Ultrafast Amplification of DNA on Plastic Microdevices for Forensic Short Tandem Repeat Analysis

The majority of microfluidic devices used as a platform for low‐cost, rapid DNA analysis are glass devices; however, microchip fabrication in glass is costly and laborious, enhancing the interest in polymeric substrates, such as poly (methyl methacrylate) (PMMA), as an inexpensive alternative. Here, we report amplification in PMMA polymerase chain reaction (PCR) microchips providing full short tandem repeat profiles (16 of 16 loci) in 30–40 min, with peak height ratios and stutter percentages that meet literature threshold requirements. In addition, partial profiles (15 of 16 loci) were generated using an ultrafast PCR method in 17.1 min, representing a ~10‐fold reduction in reaction time as compared to current amplification methods. Finally, a multichamber device was demonstrated to simultaneously amplify one positive, one negative, and five individual samples in 39 min. Although there were instances of loci dropout, this device represents a first step toward a microfluidic system capable of amplifying more than one sample simultaneously.     

High‐Throughput STR Analysis for DNA Database Using Direct PCR,

Since the Korean criminal DNA database was launched in 2010, we have focused on establishing an automated DNA database profiling system that analyzes short tandem repeat loci in a high‐throughput and cost‐effective manner. We established a DNA database profiling system without DNA purification using a direct PCR buffer system. The quality of direct PCR procedures was compared with that of conventional PCR system under their respective optimized conditions. The results revealed not only perfect concordance but also an excellent PCR success rate, good electropherogram quality, and an optimal intra/inter‐loci peak height ratio. In particular, the proportion of DNA extraction required due to direct PCR failure could be minimized to <3%. In conclusion, the newly developed direct PCR system can be adopted for automated DNA database profiling systems to replace or supplement conventional PCR system in a time‐ and cost‐saving manner.     

中国成都地区汉族群体5个STR基因座的遗传多态性

采用PCR扩增,聚丙烯酰胺凝胶电泳分析技术,调查中国成都汉族群体DIS1656、D851179、D9S302、D185535及D195253等5个STR基因座的等位基因频率分布。D1S1656检出11个等位基因,35种基因型;DSS1179检出9个等位基因,32种基因型;D95302检出12个等位基因,50种基因型;D185535检出7个等位基因,20种基因型;D195253检出8个等位基因,28种基因型。5个STR基因座基因型频率分布符合Hardy-weinberg平衡（P＞0．05）。个人识别机率（DP）为0．92～0．98。分析了二代3口之家的遗传模式,证明5个STR基因座均符合孟德尔遗传规律。5个STR基因座PCR扩增采用同一条件,方法简单、快速、灵敏、重复性好,可用于法科学亲子鉴定和个人识别。     

Concordance Between the AmpFℓSTR® MiniFiler™ and AmpFℓSTR® Identifiler® PCR Amplification Kits in the Kuwaiti Population

Abstract:  The AmpFℓSTR^® MiniFiler™ polymerase chain reaction amplification kit, developed and supplied by Applied Biosystems, complements the AmpFℓSTR^® Identifiler^® polymerase chain reaction amplification kit (Applied Biosystems, Warrington, U.K.) by improving the success rate when profiling DNA that is degraded or contains inhibitors. Before applying the MiniFiler™ kit to casework, the profiles from 200 unrelated Kuwaitis were compared to Identifiler^® profiles. Concordance was observed for 99.875% (1598 of 1600) of the compared STR loci. The two discordant profiles displayed allelic dropout: one at the D13S317 locus due to nonamplification of allele 10 in the MiniFiler™ profile, and one at the D18S51 locus due to nonamplification of allele 18 in the Identifiler^® profile.     

