Skip to main content
Key Specialties
Cardiology Diabetology Endocrinology Gastroenterology Geriatrics and Gerontology Gynecology and Obstetrics Hematology Infectious Disease Internal Medicine Nephrology Neurology Oncology Pediatrics Respiratory Medicine Rheumatology Surgery
Congress Coverage
2024 ACC Congress 2023 ESMO Congress 2023 EASD Congress 2023 ERS Congress 2023 ESC Congress
Clinical Collections
Advances in Alzheimer’s

Keynote webinar | Spotlight on medication adherence

LIVE: Thursday 27th June 2024, 18:00-19:30 (CEST)
Join our expert panel to discover why you need to understand the drivers of non-adherence in your patients, and how you can optimize medication adherence in your clinics to drastically improve patient outcomes.
ADVANCED SEARCH
Log in
Top
Forensic Science, Medicine and Pathology
Published in: Forensic Science, Medicine and Pathology 3/2015

01-09-2015 | Original Article

Reduced reaction volumes and increased Taq DNA polymerase concentration improve STR profiling outcomes from a real-world low template DNA source: telogen hairs

Authors: Dennis McNevin, Janette Edson, James Robertson, Jeremy J. Austin

Published in: Forensic Science, Medicine and Pathology | Issue 3/2015

Login to get access

Abstract

Purpose

The primary method for analysis of low template DNA (LTDNA) is known as the low copy number (LCN) method involving an increased number of PCR cycles (typically 34). In common with other LTDNA methods, LCN profiles are characterized by allelic imbalance, drop in, and drop out that require complicated interpretation rules. They often require replicate PCR reactions to generate a “consensus” profile in a specialized facility. An ideal method for analysis of LTDNA should enhance profiling outcomes without elevated error rates and be performed using standard facilities, with minimum additional cost.

Methods

In this study, we present a comparison of four method variations for the amplification of STRs from LTDNA with a widely used, commercially available kit (AmpFℓSTR® Profiler Plus®): the standard method, the standard method with a post-PCR clean up, the LCN method, and a reduced reaction volume with increased Taq DNA polymerase concentration.

Results

Using telogen hairs—a common source of LTDNA—and matched reference DNA, the LCN method produced the highest number of concordant and non-concordant (i.e., dropped-in) alleles. In comparison, the reduced reaction volume with increased Taq polymerase yielded more full and concordant DNA profiles (all alleles combined) and less off-ladder alleles from a broad range of input DNA. In addition, this method resulted in less non-concordant alleles than LCN and no more than for standard PCR, which suggests that it may be preferred over increased PCR cycles for LTDNA analysis, either with or without consensus profiling and statistical modelling.

