNA: Sequence based on two amplifications and cycle sequencing procedures. 
27

HL-60 DNA: Sequence based on two amplifications and cycle sequencing procedures in both the forward and 

total of 4 sequences 

ATPase 6: ATP synthase 6 ATPase 8: ATP synthase 8 CYTB: Cytochrome B COI: Cytochrome C Oxidase I COII: Cytochrome C Oxidase II COIII: Cytochrome C Oxidase III ND1: NADH dehydrogenase 1 ND2: NADH dehydrogenase 2 ND3: NADH dehydrogenase 3 ND4: NADH dehydrogenase 4 ND5: NADH dehydrogenase 5 ND6: NADH dehydrogenase 6 
29

Table 3. Universal Genetic Code and (Human mtDNA Differences) 

2nd Position 

5’ End 

T C A G 

3’ End 

Phe Ser Tyr Cys T 

Phe Ser Tyr Cys C 

Leu Ser TER1 TER1 (Trp) A 

T 

Leu Ser TER1 Trp G 

Leu Pro His Arg T 

Leu Pro His Arg C 

Leu Pro Gln Arg A 

C 

Leu Pro Gln Arg G 

Ile (INT)2 Thr Asn Ser T 

Ile (INT)2 Thr Asn Ser C 

Ile (Met) (INT)2 Thr Lys Arg (TER)1 A 

A 

Met (INT)2

 Thr Lys Arg (TER)1 G 

Val Ala Asp Gly T 

Val Ala Asp Gly C 

Val Ala Glu Gly A 

G 

Val Ala Glu Gly G 

1. TER: Termination codon 

2. INT: Initiation codon
30
31

Figure 1. Schematic of human mtDNA showing its circular double-stranded DNA and all the 

differences from the Cambridge Reference Sequence (Anderson et al., 1981) found in CHR 

(red), 9947A (green), HL-60 (black), GM03798 (blue), and GM10742A (purple) as numbers 

along the outside of the color-coded circle. Locations of the control region, rRNAs and genes 

(see legend to Table 2 for abbreviations) coded by human mtDNA are shown. The locations 

of the 22 tRNAs are noted by white areas in the circle and designated by their single letter 

amino acid code. Since a number of mutations found in GM10742A and HL-60 and one 

change in CHR have been associated with primary, intermediate or secondary mutations 

linked to the disease LHON, the position of these mutations plus other LHON mutations are 

shown on the inside of the circle (Wallace et al., 1997). The question mark following the np 

of the LHON mutations indicates the assignment is not confirmed. One of the primary 

mutations that have been associated with LHON, G11778A, was found in GM10742A (Table 

2) but not found in the other DNA templates examined in this research. (Modified from 

Levin et al., 1999). 
32
33

Appendix A 

SRM 2392 Certificate 
National Institute of Standards & Technology 

Certificate of Analysis 

Standard Reference Material 2392 

Mitochondrial DNA Sequencing 

(Human) 

This Standard Reference Material (SRM) is intended to provide quality control when performing the polymerase chain 

reaction (PCR) and sequencing of human mitochondrial DNA (mtDNA) for forensic identifications, medical diagnosis, 

or mutation detection. It may also be used as a control when amplifying (PCR) and sequencing any DNA. This SRM 

can also be used for quality assurance when assigning values to in-house control materials. It is certified for the 

sequences of the entire human mtDNA (16 569 base pairs) from two lymphoblastoid cell culture lines (CHR and 

GM09947A) from apparently normal individuals, plus the cloned HV1 region of CHR containing a C-stretch which is 

difficult to sequence. The SRM is packaged in a single box containing three components: (1) extracted DNA from cell 

culture line CHR (tube contains 60 μL of DNA at a concentration of 1 ng/μL); (2) extracted DNA from cell culture line 

GM09947A (tube contains 60 μL of DNA at a concentration of 1 ng/μL); and (3) cloned DNA from the CHR HV1 region 

containing the C-stretch (tube contains 10 μL of DNA at a concentration of 100 ng/μL). 

This SRM is composed of well-characterized extracted human DNA from CHR and GM09947A and cloned DNA from

the HV1 region of CHR. Table 1 contains the certified sequence information of two entire mtDNA templates (CHR and

GM09947A). Table 2 contains the reference sequences of 58 unique primer sets which were designed to amplify any 

portion or the entire human mtDNA. The sequence information of a third DNA template (GM03798) that was amplified 

and sequenced in its entirety three to four times at NIST is provided in reference 1. Although the extracted DNA from

GM03798 is not provided, the cell culture line can be obtained from NIGMS Human Genetic Mutant Cell Repository, 

Coriell Institute for Medical Research, Camden, NJ. 

Expiration of Certification: The certification of this SRM is valid until 31 May 2008, provided the SRM is handled 

and stored in accordance with the instructions given in this certificate. This certification is nullified if the SRM is 

damaged, contaminated, or modified. 

Maintenance of SRM Certification: NIST will monitor this SRM over the period of its certification. If substantive 

technical changes occur that affect the certification before the expiration of this certificate, NIST will notify the 

purchaser. Return of the attached registration card will facilitate notification. 

The overall direction and coordination of the technical measurements leading to the certification were performed by 

B.C. Levin and D.J. Reeder of the NIST DNA Technologies Group, Biotechnology Division. 

The analytical determination and technical measurements for the certification of this SRM were performed by 

B.C. Levin, H. Cheng, L.A. Tully, M.P. Jones, and D.J. Reeder of the NIST DNA Technologies Group, 

Biotechnology Division. 

The support aspects involved in the preparation, certification, and issuance of this SRM were coordinated through the 

NIST Standard Reference Materials Program by J.C. Colbert and B.S. MacDonald of the NIST Measurement Services 

Division. 

Vincent L. Viker, Chief 

Biotechnology Division 

Gaithersburg, MD 20899 John Rumble, Jr., Chief 

Certificate Issue Date: 17 June 2003 Measurement Services Division 

See Certificate Revision History on Last Page 

SRM 2392 Page 1 of 14 
NOTICE AND WARNING TO USER 

Warning: SRM 2392 IS A HUMAN SOURCE MATERIAL. SINCE THERE IS NO CONSENSUS ON THE 

INFECTIOUS STATUS OF EXTRACTED DNA, HANDLE PRODUCT AS A BIOHAZARDOUS MATERIAL 

CAPABLE OF TRANSMITTING INFECTIOUS DISEASE. 

Storage: Store frozen at a temperature of -20 o

C. DO NOT use a self-defrosting freezer because periodic cycling of 

temperatures may cause shortened shelf life of this SRM. 

INSTRUCTIONS FOR USE 

It is recommended that once thawed, each SRM component should be used in its entirety. Repeated freezing and thawing 

is NOT recommended as this might shorten the shelf life of the SRM. If it is necessary to perform repeated analyses, 

thaw the SRM and divide the tube contents into aliquots that will be kept frozen until use. Thawing can be conducted at 

refrigerator temperatures, room temperature, or at 37 o

C. Once thawed, the sample should be processed without delay. 

SOURCE AND ANALYSIS1

Source of Material: CHR DNA, both extracted and cloned, was prepared in the NIST DNA Technologies Group, 

Biotechnology Division. DNA for GM09947A was prepared by Life Technologies, Inc., Gaithersburg, MD. 

NIST Analysis: NIST extracted DNA from the CHR cell culture, PCR was used to amplify both the CHR DNA and 

GM09947A DNA with all 58 primer sets multiple times, and sequenced the PCR products with a Perkin-Elmer Applied 

Biosystems, Inc. (ABI) 373 automated sequencer or an ABI 310 sequencer. The cloned DNA was prepared at NIST as 

described in reference 1. The sequence of the CHR clone and of representative PCR products of the final CHR and 

GM09947A DNA included in SRM 2392 was reanalyzed to ensure sequence accuracy. 

Interlaboratory Analysis: An interlaboratory evaluation of the amplification, sequencing, and data analysis of the CHR

template was conducted by four laboratories, including NIST. These laboratories were: The Bode Technology Group, 

Inc., Sterling, VA; IIT Research Institute, Virginia Technology Center, Newington, VA; and Lark Technologies, Inc., 

Houston, TX. Description of the interlaboratory analyses is described in reference 1. 

Description of Components: Three components are included in each unit; all components must be stored at -20 oC. 

# 1 Extracted DNA from cell culture line CHR (tube contains 60 μL of DNA at a concentration of 1 ng/μL) 

# 2 Extracted DNA from cell culture line GM09947A (tube contains 60 μL of DNA at a concentration of 

1 ng/μL) 

# 3 Cloned DNA from the CHR HV1 region containing the C-stretch (tube contains 10 μL of DNA at a 

concentration of 100 ng/μL) 

NOTE: DNA concentrations given are nominal values and are not intended for use as concentration standards. 

 1

Certain commercial equipment, instruments, materials, or companies are identified in this paper to specify the experimental

procedure. Such identification does not imply recommendation or endorsement by NIST, nor does it imply that the materials or 

equipment identified are the best available for this purpose. 

SRM 2392 Page 2 of 14 
SRM 2392 

Table 1. Certified Human mtDNA Sequence Differences from the 

Cambridge Reference Sequence [2] Found in Two Templates at NIST 

Primer Set Amplified Region Comparison with Cambridge Reference Sequence (CRS) 

a Length of 

Amplified 

Region CRS # bp Template 

CHR 

Template 

GM09947A 

1 

(HV2) 

 15 - 484 470 

73 93 

195 204 207 214 263 309.1 309.2 315.1 

A A T T G A A 

Start 39 G - C C A - G C(ins) - C(ins) End 436 

Start 39 - G C - - G G C(ins) C(ins) C(ins) End 473 

2 361 - 921 561 

709 750 

G A 

Start 429 A G End 891 

Start 421 - G End 846 

3 756 - 1425 670 NONE Start 778 

End 1197 

Start 778 End 1278 

4 873 - 1425 553 NONE Start 931 

End 1335 

Start 928 End 1377 

5 1234 - 1769 536 

1438 1719 

A G 

Start 1279 G A End 1738 

Start 1275 G - End 1741 
Table 1. Continued 

SRM 2392 

6 1587 - 2216 630 

1719d G 

Start 1632 A End 2106 

Start 1632 - End 2106 

7 1657 - 2216 560 

1719d G 

Start 1691 A End 2170 

Start 1686 - End 2173 

8 1993 - 2216 224 NONE Start 2036 

End 2213 

Start 2018 End 2217 

9 2105 - 2660 556 NONE Start 2157 

End 2636 

Start 2150 End 2586 

10 2417 - 3006 590 

2706 A 

Start 2465 G End 2920 

Start 2458 - End 2956 

11 2834 - 3557 724 

3106/3107 C 

Start 2861 Del End 3350 

Start 2869 Del End 3373 

12 2972 - 3557 586 

3106/3107d

3423 

C G 

Start 2999 Del E End 3422 

Start 2999 Del T End 3460 

13 3234 - 3557 324 

3423d G 

Start 3265 T End 3548 

Start 3258 T End 3545 

14 3441 - 3940 500 NONE Start 3487 

End 3916 

Start 3491 End 3920 

15 3635 - 4162 528 NONE Start 3667 

End 4126 

Start 3662 End 4061 

16 3931 - 4728 798 

4135 T 

Start 3964 - End 4399 

Start 3968 C End 4427 
Table 1. Continued 

SRM 2392 

17 4183 - 4728 546 NONE Start 4208 

End 4657 

Start 4249 End 4657 

18 4392 - 4982 591 

4769 A 

Start 4449 G End 4860 

Start 4453 G End 4935 

19 4447 - 4982 536 

4769d A 

Start 4492 G End 4958 

Start 4492 G End 4921 

20 4797 - 5553 757 

4985 5186 

G A 

Start 4838 A G End 5327 

Start 4845 A - End 5324 

21 4976 - 5553 578 

5186d A 

Start 5000 G End 5516 

Start 5007 - End 5521 

22 5318 - 5882 565 NONE Start 5361 

End 5754 

Start 5360 End 5758 

23 5700 - 6262 563 NONE Start 5741 

End 6149 

Start 5744 End 6163 

24 5999 - 6526 528 

6221 6371 

T C 

Start 6043 C T End 6442 

Start 6058 - - End 6503 

25 6242 - 6526 285 

6371d C 

Start 6271 T End 6520 

Start 6302 - End 6520 

26 6426 - 7030 605 

6791 6849* 

A A 

Start 6451 G G(0.3A)h* 

End 6916 

Start 6474 - - End 6930 
Table 1. Continued 

SRM 2392 

27 6744 - 7255 512 

6849d* 

7028 

A C 

Start 6775 G(0.3A)h* 

T End 7215 

Start 6782 - - End 7221 

28 7075 - 7792 718 NONE Start 7123 

End 7602 

Start 7123 End 7601 

29 7215 - 7792 578 

7645 T 

Start 7263 - End 7722 

Start 7280 C End 7769 

30 7645 - 8215 571 

7861 T 

Start 7671 - End 8149 

Start 7666 C End 8155 

31 7901 - 8311 411 NONE Start 7960 

End 8289 

Start 7959 End 8288 

32 8164 - 8669 506 

8448 8503 

T T 

Start 8211 - C End 8646 

Start 8212 C - End 8641 

33 8539 - 9059 521 

8860 A 

Start 8581 G End 9019 

Start 8582 G End 8999 

34 8903 - 9403 501 

9315 T 

Start 8947 - End 9380 

Start 8944 C End 9381 

35 9309 - 9848 540 

9559 G 

Start 9334 C End 9823 

Start 9333 C End 9827 

36 9449 - 9995 547 

9559d G 

Start 9476 C End 9964 

Start 9485 C End 9940 
Table 1. Continued 

SRM 2392 

37 9754 - 10275 522 NONE Start 9777 

End 10225 

Start 9781 End 10251 

38 10127 - 10556 430 NONE Start 10168 

End 10534 

Start 10166 End 10536 

39 10386 - 11166 781 NONE Start 10410 

End 10899 

Start 10416 End 10916 

40 10704 - 11267 564 NONE Start 10734 

End 11223 

Start 10742 End 11197 

41 11001 - 11600 600 

11335 T 

Start 11026 C End 11461 

Start 11040 C End 11517 

42 11403 - 11927 525 

11719 G 

Start 11428 A End 11795 

Start 11432 - End 11853 

43 11760 - 12189 430 

11878 T 

Start 11784 C End 12159 

Start 11802 - End 12164 

44 11901 - 12876 976 NONE Start 11926 

End 12404 

Start 11926 End 12443 

45 12357 - 12876 520 

12612 12705 

A C 

Start 12404 G T End 12769 

Start 12391 - - End 12849 

46 12601 - 13123 523 

12705d C 

Start 12627 T End 13102 

Start 12645 - End 13045 

47 12793 - 13343 551 NONE Start 12817 

End 13295 

Start 12807 End 13307 
Table 1. Continued 

SRM 2392 

48 13188 - 13611 424 

13572 T 

Start 13238 - End 13587 

Start 13238 C End 13593 

49 13518 - 13935 418 

13572d

13702 13708 13759 

T G G G 

Start 13541 - C A - End 13910 

Start 13541 C C - A End 13921 

50 13715 - 14118 404 

13966 A 

Start 13775 G End 14094 

Start 13760 - End 14110 

51 13899 - 14388 490 

13966d

14199 14272 14365 

A G G G 

Start 13926 G T C C End 14369 

Start 13927 - T C C End 14374 

52 14189 - 14926 738 

14272d

14365d14368 14470 14766 

G G G T T 

Start 14216 C C C C E End 14699 

Start 14216 C C C - C End 14806 

53 14470 - 14996 527 

14766d T 

Start 14502 - End 14957 

Start 14513 C End 14972 

54 14909 - 15396 488 

15326 A 

Start 14941 G End 15380 

Start 14933 G End 15373 
Table 1. Continued 

SRM 2392 

55 15260 - 15774 515 

15326d A 

Start 15305 G End 15754 

Start 15293 G End15950 

56 15574 - 16084 511 NONE Start 15637 

End 16056 

Start 15599 End 16058 

57 

 (HV1) 

15971 - 16451 481 

16183 16189 16311 

A T T 

Start 16014 C C E End 16193 

Start 16011 - - C End 16430 

58 16097 - 336 809 

16183d

16189d16311d16519 

A T T T 

Start 16125 C C E E End 16193 

Start 16130 - - C C End 59 

-21M13c

cloned DNA 

16133 - 40 477 

16183d

16189d16193.1 16223 16278 16519d

A T C C T 

Start 16131 C C C(ins) T T C End 40 

ND 
Table 1. Continued 

SRM 2392 a Numbers correspond to Cambridge Reference Sequence [2]. 

E Base pair change came after the readable sequence. - Base pair same as in Cambridge Reference Sequence [2]. h* Possible heteroplasmic site * This heteroplasmy seen in the first CHR cell culture line was not seen with the second CHR cell culture line. It is the se

that is supplied in NIST SRM 2392. 

c This primer is used for sequencing the cloned DNA of the HV1 region. d Change also seen in previous primer set. Start Start of readable sequence. End End of readable sequence. CHR cells Sequence based on two amplifications and cycle sequencing procedures in first cell culture line and at least one amplific

procedure with the second cell culture line. 

