Standard for Training in Forensic DNA Amplification Methods for 

Capillary Electrophoresis Sequencing

Standard for Training in 

Forensic DNA 

Amplification Methods for 

Capillary Electrophoresis 

Sequencing

Biological Methods Subcommittee

Biology/DNA Scientific Area Committee

Organization of Scientific Area Committees (OSAC) for Forensic Science
Standard for Training in Forensic DNA Amplification Methods for 

Capillary Electrophoresis Sequencing

OSAC Proposed Standard

Standard for Training in Forensic 

DNA Amplification Methods for 

Capillary Electrophoresis Sequencing

Prepared by 

Biological Methods Subcommittee

Version: 1.0

2018

Disclaimer:

This document has been developed by the Biological Methods Subcommittee of the Organization 

of Scientific Area Committees (OSAC) for Forensic Science through a consensus process and is 

proposed for further development through a Standard Developing Organization (SDO). This 

document is being made available so that the forensic science community and interested parties 

can consider the recommendations of the OSAC pertaining to applicable forensic science 

practices. The document was developed with input from experts in a broad array of forensic 

science disciplines as well as scientific research, measurement science, statistics, law, and policy. 

This document has not been published by an SDO. Its contents are subject to change during the 

standards development process. All interested groups or individuals are strongly encouraged to 

submit comments on this proposed document during the open comment period administered by 

the Academy Standards Board (www.asbstandardsboard.org). 
Standard for Training in Forensic DNA Amplification Methods for 

Capillary Electrophoresis Sequencing

Foreword

This standard defines the minimum requirements that shall be met in a forensic DNA analyst

training program for DNA amplification for capillary electrophoresis (CE) sequencing methods. The 

aim is to provide a framework for quality training that will result in consistency in the forensic DNA 

community.

This standard was revised, prepared and finalized as a standard by the DNA Consensus Body of the 

AAFS Standards Board (ASB). The initial draft document was developed by the Biological Methods

Subcommittee of the Organization of Scientific Area Committees. All hyperlinks and web addresses 

shown in this document are current as of the publication date of this standard.

Keywords: Training, DNA, amplification for sequencing, capillary electrophoresis
Standard for Training in Forensic DNA Amplification Methods for 

Capillary Electrophoresis Sequencing

Table of Contents

1 Scope.......................................................................................................................................................1

2 Normative References.........................................................................................................................1

3 Terms and Definitions.........................................................................................................................1

4 Requirements .......................................................................................................................................2

4.1 Knowledge-based training..........................................................................................................2

4.2 Practical training ..........................................................................................................................5

4.3 Competency testing ......................................................................................................................5

5 Conformance...........................................................................................................................................6

Annex A .......................................................................................................................................................7
Standard for Training in Forensic DNA Amplification Methods for 

Capillary Electrophoresis Sequencing

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1 Scope

This standard provides the requirements of a forensic DNA laboratory’s training program to ensure 

proper training in the approved methods of DNA amplification for capillary electrophoresis (CE) 

sequencing used within the trainee’s forensic DNA laboratory.

2 Normative References

ASB Standard 022 - Standard for Forensic DNA Analysis Training Programs 1

3 Terms and Definitions

For the purpose of this document the following definitions apply:

3.1

Control region

A presumed non-coding portion of the mitochondrial DNA molecule analyzed through DNA 

sequencing, which may be used to determine an individual's mitochondrial haplotype or for 

taxonomic discrimination. The control region often contains hypervariable regions (in humans: 

HV1 and HV2) that differ in sequence among non-maternally related individuals. The control region 

encompasses the D-loop region in humans and other species.

3.2

Chloroplast DNA (cpDNA)

A circular DNA molecule, approximately 120-190kbp, which is located in the chloroplasts of plants. 

The DNA is maternally inherited and contains genes involved in the photosynthetic pathway. 

3.3

Degradation

The fragmenting, or breakdown, of DNA or protein (e.g. loss of enzymatic-activity) by chemical, 

physical, or biological means; a common occurrence when biological samples containing DNA 

encounter warm moist environments or excessive UV light.

3.4

dNTPs

Abbreviation for deoxynucleotide tri-phosphates; used in PCR as building blocks to construct new 

DNA strands.

3.5

Hypervariable region

A segment of DNA that often contains polymorphisms and thus is useful for differentiating taxa or 

unrelated individuals.

3.6

Hypervariable region 1 (HV1)

1 American Academy of Forensic Sciences Standards Board, 4200 Wisconsin Avenue, NW, Suite 106-310, 

Washington, DC 20016-2143 asb@aafs.org
Standard for Training in Forensic DNA Amplification Methods for 

Capillary Electrophoresis Sequencing

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A section of the human mtDNA control region spanning nucleotide positions 16024-16365, that 

often differs among non-maternally related individuals.