Conclusions

Overall, this study highlights the importance and benefit of optimizing PCR conditions and developing improved laboratory methods to amplify and analyze LTDNA.
Literature
1.
2.
Schneider H, Sommerer T, Rand S, Wiegand P. Hot flakes in cold cases. Int J Legal Med. 2011;125(4):543–8.PubMedCrossRef
3.
Edson J, Brooks EM, McLaren C, Robertson J, McNevin D, Cooper A, et al. A quantitative assessment of a reliable screening technique for the STR analysis of telogen hair roots. Forensic Sci Int Genet. 2013;7(1):180–8.PubMedCrossRef
4.
Horsman-Hall KM, Orihuela Y, Karczynski SL, Davis AL, Ban JD, Greenspoon SA. Development of STR profiles from firearms and fired cartridge cases. Forensic Sci Int Genet. 2009;3(4):242–50.PubMedCrossRef
5.
Alonso A, Andelinovic Š, Martín P, Sutlovic D, Erceg I, Huffine E, et al. DNA typing from skeletal remains: evaluation of multiplex and megaplex STR systems on DNA isolated from bone and teeth samples. Croat Med J. 2001;42(3):260–6.PubMed
6.
Caragine T, Mikulasovich R, Tamariz J, Bajda E, Sebestyen J, Baum H, et al. Validation of testing and interpretation protocols for low template DNA samples using AmpFlSTR identifiler. Croat Med J. 2009;50(3):250–67.PubMedCentralPubMedCrossRef
7.
8.
9.
10.
11.
Balding DJ, Buckleton J. Interpreting low template DNA profiles. Forensic Sci Int Genet. 2009;4(1):1–10.PubMedCrossRef
12.
Lowe A, Murray C, Whitaker J, Tully G, Gill P. The propensity of individuals to deposit DNA and secondary transfer of low level DNA from individuals to inert surfaces. Forensic Sci Int. 2002;129(1):25–34.PubMedCrossRef
13.
Gill P. Application of low copy number DNA profiling. Croat Med J. 2001;42(3):229–32.PubMed
14.
Gill P, Whitaker J, Flaxman C, Brown N, Buckleton J. An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA. Forensic Sci Int. 2000;112(1):17–40.PubMedCrossRef
15.
Whitaker JP, Cotton EA, Gill P. A comparison of the characteristics of profiles produced with the AmpFℓSTR® SGM Plus™ multiplex system for both standard and low copy number (LCN) STR DNA analysis. Forensic Sci Int. 2001;123(2–3):215–23.PubMedCrossRef
16.
Taberlet P, Griffin S, Goossens B, Questiau S, Manceau V, Escaravage N, et al. Reliable genotyping of samples with very low DNA quantities using PCR. Nucleic Acids Res. 1996;24(16):3189–94.PubMedCentralPubMedCrossRef
17.
Benschop CC, van der Beek CP, Meiland HC, van Gorp AG, Westen AA, Sijen T. Low template STR typing: effect of replicate number and consensus method on genotyping reliability and DNA database search results. Forensic Sci Int Genet. 2011;5(4):316–28.PubMedCrossRef
18.
Cowen S, Debenham P, Dixon A, Kutranov S, Thomson J, Way K. An investigation of the robustness of the consensus method of interpreting low-template DNA profiles. Forensic Sci Int Genet. 2011;5(5):400–6.PubMedCrossRef
19.
Smith PJ, Ballantyne J. Simplified low-copy-number DNA analysis by post-PCR purification. J Forensic Sci. 2007;52(4):820–9.PubMedCrossRef
20.
Forster L, Thomson J, Kutranov S. Direct comparison of post-28-cycle PCR purification and modified capillary electrophoresis methods with the 34-cycle “low copy number” (LCN) method for analysis of trace forensic DNA samples. Forensic Sci Int Genet. 2008;2(4):318–28.PubMedCrossRef
21.
Hanson EK, Ballantyne J. Whole genome amplification strategy for forensic genetic analysis using single or few cell equivalents of genomic DNA. Anal Biochem. 2005;346(2):246–57.PubMedCrossRef
22.
Fregeau CJ, Bowen KL, Leclair B, Trudel I, Bishop L, Fourney RM. AmpFℓSTR® Profiler Plus™ short tandem repeat DNA analysis of casework samples, mixture samples, and nonhuman DNA samples amplified under reduced PCR volume conditions (25uL). J Forensic Sci. 2003;48(5):1–21.
23.
Gaines ML, Wojtkiewicz PW, Valentine JA, Brown CL. Reduced volume PCR amplification reactions using the AmpFℓSTR Profiler Plus kit. J Forensic Sci. 2002;47(6):1224–37.PubMed
24.
Leclair B, Sgueglia JB, Wojtowicz PC, Juston AC, Fregeau CJ, Fourney RM. STR DNA typing: increased sensitivity and efficient sample consumption using reduced PCR reaction volumes. J Forensic Sci. 2003;48(5):1001–13.PubMed
25.
Hoffman NH, Fenger T. Validation of half-reaction amplification using Promega PowerPlex® 16. J Forensic Sci. 2010;55(4):1044–9.PubMedCrossRef
26.
Gill P, Kirkham A, Curran J. LoComatioN: a software tool for the analysis of low copy number DNA profiles. Forensic Sci Int. 2007;166(2–3):128–38.PubMedCrossRef
