Validation of reduced volume VeriFiler™ Express PCR Amplification Kit for buccal swab samples extracted using Prep‐n‐Go™ Buffer

Abstract

The efficiency of reduced volume PCR amplification was studied using the VeriFiler™ Express PCR Amplification Kit. Full (25 μL) and reduced (5 μL) volumes were tested in parallel to identify any differences in template DNA sensitivity and other electropherogram parameters. Both volumes produced full DNA profiles down to 0.08 ng/μL DNA concentration at 26 PCR cycles; however, reduced volume produced higher peak heights due to increased signal intensities. Significant difference (p-value ≤ 0.05) in heterozygote peak height ratios was observed between both volumes, where the reduced volume threshold was lowered to 0.6 to accommodate all data points. However, no significant difference (p-value > 0.05) was identified in the stutter ratios between both volumes. The analytical threshold for reduced volume was also determined to be 150 RFU with the presence of template DNA in PCR amplification. When the optimized reduced volume parameters were tested on DNA extracted from buccal swab samples using Prep-n-Go™ Buffer, good quality DNA profiles were produced. Overall, the reduced volume not only showed better results compared to the full volume, but also enable more samples to be processed with a PCR amplification kit, thus reduced the cost.

A systematic approach to improve downstream single‐cell analysis for the DEPArray™ technology

“…In agreement with our results, excessive off-ladder and artifact peaks occurred when the DNA input was high, and a reduction in amplification volume can increase the occurrence of artifacts [27,42]. These findings are reasonable, since higher amounts of DNA (≥62.5 pg) in small amplification volumes (e.g., 6.5 μL) might lead to overloading as reported for ≥500 pg DNA in low-volume PCR on chips [49].…”

“…DNA was extracted from a fresh venous blood sample from a healthy male donor with written consent. As it follows regular sample procedures (i.e., extraction), the biological sample was considered to be a better representative of forensic samples than commercially available human control DNA, which is also used in (validation) studies [2,27]. Extraction was conducted on a Maxwell® RSC Instrument (Promega Corporation), using Maxwell® FSC DNA IQ™ Casework Kit (Promega) with a final elution volume of 50 μL.…”

“…Amplification kits are generally not considered to be a significant factor in a profiles' dropout rate, however many studies have compared the performance of different STR kits and also at low DNA amounts [24,40,41], i.e., for a comprehensive investigation of STR amplification success for touch DNA applications [12]. Moreover, a plethora of studies [24,27,[42][43][44][45][46][47][48] indicate that the use of a reduced amplification volume increases PCR sensitivity. Tay et al [40] examined the performance of three forensic autosomal STR kits on buccal cells according to their manufacturers' recommendations (using DNA ranges from 0.008 to 2 ng).…”

Internal validation of reduced PCR reaction volume of the Qiagen Investigator® Argus X‐12 QS Kit from blood samples on FTA cards

“… Note : The highest LOQ in both controls are indicated in bold. LOQ = Average + 10 SD [17]; Analytical threshold = 2 (Max–Min peak heights) [18]. …”

“…Moreover, the peaks from alleles, forward and reverse stutter and pull‐up in positive controls were removed prior to calculations. The limit of detection (LOD) and the limit of quantitation (LOQ), as suggested by Perry et al [17], were used to determine the minimal threshold. In addition, the method suggested by SWGDAM [18] was used for calculating the analytical threshold.…”