GM09947A cells Sequence based on two amplifications and cycle sequencing procedures. Ins Insertion Del Deletion ND Not done 
SRM 2392 Page 11 of 14 

Table 2. Reference Sequences for Primer Sets Used for PCR 

Amplification of Human mtDNA 

PRIMER SET 

NUMBER PRIMER SEQUENCE 

1 

(HV2) 

F15 

R484 

CACCCTATTAACCACTCACG 

TGAGATTAGTAGTATGGGAG 

2 F361 

R921 

ACAAAGAACCCTAACACCAGC 

ACTTGGGTTAATCGTGTGACC 

3 F756 

R1425 

CATCAAGCACGCAGCAATG 

AATCCACCTTCGACCCTTAAG 

4 F873 

R1425 

GGTTGGTCAATTTCGTGCCAG 

AATCCACCTTCGACCCTTAAG 

5 F1234 

R1769 

CTCACCACCTCTTGCTCAGC 

GCCAGGTTTCAATTTCTATCG 

6 F1587 

R2216 

TGCACTTGGACGAACCAGAG 

TGTTGAGCTTGAACGCTTTC 

7 F1657 

R2216 

CTTGACCGCTCTGAGCTAAAC 

TGTTGAGCTTGAACGCTTTC 

8 F1993 

R2216 

AAACCTACCGAGCCTGGTG 

TGTTGAGCTTGAACGCTTTC 

9 F2105 

R2660 

GAGGAACAGCTCTTTGGACAC 

AGAGACAGCTGAACCCTCGTG 

10 F2417 

R3006 

CACTGTCAACCCAACACAGG 

ATGTCCTGATCCAACATCGAG 

11 F2834 

R3557 

CCCAACCTCCGAGCAGTACATG 

AGAAGAGCGATGGTGAGAGC 

12 F2972 

R3557 

ATAGGGTTTACGACCTCGATG 

AGAAGAGCGATGGTGAGAGC 

13 F3234 

R3557 

AGATGGCAGAGCCCGGTAATC 

AGAAGAGCGATGGTGAGAGC 

14 F3441 

R3940 

ACTACAACCCTTCGCTGACG 

TGAAGCCTGAGACTAGTTCGG 

15 F3635 

R4162 

GCCTAGCCGTTTACTCAATCC 

TGAGTTGGTCGTAGCGGAATC 

16 F3931 

R4728 

TCAGGCTTCAACATCGAATACG 

TTATGGTTCATTGTCCGGAGAG 
Table 2. Continued 

SRM 2392 Page 12 of 14 

17 F4183 

R4728 

TTTCTACCACTCACCCTAGCATTAC 

TTATGGTTCATTGTCCGGAGAG 

18 F4392 

R4983 

CCCATCCTAAAGTAAGGTCAGC 

GGTTTAATCCACCTCAACTGCC 

19 F4447 

R4982 

TTGGTTATACCCTTCCCGTAC 

GTTTAATCCACCTCAACTGCC 

20 F4797 

R5553 

CCCTTTCACTTCTGAGTCCCAG 

AGGGCTTTGAAGGCTCTTG 

21 F4976 

R5553 

ATTAAACCAGACCCAGCTACG 

AGGGCTTTGAAGGCTCTTG 

22 F5318 

R5882 

CACCATCACCCTCCTTAACC 

GCTGAGTGAAGCATTGGACTG 

23 F5700 

R6262 

TAAGCACCCTAATCAACTGGC 

GCCTCCACTATAGCAGATGCG 

24 F5999 

R6526 

TCTAAGCCTCCTTATTCGAGC 

ATAGTGATGCCAGCAGCTAGG 

25 F6242 

R6526 

CGCATCTGCTATAGTGGAGG 

ATAGTGATGCCAGCAGCTAGG 

26 F6426 

R7030 

GCCATAACCCAATACCAAACG 

TGGGCTACAACGTAGTACGTG 

27 F6744 

R7255 

GGCTTCCTAGGGTTTATCGTG 

TTTCATGTGGTGTATGCATCG 

28 F7075 

R7792 

GAGGCTTCATTCACTGATTTCC 

GGGCAGGATAGTTCAGACGG 

29 F7215 

R7792 

CGACGTTACTCGGACTACCC 

GGGCAGGATAGTTCAGACGG 

30 F7645 

R8215 

TATCACCTTTCATGATCACGC 

GACGATGGGCATGAAACTG 

31 F7901 

R8311 

TGAACCTACGAGTACACCGACTAC 

AAGTTAGCTTTACAGTGGGCTCTAG 

32 F8164 

R8669 

CGGTCAATGCTCTGAAATCTGTG 

CATTGTTGGGTGGTGATTAGTCG 

33 F8539 

R9059 

CTGTTCGCTTCATTCATTGCC 

GTGGCGCTTCCAATTAGGTG 

34 F8903 

R9403 

CCCACTTCTTACCACAAGGC 

GTGCTTTCTCGTGTTACATCG 
Table 2. Continued 

SRM 2392 Page 13 of 14 

35 F9309 

R9848 

TTTCACTTCCACTCCATAACGC 

GAAAGTTGAGCCAATAATGACG 

36 F9449 

R9995 

CGGGATAATCCTATTTATTACCTCAG 

AGAGTAAGACCCTCATCAATAGATGG 

37 F9754 

R10275 

AGTCTCCCTTCACCATTTCCG 

AAAGGAGGGCAATTTCTAGATC 

38 F10127 

R10556 

ACTACCACAACTCAACGGCTAC 

GGAGGATATGAGGTGTGAGCG 

39 F10386 

R11166 

GGATTAGACTGAACCGAATTGG 

CATCGGGTGATGATAGCCAAG 

40 F10704 

R11267 

GTCTCAATCTCCAACACATATGG 

TGTTGTGAGTGTAAATTAGTGCG 

41 F11001 

R11600 

AACGCCACTTATCCAGTGAACC 

CTGTTTGTCGTAGGCAGATGG 

42 F11403 

R11927 

GACTCCCTAAAGCCCATGTCG 

TTGATCAGGAGAACGTGGTTAC 

43 F11760 

R12189 

ACGAACGCACTCACAGTCG 

AAGCCTCTGTTGTCAGATTCAC 

44 F11901 

R12876 

TGCTAGTAACCACGTTCTGGTG 

GATATCGCCGATACGGTTG 

45 F12357 

R12876 

AACCACCCTAACCCTGACTTCC 

GATATCGCCGATACGGTTG 

46 F12601 

R13123 

TTCATCCCTGTAGCATTGTTCG 

AGCGGATGAGTAAGAAGATTCC 

47 F12793 

R13343 

TTGCTCATCAGTTGATGATACG 

TTGAAGAAGGCGTGGGTACAG 

48 F13188 

R13611 

CACTCTGTTCGCAGCAGTATG 

TCGAGTGCTATAGGCGCTTGTC 

49 F13518 

R13935 

CATCATCGAAACCGCAAAC 

TGTGATGCTAGGGTAGAATCCG 

50 F13715 

R14118 

GAAGCCTATTCGCAGGATTTC 

TGGGAAGAAGAAAGAGAGGAAG 

51a F13899 

R14388 

R14388 

TTTCTCCAACATACTCGGATTC 

TTAGCGATGGAGGTAGGATTCG (Old Primer) 

TTAGCGATGGAGGTAGGATTGG (New Primer) 

52 F14189 

R14926 

ACAAACAATGGTCAACCAGTAAC 

TGAGGCGTCTGGTGAGTAGTGC 
Table 2. Continued 

SRM 2392 Page 14 of 14 

53 F14470 

R14996 

TCCAAAGACAACCATCATTCC 

CGTGAAGGTAGCGGATGATTC 

54 F14909 

R15396 

TACTCACCAGACGCCTCAACCG 

TTATCGGAATGGGAGGTGATTC 

55 F15260 

R15774 

AGTCCCACCCTCACACGATTC 

ACTGGTTGTCCTCCGATTCAGG 

56 F15574 

R16084 

CGCCTACACAATTCTCCGATC 

CGGTTGTTGATGGGTGAGTC 

57 (HV1) F15971 

R16451 

TTAACTCCACCATTAGCACC 

GCGAGGAGAGTAGCACTCTTG 

58 F16097 

R336 

TACATTACTGCCAGCCACCATG 

TTAAGTGCTGTGGCCAGAAG 

-21M13 F TGTAAAACGACGGCCAGT 

a

 The reverse primer of set 51 has been changed. One should use the new primer. 

REFERENCES 

[1] Levin, B.C.; Cheng, H.; Reeder, D.J.; Human Mitochondrial DNA Standard Reference Material for Quality 

Control in Forensic Identification, Medical Diagnosis, and Mutation Detection; Genomics, Vol. 55, pp. 135-146 

(1999). 

[2] Anderson, S.; Bankier, A.T.; Barrell, B.G.; deBrujin, M.H.L.; Coulson, A.R.; Drouin, J.; Eperon, I.C.; 

Nierlich, D.P.; Roe, B.A.; Sanger, F.; Schreier, P.H.; Smith, A.J.H.; Staden, R.; Young, I.G.; Sequence and 

Organization of the Human Mitochondrial Genome; Nature, Vol. 290, pp. 457-465 (1981). 

Certificate Revision History: 17 June 2003 (This revision reports an extension in expiration date and replacement of reverse primer 51); 29 

December 1999 (Original certificate date). 

Users of this SRM should ensure that the certificate in their possession is current. This can be accomplished by 

contacting the SRM Program at: telephone (301) 975-6776; fax (301) 926-4751; e-mail srminfo@nist.gov; or via the 

Internet http://www.nist.gov/srm. 
Appendix B

SRM 2392-I Certificate 
National Institute of Standards & Technology 

Certificate of Analysis 

Standard Reference Material 2392-I 

Mitochondrial DNA Sequencing 

(Human HL-60 DNA) 

This Standard Reference Material (SRM) is intended to provide quality control when performing the polymerase 

chain reaction (PCR) and sequencing of human mitochondrial DNA (mtDNA) for forensic identification, medical 

diagnosis, or mutation detection. It may also serve as a control when amplifying (PCR) and sequencing any DNA.

This SRM can also be used for quality assurance when assigning values to in-house control materials. It is certified 

for the sequences of the entire human mtDNA (16 569 base pairs) from a promyelocytic cell line (HL-60) prepared

from the peripheral blood leukocytes from an individual with acute promyelocytic leukemia. A unit of SRM 2392-I

consists of 65 μL of extracted DNA from cell culture line HL-60 at a nominal concentration of 1.4 ng/μL, which is

contained in a vial packaged in a protective plastic box.

Certified Values: The certified sequence information of extracted human DNA from HL-60 is provided in Table 1.

Also provided in Table 1 is the certified sequence information for two additional entire mtDNA templates, CHR and 

GM09947A, which are provided in SRM 2392. SRM 2392-I only contains the HL-60 template. Table 2 contains

the sequences of 58 unique primer sets that were designed to amplify any portion or the entire human mtDNA [1]. 

Supplemental Information: The sequence information of an additional two DNA templates, (GM03798 [1] and 

GM10742A [2]), that were amplified and sequenced in their entirety multiple times at NIST are provided in

references 1 or 2. Although the extracted DNA from GM03798 and GM10742A are not provided, the cell cultures 

can be obtained from NIGMS Human Genetic Mutant Cell Repository, Coriell Institute for Medical Research, 

Camden, NJ. A schematic of the differences from the Cambridge Reference Sequence [3] found in the mtDNA

from all five templates is shown in Figure 1. 

Expiration of Certification: The certification of this SRM is valid until 31 March 2008, provided the SRM is 

handled and stored in accordance with the instructions given in this certificate. This certification is nullified if the 

SRM is damaged, contaminated, or modified. 

Maintenance of SRM Certification: NIST will monitor this SRM over the period of its certification. If 

substantive technical changes occur that affect the certification before the expiration of this certificate, NIST will

notify the purchaser. Return of the attached registration card will facilitate notification. 

The analytical determination, technical measurements and analysis of data for the certification of this SRM were 

performed by D.K. Hancock, K.L. Richie, K.A. Holland (on sabbatical from Gettysburg College, Gettysburg, PA), 

and B.C. Levin of the NIST DNA Technologies Group, Biotechnology Division.

The overall direction and coordination of the technical measurements leading to the certification was performed by 

B.C. Levin of the NIST DNA Technologies Group, Biotechnology Division. 

The support aspects involved in the issuance of this SRM were coordinated through the NIST Standard Reference 

Materials Program by B.S. MacDonald of the NIST Measurement Services Division. 

Vincent L. Vilker, Chief 

 Biotechnology Division

Gaithersburg, MD 20899 John Rumble, Jr., Chief

Certificate Issue Date: 13 June 2003 Measurement Services Division 

SRM 2392-I Page 1 of 9 
Support for the preparation and certification of this SRM was provided by the National Institute of Justice through

the NIST Office of Law Enforcement Standards.

NOTICE AND WARNINGS TO USER

Warning: SRM 2392-I IS A HUMAN SOURCE MATERIAL. SINCE THERE IS NO CONSENSUS ON THE

INFECTIOUS STATUS OF EXTRACTED DNA, HANDLE PRODUCT AS A BIOHAZARDOUS MATERIAL

CAPABLE OF TRANSMITTING INFECTIOUS DISEASE.

Permissions: The research to use HL-60 DNA in SRM 2392-I was deemed exempt from the policy of Part 27 of

Title 15 of the Code of Federal Regulations by the NIST Institutional Review Board and the Director of the 

Chemical Science and Technology Laboratory. This work fit into the exemption category described in 15 CFR 

27.101(b)(4) which states: “Research, involving the collection or study of existing data, documents, pathological

specimens, or diagnostic specimens, if, these sources are publicly available or if the information is recorded by the

investigator in such a manner that subjects cannot be identified, directly or through identifiers linked to the 

subjects.” 

ATCC also waived condition 3(c) in their Material Transfer Agreement which states that the “purchaser shall not

sell, lend, distribute or otherwise transfer the material or replicates to any others” for the use of HL-60 in the NIST 

mitochondrial DNA SRM. They stated that, in their view, “as a government agency, NIST will not be providing this 

material as a commercial product despite the collection of fees for the SRM.” 

Storage: Store frozen at a temperature of –20 o

C. DO NOT use a self-defrosting freezer because of periodic 

cycling of temperatures may shorten the shelf life of this SRM. 

INSTRUCTIONS FOR USE 

It is recommended that once thawed, each SRM component should be used in its entirety. Repeated freezing and 

thawing is NOT recommended as this might shorten the shelf life of the SRM. If it is necessary to perform repeated 

analyses, thaw the SRM and divide the tube contents into aliquots that will be kept frozen until use. Thawing can be

conducted at refrigerator temperatures, room temperature, or at 37 oC. Once thawed, the sample should be

processed without delay. DNA concentrations given are nominal values and are NOT intended for use as

concentration standards.

SOURCE AND ANALYSIS1

Source of Material: DNA from HL-60 was prepared by the Professional Services Department of the American 

Type Culture Collection (ATCC), Manassas, VA. This material was subsequently vialed at NIST into 65 μL

portions (nominal DNA concentration of 1.4 ng/μL) and labeled SRM 2392-I Component D (Components A, B, and

C are available in SRM 2392). 

 

NIST Analysis: PCR was used to amplify the HL-60 DNA in its entirety multiple times using all 58 primer sets. 

The PCR products were sequenced with an Applied Biosystems, Inc. 310 automated sequencer. The sequences of 

representative PCR products of the final HL-60 DNA included in SRM 2392-I were reanalyzed to ensure sequence

accuracy. 

Interlaboratory Analyses: An interlaboratory evaluation of the amplification, sequencing and data analysis of the

HL-60 template was conducted by four laboratories, including NIST. These laboratories were: The Armed Forces 

DNA Identification Laboratory (AFDIL), Rockville, MD; Federal Bureau of Investigation Laboratory (FBI), 

Quantico, VA; and The Georgia Bureau of Investigation (GBI), Decatur, GA. The sequences obtained by all of the 

laboratories were identical. Description of the interlaboratory analysis of HL-60 is described in reference 2. 

 1

Certain commercial equipment, instruments, or materials are identified in this certificate in order to specify adequately

the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of 

Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the

purpose.

SRM 2392-I Page 2 of 9 
Table 1. Certified Human mtDNA Sequence Differences from the Cambridge Reference Sequence 

(CRS) [3,4] Found in the Two Templates (CHR and GM09947A) in NIST SRM 2392 

and One Template (HL-60) in NIST SRM 2392-I

Comparison with the Cambridge Reference Sequence (CRS) 

 CRS 

 # a

Baseb 1981/ 

1999

Template 

CHRd 

Template 

9947Ad Template 

HL-60e

 

Amino acid 

change Region 

73 

93 

150 

152 

195 

204 

207 

214 

263*R 

295 

303-309

311-315*R

A 

A 

C 

T 

T 

T 

G 

A 

A 

C 

- 

- 

G 

- 

- 

- 

C 

C 

A 

- 

G 

- 

C (ins) 

C (ins) 

- 

G 

- 

- 

C 

- 

- 

G 

G 

- 

CC (ins) 

C (ins) 

G 

- 

T 

C 

- 

- 

- 

- 

G 

T 

- 

C (ins) 

HV2

HV2 

HV2 

HV2 

HV2 

HV2 

HV2 

HV2 

HV2 

HV2 

HV2 

HV2 

489 

709 

750 *R

T 

G 

A 

- 

A 

G 

- 

- 

G 

C 

- 

G 

HV2

12sRNA 

12sRNA 

1438*R

1719

A 

G 

G 

A 

G 

- 

G 

- 

12sRNA

16sRNA 

2706 A G - G 16sRNA

3106-3107*E CC/del del C del C del C 16sRNA 

3423*E G/T T T T Silent ND1

4135

4216

T 

T 

- 

- 

C 

- 

- 

C 

Tyr → His 

Tyr → His 

ND1 

ND1 LHON 

4769*R A G G G Silent ND2

4985*E 

5186

5228

5633

G/A 

A 

C 

C 

A 

G 

- 

- 

A 

- 

- 

- 

A 

- 

G 

T 

Silent

Silent

Silent

ND2 

ND2 

ND2 

tRNA Ala 

6221

6371

T 

C 

C 

T 

- 

- 

- 

- 

Silent

Silent

COI 

COI 

SRM 2392-I Page 3 of 9 
Comparison with the Cambridge Reference Sequence (CRS) 

 CRS 

 # a

Baseb 1981/ 

1999

Template 

CHRd 

Template 

9947Ad Template 

HL-60e

 

Amino acid 

change Region

6791

6849h

A 

A 

G 

G(0.3A)

h

- 

- 

- 

- 

Silent

Thr → Alah

COI 

COI 

7028

7476

C 

C 

T 

- 

- 

- 

T 

T 

Silent COI 

tRNA Ser 

7645 T - C - Silent COII

7861 T - C - Silent COII

8448

8503

T 

T 

- 

C 

C 

- 

- 

- 

Met → Thr

Silent

ATPase 8 

ATPase 8 

8860*R A G G G Thr → Ala ATPase 6 

9315 T - C - Phe → Leu COIII 

9559*E 

10172 

10398 

11251 

G/C 

G 

A 

A 

C 

- 

- 

- 

C 

- 

- 

- 

C 

A 

G 

G 

Arg → Pro

Silent

Thr → Ala 

Silent

COIII 

ND3 

ND3 

ND4 

11335*E T/C C C C Silent ND4

11719 G A - A Silent ND4

11878 

12071het

T 

T 

C 

- 

- 

- 

- 

C/Thet 

Silent

Phe→Leuhet 

ND4 

ND4 

12612 

12705 

A 

C 

G 

T 

- 

- 

G 

- 

Silent

Silent

ND5 

ND5 

13572 T - C - Silent ND5

13702*E 

13708 

13759 

G/C 

G 

G 

C 

A 

- 

C 

- 

A 

C 

A 

- 

Gly → Arg 

Ala → Thr 

Ala → Thr 

ND5 

ND5 LHON 

ND5 

13966 A G - - Thr → Ala ND5 

14199*E 

14272*E 

14365*E 

G/T 

G/C 

G/C 

T 

C 

C 

T 

C 

C 

T 

C 

C 

Pro → Thr

Phe → Leu 

Silent

ND6 

ND6 

ND6 

14368*E 

14470 

14569 

14766*E 

15257 

G/C 

T 

G 

T/C 

G 

C 

C 

- 

T 

- 

C 

- 

- 

C 

- 

C 

- 

A 

T 

A 

Phe → Leu 

Silent

Silent

Ile → Thr 

Asp→ Asn 

ND6 

ND6 

ND6 

ND6 

CYT B LHON 

SRM 2392-I Page 4 of 9 
Comparison with the Cambridge Reference Sequence (CRS) 

 CRS 

 # a

Baseb 1981/ 

1999

Template 

CHRd 

Template 

9947Ad Template 

HL-60e

 

Amino acid 

change Region

15326*R 

15452 

A 

C 

G 

- 

G 

- 

G 

A 

Thr → Ala 

Leu → Ile 

CYT B 

CYT B 

15812 

16069 

G 

C 

- 

- 

- 

- 

A 

T 

Val → Met CYT B LHON 

HV1 

16183 

16184-93

16189 

16193 

16223 

16278 

16311 

16362 

A 

- 

T 

C 

C 

C 

T 

T 

C 

C (ins) 

C 

- 

T 

T 

- 

- 

- 

- 

- 

- 

- 

- 

C 

- 

- 

- 

- 

T 

- 

T 

- 

C 

HV1

HV1 

HV1 

HV1 

HV1 

HV1 

HV1 

HV1 

16519 T C C - HV1

a Numbers correspond to Cambridge Reference Sequence [3] b Base found in 1981[3] Base found in 1999 [4] d SRM 2392, reference 1 e SRM 2392-I, reference 2 - Base pair same as in 1981 Cambridge Reference Sequence [3] h Possible heteroplasmic site. This heteroplasmy seen in the mtDNA from the first CHR cell culture line is not seen in the 

mtDNA from the second CHR cell culture line. The second CHR cell culture line agrees with the CRS at np 6849. It is 

DNA from the second CHR cell culture line that is supplied in NIST SRM 2392. 

