Forensic casework methodology for direct PCR amplification of blood swabs.
In conclusion, direct PCR amplification produced promising results with blood samples. The aim was to alter the sampling size as opposed to the cycle numbers when performing direct amplification. For the Identifiler® Plus kit, 29 cycle PCR was consistent for both sample sizes tested, with no changes to other cycling conditions. Half volume reactions were found to be suitable for direct amplification as indicated by successful typing of blood samples. Complete profiles were possible down to a dilution of 1/100. There was apparent inhibition in blood samples, enhanced in the presence of TE buffer, but peaks were well balanced. In the absence of TE buffer, reaction volumes would be small, with no guaranteed PCR product for re-electrophoresis, especially if 2 × 1 mm2 sample size were used. As the entire swab is not consumed with direct amplification, resampling would be a feasible option in the case of post PCR failure. Whilst mixtures were detected, they may not reflect the original ratios of contributors in the starting material. Therefore, without further thorough investigation, direct amplification of mixtures would not be recommended. If a mixture were to be obtained using direct amplification, it is recommended that the sample be re-processed using the conventional DNA profiling method. We have shown that direct quantification could be useful as a qualitative tool for high throughput Y-screening of samples and as a quality check to determine the presence of human DNA.view journal