Examining peak height ratios in low template DNA samples with and without sampling using a single-tube extraction protocol
Nguyen, Thutrang Thi
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The developments of the polymerase chain reaction (PCR) and the short tandem repeat multiplex kits increased the ease and lowered the time and sample quantity required for deoxyribonucleic acid (DNA) typing compared to previous methods. However the amplification of low mass of DNA can lead to increased stochastic effects, such as allele drop-out (ADO) and heterozygous peak height (PH) imbalance, which make it difficult to determine the true donor profile. These stochastic effects are believed to be due to: 1) pre-PCR sampling from pipetting and sample transferal of dilute samples prior to amplification resulting in unbalanced heterozygous allele templates in the amplification reaction, and 2) the kinetics of the PCR process where, when few target templates are available, there is uneven amplification of heterozygous alleles during early PCR cycles. This study looks to examine the contribution of PCR chemistry and pre-PCR sampling errors on stochastic effects by utilizing a single-tube DNA extraction and direct amplification method. Cells were collected into tubes using the McCrone and Associates, Inc. cell transfer method, which allowed for approximation of DNA mass without quantification. The forensicGEM® Saliva Kit was used to lyse the cells and inactivate nucleases without inhibiting downstream amplification. The samples were then directly amplified with the AmpFLSTR® Identifiler® Plus PCR Amplification Kit. These samples should only show the effects of PCR chemistry since pipetting and tube transferal steps prior to amplification were removed with the expectation that equal numbers of heterozygous alleles are present in the sample pre-amplification. Comparisons of PH imbalance were made to samples extracted with forensicGEM® but had one or more pipetting and tube transferal steps prior to amplification. These samples were either created through the dilution of stock DNA or from the cell transfer method where aliquots were then taken for amplification; thus these samples would exhibit the effects of both pre-PCR sampling and PCR chemistry errors and inefficiencies. The use of carrier ribonucleic acid (cRNA) was also added to cell transfer samples prior to the amplification of samples to see if it assisted with amplification and increased signal. Results show that the samples with only PCR chemistry generally have significantly higher mean peak height ratios (PHRs) than samples with both pre-PCR sampling and PCR chemistry except in cases where there were large numbers of ADOs. When compared to the diluted samples, the cell transfer samples had significantly higher mean PHR at 0.0625 ng and 0.125 ng, and higher mean PHR at 0.0375 ng when PHs from ADOs are included. Average peak heights (APHs) in the cell transfer samples were also significantly higher in these comparisons. When compared to aliquots taken from cell transfer samples, mean PHR was significantly higher at 0.0625 ng in cell transfer samples with only PCR chemistry than cell transfer samples with both pre-PCR sampling and PCR chemistry; however APH for the samples with only PCR chemistry was also significantly higher in one experiment and not significantly different in another. In a third experiment, the difference in mean PHR was not significant while APH was significantly higher in the samples with pre-PCR sampling and PCR chemistry; however there were also a large numbers of ADOs. Our results also found quantification of dilute samples unreliable but cell counting through the cell transfer method is an appropriate alternative for DNA mass approximation. Also there were no significant changes in PHR or APH in the presence or absence of cRNA.