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

Dennis MCNEVIN, Janette Edson, James ROBERTSON, Jeremy Austin

Research output: Contribution to journalArticle

6 Citations (Scopus)

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.
Original languageEnglish
Pages (from-to)326-338
Number of pages13
JournalForensic Science, Medicine and Pathology
Volume11
Issue number3
DOIs
Publication statusPublished - Sep 2015

Fingerprint

Taq Polymerase
Hair
DNA
Polymerase Chain Reaction
Alleles
Allelic Imbalance

Cite this

@article{e5c8879bc6884a18aa7d4472e22c167d,
title = "Reduced reaction volumes and increased Taq DNA polymerase concentration improve STR profiling outcomes from a real-world low template DNA source: telogen hairs",
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{\circledR} Profiler Plus{\circledR}): 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.",
keywords = "Low template DNA (LTDNA), Trace DNA, Low copy number (LCN), Reduced volume PCR, Short tandem repeat (STR)",
author = "Dennis MCNEVIN and Janette Edson and James ROBERTSON and Jeremy Austin",
year = "2015",
month = "9",
doi = "10.1007/s12024-015-9679-3",
language = "English",
volume = "11",
pages = "326--338",
journal = "Forensic Science, Medicine and Pathology",
issn = "1547-769X",
publisher = "Humana Press",
number = "3",

}

Reduced reaction volumes and increased Taq DNA polymerase concentration improve STR profiling outcomes from a real-world low template DNA source: telogen hairs. / MCNEVIN, Dennis; Edson, Janette; ROBERTSON, James; Austin, Jeremy.

In: Forensic Science, Medicine and Pathology, Vol. 11, No. 3, 09.2015, p. 326-338.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Reduced reaction volumes and increased Taq DNA polymerase concentration improve STR profiling outcomes from a real-world low template DNA source: telogen hairs

AU - MCNEVIN, Dennis

AU - Edson, Janette

AU - ROBERTSON, James

AU - Austin, Jeremy

PY - 2015/9

Y1 - 2015/9

N2 - 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.

AB - 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.

KW - Low template DNA (LTDNA)

KW - Trace DNA

KW - Low copy number (LCN)

KW - Reduced volume PCR

KW - Short tandem repeat (STR)

UR - http://www.scopus.com/inward/record.url?scp=84938955801&partnerID=8YFLogxK

U2 - 10.1007/s12024-015-9679-3

DO - 10.1007/s12024-015-9679-3

M3 - Article

VL - 11

SP - 326

EP - 338

JO - Forensic Science, Medicine and Pathology

JF - Forensic Science, Medicine and Pathology

SN - 1547-769X

IS - 3

ER -