27.
Evett IW, Gill PD, Jackson G, Whitaker J, Champod C. Interpreting small quantities of DNA: the hierarchy of propositions and the use of Bayesian networks. J Forensic Sci. 2002;47(3):520–30.PubMed
28.
Gill P, Curran J, Elliot K. A graphical simulation model of the entire DNA process associated with the analysis of short tandem repeat loci. Nucleic Acids Res. 2005;33(2):632–43.PubMedCentralPubMedCrossRef
29.
McNevin D, Wilson-Wilde L, Robertson J, Kyd J, Lennard C. Short tandem repeat (STR) genotyping of keratinised hair: part 1. Review of current status and knowledge gaps. Forensic Sci Int. 2005;153(2–3):237–46.PubMedCrossRef
30.
McNevin D, Wilson-Wilde L, Robertson J, Kyd J, Lennard C. Short tandem repeat (STR) genotyping of keratinised hair: part 2. An optimised genomic DNA extraction procedure reveals donor dependence of STR profiles. Forensic Sci Int. 2005;153(2–3):247–59.PubMedCrossRef
31.
Swango KL, Timken MD, Chong MD, Buoncristiani MR. A quantitative PCR assay for the assessment of DNA degradation in forensic samples. Forensic Sci Int. 2006;158(1):14–26.PubMedCrossRef
32.
Applied Biosystems. AmpFℓSTR® Profiler Plus® PCR Amplification Kit User’s Manual. Life Technologies Corporation; 2012.
33.
Leclair B, Fregeau CJ, Bowen KL, Fourney RM. Systematic analysis of stutter percentages and allele peak height and peak area ratios at heterozygous STR loci for forensic casework and database samples. West Conshohocken, PA: ASTM International; 2004.
34.
Bessekri MW, Aggoune A, Lazreg S, Bucht R, Fuller V. Comparative study on the effects of reduced PCR reaction volumes and increased cycle number, on the sensitivity and the stochastic threshold of the AmpFℓSTR® Identifiler® Plus kit. Forensic Sci Int Genet Suppl. 2013;4(1):e306–7.CrossRef
35.
Cave CA, Hancock K, Schumm JW. Principles of STR multiplex amplification. Forensic Sci Int Genet Suppl. 2008;1(1):102–4.CrossRef
36.
37.
38.
39.
Kozlowski B, Duceman B, Kadash K, Biega L. Validation study of the True Allele® Automated Data Review System. West Conshohocken, PA: ASTM International; 2004.
40.
Ballantyne J, Hanson EK, Perlin MW. DNA mixture genotyping by probabilistic computer interpretation of binomially-sampled laser captured cell populations: combining quantitative data for greater identification information. Sci Justice. 2013;53(2):103–14.PubMedCrossRef
41.
Taylor D, Bright JA, Buckleton J. The interpretation of single source and mixed DNA profiles. Forensic Sci Int Genet. 2013;7(5):516–28.PubMedCrossRef
42.
Institute of Environmental Science and Research (ESR). STRmix. Resolve more DNA mixtures. Institute of Environmental Science and Research (ESR); 2014. http://​strmix.​esr.​cri.​nz/​. Accessed 9 Oct 2014.
43.
Tvedebrink T, Eriksen PS, Mogensen HS, Morling N. Estimating the probability of allelic drop-out of STR alleles in forensic genetics. Forensic Sci Int Genet. 2009;3(4):222–6.PubMedCrossRef
44.
Ottens R, Taylor D, Abarno D, Linacre A. Optimising direct PCR from anagen hair samples. Forensic Sci Int Genet Suppl. 2013;4(1):e109–10.CrossRef
45.
Templeton JE, Linacre A. DNA profiles from fingermarks. BioTechniques. 2014;57(5):259.PubMed
Metadata
Title
Reduced reaction volumes and increased Taq DNA polymerase concentration improve STR profiling outcomes from a real-world low template DNA source: telogen hairs
Authors
Dennis McNevin
Janette Edson
James Robertson
Jeremy J. Austin
Publication date
01-09-2015
Publisher
Springer US
Published in
Forensic Science, Medicine and Pathology / Issue 3/2015
Print ISSN: 1547-769X
Electronic ISSN: 1556-2891
DOI
https://doi.org/10.1007/s12024-015-9679-3

Other articles of this Issue 3/2015

Forensic Science, Medicine and Pathology 3/2015 Go to the issue

Images in Forensics

Mercury embolism of the lung and right ventricle revealed by postmortem computed tomography and X-ray analytic microscopy

Original Article

Detection of decomposition volatile organic compounds in soil following removal of remains from a surface deposition site

Original Article

Prescription opioid related deaths in New York City: a 2 year retrospective analysis prior to the introduction of the New York State I-STOP law

Case Report

A case of suicide by self-injection of adrenaline

Letter to the Editor

Deaths linked to synthetic cannabinoids

Original Article

The radiographic visibility of the root pulp of the third lower molar as an age marker