*R Rare polymorphisms in Cambridge Reference Sequence discovered by reanalysis of original placenta by Andrews et al.,

1999 [4].

*E Error in Cambridge Reference Sequence discovered by reanalysis of original placenta by Andrews et al., 1999 [4].

del Deletion 

ins Insertion 

het Heteroplasmy found in HL-60 at np 12071 

HV1 Non-coding region found from 16024 and 16569

HV2 Non-coding region found from 1 and 576 

CHR DNA Sequence based on two amplifications and cycle sequencing procedures with DNA from the first cell culture line 

and at least one amplification and cycle sequencing procedure with DNA from the second cell culture line. 

GM09947A DNA Sequence based on two amplifications and cycle sequencing procedures. 

HL-60 DNA Sequence based on two amplifications and cycle sequencing procedures in both the forward and reverse

directions for a total of 4 sequences. 

ATPase 6 ATP synthase 6 

ATPase 8 ATP synthase 8 

CYTB Cytochrome B 

COI Cytochrome C Oxidase I 

COII Cytochrome C Oxidase II 

COIII Cytochrome C Oxidase III 

LHON Leber Hereditary Optic Neuropathy

ND1 NADH dehydrogenase 1 

ND2 NADH dehydrogenase 2 

ND3 NADH dehydrogenase 3 

ND4 NADH dehydrogenase 4 

ND5 NADH dehydrogenase 5 

ND6 NADH dehydrogenase 6 

SRM 2392-I Page 5 of 9 
Table 2. Reference Sequences for Primer Sets Used for PCR Amplification of Human mtDNA 

Primer Set 

Number 

Primer Sequence 

1(HV2) F15 

R484

CACCCTATTAACCACTCACG 

TGAGATTAGTAGTATGGGAG

2 F361 

R921

ACAAAGAACCCTAACACCAGC 

ACTTGGGTTAATCGTGTGACC 

3 F756 

R1425

CATCAAGCACGCAGCAATG 

AATCCACCTTCGACCCTTAAG 

4 F873 

R1425

GGTTGGTCAATTTCGTGCCAG 

AATCCACCTTCGACCCTTAAG 

5 F1234 

R1769

CTCACCACCTCTTGCTCAGC 

GCCAGGTTTCAATTTCTATCG 

6 F1587 

R2216

TGCACTTGGACGAACCAGAG 

TGTTGAGCTTGAACGCTTTC 

7 F1657 

R2216

CTTGACCGCTCTGAGCTAAAC 

TGTTGAGCTTGAACGCTTTC 

8 F1993 

R2216

AAACCTACCGAGCCTGGTG 

TGTTGAGCTTGAACGCTTTC 

9 F2105 

R2660

GAGGAACAGCTCTTTGGACAC 

AGAGACAGCTGAACCCTCGTG 

10 F2417 

R3006

CACTGTCAACCCAACACAGG 

ATGTCCTGATCCAACATCGAG

11 F2834 

R3557

CCCAACCTCCGAGCAGTACATG 

AGAAGAGCGATGGTGAGAGC 

12 F2972 

R3557

ATAGGGTTTACGACCTCGATG 

AGAAGAGCGATGGTGAGAGC 

13 F3234 

R3557

AGATGGCAGAGCCCGGTAATC 

AGAAGAGCGATGGTGAGAGC 

14 F3441 

R3940

ACTACAACCCTTCGCTGACG 

TGAAGCCTGAGACTAGTTCGG

15 F3635 

R4162

GCCTAGCCGTTTACTCAATCC 

TGAGTTGGTCGTAGCGGAATC 

16 F3931 

R4728

TCAGGCTTCAACATCGAATACG

TTATGGTTCATTGTCCGGAGAG 

17 F4183 

R4728

TTTCTACCACTCACCCTAGCATTAC 

TTATGGTTCATTGTCCGGAGAG 

18 F4392 

R4983

CCCATCCTAAAGTAAGGTCAGC

GGTTTAATCCACCTCAACTGCC 

19 F4447 

R4982

TTGGTTATACCCTTCCCGTAC 

GTTTAATCCACCTCAACTGCC 

20 F4797 

R5553

CCCTTTCACTTCTGAGTCCCAG 

AGGGCTTTGAAGGCTCTTG

21 F4976 

R5553

ATTAAACCAGACCCAGCTACG 

AGGGCTTTGAAGGCTCTTG

22 F5318 

R5882

CACCATCACCCTCCTTAACC 

GCTGAGTGAAGCATTGGACTG

23 F5700 

R6262

TAAGCACCCTAATCAACTGGC 

GCCTCCACTATAGCAGATGCG 

24 F5999 

R6526

TCTAAGCCTCCTTATTCGAGC 

ATAGTGATGCCAGCAGCTAGG

25 F6242 

R6526

CGCATCTGCTATAGTGGAGG

ATAGTGATGCCAGCAGCTAGG

26 F6426 

R7030

GCCATAACCCAATACCAAACG 

TGGGCTACAACGTAGTACGTG 

27 F6744 

R7255

GGCTTCCTAGGGTTTATCGTG 

TTTCATGTGGTGTATGCATCG 

SRM 2392-I Page 6 of 9 
Primer Set 

Number 

Primer Sequence 

28 F7075 

R7792

GAGGCTTCATTCACTGATTTCC GGGCAGGATAGTTCAGACGG 

29 F7215 

R7792

CGACGTTACTCGGACTACCC GGGCAGGATAGTTCAGACGG

30 F7645 

R8215

TATCACCTTTCATGATCACGC 

GACGATGGGCATGAAACTG 

31 F7901 

R8311

TGAACCTACGAGTACACCGACTAC 

AAGTTAGCTTTACAGTGGGCTCTAG

32 F8164 

R8669

CGGTCAATGCTCTGAAATCTGTG

CATTGTTGGGTGGTGATTAGTCG

33 F8539 

R9059

CTGTTCGCTTCATTCATTGCC 

GTGGCGCTTCCAATTAGGTG 

34 F8903 

R9403

CCCACTTCTTACCACAAGGC 

GTGCTTTCTCGTGTTACATCG 

35 F9309 

R9848

TTTCACTTCCACTCCATAACGC 

GAAAGTTGAGCCAATAATGACG 

36 F9449 

R9995

CGGGATAATCCTATTTATTACCTCAG AGAGTAAGACCCTCATCAATAGATGG

37 F9754 R10275 

AGTCTCCCTTCACCATTTCCG 

AAAGGAGGGCAATTTCTAGATC 

38 F10127 R10556 

ACTACCACAACTCAACGGCTAC 

GGAGGATATGAGGTGTGAGCG

39 F10386 R11166 

GGATTAGACTGAACCGAATTGG 

CATCGGGTGATGATAGCCAAG

40 F10704 R11267 

GTCTCAATCTCCAACACATATGG

TGTTGTGAGTGTAAATTAGTGCG 

41 F11001 R11600 

AACGCCACTTATCCAGTGAACC 

CTGTTTGTCGTAGGCAGATGG

42 F11403 R11927 

GACTCCCTAAAGCCCATGTCG 

TTGATCAGGAGAACGTGGTTAC 

43 F11760 R12189 

ACGAACGCACTCACAGTCG 

AAGCCTCTGTTGTCAGATTCAC 

44 F11901 R12876 

TGCTAGTAACCACGTTCTGGTG 

GATATCGCCGATACGGTTG

45 F12357 R12876 

AACCACCCTAACCCTGACTTCC 

GATATCGCCGATACGGTTG

46 F12601 R13123 

TTCATCCCTGTAGCATTGTTCG 

AGCGGATGAGTAAGAAGATTCC 

47 F12793 R13343 

TTGCTCATCAGTTGATGATACG

TTGAAGAAGGCGTGGGTACAG

48 F13188 R13611 

CACTCTGTTCGCAGCAGTATG 

TCGAGTGCTATAGGCGCTTGTC 

49 F13518 R13935 

CATCATCGAAACCGCAAAC 

TGTGATGCTAGGGTAGAATCCG

50 F13715 R14118 

GAAGCCTATTCGCAGGATTTC 

TGGGAAGAAGAAAGAGAGGAAG

51 

F13899 R14388 R14388 

TTTCTCCAACATACTCGGATTC 

TTAGCGATGGAGGTAGGATTGG (New Primer)

TTAGCGATGGAGGTAGGATTCG (Old Primer)

52 F14189 R14926 

ACAAACAATGGTCAACCAGTAAC 

TGAGGCGTCTGGTGAGTAGTGC

53 F14470 R14996 

TCCAAAGACAACCATCATTCC 

CGTGAAGGTAGCGGATGATTC 

54 F14909 R15396 

TACTCACCAGACGCCTCAACCG

TTATCGGAATGGGAGGTGATTC 

55 F15260 R15774 

AGTCCCACCCTCACACGATTC 

ACTGGTTGTCCTCCGATTCAGG 

SRM 2392-I Page 7 of 9 
Primer Set 

Number 

Primer Sequence 

56 F15574

R16084 

CGCCTACACAATTCTCCGATC 

CGGTTGTTGATGGGTGAGTC 

57 (HV1) F15971

R16451 

TTAACTCCACCATTAGCACC 

GCGAGGAGAGTAGCACTCTTG

58 F16097

R336

TACATTACTGCCAGCCACCATG 

TTAAGTGCTGTGGCCAGAAG

-21M13 F TGTAAAACGACGGCCAGT

HV2: Hypervariable region 2

HV1: Hypervariable region 1

F: forward primer

R: reverse primer 

These are the same primers used for SRM 2392 and reference 1 except the reverse primer of set 51 has been

changed to: TTAGCGATGGAGGTAGGATTGG. The change (C to G) occurs at np 14368 and is in bold and

underlined. Those using SRM 2392 should also use the new reverse primer 51. 

 

REFERENCES 

[1] Levin, B.C.; Cheng, H.; Reeder, D.J.; A Human Mitochondrial DNA Standard Reference Material for Quality 

Control in Forensic Identification, Medical Diagnosis, and Mutation Detection; Genomics, Vol. 55, pp. 135-

146 (1999).

[2] Levin, B.C.; Holland, K.A.; Hancock, D.K.; Coble, M.; Parsons, T.J.; Kienker, L.J.; Williams, D.W.; Jones, 

MP.; Richie, K.L.; Comparison of the Complete mtDNA Genome Sequences of Human Cell Lines - HL-60 and

GM10742A - from Individuals with Promyelocytic Leukemia and Leber Hereditary Optic Neuropathy,

Respectively, and the Inclusion of HL-60 in the NIST Human Mitochondrial DNA Standard Reference Material

- SRM 2392-I; Mitochondrion, Vol. 2, pp. 386-399 (2003). 

[3] Anderson, S.; Bankier, A.T.; Barrell, B.G.; deBrujin, M.H.L.; Coulson, A.R.; Drouin, J.; Eperon, I.C.; Nierlich,

D.P.; Roe, B.A.; Sanger, F.; Schreier, P.H.; Smith, A.J.H.; Staden, R.; Young, I.G.; Sequence and Organization 

of the Human Mitochondrial Genome; Nature, Vol. 290, pp. 457-465 (1981). 

[4] Andrews, R.M.; Kubacka, I.; Chinnery, P.F.; Lightowlers, R.N.; Turnbull, D.M.; Howell, N.; Reanalysis and 

Revision of the Cambridge Reference Sequence for Human Mitochondrial DNA; Nature Genetics, Vol. 23, 

p. 147 (1999). 

Users of this SRM should ensure that the certificate in their possession is current. This can be accomplished by

contacting the SRM Program at: telephone (301) 975-6776; fax (301) 926-4751; e-mail srminfo@nist.gov; or via 

the Internet http://www.nist.gov/srm. 

SRM 2392-I Page 8 of 9 
Figure 1. Schematic of human mtDNA showing its circular double-stranded DNA and all the differences from

Cambridge Reference Sequence (1981) found in CHR (red), 9947A (green), HL-60 (black), GM03798 (blue), and 

GM10742A (purple) as numbers along the outside of the color-coded circle. Locations of the control region, rRNAs 

and genes coded by human mtDNA are shown. The locations of the 22 tRNAs are noted by white areas in the circle

and designated by their single letter amino acid code. Since a number of mutations found in GM10742A and HL-60

and one change in CHR have been associated with primary, intermediate or secondary mutations linked to the 

disease Leber Hereditary Optic Neuropathy (LHON), the position of these mutations plus other LHON mutations

are shown on the inside of the circle (Wallace et al., 1997). The question mark following the np of the LHON 

mutations indicates the assignment is not confirmed. One of the primary mutations that have been associated with

LHON, G11778A, was found in GM10742A [2] but not found in the other DNA templates examined in this 

research. (Modified from Levin et al., 1999). 

SRM 2392-I Page 9 of 9 
Appendix C 

Journal Article: 

B. C. Levin, H. Cheng, and D. J. Reeder, “A Human Mitochondrial 

DNA Standard Reference Material for Quality Control in Forensic 

Identification, Medical Diagnosis, and Mutation Detection,” 

Genomics 55, 135-146 (1999).

(See end of Appendix C for color representation of Figure 2.)
Article ID geno.

Genomics55, 135-146 (1999) Q!) 1998.5513, available online at http://www.idealibrary.com on IDE ~ L

Human Mitochondrial DNA Standard Reference Material

for Quality Control in Forensic Identifi.cation,

Medical Diagnosis, and Mutation Detection

Barbara C. Levin

, .

1 Haiyan Cheng, t and Dennis J. Reeder*

*Biotechnology Division, Chemical Science and Technology Laboratory, National Institute of Standards and Technology,

Gaithersburg, Maryland 20899; and tGEO-CENTERS, Inc., Newton Centre, Massachusetts 02159

Received May 8. 1998; accepted August 4. 1998

A human mitochondrial DNA (mtDNA) standard reference material (SRM 2392) will provide quality control when mtDNA is sequenced for forensic identifications, medical diagnosis, or mutation detection. SRM

2392 includes DNA from two lymphoblast cell cultures

(CRR and 9947A) and cloned DNA from the CRR HV1

region, which contains a C stretch and is difficult to

sequence. The mtDNA sequence (but not the DNA) of a

third human template GM03798 is provided for comparison. Fifty-eight unique primer sets allow any area

Or the entire mtDNA (16,569 bp) to be amplified and

sequenced. While none of the differences in these

three templates correspond to published mutations

associated with specific diseases, some of these differences did result in animo acid changes compared with

that published by S. Anderson et al. (1981, Nature 290:

457-465). An interlaboratory evaluation of theamplification, sequencing, and data analysis of the CRR

template was conducted by four laboratories. Corroboration of the SRM results will provide quality assurance that any unknown mtDNA is also being amplified

and sequenced correctly. ~ 1999 Academic Press

This paper is a contribution of the U.S. National Institute of

Standards and Technology (NIST) and is not subject to copyright.

Certain commercial equipment, instruments, materials, or compa"

nies are identified in this paper to specify the experimental procedure. Such identification does not imply recommendation or endorsement by NIST, nor does it imply that the materials or equipment

identified are the best available for this purpose. Some of these data

have been presented at the following meetings: Environmental Mutagen Society 27th Annual Meeting, Victoria, British Columbia, Canada, March 1996; Seventh International Symposium on Human

Identification, Scottsdale, Arizona, September 1996; Human Identification User s Meeting, Rockville, Maryland, November 1996; The

American Association of Clinical Chemistry, Inc., San Diego, California, November 1996; The Society of Toxicology Annual Meeting,

Cincinnati, Ohio, March 1997; Genetic Toxicology Gordon Conference, New London, New Hampshire, June 1997; and the 47th Annual

Meeting of The American Society of Human Genetics, Baltimore

Maryland, October 1997. 1 To whom correspondence should be addressed at 100 Bureau

Drive, Stop 8311, National Institute of Standards and Technology,

Gaithersburg, Maryland 20899-8311. Telephone: (301) 975-6682.

Fax: (301) 330-3447. E-mail: barbara.levin~st.gov.

INTRODUCTION

Human mitochondrial DNA (mtDNA) has been completely sequenced and found to be circular doublestranded molecules containing 16,569 bp (Anderson 

al. 1981). Each human cell can have a few dozen to

several thousand molecules of mtDNA (Bogenhagen

and Clayton, 1974; King and Attardi, 1989). Sequence

analysis of mtDNA is being used by the forensic community for human identification, especially in those

cases in which genomic DNA is highly degraded or

nonexistent (Holland et al. 1993, 1995). Forensic analysis to determine the distinction between individuals is

primarily based on the considerable sequence variation

found in the two hypervariable regions (lIVl, lIV2)

located in the noncoding displacement loop (D loop).

The medical community is also using sequence analysis of mtDNA for diagnoses of diseases associated with

specific mutations and deletions (Wallace et al. 1997).

A third area of research, which is largely unexplored

and which needs sequence analysis, is the examination

of the mutagenic effects of chemical and physical

agents on mtDNA (Grossman, 1995; Ballinger et al.

1996). The objective of this research was to develop a

human mtDNA standard reference material (SRM) for

quality control in sequencing, forensic identifications

medical diagnostics, and mutation detection.

MATERIALS AND METHODS

Origin of extracted DNA. The DNA template designated CHR

came from human white blood cells that were transformed with the

Epstein-Barr virus and immortalized as a cell culture line (CHR

cells) by the American Type Culture Collection (ATCC; Rockville

MD). After transformation, the cells were grown in Iscove s modified

Dulbecco s media or RPMI 1640 media with L-glutarnine, sodium

bicarbonate, penicillin, streptomycin, and 20% fetal calf serum (Life

Technologies, Inc., Grand Island, NY). The cell cultures were grown

at 37OC in humidified atmosphere containing 5% CO2 and 95% air.

The DNA was extracted from 2 X 108 CRR cells by the Qiagen

Plasmid/Cosmid Purification Protocol (Qiagen, Inc. , Chatsworth

CA). This procedure enhanced the concentration of mtDNA and

reduced, but did not eliminate, nuclear DNA.

The CHR data presented in this paper were obtained primarily

with the above-mentioned immortalized CRR cell culture line. How135

0888-7543/99 $30.

Copyright ~ 1999 by Academic Press

All rights of reproduction in any form reserved.
136 LEVIN, CHENG, AND REEDER

ever, before production of the final SRM began, it was necessary to

obtain fresh blood from CHR and to reestablish the cell line. This

second CHR cell line was established by the ATCC as above. The

sequence of this second CHR cell line was examined and found to be

identical to that of the first CHR cell line, with the single exception

that no heteroplasmy was noted at bp6849; the second CHR cell line

agreed with Anderson at bp 6849. It is the second CHR cell line that

is included in SRM 2392.

The DNA template 9947A was obtained from Life Technologies

Inc. , who prepared it from a Epstein-Barr virus-immortalized human lymphoid cell line. DNA from 9947A is also used in the PCRbased DNA profiling standard (SRM 2391)2 designed for forensic and

paternity testing, law enforcement training, and research.

