Standard for Training in Forensic DNA Quantification Methods

Standard for Training in 

Forensic DNA Quantification 

Methods

Biological Methods Subcommittee

Biology/DNA Scientific Area Committee

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

OSAC Proposed Standard

Standard for Training in Forensic DNA 

Quantification Methods

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 Quantification Methods

Foreword

This standard defines the minimum requirements that shall be met in a Forensic DNA Analyst 

training program for DNA quantification 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, nuclear DNA, quantification, quantitative PCR/real time PCR
Standard for Training in Forensic DNA Quantification Methods

Table of Contents

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

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

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

4 Requirements.....................................................................................................................................................1

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

4.2 Practical training.......................................................................................................................................4

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

5 Conformance.......................................................................................................................................................5

Annex A ......................................................................................................................................................................6
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Standard for Training in Forensic DNA Quantification Methods

1 Scope

This standard provides the requirements for a forensic DNA laboratory’s training program in DNA

quantification.

2 Normative References

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

3 Terms and Definitions

For purposes of this document, the following definitions apply.

3.1

DNA quantification

A process by which the DNA concentration in a sample is determined.

3.2

Cycle threshold

Cycle number (in quantitative PCR) at which the fluorescence generated within a reaction exceeds a 

defined threshold; this value is converted to a DNA concentration for each sample tested using a 

standard curve developed from DNA samples of known concentrations.

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

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.

3.5

Quantitative PCR (qPCR)

A means for quantifying the amount of nucleic acid present in a sample using PCR

4 Requirements

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

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

4.1 Knowledge-based training

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

fundamental principles of the theory behind various DNA quantification methods, the function of 

the reagents and other components used in each method, the information generated by each assay, 

the limitations of each method, and the laboratory’s own DNA quantification 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 quantification

b) The laboratory’s applicable validation studies

c) Literature used to support validation

d) Applicable literature as assigned by the trainer

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 on 

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

successful training.

a) Principles of non-PCR based DNA quantification methods

i. Spectrophotometric analysis

ii. Fluorometric methods

iii. Yield gels

iv. Southern blots (e.g., slot blots)

b) Principles of quantitative PCR (qPCR) DNA quantification methods

i. qPCR assays based on fluorescence quenching using specific probes

ii. qPCR assays based on non-specific double-stranded DNA intercalating dye

iii. Digital PCR (dPCR)

iv. End-point PCR assays

v. Standards and standard curves

vi. Cycle threshold (Ct) establishment and its role in the quantification process

c) Characteristics, performance, and information provided by PCR and non-PCR based 

methods of DNA quantification
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Standard for Training in Forensic DNA Quantification Methods

i. Sensitivity (limit of detection and reliable concentration range)

ii. Specificity (non-human, higher primate, human, male/female)

iii. Single-copy vs. multi-copy qPCR targets

iv. qPCR target length and consequent performance on degraded DNA

v. qPCR multiplex capabilities

a. Limits of detection

b. Internal positive control for the co-detection of PCR inhibitors

c. Autosomal and Y chromosome co-quantification

d. Detection and characterization of degradation based on targeting fragments 

of different lengths in a multiplex reaction

d) Characteristics of results of different methods of DNA quantification

i. Amount of total DNA or target DNA

ii. Sample purity

iii. Detection of male contributor(s)

iv. Presence of PCR inhibitors

v. DNA degradation levels

vi. Suitability of DNA for downstream DNA typing methods

vii. Impact of the source of the DNA standard (e.g., cell line, single source genomic DNA, 

pooled genomic DNA) on the quantification estimate

e) Interpretation of results

i. Evaluation of the standard curve (slope, R2, Y intercept)

ii. DNA concentration in the sample

iii. Presence of PCR inhibitors (if applicable)

iv. Ratio between male and female contributors (if applicable)

v. Level of sample degradation (if applicable)

vi. Determining whether the DNA should be further purified, concentrated, or reextracted
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Standard for Training in Forensic DNA Quantification Methods

vii. Determining whether the sample has detectable amounts of DNA

viii. Determining whether it is appropriate to proceed with the amplification step

ix. Determining the amount of DNA to be added to the amplification reaction

x. Method limitations (e.g., non-targeted detection methods, specificity, sensitivity)

f) Instrumentation and reagents

i. DNA quantification instruments and parameters

ii. Software parameters associated with instruments

iii. Maintenance and calibration

iv. Storage and handling of quantification kit reagents

g) Troubleshooting

i. Spectral calibration failure

ii. Standard quantification curve failure

iii. Non-specific detection (e.g., background fluorescence, cellular debris, pigments)

iv. General equipment failure

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 DNA quantification protocols used by the laboratory.

