Emily C. Lennert
direct PCR, STR, allele, DNA, tape lift, single fiber, trace
Blackie, R.; Taylor, D.; Linacre, A. DNA profiles from clothing fibers using direct PCR. Forensic Science, Medicine, and Pathology. 2016, 12, 331-335.
The opinions expressed in this review are an interpretation of the research presented in the article. These opinions are those of the summation author and do not necessarily represent the position of the University of Central Florida or of the authors of the original article.
Direct polymerase chain reaction (PCR) is a form of DNA analysis in which the extraction process is skipped and samples are directly processed by PCR. In this type of analysis, laboratory error and exogenous DNA contamination may be reduced, as well as the time and cost of analysis. Direct PCR is applied to trace DNA samples, which may be adversely affected by the extraction process in that already minute amounts of DNA may be lost due to various tube transfers, as well as introducing additional opportunity for contamination.
Clothing recovered from crime scenes may be processed for DNA by swabbing, tape lifting, or extraction from cut sections of fabric. It is less likely that single fibers will be processed for DNA, with fibers often being analyzed microscopically instead. The authors of this study state that the ability to obtain DNA profiles from single fibers would be of benefit, particularly if microscopy should fail to provide significant information. The authors present a successful application of direct PCR to single fibers from clothing worn by an individual participant.
One female participant wore five different long-sleeved items of clothing, of various fabric compositions and colors, for 12 hours each. Each item was washed by the participant prior to wearing. Single fibers, approximately 5 mm in length, were cut from inside the right cuff of the article of clothing at time 0, 2, 4, 6.5, 9, and 11 hours. At 12 hours a tape-lift was conducted inside the right cuff of the garment. Fibers from time points 0, 2, 4, 6.5, and 9 hours were subjected to direct PCR, as well as the tape lift samples. Fibers were not processed in any way prior to direct PCR amplification. For tape lift samples, a 1 x 1 cm square was cut from the center of the tape and placed in a 0.2 mL tube for direct PCR. Fibers from the 11 hour time point were subjected to standard DNA extraction and subsequent PCR amplification. Reference buccal swabs were obtained from the participant and the participant’s co-habiting partner.
Direct PCR was carried out by placing the sample in a 0.2 mL tube with 10 μL of PCR master mix, obtained from the AMPFlSTR® NGM ™ kit, 5 μL of primr mix, 1 μL of AmpliTaq Gold® DNA polymerase, and 9 μL of sterile water. A GeneAmp® System 9600 thermal cycler was used for amplification. For the 11 h samples, extractions were performed prior to PCR amplification. Extracts were performed using a QIAamp® DNA Mini Kit, following manufacturer instructions for buccal swabs, since swabs are made from fibers. From the extracts, 10 μL was then amplified by PCR following the same conditions as used for direct PCR. Samples were processed by capillary electrophoresis and analyzed using GeneMapper® software.
All samples from clothing item A4 produced no alleles; it was concluded that the likely cause was a PCR inhibitor, likely the dye, which prevented successful amplification of DNA. All but two fiber samples produced mixed DNA profiles with major and minor contributors. All observed alleles, except for one single allele present in one sample, could be attributed to the participant or their co-habiting partner. Table 1 within the study summarizes the total number of alleles obtained for each sample at each time point. No distinguishable pattern was observed with regard to allelic increase over time. All samples subjected to extraction, i.e. time point 11 h, resulted in the recovery of no alleles. Full DNA profiles of the participant, i.e. 15 participant alleles, were recovered from two samples, and nine samples produced partial profiles for the participant. All tape lift samples produced profiles containing 15 alleles or more for the participant and co-habiting partner. Overall, the authors report that 81% of samples produced useable DNA profiles, i.e. 14 alleles or more, from either the participant or their cohabiting partner. Only three single fiber samples, excluding sample A4, did not produce any alleles.
Direct PCR of single fibers resulted in DNA profiles that were considered useable in 81% of samples tested.
- Single fibers that were subjected to standard extraction failed to produce any alleles.
- One item tested, A4, contained direct PCR inhibitors.
Direct PCR allows for the analysis of trace DNA samples which may not be detectable through standard extraction methods.
A successful method is described for direct PCR of single fibers and tape lifts.