A third DNA template was extracted from an apparently normal

human lymphoblastoid cell culture (GM03798) obtained from

NIGMS3 and grown in the same manner as the CHR cells. The DNA

was extracted using DNA NOW, a phenol-free DNA isolation reagent

(BIOGENTEX, Seabrook, TX). The information on this template is

included for informational purposes only; the DNA is not included as

part of this SRM.

Isolation and cloning of mtDNA containing the C stretch. Confluent CHR cells were harvested by centrifugation at 1500 rpm for 5

min. The mtDNA was isolated using the Qiagen Plasmid/Cosmid

Purification Protocol (Qiagen, Inc.). Following isolation, the mtDNA

was digested with restriction enzymes Sac! and KpnI (New England

Biolabs, Inc., Beverly, MA) into fivefragments which were separated

on a 0.7% low-melting-agarose gel. Bands of the size of the fragment

containing the HV1 region were cut from the gel and melted at 65O

DNA was extracted with phenol twice and precipitated by adding

sodium chloride (150 mM) and 2 vol of 100% ethanol. The final

product was resuspended in Tris~EDTA (TE) buffer.

The cloning vector, M13mp18, was also digested with Sac! and

KpnI treated with calf intestinal alkaline phosphatase (New England Biolabs, Inc.), extracted with phenol, and precipitated with

NaCl and ethanol as described above. The vector was incubated with

the mtDNA product and T. DNA ligase (Life Technologies, Inc.) at

C overnight. An overnight culture of Escherichia coli host TGcells was diluted and grown at 37OC in LB media (Sambrook et al.

1989) until the ODs5o reached 0.4~ 5. The cells were harvested by

centrifugation at 1500 rpm for 5 min. The cell pellet was resuspended in 10 ml calcium chloride (50 mM) and incubated for 1 h on

ice, centrifuged, and resuspended in 1 ml calcium chloride (50 rnM)

and incubated for 30 min on ice. The treated TG-1 cell suspension

(0.3 m!) was incubated for 30min on ice with 20 p.l of a ligation

mixture containing the isolated mtDNA fragment, the cloning vector

that had been treated overnight, T. ligase, and ligation buffer (Life

Technologies, Inc.). This mixt1lI'e was then exposed to a heat shock of

42OC for 2 min; mixed with 0.2 ml of untreated TG-1 cells (from the

overnight culture), 4 p.l of 1 M isopropylthio-(3-D-galactoside, 40 p.lof

20 mg/ml 5-bromo-4-chloro- indolyl-(3-D-galactopyranoside, and 3

ml of melted (55OC) top agar; and spread on the surface of freshly

prepared LB agarose plates (Sambrook et al. 1989). The plates were

incubated at 37OC overnight. Both colorless and blue plaques were

visible in the morning. The colorless plaques indicate that insertion

of the vector has occurred, whereas the blue plaques have no insertion of the vector.

Bacteriophage DNA isolation and sequencing. Single, well-isolated colorless plaques from the above LB plates were each placed in

a sterile tube with 1.5 rnl of a TG-l/LB cell suspension that contained

TG-1 cells that were grown overnight and diluted 1/100 in LB media

and grown for 1 h at 37OC. The plaques and the TG-l/LB cell suspension were grown at 37OC for 5 h. Cell debris WaS removed by

centrifugation at 15 000 rpm for 5 min. The supernatant containing

the bacteriophage was incubated with 0.2 ml polyethylene glycol

2 SRM 2391 may be obtained from the Standard Reference Material Program, NIST, Gaithersburg, Maryland 20899. 3 NIGMS Human Genetic Mutant Cell Repository, Coriell Institute

for Medical Research, 401 Haddon Avenue, Camden, New Jersey

08103.

(20% PEG in 2.5 M NaC!) overnight at 4O , and the resultant

precipitate containing the DNA was pelleted by centrifugation at

000 rpm for 15 min. The bacteriophage DNA was isolated by

phenol extraction and NaClIethanol precipitation as described above

and then dissolved in 25 p.l ofTE buffer. The bacteriophage DNA was

cycle sequenced with AmpliTaq DNA polymerase, FS, and the ~

M13 primer: 5' TGTAAAACGACGGCCAGT-3' according to the protocol in the ABI PRISM Dye TerIninator Cycle Sequencing Ready

Reaction Kit (Perkin-Elmer, Foster City, CA). The cycle sequencing

was conducted in a Perkin~Elmer Model .9600 thermocycler by first

heating the DNA reaction mixture at 96OC for 1 min and then

subjecting the mixture to 25 cycles of 96OC for 10 s, 500C for 5 s, and

600C for 2 min. The cycle sequencing product was purified using a

Centri-Sep spin column (Princeton Separations, Inc., Adelphia, NJ).

The DNA pellet was rinsed with 70% ethanol, vacuum dried, resuspendedin loading buffer prepared by combining deionized formamide and 25 mM EDTA (pH 8.0) in a ratio of 5:1, loaded onto a

75% acrylamide gel, and electrophoresed on an ABI 373 DNA

sequencer. One of the clones containing the C stretch sequence was

used as the source of the cloned DNA for the SRM.

mtDNA primers. Fifty-eight sets of unique primers (19-28 bp)

for sequencing the entire mtDNA (16 569 bp) were computer-designed using Gene Runner for Windows (Hastings Software, Inc.

Hastings, NY) and custom made by Bio-Synthesis, Inc. (Lewisville

TX). The -21M13 primer was used to sequence the cloned HV1

region of the DNA from the CHR template. The sequences of all the

primers are shown in Table 1.

Polymerase chain reaction (PCR). Extracted DNA WaS resuspended in TE buffer (pH 7.5) containing 10 mM Tris and 1 mM

EDTA. The PCR mixture contained DNA (1 p.!), Taq DNA polymerase (0.5 p.l or 2.5 units) (Boehringer Mannheim), and lOX buffer (5

p.!) (Boehringer Mannheim), dNTP's (0.2 mM each) (Life Technologies, Inc.), forward and reverse primers (0.4 p.M each), plus H2O to a

final volume of 50 p.l. The lOxbuffer (pH 8.3) contained Tris-HCl

(100 mM), MgCl2 (15 mM), and KCl (500 mM).

Thermal cycling Was conducted in a Perkin-Elmer Model 9600

thermocycler and consisted of 1 min at 96OC; followed by 32 cycles of

15 s at 94OC (denaturation), 30 s at 56OC (annealing), and 15 s at

72OC (extension); and ending with a final extension of 7 rnin at 72O

A sample of the amplified DNA was electrophoresed in 0.7% agarose and stained with ethidium bromide to assess the purity and size

of the PCR product. Before sequencing, extraneous materials were

removed from the PCRproduct with a QIAquick PCR Purification

Kit (QIAGEN, Inc.

Sequencing. Cycle sequencing using fluorescent dye-labeled terminators was performed with an ABI PRISM Dye Terminator Cycle

Sequencing Kit with AmpliTaq DNA polymerase, FS (Perkin-Elmer

Foster City, CA). Thermal cycling was conducted ip. a Perkin~Elmer

Model 9600 thermocycler and started with 1 min at 96OC. The reaction then underwent 25 cycles of 96OC for 15 s (denaturation), 500

for 5 s (annealing), and 600C for 2 min(extension). The DNA product

was purified by passage through a Centri-Sep spin column (Princeton Separations, Inc.

Electrophoresis and sequencing of the fluorescently labeled purified DNA were performed with a 373 ABI Sequencer (Perkin-Elmer)

using a 4.75% acrylamide gel. Data analysis was executed with the

Sequence Navigator software package (Perkin-Elmer).

Interlaboratory evaluation. Three laboratories in addition to

NIST participated in an interlaboratory evaluation of the CRR template. These laboratories were The Bode Technology Group, Inc.

(21515 Ridgetop Circle, Suite 140, Sterling, VA 20166), IIT Research

Institute (Virginia Technology Center, 8510 Cinderbed Road, Suite

300, P.O. Box 899, Newington, VA 22122), and Lark Technologies

Inc. (9545 Katy Fwy, Suite 465, Houston, TX 77024).

Each laboratory was sent:

1. Two tubes of DNA from the first CHR cell culture line. One tube

contained extracted DNA ready for PCR amplification of the entire

mtDNA. The other contained the cloned DNA ready for cycle sequencing of the HV1 region (this DNA did not need to be PCR amplified).
HUMAN mtDNA STANDARD REFERENCE MATERIAL

2. Fifty-eight sets of primers labeled with either F# (forward

primer) or R# (reverse primer). Forward and reverse primers with

the same number were paired and numbered from the 5' end. Primers were diluted to 10 J.Ll and ready for use. Also enclosed was the

-21M13 primer for the sequencing of the cloned HV1 region of the

CHR DNA, which covered basepairs 16133 to 40.

3. The protocol used at NIST to amplify and sequence the DNA.

The laboratories, however, were free to use any protocol with which

they were familiar and felt comfortable.

4. A form table to record the results. This table provided the

number of the primer set, the region that each primer set amplified,

and the length of the amplified region. We requested that the laboratory fill in the differences found when they compared the sequence

that they determined for the SRM with that of Anderson et al. (1981).

5. The following cautionary note:

WARNING: The DNA and cells were derived from a cell culture

line from an apparently healthy human subject. The cell culture

line has been tested and found to be nonreactive for hepatitis B

surface antigen and HIV. However, no test method can enSure

that a product derived from human blood does not contain HIV

hepatitis or other infectious agents. HANDLE AS IF CAPABLE

OF TRANSMITTING DISEASE. (The second CHR cell culture

line generated from the same individual was not tested again for

hepatitis or HIV. Normal precautions should be used.

Differences in methodology used by laboratories in interlaboratory

evaluation. The Bode Technology Group, Inc., essentially followed

the NIST protocol except that they used a 6% acrylamide/8.3 M urea

gel for the sequencing electrophoresis instead of a 4.75% acrylamide.

IIT Research Institute also followed the NIST protocol except that

they used Taq Gold (Perkin-Elmer) for the amplification reaction

which was modified to include a hot start of 95OC for 11 min. Microcon 100 rnicroconcentrators (Arnicon, Inc., Beverly, MA) were used to

purify the PCR products. The quantities of DNA were detemined by

capillary electrophoresis (CE) with a Beckman PlACE 5010 System

(Beckman Instruments, Inc., Fullerton, CA) as follows: 1 J.Ll of the

amplified product was mixed with 25 J.Ll of sterile deionized H2

containing 0.52 ng/pJ of a 200-bp internal standard (GenSura Laboratories, Inc., Del Mar, CA) and run on the CEo One determines the

quantity of the amplified product from the ratio of the PCR product

peak area to the internal standard peak area multiplied by a migration standard. A 6% acrylamide gel was used for the sequencing

electrophoresis instead of a 4.75% acrylamide.

Lark Technologies, Inc., followed the NIST protocol with the following differences: AmpliTaq DNA polymerase (Perkin-Elmer) was

used to amplify the DNA; the dNTP's were purchased from Pharma"

cia Biotech, Inc. (Piscataway, NJ); the products were purified with

Qiaquick PCR purification kit (Qiagen); in the sequencing reactions

the amount of PCR product used varied from 1 to 3 J.Ll based on the

concentration estimated from agarose gels; cycling conditions were

95OC for 1 rnin followed by 25 cycles of 96OC for 15 s, 50oC for 15 s

and 60oC for 4 min; the sequence reactions were cleaned up by

ethanol precipitation; a 4.25% polyacrylamide gel was used for the

sequencing electrophoresis instead of 4.75% acrylamide; and an ABI

377 was used instead of the ABI 373. Electropherograms were

printed for each reaction, and the sequences were manually edited

based on the electropherogram patterns. Printed electropherograms

and a floppy disk with the sequence data were sent to NIST where

the data were compared to the Anderson sequence.

RESULTS AND DISCUSSION

SRM templates. Two DNA templates CHR and

9947 A, are included in the NIST human mtDNA sequencing SRM 2392. Both of these DNA samples come

from human cell culture lines that were developed from

apparently normal individuals. The DNA from 9947 A

is the total extracted DNA, which also includes nuclear

DNA. The DNA from CHR was isolated in a manner

that enhanced the concentration of the mtDNA, but did

137

not totally eliminate the nuclear DNA. The SRM also

provides cloned DNA from the HVI region of the CHR

template, which contains a C stretch. In most people,

the HVI region has a string of cytosine (C) residues

interrupted by a thymine(T) at nucleotide position

16189.4 In some individuals, however, a transition that

changes the T to a C occurs, producing a long string of

Cs called the C stretch. When this happens, sequencing

beyond the C stretch becomes very difficult, if not impossible. Clones of the HVI region containing the C

stretch indicated that the number of Cs differed among

the different clones and the difficulty in sequencing

was due to frameshifts that resulted from the simultaneous sequencing of templates with differing numbers

of Cs (Bendall and Sykes , 1995; Levin et al. 1995

1997). We found, however, that one could sequence

through the entire HVI region including the C stretch

without problems if one used the clone of the area.

Therefore, we have included the cloned HVI region of

the CRR DNA template in the SRM.

In addition to templates CHR and 9947 A, we have

included all the information regarding a third template

from a lymphoblastoid cell line (GM03798) that was

obtained from the NIGMS Human Genetic Mutant Cell

Repository and that was completely sequenced at NIST

three times. The DNA from this .cellline is not part of

the SRM, and the data are included for information

only.

Primers. The 58 sets of unique primers were designed to allow the amplification and sequencing of any

region or the .entire 16 569 bp that comprise human

mitochondrial DNA. The sequences of both the forward

and the reverse primers that are in each set are shown

in Table 1. The numbers indicate the 5' end of the

primer. They .are all between 19 and 28 bp long, and

the criteria that were used to choose these primers

were primer m = 50-65O , primer length = 15-30 bp,

and PCR product length = 400-850 bp. The primers

were designed to produce sequences that overlapped

with both the previous and the following regions to

allow those areas in the beginning and end of electropherograms, which are difficult to sequence, to become

readable. Table 2 shows the number of the base where

the readable sequence begins and ends (indicated in

the table as "start" and "end"). The readable region is

always smaller than the amplified region.

In addition to the designed primers, we also used

the -21M13 primer (Table 1) to sequence the cloned

DNA from the HVI region of the CHR template that

contained the C stretch. The PCR products produced

single, distinct bands for all 58 primer sets (Figs. lA

and IB).

Differences between the SRM templates and the Anderson sequence. Anderson and his co-workers completely

sequenced human mtDNA in 1981 (Anderson et al.

1981). All investigators who subsequently examined human mtDNA have used the numbering system of Ander4 All nucleotide numbers referred to in this paper are based on the

numbering system of Anderson et al. (1981).
138 LEVIN, CHENG, AND REEDER

TABLE 1

Primer Sets Used for PCR Amplification of Human mtDNA

Primer set Primer sequence Primer set Primer sequence

1 (HV2) F15 CACCCTATTAACCACTCACG R8215 GACGA TGGGCATGAAACTG

R484 TGAGATTAGTAGTATGGGAG F7901 TGAACCT ACGAGTACACCGACT AC

F361 A C AAAGAAC C CT AA CAC C A GC R8311 AA GTT A GC TTT A CAG TGGGCTCT A G

R921 A CTTGGG TTAA TC G TG TGA C C F8164 C GG TCAATGCTCTGAAA TCTG TG

F756 CATCAAGCACGCAGCAATG R8669 CA TTG TTGGGTGGTGATT AGTCG

R1425 AA TCCACCTTCGACCCTT AAG F8539 CTGTTCGCTTCA TTCA TTGCC

F873 GG TTGG TC AA TTTC GTGC CA G R9059 GTGGCGCTTCCAATTAGGTG

R1425 AATCCA CCTTC G ACCCTTAA G F8903 CCCACTTCTTACCACAAGGC

F1234 CTCA C C A C CTCTTGCTCAGC R9403 GTGCTTTCTCGTGTT ACA TCG

R1769 GCCAGGTTTCAATTTCT A TCG F9309 TTTCACTTCCACTCCA T AACGC

F1587 TGCACTTGGACGAACCAGAG R9848 GAAAGTTGAGCCAA T AA TGACG

R2216 TG TTGA GCTTG AA CGCTTTC F9449 CGGGA T AA TCCT A TTT A TTACCTCAG

F1657 CTTG A CC GCTCTG A GCT AAA C R9995 AGAGT AAGACCCTCA TCAA T AGATGG

R2216 TG TTG A GCTTGAA C GCTTTC F9754 A G TCTC C CTTCA CC A TTTCCG

F1993 AAACCTACCGAGCCTGGTG R10275 AAAGGAGGGCAA TTTCTAGATC

R2216 TG TTG AGCTTGAA CGCTTTC F10:!.27 ACT ACCACAACTCAACGGCT AC

F2105 G A GG AAC A GCTCTTTGG ACA C R10556 GGAGGA T A TGAGGTGTGAGCG

R2660 A GAGA CA GCTGAA CCCTC G TG F10386 GGATTAGACTGAACCGAATTGG

F2417 CACTGTCAACCCAACACAGG Rl1166 CATCGGGTGATGATAGCCAAG '

R3006 A TGTCCTGA TCCAACA TCGAG F1O704 GTCTCAATCTCCAACACA T A TGG

F2834 CCCAACCTCCGAGCAGT ACA TG R11267 TG TTGTG A G TGT AAA TT A G TGCG

R3557 AGAAGAGCGATGGTGAGAGC F11001 AACGCCACTT A TCCAGTGAACC

F2972 AT AGGGTTT ACGACCTCGA TG R11600 CTG TTTG TC GT AGGC A GATGG

R3557 AGAAGAGCGATGGTGAGAGC F11403 GACTCCCT AAAGCCCA TGTCG

F3234 AGA TGGCAGAGCCCGGT AA TC R11927 TTGA TCAGGAGAACGTGGTT AC

R3557 A GAAG A GCGA TGGTGA GA GC F11760 ACGAACGCACTCACAGTCG

F3441 ACTACAACCCTTCGCTGACG R12189 AA GCCTCTGTTG TCAGA TTC A C

R3940 TGAA GC CTG AGA CT A G TTC GG F11901 TGCT AGT AACCACGTTCTGGTG

F3635 GCCT AGCCGTTT ACTCAA TCC R12876 GATATCGCCGATACGGTTG

R4162 TGAGTTGGTCGT AGCGGAA TC F12357 AA C CA CC CT AA CC CTGA CTTCC

F3931 TCAGGCTTCAACATCGAA T ACG R12876 GATATCGCCGATACGGTTG

R4728 TT A TGGTTCA TTGTCCGGAGAG F12601 TTCA TCCCTGT AGCA TTGTTCG

F4183 TTTCT ACCACTCACCCT AGCA TT AC R13123 AGCGGATGAGT AAGAAGA TTCC

R4728 TT ATGGTTCA TTGTCCGGAGAG F12793 TTGCTCA TCAGTTGATGA T ACG

F4392 CCCA TCCT AAAGT AAGGTCAGC R13343 TTGAA GAAGGC GTGGG T A CA 

R4983 GG TTT AA TC CACCTCAACTGCC F13188 CACTCTGTTCGCAGCAGT A TG

F4447 TTGGTTA T ACCCTTCCCGT AC R13611 TCGAGTGCT AT AGGCGCTTGTC

R4982 G TTT AA TC CA C CTC AA CTGC C F13518 CATCATCGAAACCGCAAAC

F4797 C C CTTTCA CTTCTG A G TC C CAG R13935 TGTGA TGCT AGGGT AGAA TCCG

R5553 AGGGCTTTGAAGGCTCTTG F13715 GAAGCCT A TTCGCAGGA TTTC

F4976 A TT AAACCAGACCCAGCT ACG R14118 TGGGAAGAAGAAAGAGAGGAAG

R5553 AGGGCTTTGAAGGCTCTTG F13899 TTTCTC CAA CAT A CTCGG A TTC

F5318 CACCATCACCCTCCTTAACC R14388 TTAGCGA TGGAGGT AGGA TTCG

R5882 GCTGA G TG AA GC A TTGGACTG F14189 ACAAACAA TGGTCAACCAG T AAC

F5700 T AAGCACCCT AA TC AA CTGGC R14926 TG AGGC GTCTGG TG AG TAG TGC

R6262 GCCTCCACT A T AGCAGA TGCG F14470 TC CAAA GA CAAC C ATCA TTCC

F5999 TCT AAGCCTCCTTA TTCGAGC R14996 CGTGAAGGT AGCGGATGA TTC

R6526 AT AGTGA TGCCAGCAGCT AGG F14909 T A CTCA CCA G A C GCCTCAA C CG

F6242 CGCATCTGCTATAGTGGAGG R15396 TT A TCGGAA TGGGAGGTGA TTC

R6526 AT AG TGATGCCAGCAGCTAGG F15260 AGTCCCACCCTCACACGA TTC

F6426 GCCA T AACCCAA T ACCAAACG R15774 A CTGG TTG TCCTC CG A TTCAGG

R7030 TGGGCT ACAACGT AGT ACGTG F15574 CGCCT ACACAATTCTCCGA TC

F6744 GGCTTCCT AGGGTTT A TCGTG R16084 CGGTTGTTGATGGGTGAGTC

R7255 TTTCATGTGGTG T A TGCA TCG 57 (HV1) F15971 TTAACTCCACCATTAGCACC

F7075 G A GGCTTC A TTCACTGA TTTC C R16451 GCG A GGA GAG T A GCA CTCTTG

R7792 GGGCAGGATAGTTCAGACGG F16097 T ACATT ACTGCCAGCCACCA TG

F7215 CGACGTTACTCGGACTACCC R336 TTAAGTGCTGTGGCCAGAAG

R7792 GGGCAGGATAGTTCAGACGG 21M13 TGTAAAACGACGGCCAGT

F7645 TATCACCTTTCA TGA TCACGC

son .and have compared their sequence findings to those would be available. Table 2 shows the mtDNA differences

described by Anderson. However, the DNA sequenced by compared to the Anderson sequence that were found at

