Investigating the origin of stochastic effects in low-template DNA samples by developing a single-tube extraction protocol
Kaeser, Jasmin Christine
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The use of polymerase chain reaction (PCR) has revolutionized DNA typing in forensic laboratories. Producing a deoxyribonucleic acid (DNA) profile now requires less time and less DNA than before. However, not all evidence samples can be reliably profiled, particularly those with low masses of DNA. These samples often exhibit stochastic effects such as allele dropout, elevated stutter and peak height imbalance, which are challenging to separate from true donor alleles. Several scholarly articles have documented these difficulties and suggest that these stochastic effects are due to uneven amplification of heterozygous alleles in early PCR. However, in early PCR all reaction components are at their maximum concentrations and should be able to amplify all alleles in a sample proportionate to their original concentrations. If both alleles are present in the sample at equal concentrations prior to PCR, both alleles should theoretically be amplified with the same efficiency; the fact that this is not the case suggests that there may already be variation within the sample. One possible reason is that pre-PCR sampling error from pipetting and sample transfers results in an uneven number of allele copies in the sample prior to amplification. Thus, it may not be PCR chemistry alone that contributes to stochastic effects, but also sampling error, which creates unequal allele concentrations prior to PCR. In order to separate and study these possibilities, a single-tube DNA extraction method was developed. The forensicGEM™ Saliva kit developed by ZyGEM provides an extraction method that utilizes a thermostable proteinase found in a proprietary Bacillus species to lyse the cell and destroy nucleases without inhibiting downstream amplification. Combining this extraction protocol with the McCrone and Associates, Inc. cell transfer method allowed for the addition of cells directly to the PCR tube, giving an approximate DNA mass without quantitation. These samples should show the effects of PCR chemistry alone, with pipetting and tube transfer steps prior to amplification removed. For comparison, samples of bulk DNA extracted with forensicGEM™ Saliva were diluted down to a comparable concentration and subjected to multiple transfer steps in an effort to identify both pre-PCR sampling error and any error due to PCR chemistry. Results show that the single-tube extraction method gives reliable results, with forensicGEM™ Saliva showing comparable peak heights (PH) and peak height ratios (PHR) to the Qiagen QIAmp DNA Investigator kit and the cell transfer method providing accurate DNA concentrations with minimal PCR inhibition. Comparison of the cell transfer-generated samples to the diluted bulk DNA samples showed that the cell transfer samples had higher average PHRs at 0.0625 ng of target DNA when amplified with Identifiler® Plus, but showed no significant difference between the sample types at 0.125 ng of target DNA. The cell transfer samples were also shown to have lower overall PHs at both concentrations and a higher occurrence of allelic dropout, but only when amplified with the Identifiler® kit; when amplified with Identifiler® Plus, the occurrence of dropout was low for cell transfer and bulk DNA samples at both concentrations. These results suggest that as DNA mass decreases, pre-PCR sampling error may contribute to the development of stochastic effects; however, the vast majority of stochastic effects are due to the PCR chemistry itself. As the PCR chemistry improves and the prevalence of stochastic effects decreases, the importance of pre-PCR sampling error may increase.
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