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Validation of reduced volume VeriFiler ™ Express PCR Amplification Kit for buccal swab samples extracted using Prep‐n‐Go ™ Buffer
Author(s) -
Perry Jessica,
Munshi Tasnim,
Haizel Thomas,
Iyavoo Sasitaran
Publication year - 2022
Publication title -
journal of forensic sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.715
H-Index - 96
eISSN - 1556-4029
pISSN - 0022-1198
DOI - 10.1111/1556-4029.15084
Subject(s) - volume (thermodynamics) , microbiology and biotechnology , dna extraction , chromatography , dna , polymerase chain reaction , chemistry , buccal swab , real time polymerase chain reaction , analytical chemistry (journal) , biology , gene , biochemistry , physics , quantum mechanics
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.