Anderson is not available for use as a positive control NlST with all three template&-CHR, 9947 A, and

during actual experiments, whereas NlST SRM 2392 GMO3798. In all three templates, all 58 areas comprising
HUMAN mtDNA STANDARD REFERENCE MATERIAL 139

TABLE 2

Primer Sets Used for PCR Amplification of Human mtDNA and Differences with the Anderson Sequence

Found in Three Templates at NIST

Comparison with Anderson

Length of

Amplified amplified Anderson Anderson Template Template Template Amino acid Primer set region region No. CHR 9947A GM03798 change

1 (HV2) 15-484 470 Start 39 Start 39 Start 55

195

204

207

214

263

309. C(ins) C(ins)

309. C(ins)

315. C(ins) C(ins) C(ins)

End 436 End 473 End 454

361-921 561 Start 429 Start 421 Start 415

709

750

End 891 End 846 End 834

756-1425 670 None Start 778 Start 778 Start 818

End 1197 End 1278 End 1146

873-1425 553 None Start 931 Start 928 Start 938

End 1335 End 1377 End 1323

1234-1769 536 Start 1279 Start 1275 Start 1295

1438

1719

End 1738 End 1741 End 1654

1587-2216 630 Start 1632 Start 1632 Start 1649

1719b

End 2106 End 2106 End 2031

1657-2216 560 Start 1691 Start 1686 Start 1715

1719b

End 2170 End 2173 End 2097

1993-2216 224 None Start 2036 Start 2018 Start 2069

End 2213 End 2217 End 2212

2105-2660 556 None Start 2157 Start 2150 Start 2161

End 2636 End 2586 End 2560

2417-3006 590 Start 2465 Start 2458 Start 2483

2706

End 2920 End 2956 End 2915

2834-3557 724 Start 2861 Start 2869 Start 2888

3010

3106/3107 Del Del Del

End 3350 End 3373 End 3243

2972-3557 586 Start 2999 Start 2999 Start 3031

31O6/31O7b Del Del Del

3423 Silent

End 3422 End 3460 End 3425 

3234-3557 324 Start 3265 Start 3258 Start 3292

3423b Silentb

End 3548 End 3545 End 3541

3441-3940 500 None Start 3487 Start 3491 Start 3499

End 3916 End 3920 End 3847

3635-4162 528 None Start 3667 Start 3662 Start 3725

End 4126 End 4061 End 4044

3931-4728 798 Start 3964 Start 3968 Start 3987

4135 Try ... His

End 4399 End 4427 End 4436

4183-4728 546 None Start 4208 Start 4249 Start 4208

End 4657 End 4657 End 4642

4392-4982 591 Start 4449 Start 4453 Start 4440

4769 Silent

End 4860 End 4935 End 4877

4447-4982 536 Start 4492 Start 4492 Start 4492

4769b Silent
140 LEVIN, CHENG, AND REEDER

TABLE Continued

Comparison with Anderson

Length of

Amplified amplified Anderson Anderson Template Template Template Amino acid

Primer set region region No. CHR 9947A GM03798 change

End 4958 End 4921 End 4931

4797-5553 757 Start 4838 Start 4845 Start 4838

4985 Silent

5186 Silent

End 5327 End 5324 End 5215

4976-5553 578 Start 5000 Start 5007 Start 5016

5186. Silentb

End 5516 End 5521 End 5400

5318-5882 565 None Start 5361 Start 5360 Start 5371

End 5754 End 5758 End 5800

5700-6262 563 None Start 5741 Start 5744 Start 5754

End 6149 End 6163 End 6136

5999-6526 528 Start 6043 Start 6058 Start 6047

6221 Silent

6371 Silent

End 6442 End 6503 End 6456

6242-6526 285 Start 6271 Start 6302 Start 6293

6371. Silent.

End 6520 End 6520 End 6520

6426-7030 605 Start 6451 Start 6474 Start 6487

6791 Silent

6849* G (0.3A)h* Thr .. Ala

End 6916 End 6930 End 6885

6744-7255 512 Start 6775 Start 6782 Start 6801

6849b. G (0.3A)h* Thr .. Ala

7028 Silent

End 7215 End 7221 End 7177

7075-7792 718 None Start 7123 Start 7123 Start 7130

End 7602 End 7601 End 7547

7215-7792 578 Start 7263 Start 7280 Start 7273

7645 Silent

End 7722 End 7769 End 7706

7645-8215 571 Start 7671 Start 7666 Start 7701

7861 Silent

End 8149 End 8155 End 8156

7901-8311 411 None Start 7960 Start 7959 Start 7960

End 8289 End 8288 End 8258

8164-8669 506 Start 8211 Start 8212 Start 8230

8448 Met.. Thr

8503 Silent

End 8646 End 8641 End 8637

8539-9059 521 Start 8581 Start 8582 Start 8581

8860 Thr .. Ala

End 9019 End 8999 End 8991

8903-9403 501 Start 8947 Start 8944 Start 8951

9315 Phe .. Leu

End 9380 End 9381 End 9370

9309-9848 540 Start 9334 Start 9333 Start 9333

9559 Arg.. Pro

End 9823 End 9827 End 9800

9449-9995 547 Start 9476 Start 9485 Start 9479

9559b Arg .. Pro

End 9964 End 9940 End 9911

9754-10275 522 None Start 9777 Start 9781 Start 9808

End 10225 End 10251 End 10184

10127-10556 430 None Start 10168 Start 10166 Start 10180

End 10534 End 10536 End 10524

10386-11166 781 None Start 10410 Start 10416 Start 10439

End 10899 End 10916 End 10865

10704-11267 564 None Start 10734 Start 10742 Start 10758

End 11223 End 11197 End 11167

11001-11600 600 Start 11026 Start 11040 Start 11059

11335 Silent

End 11461 End 11517 End 11497
HUMAN mtDNA STANDARD REFERENCE MATERIAL 141

TABLE Continued

Comparison with Anderson

Length of

Amplified amplified Anderson Anderson Template Template Template Amino acid Primer set region region No. CHR 9947A GM03798 change

11403-11927 525 Start 11428 Start 11432 Start 11456

11719 Silent

End 11795 End 11853 End 11855

11760-12189 430 Start 11784 Start 11802 Start 11802

11878 Silent

End 12159 End 12164 End 12163

11901-12876 976 None Start 11926 Start 11926 Start 11961

End 12404 End 12443 End 12397

12357-12876 520 Start 12404 Start 12391 Start 12391

12612 Silent

12705 Silent

End 12769 End 12849 End 12775

12601-13123 523 Start 12627 Start 12645 Start 12643

12705b Silentb

End 13102 End 13045 End 13024

12793-13343 551 None Start 12817 Start 12807 Start 12816

End 13295 End 13307 End 13266

13188-13611 424 Start 13238 Start 13238 Start 13244

13572 Silent

End 13587 End 13593 End 13590

13518-13935 418 Start 13541 Start 13541 Start 13571

13572b Silentb

13702 Gly ... Arg

13708 Ala ... Thr

13759 Ala ... Thr

End 13910 End 13921 End 13900

13715-14118 404 Start 13775 Start 13760 Start 13760

13966 Thr ... Ala

End 14094 End 14110 End 14104

13899-14388 490 Start 13926 Start 13927 Start 13961

13966b Thr ... Ala

14199 Pro'" Thr

14272 Phe ... Leu

14365 Silent

End 14369 End 14374 End 14342

14189-14926 738 Start 14216 Start 14216 Start 14240

14272b Phe ... Leu

14365b Silentb

14368 Phe ... Leu

14470 Silent

14766 lIe ... Thr

End 14699 End 14806 End 14698

14470-14996 527 Start 14502 Start 14513 Start 14527

14766b lIe ... Thrb

End 14957 End 14972 End 14956

14909-15396 488 Start 14941 Start 14933 Start 14950

15326 Thr ... Ala

End 15380 End 15373 End 15359

15260-15774 515 Start 15305 Start 15293 Start 15287

15326b Thr ... Ala. 

15646 Silent

End 15754 End 15950 End 15723

15574-16084 511 Start 15637 Start 15599 Start 15601

15646b Silentb

End 16056 End 16058 End 16030

57 (HV1) 15971-16451 481 Start 16014 Start 16011 Start 16004

16183

16189

16311

16357

End 16193 End 16430 End 16403

16097-336 809 Start 16125 Start 16130 Start 16151

16183b

16189b
142 LEVIN, CHENG, AND REEDER

TABLE Continued

Length of

amplified Anderson Anderson Template Template Template Amino acid

region No. CHR 9947A GM03798 change

1631lb

16357b

16519

End 16193 End 59 End 103

477 Start 16131

16183b

16189b

16193. C(ins)

16223

16278

16519b

End 40

Primer set

Amplified

region

-21M13C

cloned DNA 16133-40

Comparison with Anderson

Note. , basepair change came before the readable sequence; E., basepair change came af'kr the readable sequence; -, basepair same as

in Anderson sequence; h*, possible heteroplasmic site. * This heteroplasmy seen in the first CHR cell culture line was not seen with the

second CHR cell culture line. It is the second CHR cell culture line that is supplied in NIST SRM 2392; Start, start of readable sequence; End,

end of readable sequence; CHR cells, sequence based on two amplifications and cycle sequencing procedures in first cell culture line and at

least one amplification and cycle sequencing procedure with the second cell culture line; 9947A cells, sequence based on two amplifications

and cycle sequencing procedures; GM03798, sequence based on three to four amplifications and cycle sequencing procedures; ins, insertion;

Del, deletion; ND, not done.

" Numbers correspond to Anderson sequence (Anderson et al. 1981).

Change also seen in previous primer set.

This primer is used for sequencing the cloned DNA of the HV1 region.

the entire mtDNA were completely amplified and sequenced at least twice (GM03798 was done three

times). There were 13, 9, and 4 differences in the noncoding regions of templates CHR, 9947A, and

GM03798, respectively, and 33, 23, and 19 differences

in the coding regions of templates CHR, 9947 A, and

GM03798, respectively. All of the differences from

Anderson found in these three templates are shown in

Fig. 2 along with many of the diseases that have been

noted in the literature (Wallace et al. 1997). None of

the basepair changes found in the coding regions of the

three templates sequenced at NIST correlate with any

of the changes found associated with these published

disease states.

Meaning of the differences from Anderson. Since all

three templates had come from apparently normal individuals, it was of interest to determine if the differences in the coding regions would actually cause amino

acid changes in the resultant protein structures. The

genetic code for human mtDNA is slightly different

from the universal genetic code (Anderson et al. 1981).

One needs to consider these .differences in the universal genetic code when determining the amino acid sequence designated by the 3-bp codons in mtDNA. Many

of the differences from the Anderson sequence were in

the third position wobble and did not affect the amino

acid sequence (silent changes) (Table 2). However

CHR, 9947A, and GM03798 had 10 (9 without the

heteroplasmyat bp 6849), 12, and 8 different basepairs, respectively, that would result in a different

amino acid from that designated by the Anderson sequence (Table 2). However, data from the literature

indicate that perhaps 14 basepair designations in the

consensus sequence of Anderson may not be the sequence found in the majority of normal individuals

(Howell et al. 1992; Marzuki et al. 1992). Our results

agree with 11 of these 14 new designations (Table 3).

Examination of our results using these new designations indicates that only 4 (3 without the heteroplasmy

at bp 6849), 6, and 2 differences for CHR, 9947A, and

GM03798, respectively, would result in amino acid

changes. These structural changes, however, do not

necessarily mean a functional change has occurred in

the protein. To determine if a functional change has

occurred, one still needs to decipher whether the amino

acid change is in an active site on the protein.

The interlaboratory evaluation of the CRR template.

An interlaboratory evaluation was conducted by four

laboratories including NIST. All of the laboratories

essentially followed the NIST protocol sent with the

DNA from the first CHR cell culture line and the prim~

ers. Any changes to the protocol are listed under Materials and Methods. Each laboratory was instructed to

amplify and sequence the 58 areas designated by the

58 primer sets and also to sequence the cloned DNA for

the HVI region. Laboratory 1 amplified and sequenced

each area at least twice. The other labs amplified and

sequenced the areas from one to six times. Laboratories 1, 2, and 3 found essentially the same polymorphisms. Laboratory 4 had less experience with sequencing mtDNA and did find differences that the

other laboratories did not observe. Data were .excluded
HUMAN mtDNA STANDARD REFERENCE MATERIAL 143

FIG. 1. Agarose gel electrophoresis of PCR products from 58 primer sets designed for human mitochondrial DNA. (A) Lane L, 123- ladder from Gibco BRL; lane c1, negative control with no primers; lane c2, negative control with no DNA; lanes 1-28, PCR products from primer sets 1-28. (B) Lane L, 123-bp ladder from Gibco BRL; lanes 29-58, PCR products from primer sets 29-58.

from the analysis of this interlaboratoy evaluation if

the following conditions were observed: (1) The computer results were ambiguous as indicated by calling a

peak "N" rather than A, C, G, or T. (2) The differences

from Anderson were not consistently seen within a

laboratory, i.e., if the laboratory sequenced in both the

forward and the reverse directions and one direction

agreed with Anderson and the other direction did not

agree, we assumed the results that agreed with Anderson were correct. (3) Within anyone laboratory, the

difference from Anderson was seen with one primer

set, but not in the overlapping sequences seen in the

previous or subsequent primer sets. Even with these

exclusions, Laboratory 4 had many differences that

were not seen by the other labs. One problem was that

they did not provide data from primer sets 29, 39, and

, and we were unable to check those overlapping

sequences. Laboratory 2 was unable to sequence the

clone, which was not a problem for the other laboratories. Laboratory 3 was missing data from primer sets

36 and 48. Laboratories 1 and 3 noted a heteroplasmy

5 In final preparation of this SRM, anew blood sample was obtained from CHR and a new cell culture line was established. The

sequence analysis of the new CHR was identical to the first cell line

except no heteroplasmy was found at bp 6849. Therefore, the cell line

supplied with this SRM does not have this heteroplasmy. The data

on the first cell line are included in the text to indicate the agreement

at base number 6849 (Anderson found an A at this

site). Laboratory 1 found a G at this site, but closer

examination of the electropherogram showed that an A

peak existed under the G peak. Laboratory 3 also noted

the A/G heteroplasmy at this site. Laboratory 4 did not

note the heteroplasmy, but when their electropherograms were .examined at NIST, the A/G heteroplasmy

was noted. NIST did not have the electropherograms of

Laboratory 2, but on questioning them, they agreed

that the heteroplasmy was there, but that they had

missed it. One of the problems with finding heteroplas.

mic sites is that if the computer call is the same as

Anderson, one would not necessarily examine that site

more closely. If the computer call is different from

Anderson, one would look more closely at the electropherogram and then note the presence of a smaller

peak under the main peak.

With the exceptions of the differences noted here

the interlaboratory evaluation was successful in that

most of the laboratories found the same results. The

many differences noted by Laboratory 4, which was

less experienced at sequencing mtDNA, confirm and

emphasize the need for a standard reference matein the interlaboratory evaluation that was done with the first cell

line.
4135 :1 512

10. kb deletion

Complex I genes

(NADH dehydrogenase)

Complex III genes

(ubiquinoJ: cytochrome c

oxidoreductase) DTransfer RNA genes

Complex IV genes

(cytochrome c oxidase)

Complex IV genes

(ATP synthase) Ribosomal RNA genes

FIG. 2. Human mitochondrial DNA (color-coded circle) showing positions of genes, diseases, and deletions (Wallace, 1992; Wallace et al. 1997)

and the polymorphisms found in three apparently normal individuals examined for this study. The pink (CHR), green (9947A), and blue (GMO3798)

numbers and lines along the outside of the color-coded circle indicate the locations of differences from Anderson found in this study. Locations of

disease base substitutions are shown on the inside of the circle. Abbreviations: ADPD, Alzheimer disease and/or Parkinson disease; DEAF

neurosensory hearing loss; LDYT, LHON plus dystonia;LHON, Leber hereditary optic neuropathy; MELAS, mitochondrial encephalomyopathy,

lactic acidosis, and stroke-like episodes; MERRF, myoclonic epilepsY and ragged-red fiber disease; MMC, myopathy and cardiomyopathy; NARP

neurogenic muscle weakness, ataxia, and retinitus pigrnentosum. Large deletions are shown by the concentric black semicircles along the outside

of the colored circle. tRNA genes (white .areas in color-coded circle) are indicated by their amino acid single-letter abbreviation. Modified with

permission of the publisher from Wallace (1992) where additional information on this figure may be found. 6849*, see legend to Table 2.
HUMAN mtDNA STANDARD REFERENCE MATERIAL 145

TABLE 3

Errors vs Polymorphisms in mtDNA Sequence Determined by Anderson

Basepair

Anderson designation ...

literature designation

(No. foundINo. examined)

1438

3423

4769

4985

8860

11335

11719

12308

13702

14199

14272

14365

14368

15326

A... Ga

G... T (87/87)b

A... G (28/3W

G... A (9/W

A... Ga

T ... C (8/8)b

G'" A (26/37)b

A ... G (3/W

G ... C (105/105)b

G... T(9/W

G ... C (9/W

G ... C (9/W

G ... C (9/W

A... G (6/6)b

Anderson designation ...

