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Paper Details New Pre-PCR Method for STR Analysis of Forensic Samples

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An Australian duo has developed a pre-PCR technique to improve results in forensic DNA analysis. The method, published this month in BioTechniques, is designed to eliminate stochastic sampling effects resulting from the miniscule amounts of starting material and possible contamination common in crime scene analysis. This is accomplished by initially increasing low-abundance template copy number in a linear, rather than exponential, manner.

Most multiplex single tandem repeat kits are validated for between 200 picograms and 2.5 nanograms of template. In that range, stochastic effects like heterozygous allele imbalance, allele drop out, locus drop out, and stutter, are not much of a problem. Forensic analyses, however, sometimes have less than 200 picograms of DNA to work with. Samples may also be a mix of DNA from more than one source.

Kelly Grisedale and Angela van Daal, researchers at Bond University in Australia, had most recently published a study in Investigative Genetics comparing low template STR with and without consensus profiling. In that study, they supported previous work which showed that increasing the number of PCR cycles increases sensitivity of detection. But with less than 100 picograms of DNA they began to see alleles drop in, as well as loci and alleles drop out. While consensus profiling ought to correct for this problem, the authors concluded that too much information is lost when a low-abundance sample is split. Allele drop-in could be particularly detrimental, said the authors, in forensic cases since it could lead to inappropriate inclusion or exclusion of suspects, or false matches in a database search.

In the BioTechniques study, extracts from buccal swabs were diluted to low template levels ─ ranging from 100 down to 6.25 picograms per microliter. The pre-PCR reaction was performed using the PowerPlex ESI 16 kit from Promega. In this case, the volumes were halved, and half of the template was amplified with forward, half with reverse unlabled primers for the loci targeted in the ESI 16 kit. These primers were also from Promega. Amplification was performed on a LifeTech GeneAmp 9700. Then the forward and reverse reactions were pooled prior to the second amplification, using the ESI kit according to the manufacturer's instructions.

The pre-PCR procedure improved the total number of alleles recovered, as well as peak height ratio, usually a measure of multiple DNA sources in the sample. Although the authors saw more variable results below 50 picograms of template, they concluded the procedure "provides more template copies for STR analysis without introducing significant peak height imbalances," according to the study. Allele drop-in was also minimal.

Low template PCR, specifically STR analysis kits created to meet new US and European forensics standards, will be a workshop topic at the 2014 International Symposium on Human Identification in Phoenix in late September.

In addition to forensic analysis, another realm where this technique may be useful is in archaeological studies, which also have issues of low abundance and oft-contaminated samples.

As covered in GWDN, a 2008 study highlighted contamination issues in the field, and showed the benefit of taking samples for PCR before archaeologists handled materials. In this study, a Danish team donned full body suits, hairnets, gloves, shoe covers, and face masks; the moment the last layer of soil was removed at a Viking era burial site, they extracted two teeth from the remains. They compared PCR results of dental pulp mitochondrial DNA in these specially pulled teeth versus ones obtained via the standard chain of custody, and showed half of the teeth handled first by archaeologists had evidence of modern DNA contamination.

PCR in general appears to be becoming increasingly important in archaeology. A recent paper, for example, determined that sexing medieval skeletal remains by a dedicated PCR multiplex, called Genderplex, may be more reliable than tried-and-true morphological analyses. And last year, remains thought to belong to Richard III were identified using an mtDNA PCR assay.

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