3.7

Hypervariable region 2 (HV2)

A section of the human mtDNA control region spanning nucleotide positions 73-340, that often 

differs among non-maternally related individuals.

3.8

Inhibitor

As related to the polymerase chain reaction (PCR), any substance that interferes with or prevents 

the synthesis of DNA during the amplification process.

3.9

Mitochondrial DNA (mtDNA)

An often small (~16,500 bp in humans), circular DNA molecule located in eukaryotic mitochondria 

that is typically maternally inherited; the resistance to degradation and presence of 

multiple copies of mtDNA in each cell make it useful with samples originating from limited or 

damaged biological material.

3.10

Polymerase chain reaction (PCR)

An enzymatic process by which a specific region of DNA is replicated during repetitive cycles that 

consist of the following: denaturation of the template; annealing of primers to complementary 

sequences at an empirically determined temperature; and extension of the bound primers by a DNA 

polymerase. The goal of the PCR process is to generate many copies (termed products or 

amplicons) of a specific region of DNA for further analysis.

3.11

PCR product quantification

An analytical procedure to determine the amount of amplicon obtained after DNA amplification.

3.12

Primer

A short polynucleotide chain, usually 18-30 bases long, which targets a specific region of the 

template DNA and allows a DNA polymerase to initiate synthesis of a complementary strand; two 

primers are required for amplification of a particular section of DNA. The primers are 

complementary to opposite strands and are designed to bracket the region of interest for 

amplification. Polymerization is initiated at the 3’ end of the primer and extends in a 5 ́ to 3 ́ 

manner.

4 Requirements

4.1 Knowledge-based training component 

The laboratory’s training program shall provide the trainee with an understanding of the 

fundamental principles of the theory behind DNA amplification for CE sequencing, the function of 
Standard for Training in Forensic DNA Amplification Methods for 

Capillary Electrophoresis Sequencing

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the amplification reagents and other components used in the method, the limitations of 

amplification, and the laboratory’s own DNA amplification guidelines.

4.1.1. At a minimum, the knowledge-based portion of the training program shall require review of 

the following:

a) The laboratory’s protocols for DNA amplification for CE sequencing

b) The laboratory’s applicable validation studies

c) Literature used to support validation

d) Applicable literature as assigned by the trainer (e.g., see references in Annex A)

4.1.2 At a minimum, the knowledge-based portion of the training program shall cover the 

following topics:

NOTE: Knowledge of historical methods is intended to provide an educated perspective. 

In-depth understanding of these methods may not be required for successful training.

a) Principles of PCR-based DNA amplification methods for CE sequencing

i. Reaction components

a. dNTPs

b. Primers

c. Buffer

d. Polymerase

e. MgCl2

f. PCR enhancers (if applicable, e.g. bovine serum albumin)

ii. Primer design

iii. Amplification parameters

iv. PCR inhibitors

v. DNA degradation

b) Primer selection for specific target regions of DNA amplification (as applicable to the 

laboratory)

i. Mitochondrial DNA

a. Control region
Standard for Training in Forensic DNA Amplification Methods for 

Capillary Electrophoresis Sequencing

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b. Portions of control region

c. Whole genome

ii. Chloroplast DNA

a. Coding regions

b. Transgenic spacer regions

iii. Nuclear/genomic DNA

a. Coding regions

b. Non-coding regions

iv. Primer binding site mutations

v. Quality and source of DNA

c) Contamination control and prevention

i. Use of personal protective equipment

ii. Ultraviolet cross-linking of consumables and utensils

iii. Decontamination of work surfaces (e.g. sodium hypochlorite, UV light)

iv. Workflow set-up

d) Quality controls used for DNA amplification

e) Purification of amplification product

f) Post-amplification quantification

i. Evaluation of the controls

ii. Determination of DNA concentration 

g) Instrumentation and reagents

i. Thermal cycling instruments and parameters

ii. Software parameters associated with instruments 

iii. Maintenance and calibration

iv. Storage of amplification reagents

h) Troubleshooting
Standard for Training in Forensic DNA Amplification Methods for 

Capillary Electrophoresis Sequencing

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i. Amplification and post-amplification quantification failure

ii. Instrument failure and recovery

4.2 Practical training 

The laboratory’s training program shall provide the trainee with sufficient practical instruction for 

the trainee to obtain the skills for performing the DNA amplification for CE sequencing protocols 

used by the laboratory. 