NIST mt SRM designation

(No. foundINo. examined) Change

A"'G(3/3)

G-+T (3/3)

A"'G (3/3)

G"'A (3/3)

A"'G (3/3)

T"'C (3/3)

G...A (1/3)

Change not found

G"'C (3/3)

G"'T (3/3)

G"'C (3/3)

G"'C (3/3)

G"'C (3/3)

A"'G (3/3)

12 s rRNA

SILENT

SILENT

SILENT

Thr ... Ala

SILENT

SILENT

tRNA'eu

Gly ... Arg

Pro'" Thr

Phe ... Leu

SILENT

Phe ... Leu

Thr ... Ala

Note. Anderson et at. (1981) seqllenced mtDNA mainly from a single h1lIIlan placenta, although some regions were from HeLa cells. Five ambiguous regions (bp 10, 934, 935, 14272, 14365) were assumed by Anderson to be same as that found in bovine mtDNA. a Marzllki et al. 1992.

Howell et al., 1992.

rial for sequencing mtDNA. If Laboratory 4 had the

NIST mtDNA SRM 2392 and had run it alongside their unknown sample, they would have realized

that they were finding an undue number of differences and could have reexamined their procedures to

try to determine the reason for these excessive

changes.

CONCLUSIONS

A NIST standard reference material (SRM 2392)

that allows one to sequence any region or the .entire

569 bp that comprise human mtDNA has been prepared. Fifty-eight pairs of unique primers have been

designed, tested, and shown to work well in the amplification and sequencing procedures. The two DNA templates (CHR and 9947A) included in the SRM have

characteristic polymorphisms throughout the noncoding and coding regions of the DNA and, therefore, can

serve as positive controls during PCR amplification

and sequencing. None of thesepolymorphisms correspond to any of the published basepair changes that

have been correlated with specific diseases.

Compared to the Anderson sequence, CRR mtDNA

had 13 differences in the noncoding regions and 33

differences in the coding regions, and the 9947 

mtDNA had 9 differences in the noncoding regions and

23 differences in the coding regions. GMO3798, whose

data are included for comparison and information, had

4 differences in the noncoding regions and 19 differences in the coding regions. These differences in the

coding regions do result in some amino acid changes in

the proteins coded for by mtDNA. Four laboratories

participated in an interlaboratory evaluation of the

CHR template; some differences between laboratories

were noted, but, in general, agreement was good. The

use of NIST SRM 2392 will provide quality control to

the scientific and medical communities when they sequence human mtDNA.

ACKNOWLEDGMENTS

The allthors are grateful to The Bode Technology Group, Inc.

(Sterling, VA), lIT Research Institute (Newington, VA), and .Lark

Technologies, Inc. (Houston, TX) for their participation in the interlaboratory evaluation. The authors also acknowl~dge the funding

received from the National Institute of Justice throllgh the NIST

Office of Law Enforcement Standards and the support from the

Advanced Technology Program at NIST. Th~ authors thank Prasad

Reddy for his help and advice on cloning, MaryPat Jones for confirming the sequences of some areas of the second CHR cells, and

Catherine O'Connell for the use of her compllter with the Gene

Runner Program and her advice in the use of the program.

REFERENCES

Anderson, S. , Bankier, A. T., Barrell, B. G., deBrujin, M. H. L.

Coulson, A. R., Drouin, J., Eperon, 1. C., Nierlich, D. P., Roe, B. A.,

Sanger, F., Schreier, P. H., Smith, A. J. H., Staden, R, and Young,

1. G. (1981). Sequence and organization of the human mitochondrial genome. Nature 290: 457-465.

Ballinger, S. W., Bouder, T. G., Davis, G. S., Judice, S. A., Nicklas

J. A. , and Albertini, R. J. (1996). Mitochondrial genome damage

associated with cigarette smoking. Cancer Res. 56: 5692-5697.

Bendall, K. E., and Sykes, B. C. (1995). Length heteroplasmy in the

first hypervariable segment of the human mtDNA control region.

Am. J. Hum. Genet. 57: 248-256.

Bogenhagen, D., and Clayton, D. A. (1974). The number ofmitochondrial deoxyribonucleic acid genomes in mouse L and human HeLa

cells. J. Biol. Chem. 249: 7991-7995.

Grossman, L. 1. (1995). Mitochondrial mutations and human disease.

Environ. Mol. Mutagen. 25(Suppl. 26): 30-37.

Holland, M. M., Fisher, D. L., Mitchell, .L. G., Rodriquez, W. C.,

Canik, J. J., Merril, C. R., and Weedn, V. W. (1993). Mitochondrial

DNA sequence analysis of h1lIIlan skeletal remains: Identification

of remains from the Vietnam War. J. Forensic Sci. 38: 542-553.
146 LEVIN, CHENG, AND REEDER

Holland, M. M., Fisher, D. L., Roby, R. K Ruderman, J., Bryson, C.

and Weedn, V. W. (1995). Mitochondrial DNA sequence analysis of

human remains. Crime Lab. Dig. 22: 109-115.

Howell, N., McCullough, D. A, Kubacka, 1., Halvorson, S. , and

Mackey, D. (1992). The sequence of human mtDNA: The question

of errors versus polymorphisms. Am. J. Hum. Genet. 50: 1333-

1337.

King, M. P. , and Attardi, G. (1989). Human cells lacking mtDNA:

Repopulation with exogenous mitochondria by complementation.

Science 246: 500-503.

Levin, B. C., Cheng, H., O'Connell, C., Reeder, D. J., and Holland, M.

(1995). Evaluation of alternative enzymes and additives to improve

sequencing of mitochondrial DNA Proc. Fifth Int. Symp. on Human

Identification, Scottsdale, Arizona, October 8- 1994, p. 170.

Levin, B. C. , Cheng, H., Holland, M., and Reeder, D. J. (1997).

Heteroplasmy responsible for difficulty experienced in sequencing

the human mitochondrial DNA HV1 region containing the Cstretch. Proc. Seventh Int. Symp. on Human Identification, Scottsdale, Arizona, September 19- , 1996, p. 166.

Marzuki, S., Lertrit, P., Noer, A. S., Kapsa, R. M. 1., Sudoyo, H.

Byrne, E. , and Thyagarajan, D. (1992). Reply to Howell et at. The

need .for a joint effort in the construction of a reference data base

for normal sequence variants of human mtDNA Am. J. Hum.

Genet. 50: 1337-1340.

Sambrook, J. Fritsch, E. F. , and Maniatis, T. (1989). "Molecular

Cloning: A Laboratory Manual " 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Wallace, D. C. (1992). Mitochondrial genetics: A paradigm for aging

and degenerative . diseases. Science 256: 628-632.

Wallace, D. C., Brown, M. D., and Lott, M. T. (1997). Mitochon"

drial genetics. In Emery and Rimoin s Principles and Practice

of Medical Genetics" (D. L. Rimoin, J. M. Connor, and R. E.

Pyeritz, Eds.), 3rd ed., Vol. 1

, pp.

277-332, Churchill Livingstone, New York.

Appendix D

Journal Article: 

B. C. Levin, K. A. Holland, D. K. Hancock, M. Coble, T. J. Parsons, L. 

J. Kienker, D. W. Williams, MP. Jones, and K. L. Richie, 

“Comparison of the Complete mtDNA Genome Sequences of 

Human Cell Lines - HL-60 and GM10742A - From Individuals with 

Pro-Myelocytic Leukemia and Leber Hereditary Optic Neuropathy, 

Respectively, and the Inclusion of HL-60 in the NIST Human 

Mitochondrial DNA Standard Reference Material - SRM 2392-I,” 

Mitochondrion 2, 387-400, (2003).

(See end of Appendix D for color representation of Figure 1.) 
lIitochondrion

ELSEVIER Mitochondrion 2 (2003) 387-.400

www.elsevier.comllocate/mito

Comparison of the complete mtDNA genome sequences of human

cell lines - HL-60 and GMIO742A - from individuals with

pro-myelocytic leukemia and leber hereditary optic neuropathy,

respectively, and the inclusion of HL-60 in the NIST

human mitochondrial DNA standard reference

material - SRM 2392-

Barbara C. Levin , Koren A: Hollanda , Diane K. Hancocka

Michael Coble , Thomas J. Parsons , Laura J. Kienker

Diana W. Williams , MaryPat Jones , Kristy L. Richie

NationalInstitute of Standards and Technology, lOa Bureau Drive, Mail Stop 83Jl, Gaithersburg, MD 20899-83JI. USA

Gettysburg College, Gettysburg, FA, USA

Armed Forces DNA Identification Laboratory, Rockville, MD, USA

The George Washington University, Washington, DC, USA

Federal Bureau of Investigation Laboratory, Quantico, VA, USA

Georgia Bureau of Investigation, Decatur, GA, USA

gNational Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA

Received 12 December 2002; received in revised form 26 January 2003; accepted 28 January 2003

* This paper is a contribution of the U.S. National Institute of Standards and Technology (NIST), Armed Forces DNA Identification

Laboratory (AFDIL), Federal Bureau of Investigation (FBI), the Georgia Bureau of Investigation (GBI) and the National Human Genome

Research Institute (NHGRI), National Institutes of Health (NIH) and is not subject to copyright Certain commercial equipment, instruments,

materials, or companies may be identified in this paper to specify the experimental procedure. Such identification does not imply

recommendation or endorsement by the authors, nor does it imply that the materials or equipment identified are the best available for this

purpose.

** Orders and requests for information concerning this SRM should be directed to the Standard Reference Materials Program, National

Institute of Standards and Technology, 100 Bureau Drive, Stop 3222, Gaithersburg, MD 20899-2322, USA. Tel.: + 1-301 "975-6776; fax: + I"

301-948-3730. E-mail: srminfoC1Ynistgov, web address http://www.nistgov/srm.

* Some of the data in this paper has been presented at the following meetings: The 12th International Symposium on Human Identification

Biloxi, MS, October, 2001; the American Academy of Forensic Sciences Annual Meeting, Atlanta, GA, February, 2002; the Third Annual

Biotechnology Retreat, Shepherdstown, WV, May, 2002; the United Mitochondrial Disease Foundation Milo Dallas 2002 Symposium and

Mitochondrial Standards Workshop, Dallas, TX, June, 2002; the Third Annual DNA Grantees' Workshop, National Institute of Justice, June

2002; and the 13th International Symposium on Human Identification, Phoenix, AZ, October, 2002.

* Corresponding author. Tel.: + 1-301"975-6682; fax: + 1-301-975-8505.

E-mail address: barbara.levinC1Ynist.gov (B.c. Levin).

1567-7249103/$20.00 ~ 2003 Published by Elsevier Science B.V. on behalf of Mitochondria Research Society.

doi: 10. 10 16/S 1567-7249(03)000 I 0-
388 c. Levin et at. Mitochondrion (2003) 387-400

Abstract

Forensic and clinical laboratories benefit from DNA standard reference materials (SRMs) that provide the quality control and

assurance that their results from sequencing unknown samples are correct. Therefore, the mitochondrial DNA (mtDNA)

genome of HL-6Q, a promyelocytic leukemia cell line, has been completely sequenced by four laboratories and will be available

to the forensic and medical communities in the spring of 2003; it will be called National Institute of Standards and Technology

(NIST) SRM 2392-1. NIST human mtDNA SRM 2392 will continue to be available and include the DNA from two apparently

healthy individuals. Both SRM 2392 and 2392-1 contain all the information (e.g. the sequences of 58 unique primer sets) needed

to use these SRMs as positive controls for the amplification and sequencing any DNA. Compared to the templates in SRM 2392

the HL-60 mtDNA in SRM 2392-1 has two tRNA differences and IIlore polymorphisms resulting in amino acid changes. Four of

these HL-60 mtDNA polymorphisms have been associated with Leber Hereditary Optic Neuropathy (LHON), one as an

intermediate mutation and three as secondary mutations. The mtDNA from a cell line (GM1O742A) from an individual with

LHON was also completely sequenced for comparison and contained some of the same LHON mutations. The combination of

these particular LHON associated mutations is also found in phylogenetic haplogroup J and its subset, J2, and may only be

indicative that HL-60 belongs to haplogroup J, one of nine haplogroups that characterize Caucasian individuals of European

descent or may mean that haplogroup J is mOre prone to LHON. Both these mtDNA SRMs will provide enhanced quality

control in forensic identification, medical diagnosis, and single nucleotide polymorphism detection.

~ 2003 Published by Elsevier Science B.V. on behalf of Mitochondria Research Society.

Keywords: Forensic identification; GMI0742A; Haplogroup J; HL-60; Leber hereditary optic neuropathy (LHON); Medical diagnosis;

Mitochondrial DNA sequence; Single nucleotide polymorphism (SNP); Standard reference material (SRM)

1. Introduction

On July 15, 1998, the Federal13ureau of Investigation (FBI) Director signed Standard 9.5, which

stated "The laboratory shall check its DNA procedures annually or whenever substantial changes are

made to the protocol(s) against an appropriate and

available National Institute of Standards and Tech~

nology (NIST) standard reference material or standard

traceable to a NIST standard" . At the present time

there are a number of human DNA standard reference

materials (SRMs) available from NIST (Levin et aI.

2001). One of these SRMs on mitochondrial DNA

(mtDNA SRM 2392) is used by the forensic community for human identification .and by the medical

community for diagnoses of a number of human

diseases now known to be associated with specific

mutations, insertions and deletions of mtDNA (Levin

et aI., 1999). This SRM was prepared by NIST to

provide quality control to the scientific community

when they amplify and sequence human mtDNA or

any DNA and includes DNA from two apparently

healthy individuals (CHR and 9947A) plus cloned

DNA of an area in CRR containing a C-Stretch

beyond which sequencing becomes difficult. All the

information necessary to successfully conduct the

polymerase chain reaction (PCR) amplification process, cycle sequencing steps, gel separation, and data

analysis to obtain the final DNA sequence is provided

as well as the information on the sequence of 

unique primer sets that allow the sequencing of any

specific portion or the entire mtDNA (16,569 bp)

without any gaps. Following an interlaboratory evaluation, SRM 2392 became available to the public in

December 1999.

The FBI acknowledges the utility of DNA SRMs to

provide the quality control and quality assurance that

the results from forensic laboratories that are sequencing unknown samples are correct. One of the FBI's

Combined DNA Index Systems now includes mtDNA

from unidentified remains, as well as from relatives of

missing persons. In order for authorized laboratories

to contribute to these indices, certain quality standards

must be met. These include the use of DNA from the

human cell line HL-60 as a positive control to be run

in conjunction with the unknown samples. HL-60 was

chosen as the positive control because of several

features present in HL-60 but not in the cell lines

currently available in NIST SRM 2392. Some of the

advantageous features of HL-60 are well-spaced

polymorphisms throughout the non-coding hypervariable regions, HV1 and HV2, of the mtDNA control
13. C. Levin et at. Mitochondrion (2003) 387-400 389

region, and no insertions at the HV2 C-stretch area

(positions 303~309). One of the current templates in

SRM 2392, the CHR DNA has a C-stretch in the HV1

region caused by .a T to Cchange at position 16189

that produces a length heteroplasmy (Bendall and

Sykes, 1995; Butler and Levin, 1998). Sequencing

through this C-stretch is difficult and time-consuming

and results in the need to perform additional

sequencing reactions to resolve this region and the

area following the C-stretch. The CHR template was

chosen by NIST specifically for the C-stretch region

since some laboratories wanted the opportunity to

address this difficult sequencing problem and try to

resolve it. The DNA from the other .current SRM

DNA template, 9947 A, has only two polymorphisms

in the HV1 region with respect to the Cambridge

Reference Sequence (Anderson etaI., 1981; Andrews

et aI. , 1999) and those polymorphisms are at common

sites. For the work that the forensic laboratories are

doing on human identification, several evenly spaced

polymorphisms within. the HV1 region are more

desirable to differentiate the positive control from the

test sample. Thus, the FBI suggested to NIST that the

development of a SRM containing the HL-60 DNA

template would be of great utility to the forensic

community. All of the research and the interlaboratory

evaluation of theHL-60 template necessary to produce this new SRM containing HL-60 have been

completed and are described in this paper. Both SRM

2392 (containing two DNA templates) and SRM

2392-1 (containing one DNA template) should fulfill

the needs of forensic laboratories by providing

additional quality control when sequencing human

mtDNA. Corroboration of the SRM results will

provide assurance that the techniques being used for

amplification and sequencing unknown DNA are

being conducted correctly.

The DNA templates in SRM 2392 came from

apparently healthy individuals; however, HL-60 in

SRM 2392-1 is from a promyelocytic cell line from

peripheral blood leukocytes provided by a 36-year-old

Caucasian female with acute promyelocytic leukemia.

Therefore, it was of interest to determine if the HLDNA contained specific mutations that may be

characteristic of this disease. Although more DNA

samples from leukemic patients would have to be

examined to associate specific mutations with this

disease, we did find four polymorphisms that are

considered either intermediate or secondary mutations

associated with leber hereditary optic neuropathy

(LHON) (Wallace et aI., 1997). We, therefore,

sequenced the mtDNA from cell line GM1O742A

that was prepared from a patient with LHON for

comparison with the HL-60 mtDNA sequence. Two

of the four LHON-associated mutations in HLwere also found in GM10742A. Two additional

mutations associated with LHON were found in

GM10742A, one primary and one secondary mutation. Many of the polymorphisms common to both

HL-60 and GM10742A (some of which are associated

with LHON) characterize the phylogenetic haplogroup J and sub-haplogroup Jz, a subset of J (FinniHi

et aI., 2001; Helgason et aI., 2001; Herrnstadt et aI.

2002). Haplogroup J is one of nine haplogroups .that

characterize Caucasian populations of European

decent. A number of papers in the literature indicate

that individuals with LHON usually belong to haplogroup J and suggest that those in haplogroup J may

therefore be mC?re prone to this mtDNA disease than

those in other haplogroups (Brown et aI., 2002;

Herrnstadt et aI., 2002; Torroni et aI., 1997).

2. Materials and methods

1. HL-60 DNA

DNA from the HL-60 cell culture was extracted,

isolated, and quantified by the Professional Services

Department of the American Type Culture Collection

(A TCC, Manassas, VA).

DNA was isolated from the HL-60 cell culture

using the QIAamp DNA Blood Mini Kit (Qiagen

Inc., Valencia, CA). Quantification was determined

by the Quantiblot Human DNA Quantification Kit

(Applied Biosystems, Foster City, CA). The final

concentration, 1.4 ng/lJ.l, is based on 12 replicate tests.

The DNA purity (A260:A280 = 1.9) was determined spectrophotometrically. The integrity of DNA

was determined electrophoretically using 0.4%

agarose gels.

1. GM1O742A DNA

The lymphoblast cell line GM1O742A was purchased from NIGMS Human Genetic Mutant Cell

Repository, Coriell Institute of Medical Research
390 c. Levin et at. Mit9chondrion (2003) 387-400

Camden, NJ. The cells were grown at NIST in RPMI

1640 plus L-glutamine and sodium bicarbonate.

growth media (Sigma, St. Louis, MO) plus fetal calf

serum (20%) (Sigma) plus the antibiotics Strepto"

mycin and Penicillin (final concentration: 100 U/ml)

(Sigma). DNA was extracted using the QIAGEN

Plasmid Kit following the Plasmid Mini Purification

Protocol.

1.2. mtDNA primers

Fifty-eight sets of unique primers (19-28 bp)

for sequencing any portion or the entire human

mtDNA (16 569 bp), including both the HV1 and

ffV2 regions, were computer-designed using GENE

RUNNER FOR WINDOWS (Hastings Software, Inc.

