Applied 

Genetics

Alternative Methods for Human Identification: 

Mitochondrial DNA Base Composition Profiling 

by ESI-TOF Mass Spectrometry

Kevin Kiesler

Research Biologist, Applied Genetics Group

Forensics@NIST 2012 Meeting

Gaithersburg, MD

November 28, 2012
Applied 

Genetics Outline

• Mitochondrial DNA typing

• Why use Mass Spectrometry?

• Abbott / Ibis Biosciences PLEX-ID Instrument

• PLEX-ID mtDNA 2.0 Assay

• Evaluation Experiments

• Future directions
Applied 

Genetics Mitochondrial DNA

• Mitochondria are organelles within cells

– Produce energy via the Krebs Cycle

• Separate genome from the nucleus 

– ≈ 16,569 bp

• Human cells have hundreds of mitochondria

• Each mitochondrion has between 2 – 10 genome copies

– One cell = 2 nuclear genome copies ≈ 1000 mtDNA copies

• High copy number of mtDNA can be useful for PCR 

amplification

– Sometimes quantity of forensic evidence is a limitation

– Trace evidence (hair & bone)

– When nuclear STR profile fails, can often obtain mtDNA results 

http://remf.dartmouth.edu/images/mammalianLungTEM/source/8.html
Applied 

Genetics mtDNA Genotyping for Human I.D.

• Mutations in mtDNA occur naturally & accumulate over generations

– Mutations allow for differentiating people based on DNA sequence

– mtDNA is passed on only from mothers to children (maternal lineage)

– Can only be used for lineage identification, not individual I.D.

• Brothers and sisters (& some cousins) will have the same mtDNA sequence

• Non-coding “hypervariable region” is used for HID

– Nucleotides 16,024 – 574

– Approximately 1122 bp

• Assayed by Sanger DNA sequencing

– Gold standard for accuracy

– Fluorescent dye terminator bases

– Capillary electrophoresis
Applied 

Genetics Sequencing Results are Different From 

Mass Spectrometry – “Base Composition”

• Sequencing gives an ordered string of bases

• Mass spectrometry only gives a mass measurement

– We know the masses of nucleotides

– Base composition of a DNA molecule can be inferred

– An empirical formula of numbers of A, G, C, and T residues

– Positional information is lost

A6 G4 C5 T3

• Base composition result is almost equally as informative as sequence
Applied 

Genetics Why Use Mass Spectrometry?

DNA Extraction

PCR Amplification

Mass Determination

Cleanup PCR Cleanup PCR

Sequencing Reaction

Sequencing Cleanup

Sequence Alignment

Data Review

C. E.

Data Review

Mass Spec

Cost $180

Time 4 hours

Sequencing

Cost $240+

Time 10+ hours

Example:

One sample mtDNA typing

• Simplified workflow vs Sanger Sequencing

– PCR product is analyzed on a fully automated system: PLEX-ID

– Reduced cost through savings in labor (wet lab and analysis)

– Faster turnaround
Applied 

Genetics Why Use Mass Spectrometry?

• Simplified workflow vs Sanger Sequencing

– PCR product is analyzed on a fully automated system: PLEX-ID

– Reduced cost through savings in labor (wet lab and analysis)

– Faster turnaround

DNA Extraction

PCR Amplification

Mass Determination

Cleanup PCR Cleanup PCR

Sequencing Reaction

Sequencing Cleanup

Sequence Alignment

Data Review

C. E.

Data Review

Mass Spec

Cost $180

Time 4 hours

Sequencing

Cost $240+

Time 10+ hours

Example:

One sample mtDNA typing

= Automated step
Applied 

Genetics The PLEX-ID Instrument

• Mass spectrometer 

designed solely for analysis 

of DNA (PCR)

• Fully automated 

– Plate stacker holds up to 15 

PCR plates

– Desalting by magnetic bead 

cleanup 

• Cleanup reagents stored 

onboard

– Fluidics system handles all 

sample transfers including 

injection into mass 

spectrometer

• Data analysis on separate 

computer
Applied 

Genetics Electrospray Ionization Time-of-Flight 

Analysis

• Soft ionization method

• Does not fragment molecules

• DNA strands of PCR product 

are dissociated on injection

• DNA molecular masses are 

measured

– Forward and reverse strands 

measured separately

• Mass is converted to a result 

by comparing to reference 

database of known masses

• Results:

– mtDNA base composition profile

– STR profile

– SNP genotypes PLEX-ID
Applied 

Genetics mtDNA 2.0 Assay Plate Layout

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Control

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Control

• 96-well plate contains all reagents

– Just add DNA (5 μL per well)

• Each sample is run in a single 

column of a plate

• Hypervariable region is amplified by 

24 PCR amplicons

– Eight triplex PCRs
Applied 

Genetics mtDNA 2.0 Assay - Result

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Negative 

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Positive 

Control

Forward strand

Reverse strand
Applied 

Genetics Evaluation Experiments

• Sensitivity

– Dilution series of three templates

– (4, 8, 20, 40) pg total DNA input

– Average % of amplicons detected

• 72.4% at 4 pg DNA input

• 85.1% at 8 pg DNA input

• 96.0% at 20 pg DNA input

• 98.8% at 40 pg DNA input

– Manufacturer recommends 200 pg 

DNA input

• Contamination

– Plate layout designed to evaluate 

reagents, fluidics, and cleanup 

carousel

– Run twice per month for six months

– No contamination detected

• Concordance

– Comparing M.S. to sequencing

– 711 templates analyzed

– 99.3 % concordance rate (706/711)

• Mixtures

– Two-component mixtures generated

– Ratios - 99:1, 19:1, 9:1, 3:1, and 1:1

– 3:1 mixture was limit of minor 

component detection
Applied 

Genetics Full Report Available Online

• http://www.cstl.nist.gov/strbase/NISTpub.htm
Applied 

Genetics Abbott Product Recall

• The PLEX-ID system is being voluntarily recalled

– Due to reliability issues reported by clinical users

• Clinical labs cannot tolerate down time

– Instruments are being removed from the field

– New more robust instrument under development

• Estimated to be several years to re-release

• Our experiments support the viability of mass 

spectrometry technology for DNA based human 

identification
Applied 

Genetics Future Directions – New Technology

• Ultra high throughput sequencing

– For deep sequencing of entire mtDNA genome

– Can generate hundreds of millions of bases of sequence

– Run completes in 5 hours

• Trained on Life Technologies instrument

– Ion Torrent Personal Genome Machine (PGM)

– Bench-top scale next-generation sequencer

PGM
Applied 

Genetics Pilot Studies With Next-Gen Sequencing

• Mitochondrial sequencing standards

– SRM 2392 and 2392-I

– Sequenced these three mtDNA genomes on one PGM run

– 150 million aligned bases

– Average coverage depth 1427.5 x

– Now comparing to certified sequence (Sanger method)

SRM 2392 SRM 2392-I
Applied 

Genetics Acknowledgments

Contact Info: 

Kevin.Kiesler@nist.gov

301-975-4306

Pete Vallone

Funding from the FBI 

Biometrics Center of 

Excellence ‘Forensic 

DNA Typing as a 

Biometric Tool’ 

NIST Team for This Work

Erica Butts

NIST Disclaimer: Certain commercial equipment, instruments and materials are identified in order to specify experimental procedures as 

completely as possible. In no case does such identification imply a recommendation or it imply that any of the materials, instruments or 

equipment identified are necessarily the best available for the purpose.

Points of view are those of the presenters and do not necessarily represent the official position of the National Institute of Standards and 

Technology or the U.S. Department of Justice.
Applied 

Genetics Thank you for your attention!