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

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

processed by the laboratory. These include:

a) DNA quantification methods to be utilized by the trainee

b) Documentation of the process

4.2.2 Practical exercises representative of the range, type, and complexity of routine casework or 

database samples processed by the laboratory. These shall include

a) DNA quantification methods to be utilized by the trainee

b) Documentation of the process
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Standard for Training in Forensic DNA Quantification Methods

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

demonstrate the ability to follow the laboratory’s DNA quantification protocol(s) and to 

produce reliable and accurate results.

4.3 Competency

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

in the application of DNA quantification. The format of the test(s) shall meet section 4.3 of ASB 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 quantification methods. The test(s) shall cover, at a minimum:

a) The theoretical and scientific bases of DNA quantification

b) The function of the reagents and other components used in each method

c) The proper application of each method

d) The quality control steps pertaining to DNA quantification

e) The laboratory’s analytical procedures pertaining to DNA quantification methods

4.3.2 Practical competency

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

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 quantification

b) Apply the laboratory’s analytical procedures to a variety of evidentiary casework- or databasetype samples

c) Operate relevant equipment and instrumentation used in the laboratory

d) 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 022.
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Standard for Training in Forensic DNA Quantification Methods

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

chapter 3, 49-67.

2) Huss, V., et al. (1983). Studies on the spectrophotometric determination of DNA hybridization 

from renaturation rates. Systematic and Applied Microbiology 4.2, 184-192.

3) Holden, M. J., et al. (2009). Factors affecting quantification of total DNA by UV spectroscopy and 

PicoGreen fluorescence. Journal of agricultural and food chemistry, 57.16, 7221-7226.

4) Teare, J. M., et al. (1997). Measurement of nucleic acid concentrations using the DyNA Quant 

and the GeneQuant. Biotechniques 22.6, 1170-1174.

5) Nicklas, J. A., and Buel, E. (2003). Quantification of DNA in forensic samples. Analytical and 

Bioanalytical Chemistry, 376, 1160–1167.

6) Ahn, S. J., et al. (1996). PicoGreen quantitation of DNA: Effective evaluation of samples pre- or 

post-PCR. Nucleic Acids Research, 24, 2623–2625.

7) Budowle, B., et al. (1995). DNA protocols for typing forensic biological evidence: 

Chemiluminescent detection for human DNA quantitation and restriction fragment length 

polymorphism (RFLP) analyses and manual typing of polymerase chain reaction (PCR) 

amplified polymorphisms. Electrophoresis, 16, 1559–1567.

8) Walsh, P. S., et al. (1992). A rapid chemiluminescent method for quantitation of human DNA. 

Nucleic Acids Research, 20, 5061–5065.

9) Greenspoon, S. A., et al. (2006). Automated PCR setup for forensic casework samples using the 

Normalization Wizard and PCR Setup robotic methods. Forensic Science International, 164, 

240–248.

10)Hayn, S., et al. (2004). Evaluation of an automated liquid hybridization method for DNA 

quantitation. Journal of Forensic Sciences, 49, 87–91.

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Standard for Training in Forensic DNA Quantification Methods

13) Zipper, H., et al. (2004). Investigations on DNA intercalation and surface binding by SYBR Green 

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14)Higuchi, R., et al. (1992). Simultaneous amplification and detection of specific DNA sequences. 

Biotechnology, 10, 413–417.

15)Higuchi, R., et al. (1993). Kinetic PCR analysis: Real-time monitoring of DNA amplification 

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16)Nicklas, J. A., & Buel, E. (2003). Development of an Alu-based, real-time PCR method for 

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number in forensic and ancient DNA studies. Forensic Science International, 139,141–149.

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Inhibitors." Journal of forensic sciences 51.4 (2006): 795-804.

20) Andreasson, H., et al. (2002). Real-time DNA quantification of nuclear and mitochondrial DNA in 

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