Hastings, NY) and were the same as those used for

SRM 2392 (Levin et aI., 1999) with the exception of

the reverse primer of set 51. During the course of this

study, this primer was changed since it contained a C

at nucleotide position (np) 14 368. Since this is the

reverse primer, it would bind to a G at np 14 368.

Andrews et al. (1999) in their reevaluation of the

placenta originally used to sequence human mtDNA

in 1981 (Anderson eta1., 1981) found that np 14 368

should have a C at that position. The new reverse

primer 51 is 5'- TT AGCGA TGGAGGT AGGATf

3' (np 14 368 is in bold and underlined) which binds

to the C at position 14 368. The 5' end is np 14, 388

and the 3' end is 14 367. The 58 sets of primers were

custom-made by Bio-Synthesis, Inc. (Lewisville

TX); the new reverse primer 51 was obtained from

Invitrogen (Carlsbad, CA). Those laboratories using

either SRM 2392 or SRM 2392-1 should use the new

reverse primer 51.

1.3. Polymerase chain reaction (PCR) at NIST

The PCR mixture contained: HL-60 DNA (1 j.Ll;

1.4 ng), AmpliTaq Gold(jj) DNA polymerase, (0.5j.Ll;

5 units) (Applied Biosystems), 10 buffer (5 j.Ll)

containing 100 mmolll Tris-HCl, pH 8.3, 500 mmol/l

KCl, 15 mmolll MgCl2 and 0.01 % (w/v) gelatin

(Applied Biosystems), 10 mmolll dNTP mix (1 j.Ll)

(Invitrogen), 10 j.Lmolll forward and reverse primers

(lor 2 j.Ll), plus water to make a final volume of 50 j.L1.

Thermal cycling was conducted in a PerkinElmer

thermocycler Model 9700 and started with 10 min

at 95OC, followed by 35 cycles of 20 s at 94O

(denaturation), 10 s at 56OC (annealing), 30 sat no

(extension) and ended with a final extension of 7 min

at no

Amplified DNA was purified with a QlAquick

PCR Purification Kit (Qiagen) and the purity and size

of the PCR product was determined by electrophoresis

in 2% agarose gels in 1 X TBE buffer containing

5 j.Lg/ml ethidium bromide (Sigma).

1.4. Sequencing at NIST

Cycle sequencing using fluorescent dye-labeled

terminators was performed with an ABI PRISM

(jj)

BigDye

(jj) 

Terminator Cycle Sequencing Ready Reac"

tion Kit with AmpliTaq(jj) DNA Polymerase, FS

(Applied Biosystems).

Cycle sequencing reactions in both the forward

and reverse modes were conducted with a 9700

PerkinElmer thermal cycler and started with 1 min at

96Oe. The reaction then underwent 25 cycles of 96O

for 15 s (denaturation), 50oC for 5 s (annealing), and

60oC for 2 min (extension). The DNA products were

purified using Edge Gel Filtration Cartridges (Edge

BioSystems, Gaithersburg, MD).

Sequencing and data analysis of the purified

DNA were performed using an Applied Biosystems

PRISM

(jj) 

Model 310 Genetic Analyzer with POP-6TM

polymer system and 47 cm X 50 j.Lm capillaries

(Applied Biosystems). Sequence data were analyzed

with Sequencing Analysis Software 3. , and com~

parisons to the Cambridge Reference Sequence

(1981) were performed with Sequence Navigator

Software 1.01.

2. Interlaboratory evaluation of HLThree laboratories, in addition to NIST, participated in an interlaboratory evaluation of the sequence

of HL-60 DNA. These laboratories included the FBI

Laboratory, FBI Academy, Quantico, V A 22135;

Armed Forces DNA Identification Laboratory

(AFDIL), Armed Forces Institute of Pathology,

Rockville, MD 20850; and the Georgia Bureau of

Investigation (GBI), Decatur, GA 30034. Each

laboratory was asked to amplify and sequence the

entire mtDNA ofHL-60. NIST provided: (1) a tube of

DNA containing the extracted DNA ready for PCR

amplification; (2) the 58 sets of primers labeled with

either F# (forward primer) or R# (reverse primer); (3)

a table to record the results; and (4) any other supplies
c. Levin et at. Mitochondrion (2003) 387-400 391

that were needed and requested by the participants.

They were allowed to use any protocol for amplification or sequencing that they wished, but were

requested to provide a copy of that protocol to NIST.

NIST also requested copies of the electropherograms

to enable us to resolve any discrepancies; although as

it turned out, there were no discrepancies.

1. Differences in methodology used by the

laboratories in the interlaboratory evaluation

1. Armed forces DNA identification laboratory

(AFD1L). AFDIL has developed a high-throughput

automated sequencing procedure using 12 primer sets

that produce overlapping PCR products ranging from

825 to 1886 bp. The primers used to amplify the first

11 products were based on those published in Levin

et al. (1999) and are: AmpOl - F361/R2216; Amp02

- F1993/R3557; Amp03 - F3441/R4983; Amp04 -

F4797/R6526; Amp05 - F6426/R83 11; Amp06 

F8164/R9848; Amp07- F9754/R11600; Amp08 -

Fl1403/R13123; Amp09 - F12793/R14388; Amp10

- F14189/R15396; Amp11 - F15260/R16084. The

primers used to amplify the control region were

developed at AFDIL and are Amp12 - F15878/R649

(Fl5878 is TTAACTCCACCATTAGCACC and

R649 is TTTGTTT A TGGGGTGATGTGA).

2.1.1. 1. AFD1L PCR amplification. The PCR

mixture contained HL-60 DNA (1 j.Ll), AmpliTaqGoldi!9 DNA polymerase (1 j.Ll) (Applied Biosystems),

10 X PCR buffer (5 j.L1) (Applied Biosystems),

dNTP' s (0. 2 nunol/l) (Invitrogen), 2 j.Ll of forward

and reverse primers (10 j.Lmol/l) (MWG Biotech

High Point, NC) plus dHzO to a final volume of

50 .j.Ll. The lOx PCR buffer was the same as that used

by NIST. Thermal cycling was conducted in a

PerkinElmer 9700 thermocycler with the following

conditions: 10 min at 96OC (activation of AmpliTaq

Goldi!9), plus 40 cycles of 94OC for 15 s, 56OC for 30 s,

and noc for 1 min. The purity and size of the PCR

products were assessed by electrophoresis in a 0.

agarose gel containing 0.3 j.Lg/ml of ethidium bromide. The PCR products' were purified with Shrimp

Alkaline Phosphatase/Exonuclease I (Amersham

Pharmacia, Piscataway, NJ). Five j.Ll of exonuclease

1(10 U/j.Ll) and 10 j.Ll of Shrimp Alkaline Phosphatase

(1 U/j.Ll) was added to each tube containing PCR

product. The tubes were heated at 37OC for 15 min

followed by 94OC for 15min in a perkinElmer 9700

thermocycler.

1.1. 2. AFD1L sequencing. Cycle sequencing

was performed with the ABI PRISMi!9 BigDye i!9

terminators (original version) cycle sequencing kit

(Applied Biosystems). The sequencing mixture contained 9 j.Ll dHzO, 6 j.Ll BigDye

i!9 dilution buffer

(400 mmolll TRIS, 10 mmol/l MgClz, pH 9.0), 2 j.Ll

BigDyei!9 terminator reaction mixture, 1 j.Ll of forward

or reverse primer (10 j.Lmolll each) and 2 j.Ll of HLPCR product for a total vol of 20 j.Ll. A few of the

sequencing primers (e.g. R649) required the use of the

Am PRISMi!9 dGTP BigDye i!9 terminator kit (Applied

Biosystems). Thermal cycling was conducted in a

PerkinElmer 9700 thermocycler at the following

conditions: an initial 1 min denaturation at 96O

followed by 25 cycles of 94OC for 15 s, 50oC for 5 s,

and 60oC for 2 min. The DNA product was purified by

filtration through a spin column matrix (Edge

BioSystems, Gaithersburg, MD). Electrophoresis

and sequencing were performed with an Am PRISMi!9

3100 Genetic Analyzer (Applied Biosystems) using

POP-6TM polymer (Applied Biosystems) with a 50 

capillary. Data analysis was executed using

Sequencher Plus 4. 5b11 (Gene Codes, Ann Arbor,

MI). The HL-60 sequence differences were identified

by comparison to the Cambridge Reference Sequence

as revised by Andrews et aI. (1999). In most cases

sequence information was acquired for both the

forward and reverse directions. In some regions, two

separate reactions using the same primer were

routinely conducted (indicated by ' 2 X ' in the

following list of primers) to achieve full sequence

confirmation. A total of 95 sequencing reactions plus

one pGEM reaction were conducted in a 96 well

format. If the primer failed the first trial, the reaction

was repeated. The finding of a heteroplasmy at

nucleotide position (np) 12,071 was also confirmed

by an additional PCR and sequencing reaction.

The following primers from Levin et al. (1999)

were used to sequence the 12 PCR amplicons:

AmpOl (F3611R2216): F361 , R921, F1234,

R1425, F873, R2216, F1657, R1769

Amp02 (F1993/R3557): F1993 , R2660, R2834,

R3557, F2417, R3006, F3234

Amp03 (F3441/R4983): F3441, R3940, F3931

(2 x), R4982, F4392, R4162
392 B.c. Levin et al. Mitochondrion (2003) 387-400

Amp04 (F4797/R6526): F4797 (2 X), R6526,

F5700 (2 X), F5318, R5882, F6242

Amp05 (F6426/R83 11): F6426 (2 X), R7255,

F7645 (2X), R8311, F7075, R7792

Amp06 (F8164/R9848): F8164 (2 X), R9059,

F8903, R9848, F8539, R9403, F9309

Amp07 (F9754/Rl1600): F9754 (2 X), R1O556

F11001 (2 X ), R11600, F1O386, R11267

Amp08 (F11403/R13123): F11403 (2 X), F12357

R13123, F11901 (2 X ), F12601, R12876

Amp09 (F12793/RI4388): F12793 , R13611

F13518 (2 X), R14388, F13188, R13343,

F13899, R13935

Amp 10 (F14189/R15396): F14189 (2 X), R15396,

F14909, R14996, F14470

Amp11 (F15260/R16084): F15260, R16084

F15574, R15774

Amp12 (F15878/R649): F15971 , R16175 (2 X),

F16450 (2 X), R274, F314 (2 X), R649 (2 X),

F16190, Rl6400.

In Amp12, F15971 came from Levin et al. (1999).

The other primers were designed by AFDIL and were

as follows:

R16175: TGGATTGGGTTTTTATGTA

F16450: GCTCCGGGCCCATAACACTTG

R274: TGTGTGGAAAGTGGCTGTGC

F314: CCGCTTCTGGCCACAGCACT

R649: TTTGTTTA TGGGGTGA TGTGA

F16190: CCCCATGCTTACAAGCAAGT

R16400: GTCAAGGGACCCCT A TCTGA.

1.2. Georgia Bureau of Investigation (GBI). The

GBI used the same protocol as that used by NIST with

the following exceptions: (1) a PerkinElmer model

9600 was used for thermal cycling; (2) amplified

DNA was electrophoresed in 2.75% agarose gels; (3)

the cycle sequencing was performed with a Perkin~

Elmer model 9600 using the program provided with

the BigDye4\) kit 

(25 cycles of 96OC for 10 s, 50oC for

5 s, and . 60oC for 4 min); (4) some samples that

needed 'to be cycle sequenced again using more

amplicon were not purified before precipitation; (5)

the samples were precipitated u.sing isopropanol

precipitation as per the BigDye4\) instructions; and

(6) comparison of the sequence data was performed

with Sequencer 3. 1.1 software (Gene Codes, Ann

Arbor, MI).

1.3. Federal Bureau of Investigation (FBI)

1.3. 1. FBI Polymerase chain reaction (PCR).

The PCR mixture contained from 0. 1 to 1.4 ng HL-60,

AmpliTaq Gold4\) Polymerase (2.5 units) (Applied

Biosystems), 10 X PCR buffer (5 fJ-l) (Applied

Biosystems), GeneAmp4\) dNTPs (0.2 mmol/l each)

(Applied Biosystems), forward and reverse primers

(0.4 fJ-mol/l each), plus dH2O to a final volume of

50 fJ-l. The 10 X buffer (pH 8.3) was the same as used

by NIST. Thermal cycling was conducted in a

GeneAmp4\) PCR System 

97QO (PerkinElmer) and

consistedof lO min at 95OC followed by 35 cycles of

94OC for 20 s, annealing temperatures of 50oC (primer

set 49), 51OC (primer sets 1 , 7, 44, 57), 52OC (primer

sets 6, 8 , 30, 45), 53OC (primer set 53) and 56OC (all

other primer sets) for 10 s, and 72OC for 30 sand

ending with a final extension of 7 min at 72O

Amplified products were purified by treatment with

Exo-SAP-IT (5 fJ-l for every 25 fJ-I of PCR product)

(USB Corp., Cleveland, OH). Samples of the PCR

products were electrophoresed in 1.2% agarose gels

containing ethidium bromide to assess the purity, size

and quantity of the PCR products.

1.3. 2. FBI sequencing. Cycle sequencing using

fluorescent dye~ labeled terminators was performed

irith an ABI PRISM4\) dRhodamine Terminator Cycle

Sequencing Ready Reaction Kit with AmpliTaq4\)

DNA Polymerase, FS (Applied Biosystems). Thermal

cycling was conducted in a GeneAmp4\) PCR System

9700 (Applied Biosystems) and started with 1 min at

96OC followed by 25 cycles of 96OC for 15 s, 50oC for

1 s and 60oC for 1 min and ended with a final

incubation at 15OC for 10 min. These products were

purified with Centri-SepTM spin columns (Princeton

Separation, Inc., Adelphia, NJ) and electrophoresed

on an ABI PRISM4\) 310 Genetic Analyzer (Applied

Biosystems) using POP-6TM and either a 47 or 61 

capillary. Comparison of the sequence data was

performed with Sequencer 4. 1 software (Gene

Codes).

The FBI also discovered the problem with original

primer set 51 (see Section 2). The reverse primer

incorporated a G at position 14,368 (an error in the

original Anderson et al. (1981) sequence that was

found and corrected by Andrews et aI. (1999)).
B.c. Levin et at. Mitochondrion (2003) 387-400 393

However, sequencing with primer set 52 showed a C

at position 14 368. They prepared a new set of primers

(primer set 51.5) to amplify approximately a 400 bp

fragment that encompassed np 14 368. The new

primers were: F14217: 5' CTAATCAACGCCCATAATCATAC-3' and R14620: 5' GTTTTCTTCTAAGCCTTCTCC- . The new primer set

confirmed that the correct base at position 14 368

was a C.

2.3. Permissions

The research to prepare SRM 2392-1 containing

HL-60 DNA was deemed exempt from the policy of

Part 27 of Title 15 of the Code of Federal Regulations

by the NIST Institutional Review Board and the

Director of the Chemical Science and Technology

Laboratory. This work fit into the exemption category

described in 15 CFR 27. 1O1(b)(4) which states:

Research, involving the collection or study of

existing data, documents, pathological specimens, or

diagnostic specimens, if, these sources are publicly

available or if the information is recorded by the

investigator in such a manner that subjects cannot be

identified, directly or through identifiers linked to the

subjects . ATCC also waived condition 3(c) in their

Material Transfer Agreement which states that .the

purchaser shall not sell, lend, distribute or otherwise

transfer the material or replicates to any others" for

the use of HL-60 in the NIST mitochondrial DNA

SRM 2392-1. They stated that, in their view

, 

as a

government agency, NIST will not be providing this

material as a commercial product despite the collection of fees for the SRM"

3. Results and discussion

HL-60 is a promyelocytic cell line from the peripheral blood leukocytes of a Caucasian female (age

36) with acute promyelocytic leukemia. HL-60 DNA

will be available in SRM 2392~1. The two DNA

templates available in SRM 2392 are from apparently

healthy individuals (both the DNA from CHR and

9947 A are from Caucasian females in their twenties

and thirties). The entire mtDNA of GM03798 (a

lO-year-old apparently healthy Caucasian male) was

amplified and sequenced at NIST, discussed in a

previous publication (Levin et aI., 1999), and is shown

in Fig. 1 in this paper; however, that DNA is not

included in SRM 2392 or SRM 2392-1. Another

mtDNA template (GM1O742A) from a 30-year-old

Caucasian male patient with LHON was also

sequenced at NIST; that sequence is shown in

Table 1 and Fig. 1, but the DNA is not included in

SRM 2392 or 2392-1. Both GM03798 and

GM1O742A can be obtained from the National

Institute of General Medical Sciences Human Genetic

Mutant Cell Repository, Coriell Institute for Medical

Research, Camden, NJ. At NIST, 58 primer sets

(Levin et al, 1999) were used to amplify the entire

HL-60 mtDNA and GMI0742A at least twice. In the

case of HL-60, all the PCR products were sequenced

in both the forward and reverse directions for a total of

four sequences for each amplicon. During this study,

reverse primer 51, which contained np 14 368, was

changed (see Section 2) to accommodate the difference found at that position by Andrews et aI. (1999)

when they resequenced the original placenta used by

Anderson et aI. (1981). Any sequence ambiguities

between experiments at NIST were resolved by

additional PCR reactions and sequencing.

The NIST sequencing results from the mtDNA of

HL-60, CHR, 9947A and GM1O742A are shown in

Table 1; all of these sequences plus GMO3798 are

shown in Fig. 1. Table 1 also compares the CHR,

9947A, HL-60 and GMI0742A templates to the

original Cambridge Reference Sequence (Anderson

et al., 1981) and the revised sequence (Andrews et aI.

1999). In Fig. 1, the HL-60 results are shown in black

numbers surrounding the circular depiction of human

mtDNA; the CHR results are shown in red; 9947A

results are shown in green; GMO3798 results are

shown in blue; and GM1O742A are in purple. At the

present time, GM03798 and GM1O742A are not part

of SRM 2392 or SRM 2392- , and the results are

presented for information and comparison only.

The numbering system in Table 1 and Fig. 1 is that

of the original Cambridge Reference Sequence

(1981). The Cambridge Reference Sequence (1981)

was based on a consensus analysis of a placenta, the

HeLa cell line and the bovine sequence (the Dovine

sequence was used in five ambiguous human sites).

The 1981 results were reexamined in 1999 by

Andrews et aI. who resequenced the original placenta.

The original Cambridge Reference Sequence was
394 B.C. Levin et at. Mitochondrion (2003) 387-400

'216--

__.

4216

ATPase 8

Fig. I. HL-60 DNA polymorphic differences from the Cambridge Reference Sequence and positions of LHON mutations are added to the figure

from Levin et al., 1999. This figure is a schematic of human mtDNA showing its circular double-stranded DNA and all the differences from

Cambridge Reference Sequence found in CHR (red), 9947A (green), GM03798 (blue), HL-60 (black), and GM1O742A (purple) as numbers

along the outside of the color-coded circle. Locations of the control region, rRNAs and genes (see footnote to Table I for abbreviations) coded

by human mtDNA are shown. The locations of the 22 tRNAs are noted by white areas in the circle and designated by their single letter amino

acid code. (Modified from Levin et aI. , 1999).

found to have a number of rare polymorphisms and

errors, which are noted in Table 1 with an asterisk.

