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FIU Researchers Show Value of qPCR to Evaluate PCR Inhibition, Improve STR Amplification


NEW YORK (GenomeWeb) — Using Promega's quantitative PCR and short tandem repeat amplification kits, researchers at Florida International University recently characterized the changes in melt curves and amplification efficiencies that result from varying concentrations of eleven different PCR inhibitors.

In a study published online last week in the Journal of Forensic Sciences, the researchers found that PCR inhibitors had unique signatures, with those known to bind DNA affecting melt curves and cycle threshold takeoff points, while those acting on Taq polymerase tending to affect the slope of the PCR amplification curve.

Bruce McCord, associate director of FIU's International Forensic Research Institute and lead author on the study, said the work could potentially help scientists doing criminal justice labwork.

"In forensic analyses ... a jury has got to decide whether or not an individual may have committed a crime; if you have an incomplete result due to inhibition, it's very important to be able to explain it, and why it occurs, to a jury," McCord told PCR Insider this week.

The group chose the Plexor method because it is a qPCR technique that doesn't involve intercalation or otherwise interfere with the DNA, McCord said.

The team had previously released a study in March in Forensic Science International: Genetics that described the way internal control sequences might be affected by PCR inhibitors

"If you use an internal sequence and it doesn't [amplify], then you know you have a problem," McCord said. That work found that inhibitors had strong effects based on the length of the internal control sequence, and much weaker effects based on the sequence itself, regardless of inhibitor type.

McCord's latest work can now provide insights to help forensic investigators determine whether problems with PCR — such as losses of alleles or imbalances in STR peaks — are due to inhibition, degraded sample, or just small sample amounts.

Each potential cause might require different trouble-shooting, McCord explained. With a "degraded sample, you might use shorter PCR amplicons to try to recover more of your sample ... but if it is inhibited, that may not be possible," he said. Small amounts of uninhibited sample, meanwhile, might benefit from other techniques, such as a pre-PCR step.

A growing body of work

Also a professor of biochemistry at FIU, McCord has long been interested in the ways inhibitors distort PCR. His previous investigations, described in a Q&A with PCR Insider in 2010, showed similar results using other methods.

"It was worth a look to see if there were changes [with Plexor compared to the previous studies]. We also wanted to expand the numbers and types of inhibitors, and compare the results with the STR typing [and use] a more advanced, more inhibition-resistant kit for the STR typing with this procedure," he said.

The initial analyses of inhibitors had been done with "kind of a homebrew SYBR Green procedure," McCord said. One of the concerns was whether this intercalating dye would have some effect on the melt curves. "Plexor is a quenching assay, and the amount of fluorescence actually decreases with the number of molecules [in the PCR] … and it doesn't include intercalating dyes, so that makes it kind of interesting."

Using the Plexor HY qPCR kit, the researchers monitored the effect of varying concentrations of known PCR inhibitors on amplification efficiency and slope, Ct value, and melt curve. It found bile salts, humic acid, hematin, melanin, and EDTA predominantly inhibited DNA binding. Tannic acid and calcium inhibited Taq polymerase itself. A combined effect, or mixed mode, was seen with phenol, collagen, urea, and guanidinium.

In general, the results were about the same with Plexor as with prior studies, McCord said. "The melt curves were a little less pronounced, but I think that was because this is a quenching assay with a single dye at the very end of the molecule, so the effect isn't quite as strong ... [as] the SYBR green."

However, the current study also took the analysis to the next level, employing multiple modeling algorithms, to try to get at the aspect of PCR most impacted by each inhibitor. It also compared these results to subsequent STR amplification using Promega's PowerPlex 16 system, to show that the real-time PCR data could predict the effects of inhibitors on amplification of buccal swab DNA.

The current work may be particularly useful to his colleagues in forensic sciences, McCord suggested, because Plexor is "a commercial kit, and it's used worldwide for forensic assays." Researchers who have a small amount of template to work with, and who may be seeing the effect of inhibitors, might use this analysis to predict which inhibitor might be impacting their results, and try to find a specific way to remove the inhibitor without losing sample.

Studies delineating the scope of PCR inhibition seem to be impacting the field of forensics more and more, McCord suggested.

"You're starting to see a lot more labs doing robotic extractions, manufacturers increasing inhibition resistance [of buffers], all based on the understanding that this is a problem — forensic samples aren't clean, and we need to be sure that we get good results, particularly in the case of mixtures," he said.

McCord will be presenting a workshop at the American Academy of Forensic Sciences' annual meeting in February, he said, and "some of this work will be discussed at that time."

As covered in GWDN, McCord also received Department of Justice funding in 2012 for work with Pressure Bio to improve upon a commonly used rape kit. That project is now coming to fruition, he said, with a study showing they can "selectively extract sperm versus female epithelial cells by a combination of alkaline treatment, temperature and high pressure" to be published in the near future. McCord said they have also filed for a patent on this method, which improves both the recovery and selectivity of swabs from rape kits.