The effects of extra PCR cycles when amplifying skeletal samples with the GlobalFiler® PCR Amplification Kit
When samples with low amounts of DNA are amplified using short tandem repeats (STRs), stochastic effects such as allele and locus dropout or drop-in, allele imbalance, and increased stutter often occur making data interpretation more difficult. The most common approach to improving STR results from low template samples is to increase the number of PCR cycles. Although more alleles may be recovered, stochastic effects may be exaggerated resulting in more complicated STR profiles. This work reports the effect of additional PCR cycles (29 vs. 30, 31, and 32) on STR success from environmentally challenged bone and tooth samples using the GlobalFiler® DNA Amplification Kit (Thermo Fisher Scientific). In addition, we compared the efficiency of two DNA extraction kits for skeletal samples: QIAamp® DNA Investigator (QIAGEN) and PrepFiler® BTA™ Forensic DNA Extraction (Thermo Fisher Scientific) kits. Results showed that more DNA was recovered from samples using the PrepFiler® BTA™ kit; but regardless of the extraction method, the number of alleles detected and the peak heights both increased with an increase in PCR cycle number. Although more alleles were reported in almost all samples, the most notable improvement was observed in samples with the DNA template < 120 pg. A general increase in the number of PCR artifacts was detected in STR profiles generated using 30–32 cycles. Overall, this study provides supporting evidence that STR profile completeness and quality may be improved when low template skeletal samples are amplified with extra PCR cycles (up to 32 cycles) using the GlobalFiler® DNA Amplification Kit.
KeywordsLow template DNA Skeletal Low copy number GlobalFiler®
We would like to thank Rachel Houston and Carrie Mayes for assistance with sample processing, and the Applied Anatomical Research Center (AARC) for providing the skeletal samples used in this study. Most importantly, we are grateful to those who donated their loved ones for scientific research. We would also like to thank Thermo Fisher Scientific for providing many of the reagents used in this work.
Compliance with ethical standards
Conflict of Interest
The authors declare that they have no conflict of interest.
- 1.Ghatak S, Muthukumaran RB, Nachimuthu SK (2013) A simple method of genomic DNA extraction from human samples for PCR-RFLP analysis. J Biomol Tech 24:224–231Google Scholar
- 3.Miloš A, Selmanović A, Smajlović L, Huel RL, Katzmarzyk C, Rizvić A, Parsons TJ (2007) Success rates of nuclear short tandem repeat typing from different skeletal elements. Croat Med J 48:486–493Google Scholar
- 5.Abuidrees AS, Alhamad NA, Alsaadany K (2016) A suitable method for dna extraction from bones for forensic applications: a case study. Arab J Forensic Sci Forensic Med 1:346–352Google Scholar
- 6.Hochmeister MN, Budowle B, Borer UV, Eggmann U, Comey CT, Dirnhofer R (1991) Typing of deoxyribonucleic acid (DNA) extracted from compact bone from human remains. J Forensic Sci 36:1649–1661Google Scholar
- 8.Gill P (2001) Application of low copy number DNA profiling. Croat Med J 42:229–232Google Scholar
- 14.Kloosterman A, Kersbergen P (2003) Efficacy and limits of genotyping low copy number DNA samples by multiplex PCR of STR loci. International Congress Series. Elsevier, pp 795–798Google Scholar
- 17.Johnson BM, Kemp BM (2017) Rescue PCR: reagent-rich PCR recipe improves amplification of degraded DNA extracts. J Archaeol Sci Rep 11:683–694Google Scholar
- 27.van Daal A (2010) LCN DNA analysis: limitations prevent ‘general acceptance’. Promega Corporation Web siteGoogle Scholar
- 28.Butler J, Hill C (2010) Scientific issues with analysis of low amounts of DNA. Profiles DNA 13Google Scholar
- 30.Service CP (2008) Review of the use of Low Copy Number DNA analysis in current cases: CPS statement. http://www.cps.gov.uk/news/pressreleases/101_08.html
- 31.Caddy B, Taylor G, Linacre A (2008) A review of the science of low template DNA analysis. UK Home Office ReportGoogle Scholar
- 32.QIAGEN (2012) QIAamp® DNA Investigator handbookGoogle Scholar
- 33.Scientific TF (2012) PrepFiler® and PrepFiler® BTA Forensic DNA Extraction Kits user guideGoogle Scholar
- 34.Scientific TF (2017) Quantifiler™ HP and Trio DNA Quantification Kits user guideGoogle Scholar
- 35.Scientific TF (2016) GlobalFiler™ PCR Amplification Kit user guideGoogle Scholar
- 36.TIBCO (2017) Statistica (data analysis software system). TIBCO Software Inc.Google Scholar
- 37.Moore D, Dowhan D (2002) Purification and concentration of DNA from aqueous solutions. Curr Protoc Mol Biol 2.1. 1-2.1. 10Google Scholar