Evaluation of the effects of trehalose on the amplification of the 15 short tandem repeats loci of the AmpFℓSTR Identifiler Plus PCR Amplification Kit
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It is of great importance to be able to unambiguously interpret deoxyribonucleic acid (DNA) profiles, especially with Low Template (LT) DNA and mixture DNA that may contain major and minor contributors. Reducing stochastic effects, such as heterozygote peak imbalance, dropouts, and stutter artifacts have been studied by scientists in order to improve the evaluation of low quality DNA profile. There has been much research on a compatible solute, trehalose, in its effectiveness in enhancing the polymerase chain reaction (PCR), especially with GC-rich templates of DNA, and thermal stabilizing Thermus Aquaticus (Taq) DNA polymerases. Based on previous research, the effect of trehalose on peak heights, peak height ratios, and stutter ratios (n-1) from 15 short tandem repeats (STR) loci of the AmpFℓSTR® Identifiler® Plus PCR Amplification Kit was evaluated with 0.025ng, 0.05ng, 0.1ng, and 1ng of DNA, through the addition of 0M (control), 0.2M, and 0.4M of trehalose for each quantity of DNA. Although there was an observation regarding changes in average peak heights at 1ng of DNA with the addition of 0.2M, and 0.4M of trehalose, no conclusions could be made with the average peak heights for 0.025ng, 0.05ng, 0.1ng, and 1ng of DNA. The reason is that the propagation of pipetting error during the preparation of each batch could have contributed to the difference in the amount of DNA between each conditions which can be directly reflected in peak heights. Furthermore, unexpected discrepancy between the average peak heights for 0.1ng of DNA from the first and the second experiments rendered 0.1ng of DNA incompatible for comparison. With regards to average peak height ratios for 0.025ng, 0.05ng, 0.1ng, and 1ng of DNA, and average reverse stutter ratios for 0.1ng, and 1ng of DNA, there were no evidence to suggest that 0.2M or 0.4M of trehalose had any effects. Consistent trends for 0.1ng (Exp. 1 and 2) and 1ng of DNA from a statistical analysis through one-way ANOVA of individual loci, suggested that trehalose may have varying effects on certain loci. However, this observation must be approached with caution as it is uncertain whether unique trends across each data sets for certain loci were observed by chance due to small sample sizes or due to mechanisms of stutters and trehalose that are currently unknown. Future studies regarding the effect of trehalose on peak heights should be done with more precision through minimizing pipetting error, which can be accomplished by preparing one batch from which aliquots are taken. The result of the research does not show enough evidence to prove the usefulness of trehalose since the addition of trehalose does not yield consistently higher average peak heights and peak height ratios, and lower average reverse stutter ratios across 15 STR loci. Therefore, our results do not support that 0.2M and 0.4M of trehalose are useful within the parameter of forensic DNA analysis as they do not enhance the polymerase chain reaction (PCR) and improve stochastic effects for DNA profiles.
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