Validation of the Short Amplicon Multiplex Q8 Including the German DNA Database Systems*

Abstract: We have developed a concept to enable the analyzing of degraded stains with limited DNA template quantity. Therefore we have constructed a short tandem repeat (STR) multiplex including the German DNA database systems (Q8). The amplicon lengths are smaller than 280 bp. For the validation of Q8 over 50 degraded samples were investigated. Amplifications were performed with “low copy number” PCR, the number of PCR cycles was increased to 33 and the reaction volume was decreased to 12.5 μL. Compared with the MPX2 and Nonaplex kit, the average success rate was increased using the Q8 kit by approximately 20% and 30%, respectively. The efficiency of a sensitive STR multiplex with reduced amplicon lengths was confirmed in comparing the success rates of Q8 for typing degraded samples and samples with limited amount of DNA template while partial profiles were observed with the majority of the samples using commercially available kits.     

常染色体STR突变基因座父权指数计算

目的概括归纳常染色体STR突变基因座的父权指数计算方法,以在实际检案中应用推广。方法根据目前对常染色体STR基因座突变的认识,用经验递减模型从基因座突变率计算等位基因突变率,分别推导在标准三联体和二联体亲子鉴定中STR突变基因座的父权指数计算式,并举例进行演算。结果总结了在标准三联体鉴定中,只允许假设父突变和既允许假设父也允许母发生突变时,以及二联体鉴定时X和Y的计算式。结论STR基因座发生突变时计算得的父权指数明显低于未发生突变时,提示要检测更多的遗传标记才能使累积父权指数达到认定亲权关系的标准。     

Genetic identification of the mummified fetus

During executing some activities by the police, a mummified human fetus was accidentally revealed on a scrap of paper. It came from pregnancy considerably not carried to term, lasting about 2.5 lunar months. Medical examination did not give evidence of mechanical manipulation such as abortion or disintegration. The aim of the study was the genetic identification of the mummified fetus and an answer to the question if an indicated supposed mother was the mother of the fetus. In the study a relevant factor was maternity confirmation by means of statistic calculations.     

Mutation rates at 14 STR loci in the population from Pernambuco, Northeast Brazil

Definition about mutation rates of short tandem repeats (STRs) loci used in forensic analysis are useful for the correct interpretation of resulting genetic profiles and the definition of criterions for exclusion in paternity testing. Germline mutation of 14 STR loci was studied for 54,105 parent–child allelic transfers from 2575 paternity testing cases carried out during 2000–2007 from the Pernambuco State, Northeast Brazil. The parenthood in each of these cases was highly validated (probability > 99.99%). We identified 43 mutations at 12 loci. Locus-specific mutation rate estimates varied between 2 × 10⁻⁴ and 2 × 10⁻³, and the overall mutation rate estimate was 8 × 10⁻⁴. Mutation events in the male germline were more frequent than in the female germline. The majority of the mutations could be explained by losses or gains of one repeat unit and there was no evidence for selection between insertion or deletion changes. Our data were compared with those of Portuguese and North-American populations for CSF1PO, D18S51, D21S11, D7S820, TH01, TPOX and demonstrated, despite the great difference in the size of the sample, that mutation rates of STR loci in a mixed population do not differ from that encountered in different populations.     

The Upper Silesia (Poland) population and forensic usefulness of 15 autosomal STR loci

Allele frequencies, forensic parameters for the 15 STR loci in the AmpFlSTR^® Identifiler Kit (Applied Biosystems), D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D19S433, VWA, TPOX, D18S51, D5S818 and FGA were determined in a sample of 150 unrelated dead and alive adults from the Upper Silesia region (Poland). The values of heterozygosity (Ht), polymorphic information content (PIC), power of discrimination (PD), matching probability (PM), mean exclusion chance (MEC) and mean exclusion probability (MEP) were calculated. Possible divergence from HWE was determined. Comparison of allele frequencies for examined STR loci between the Upper Silesia population and other Polish populations was carried out.     

Genetic polymorphism of 17 STR loci for forensic use in Chinese population from Shanghai in East China

Allele frequencies for 17 STR loci found in Identifier kit and PowerPlex^®16 Monoplex System were determined in a sample of 1000 unrelated individuals living in Shanghai in East China. The values of observed heterozygosity (Ho), power of discrimination (PD), probability of paternity exclusion (PE) and polymorphism information content (PIC) were calculated. All loci were in accordance with Hardy–Weinberg equilibrium (p < 0.05). The obtained frequency distributions were compared with other previously reported population data.