[Analysis of appropriate amount of template DNA for sinofiler kit by real time quantitative PCR technique].

Shanghai Key Laboratory of Forensic Medicine, Institute of Forensic Science, Ministry of Justice, P.R. China.
Fa yi xue za zhi 05/2008; 24(2):129-30, 133.
Source: PubMed


To explore the appropriate amount of template DNA for Sinofiler Kit.
The DNA samples with ideally genotyped results by Sinofiler Kit were detected by real-time quantitative PCR assay.
It was shown that 1.29-1.51 ng of template DNA in 12.5 microL reaction volume was optimal for STR genotyping with Sinofiler Kit.
Real time quantitative PCR is an accurate and necessary technique for detection of appropriate amount of template DNA for different kits.

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    ABSTRACT: In this study, a new STR 25-plex typing system, including 23 autosomal STRs (D1S1656, D2S1338, D2S441, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, D22S1045, CSF1PO, FGA, Penta D, Penta E, TH01, TPOX, vWA) and a Y-STR locus of DYS391 and amelogenin, was developed. The included 24 STRs belonged to the main international DNA databases (CODIS, ISSL, ESS-extended, UCL, GCL and NCIDD) except D6S1043 (specially chosen for Chinese population). Developmental validation indicated that the STR 25-plex typing system was reproducible, accurate, sensitive and robust. The sensitivity testing of the system was such that a full profile was obtainable even with 125pg of human DNA. Specificity testing was demonstrated by the lack of cross-reactivity with a variety of commonly encountered animal species and microbial pool. For the stability testing, full profiles can been obtained with humic acid concentration≤60ng/μL and hematin<500μM. Also, this multiplex system is suitable for mixture study. All of the minor alleles were called for ratios of 1:1, 1:3 and 3:1 of the mixture with the system. In addition, the whole PCR amplification can finish within 1h, making the system suitable for fast-detection. For the forensic evaluation of the multiplex system, 23 autosomal STRs included followed the Hardy-Weinberg equilibrium. A total of 268 alleles were detected for the 23 autosomal STR loci among 200 individuals. Since 23 autosomal STRs were independent from each other, CMECduo was 0.99999916563607 and CMECtri was 0.99999999986525. All the forensic efficiency parameters demonstrated that this multiplex system is highly polymorphic and informative in the Han population of China. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
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