4.2.1 At a minimum, the practical portion of the training program shall include the observation of 

the processes at least once and until clearly understood. These include:

a) DNA amplification for CE sequencing methods to be utilized by the trainee

b) Documentation of the process

4.2.2 At a minimum, the practical portion of the training program shall include exercises 

representative of the range, type, and complexity of routine casework and/or database 

samples processed by the laboratory. These include:

a) DNA amplification for CE sequencing methods to be utilized by the trainee

b) Documentation of the process

c) NOTE: The number and quality of samples processed by the trainee shall be appropriate to 

demonstrate the ability to follow the laboratory’s DNA amplification for CE sequencing 

protocol(s) and to produce reliable and accurate results.

4.3 Competency testing

The laboratory’s training program shall include knowledge-based and practical competency testing

in the application of amplification of DNA for CE sequencing methods. The format of the test(s) 

shall meet Section 4.3 of the ASB Standard 022.

4.3.1 Knowledge-based competency

The trainee shall successfully complete a knowledge-based test covering the critical information 

obtained during the training of DNA amplification for CE sequencing methods. The test(s) shall 

cover, at a minimum:

a) The theoretical and scientific bases of DNA amplification for CE sequencing

b) The function of the reagents and other components used for DNA amplification for CE 

sequencing

c) The quality control steps pertaining to DNA amplification for CE sequencing
Standard for Training in Forensic DNA Amplification Methods for 

Capillary Electrophoresis Sequencing

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d) The laboratory’s analytical procedures pertaining to DNA amplification for CE sequencing 

methods

4.3.2 Practical competency

The trainee shall successfully complete a practical test covering each of the DNA amplification for 

CE sequencing protocol(s) for which he or she will be independently authorized. The trainee shall 

be able to satisfactorily perform the following, as applicable:

a) Properly and accurately execute the analytical procedures related to DNA amplification for 

CE sequencing

b) Apply the laboratory’s analytical procedures to a set of samples representing the range of 

DNA quality and quantity expected to be encountered in the laboratory

b) Operate relevant equipment and instrumentation used in the laboratory

c) Document work performed in accordance with laboratory procedures

5 Conformance

In order to demonstrate conformance with this standard, the laboratory shall meet Section 5 of the 

ASB Standard 022.
Standard for Training in Forensic DNA Amplification Methods for 

Capillary Electrophoresis Sequencing

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Annex A

(informative)

Bibliography

The following information provides a list of the literature resources that may assist the DNA 

technical leader in defining the breadth and scope of the materials to be reviewed by the trainee. 

This list is not meant to be all inclusive. The laboratory shall develop a list tailored to its specific 

needs. Updated references shall be added to the laboratory’s list as new methods or technologies 

are incorporated into the laboratory’s protocols. 

1) Anderson S, Bankier AT, Barrell G, de Bruijn MH, Coulson AR, Drouin J et al. Sequence and 

organization of the human mitochondrial genome. Nature 1981;290(5806):457 – 65.

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

Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA

Nat. Genet. 23 (2), 147 (1999)

3) Berger, C., and Parson, W. (2009). Mini-midi-mito: Adapting the amplification and sequencing 

strategy of mtDNA to the degradation state of crime scene samples. Forensic Science 

International: Genetics, 3, 96-103.

4) Butler, J. M. (2012) Advanced Topics in Forensic DNA Typing: Methodology. Academic Press, 

chapter 14, 405-456.

5) CBOL Plant Working Group. A DNA barcode for land plants. Proceedings of the National 

Academy of Sciences Aug 2009, 106 (31) 12794-12797; DOI:10.1073/pnas.0905845106

6) Dormontt, E., Boner, M., Braun, B., Breulmann, G., Degen, B., Espinoza, E., Lowe, A. (2015).

Forensic timber identification: it's time to integrate disciplines to combat illegal

logging. Biological Conservation, 191, 790-798.

7) Eckert, K.A., and Kunkel, T.A. (1990). High fidelity DNA synthesis by the Thermus aquaticus 

DNA polymerase. Nucleic Acids Research, 18, 3739-3744. 

8) Eckert, K.A. and Kunkel, T.A. (1991). DNA polymerase fidelity and the polymerase chain 

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9) Edson, S.M., et al. (2004). Naming the dead – confronting the realities of rapid identification of 

degraded skeletal remains. Forensic Science Review, 16, 63-90.

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amplicons for compound sequence analysis of the entire mtDNA control region in highly

degraded samples. International Journal of Legal Medicine, 122, 385-388.
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Capillary Electrophoresis Sequencing

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Ovouthan K, Johnson RN. An internationally standardized species identification test for use on 

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Capillary Electrophoresis Sequencing

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24)Wesselink, Monique et al. Species identification of botanical trace evidence using molecular 

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25)Wilson, M.R. et al. (1995). Extraction, PCR amplification and sequencing of mitochondrial DNA 

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