Table 1 also shows the unique (D) coding region

polymorphisms (either silent with no amino acid

change or resulting in an amino acid change) in

CRR, 9947 A, HL-60 and GM1O742A that were

not found in the MITOMAP web site database

(http://www.rnitornap.org). This is a very extensive

internet database on the mitochondrial DNA polymorphisms found in the literature. CRR has five

polymorphisms (one produces an amino acid change),

9947 A has four polymorphisms (two producing amino

acid changes), HL-60 has two polymorphisms (one

producing an amino acid change and is heteroplasmic), and GM1O742A has two (one in the

16sRNA and one that is silent) that were not found

in the MITOMAP database.

Table 2 shows the number of differences compared

to the Cambridge Reference Sequence found in CRR,

9947 A, HL-60 and GM10742A. There are 13, 9, 

and 12 differences in the non-coding regions of CRR

9947A, HL-60, and GM10742A, respectively. The

non-coding region is the primary area used by the

forensic community for human identification. There
B.C. Levin et al. Mitochondrion (2003) 387-400

are 33, 33 and 31 differences ~n the coding regions

of CHR, 9947 A, HL-60, and GM1O742A, respectively. These differences would have resulted in 11 , 12

15 and 14 amino acid changes in their respective

proteins if one compares the results to the original

Cambridge Reference Sequence published by

Anderson et aI. (1981). If compared to the revised

Cambridge Reference Sequence (Andrews et aI.

1999), the actual amino acid changes would be

reduced to 3 in CHR, 4 in 9947 A, and 7 in both

HL-60 and GMI0472A (the non-normal cell lines

sequenced). HL-60 also has a change in the tRNA for

alanine and the tRNA for serine. It would be of

interest to examine other cell lines from patients with

acute promyelocytic leukemia or LHON to see if they

have any of these same mutations.

Table 3 shows the HL-60 and GMI0742A polymorphisms in the coding regions that (with the

exception of one CHR mutation included because

of its association with LHON) were not found in

the other templates in SRM 2392. This table also

indicates if the mutation has been associated with a

disease according to the MITOMAP web site

database. At least four HL-60 mutations (T4216C in

NADH dehydrogenase 1 , G13708A in NADH dehydrogenase 5 , and G15257A and G15812A in Cytochrome B) were found to be associated with LHON

(Wallace et aI., 1997). Four mutations associated with

LHON were also found in GM1O742A; these are

T3394C and T4216C in NADH dehydrogenase 1

G 11778A in NADH dehydrogenase 4, and G 13708A

in NADH dehydrogenase 5. Two of these mutations

T4216C and G13708A, were found in both HL-60 and

GM1O742A. The mutation G13708A was also seen in

the CHR template. LHON causes central vision loss in

patients in their twenties or thirties although the onset

of symptoms can occur both earlier and later. The

disease is inherited like other mitochondrial DNA

diseases through the maternal lineage. LHON was

first associated with a G 11778A mtDNA mutation

(Wallace et aI. , 1988) that results in an arginine to

histidine change in NADH dehydrogenase 4 at position 340 in a protein that contains 460 amino acids

(Lee and Levin, 2002). G 11778A is considered a

primary mutation causing LHON and was seen in

GMI0742A, a cell line from a patient who experienced the sudden onset of blindness at the age of 24.

Since 1988, 27 missense mutations and one mutation

395

in tRNA for leucine have been associated with LHON

(http://www.mitomap.org). Some of these mutations

are shown inside the inner circle of Fig. 1. The four

mutations considered 'primary' in causing LHON are

G14459A, Gl1778A, G3460A, and T14484C and are

presented in order of decreasing severity (Wallace

et aI., 1997). The mutations associated with LHON

found in this study are considered 'primary

(Gl1778A is in GM1O742A), 'intermediate

(G 15257 A is in HL-60) or 'secondary' (T3394C in

GMI0742A; T4216C in HL-60 and GMI0742A;

Gl3708A in HL- , CHR and GMI0742A;

G15812A in HL-60). According to Wallace et aI.

(1997), the intermediate or secondary mutations may

increase the probability of having LHON or may be

linked to one of the primary mutations. It is interesting

that GMI0742A has two of the secondary mutations

also found in HL- , namely T4216C and G13708A.

CHR also has the G 13708A mutation. Torroni et aI.

(1997) found that the combination of the np 4216 and

708 mutations were more frequent among the

LHON patients containing the mutation at 11 778 than

among the controls.

Characteristic sets of polymorphisms in human

mtDNA are being used to distinguish various groups

and to trace their maternal genealogy (Macaulay et aI.

1999). Haplogroup J and sub-haplogroup Jz have

specific polymorphisms that distinguish this group of

Caucasians with European ancestry from the other

eight haplogroups (H, I, K, T, U, V, Wand X) that

have also been found to characterize those with a

European background (Torroni et aI. , 1997). Both

HL-60 and GM1O742A have a number of identical

polymorphisms that place them in Haplogroup J and

Sub-haplogroup Jz (Table 4).

In the interlaboratory evaluation of the entire

sequence of HL-60 that was conducted by three

laboratories plus NIST, all four laboratories found the

same sequence with the exception of the Georgia

Bureau of Investigation who had the problem noted

in Section 2 with primer set 51 and was unable to

examine that amplicon and therefore, did not find the

polymorphism at 14 199.

In conclusion, NIST has sequenced the entire

mtDNA 06 569 bp) from the HL-60 cell line multiple

times and compared these results with those from

AFDIL, FBI, and GBI who participated in the

interlaboratory evaluation. All four laboratories
396 B.c. Levin et al. Mitochondrion (2003) 387-400

Table 1

Cambridge Reference Sequence nucleotide differences (some unique) found at NIST in the two DNA templates, CHR and 9947 A included in

SRM 2392; HL-60 included in SRM 2392-1 and GM1O742A DNA"

Comparison with Cambridge Reference Sequence (CRS)

CRS Base Template Template Template Template Amino acid Region

19811199ge CHR 9947Ab HL,60c GMI0472A change

HV2

HV2

150 HV2

152 HV2

185 HV2

195 HV2

204 HV2

207 HV2

214 HV2

228 HV2

263*Rg HV2

295 HV2

303-309 C (ins) CC (ins) HV2

311-315*R C (ins) C (ins) C (ins) C (ins) HV2

462 HV2

482 HV2

489 HV2

709 12sRNA

750*R 12sRNA

1438*R 12sRNA

1719 16sRNA

2706 16sRNA

2841 A(U) 16sRNA

3010 16sRNA

31O6- 31O7*Eh C/del del del del del 16sRNA

3394 Tyr -+ His NDI LHON

3423*E GIT Silent NDI

4135 C(U) Tyr -+ His NDI

4216 Tyr -+ His NDI LHON

4769*R Silent ND2

4985*E G/A Silent ND2

5186 G (U) Silent ND2

5228 G (U) Silent ND2

5633 tRNA Ala

6221 Silent cor

6371 T(U) Silent cor

6791 Silent cor

6849i G(0. Thr -+ Ala cor

7028 Silent cor

7476 tRNA Ser

7645 Silent COIl

7861 C (U) Silent COIl

8020 Silent COIl

8448 Met -+ Thr ATPase 8

8503 Silent ATPase 8

8860*R Thr -+ Ala ATPase 6

9315 C (U) Phe -+ Leu COllI

9559*E G/C Arg -+ Pro COllI

172 Silent ND3

10,398 Thr -+ Ala ND3

11,251 Silent ND4

287 C (U) Silent ND4
c. Levin et at. Mitochondrion (2003) 387-400

Table I (continued)

Comparison with Cambridge Reference Sequence (CRS)

397

11,335*E

719

11,778

II ,878

071 het

612

705

13,572

13,702*E

708

13,759

966

14, 199*E

14,272*E

365*E

14,368*E

470

569

766*E

798

15,257

15,326*R

15,452

15,812

16,069

16,126

16,183

16, 184-

16, 189

16, 193

16,223

16,278

16,292

16,311

16,362

16,519

TIC

G/C

GIT

G/C

G/C

G/C

TIC

ClThet (u)

Silent

Silent

Arg..... His

Silent

Phe ..... Leuhet

Silent

Silent

Silent

Gly -+ Arg

Ala -+ Thr

Ala -+ Thr

Thr -+ Ala

Pro -+ Thr

Phe -+ Leu

Silent

Phe -+ Leu

Silent

Silent

Ile -+ Thr

Phe -+ Leu

Asp -+ Asn

Thr -+ Ala

Leu -+ Ile

Val..... Met

ND4

ND4

ND4 LHON

ND4

ND4

ND5

ND5

ND5

ND5

ND5 LHON

ND5

ND5

ND6

ND6

ND6

ND6

ND6

ND6

ND6

CYTB

CYT B LHON

CYTB

CYTB

CYT B LHON

HVI

HVI

HVI

HVI

HVI

HVI

HVI

HVI

HVI

HVI

HVI

HVI

C (U)

C (U)

" ATPase 6, ATP synthase 6; ATPase 8, ATP synthase 8; CYTB, Cytochrome B; CO!, Cytochrome C Oxidase I; COIl, Cytochrome C

Oxidase II; com, Cytochrome C Oxidase III; del, deletion; het, heteroplasmy found in HL-60 at np 12 071; HVl , non-coding region found

from 16,024 and 16,569; HV2, non-coding region found from I and 576; ins, insertion; nd, area not amplified or sequenced; NDl, NADH

dehydrogenase I; ND2, NADH dehydrogenase 2; ND3, NADH dehydrogenase 3; ND4, NADH dehydrogenase 4; ND5, NADH dehydrogenase

5; ND6, NADH dehydrogenase 6; (U), Unique polymorphisms found in coding regions of CHR, 9947 A, fIL-60and GM1O742A determined by

comparison with the MITOMAP database (http://www.mitomap.org).

b Levin et al. (1999). CHR DNA: Sequence based on two amplifications and cycle sequencing procedures with DNA from the first cell culture

line and at least one amplification and cycle sequencing proc'edure with DNA from the second cell culture line. 9947 A DNA: Sequence based on

two amplifications and cycle sequencing procedures.

c HL-60 DNA: Sequence based on two amplifications and cycle sequencing procedures in both the forward and reverse directions for a total of

four sequences.

d Numbers correspond to Cambridge Reference Sequence (Anderson et aI.

, 1981).

e Base found in 1981 (Anderson 

et aI., 1981)/base found in 1999 (Andrews et al., 1999).

f Base pair same as in 1981 Cambridge Reference Sequence.

g *R: Rare polymorphisrns in Cambridge Reference Sequence discovered by reanalysis of original placenta by Andrews et al. (1999).

h *E: Error in Cambridge Reference Sequence discovered by reanalysis of original placenta by Andrews et at (1999).

i Possible heteroplasmic site. This heteroplasmy seen in the mtDNA from the first CHR ceUculture line is not seen in the mtDNA from

the second CHR cell culture line. It is DNA from the second CHR cell culture line that is supplied in NIST SRM 2392.

C (ins)
398 c. Levin et at. Mitochondrion (2003) 387-400

Table 2

Number of differences from the Cambridge Reference Sequence

DNA template Non-coding Coding Amino Real change

regions region acid

changes

CHR

9947Ad

HLGMlO742A

7 + 2 tRNA

" The non-coding regions cover the areas from 16,024 to 16,569

and I to 576.

b Amino acid changes compared to original 1981 Cambridge

Reference Sequence.

C Amino acid and tRNA changes compared to revised 1999 Cambridge Reference Sequence and not counting those changes

considered a rare polymorphism.

d Levin et al. (1999).

found identical results in all amplified regions. SRM

2392-1 containing HL-60 will be available in the

spring of 2003. SRM 2392 that currently includes the

CHR and 9947A templates also will continue to be

available. The CHR and 9947A DNAs come from

apparently normal individuals, but the HL-60 DNA

comes from an individual who has acute promyelocytic leukemia. Since this is the first time that the

entire mtDNA from HL-60 has been sequenced, it

would be interesting to determine if the differences

found in HL-60 are also found in other leukemia

patients. Another interesting finding is the four

differences in HL-60 that have been associated with

LHON disease. For comparison, we also sequenced

GM1O472A, a cell line from a patient with LHON

and found many of the same polymorphisms and

mutations. Analysis of these polymorphisms places

both HL-60 and GM1O742A in Haplogroup J. These

results support the premise that individuals in Haplogroup J may be more prone to LHON. At this time,

Table 3

HL-60 and GMI0472A Polymorphisms in Coding Regions and not found in original SRM 2392"

Nucleotide position Nucleotide change Region

2841GM T-+A 16sRNA

30lOGM G-+A 16sRNA

3394 T-+C NDI

4216HL60;GM T-+C NDI

5228HL60 C-+G ND2

5633HL60 C-+T tRNA ala

7476HL60 C-+T tRNA ser

8020 G-+A COIl

HL6o G-+A ND3

1O,398HL60;GM A-+G ND3

251HL60;GM A-+G ND4

1l,287 T-+C ND4

778 G-+A ND4

I 2,071 HL60 T-+ CIT ND4

13,708HL60;GMC G-+A ND5

14,569HL60 G-+A ND6

14,798GM T-+C CYTB

257HL6o G-+A CYTB

15,452HL60;GM C-+A CYTB

812HL6o G-+A CYTB

Amino acid difference Associated disease Reference

Tyr -+ His

Tyr -+ His

Silent

MM,

MM,

MM,

MM,

MM, MA, MtMM,

MM,

MM,

MM,

MM,

MM,

LHON, NIDDM

LHON

Silent

Silent

Thr -+ Ala

Silent

Silent

Arg -+ His

Phe -+ Leu

Ala -+ Thr

Silent

Phe -+ Leu

Asp -+ Asn

Leu -+ Ile

Val-+ Met

LHON

LHON

LHON

LHON

" GM, GMIO742A;NIDDM, non-insulin dependent diabetes mellitus; MM http:llwww.mitomap.org; MA, http://www.cstI.nistgovlbiotechl

strbaselmitoanalyzer.html; Mt- , Levin, BC, Sekiguchi, K, Tully, LA, Chen, JT, and Gropman, A. A patient with chronic progressive external

ophthalmoplegia reexamined 30 years later. (Manuscript In preparation, 2003).

b See footnote to Table 1. 

C Also seen in CHR as well as HL-60 and GMI0742A.
B.c. Levin et at. Mitochondrion (2003) 387~OO 399

Table 4

Polymorphisms common to haplogroup J and sub-haplogroup Jz found in the mtDNA of HL-60 and GM1O742A"

Polymorphisms Polymorphisms Found Found in

characteristic of characteristic of in HL- GM1O742A

haplogroup J sub-haplogroup Jz

A73G Yes Yes

CI50T Yes

Tl52C Yes

GI85A Yes

Tl95C

G228A Yes

C462T Yes

T489C Yes Yes

G3010A Yes

T4216C Yes Yes

A1O398G Yes Yes

A1l251G Yes Yes

Gll719A Yes Yes

A12612G Yes Yes

Gl3708A Yes Yes

Tl4798C Yes

CI5452A Yes Yes

CI6069T Yes Yes

TI6126C Yes

Tl6189C

CI6278T Yes

Tl631lC

C295T Yes Yes

C5633T Yes

C7476T Yes

GI0172A Yes

GI5257A Yes

Gl5812A Yes

CI6193T Yes

Also found in Disease associated

CHR

CHR 9947A

LHON (secondary)

CHR

CHR

CHR LHON (secondary)

CHR

CHR

9947A

LHON (intermediate)

LHON (secondary)

" References: Torroni et al. (1997); and FinniHi et al. (2001).

since the linkage of HL-60 to the actual donor has

been broken, there is no way to determine if this

patient actually had LHON.

Acknowledgements

This research was supported by Interagency

Agreement number 1999-IJ- O94 from the National

Institute of Justice. Points of view are those of the

authors and do not necessarily represent the position

of the U.S. Department of Justice. The authors are

grateful to Gettysburg College for the partial funding

of Koren A. Holland during her sabbatical year at

NIST. We also acknowledge and thank Kathryn

Kapfer for help in sequencing GMI0742A at NIST.

References

Anderson, S., Bankier, AT., Barrell, B.G., et aI., 1981. Sequence

and organization of the human mitochondrial genome. Nature

290, 457 -465.

Andrews, R.M., Kubacka, I., Chinnery, P.F., Lightowlers, R.N.

Turnbull, D.M., Howell, N., 1999. Reanalysis and revision of

the Cambridge Reference Sequence for human mitochondrial

DNA. Nat Genet 23, 147.

Bendall, K.E., Sykes, B.C., 1995. Length heteroplasmy in the first

hypervariable segment of the human mtDNA control region.

Am. J. Hum. Genet 57, 248-256.
400 G. Levin et al. Mitochondrion (2003) 387-400

Brown, M. , Starikovskaya, E., Derbeneva, 0., et aI., 2002. The

role of mtDNA background in disease expression: a new

primary LHON mutation associated with Western Eurasian

haplogroup. J. Hum. Genet 110, 130~ 138.

Butler, J. , Levin, B. , 1998. Forensic applications of mitochondrial DNA Trends Biotechnol. 16, 158- 162.

Finnilii, A., Lehtonen, M. , Majamaa, K, 2001. Phylogenetic

network for European mtDNA Am. J. Hum. Genet 68,

1475~ 1484.

Helgason, A., Hickey, E., Goodacre, S., et ai., 2001. MtDNA and

the islands of the North Atlantic: estimating the proportions

of Norse and Gaelic Ancestry. Am. J. Hum. Genet 68,

723- 737.

Herrnstadt, c., Elson, J. , Fahy, E., et aI. , 2002. Reduced-mediannetwork analysis of complete mitochondrial DNA codingregion sequences for the major African, Asian, and European

haplogroups. Am. J. Hum. Genet 70, 1152~ 1171.

Lee, M. , Levin, B.C., 2002. MitoAnalyzer, a computer program

and interactive web site to determine the effects of single

nucleotide polymorphisms and mutations in human mitochondrial DNA Mitochondrion I, 321~326.

Levin, B.c., Cheng, H., Reeder, DJ., 1999. A human mitochondrial

DNA Standard Reference Material for quality control in

forensic identification, medical diagnosis, and mutation detec"

tion. Genomics 55, 135- 146.

Levin, B. , Cheng, H., Kline, M.c., Redman, J.W., Richie, KL

2001. A review of the DNA standard reference materials

developed by the National Institute of Standards and Technology. Fresenius J. Anal. Chem. 370, 213-219.

Macaulay, V., Richards, M., Hickey, E., et aI., 1999. The emerging

tree of West Eurasian mtDNAs: a synthesis of control region

sequences and RFLPs. Am. J. Hum. Genet 64, 232- 249.

Torroni, A, Petrozzi, M., D' Urbano, L, et aI., 1997. Haplotype and

phylogenetic analyses suggest that one European-specific

mtDNA background plays a role in the expression of Leber

Hereditary Optic Neuropathy by increasing the penetrance of

the primary mutations 11,778 and 14,484. Am. J. Hum. Genet

, 1107- 1121.

Wallace, D. , Singh, G., Lott, M.T., etal., 1988. Mitochondrial

DNA mutation associated with Leber Hereditary Optic Neuropathy. Science 242, 1427~ 1430.

Wallace, D,c., Brown, M.D., Lott, M.T., 1997. Mitochondrial

genetics. In: Rimoin, D. , Connor, J.M., Pyeritz, RE. (Eds.

Chapter 13 in Emery and Rimoin s Principles and Practice of

Medical Genetics, Churchill Livingstone, New York, Edinburgh, London, pp. 277-332.