Developmental validation of the AmpF?STR SEfiler Plus™ PCR amplification kit: An improved multiplex with enhanced performance for inhibited samples

Since its introduction in 2002, the AmpF?STR^® SEfiler™ kit has provided a highly discriminating DNA profiling option to German forensic laboratories by combining the widely used SGM Plus^® Kit loci with the SE-33 locus required for the German DNA Database. Whilst proving successful on database samples, laboratories using the SEfiler™ kit have reported the need for chemistry better able to handle the ever-increasing number of casework samples.The new AmpF?STR^® SEfiler Plus™ kit contains the same loci and primer sequences as the SEfiler™ kit but uses improved synthesis and purification processes to minimize the presence of dye-labeled artifacts. Other improvements include modified PCR cycling conditions for enhanced sensitivity and a new buffer formulation that improves performance with inhibited samples when compared to the original SEfiler™ kit.Validation studies demonstrating the effectiveness of the multiplex are presented with emphasis on the models of inhibition and casework samples.     

荧光标记STRs复合扩增分析混合血样品

探讨应用荧光标记STRs复合扩增技术能够检测出混合血样品中较少个体成份的最低检出量及所占的比例多少与基因型的峰值高低是否存在一定的剂量反应关系。采用荧光标记STRs复合扩增技术 ,分析 4对两无关男 /女的混合血样品。扩增基因座包括D8S1179、D2 1S11、D18S5 1、D3S135 8、vWA、FGA、D5S818、D13S317、D7S82 0及性别Amelogeine。结果表明 ,对经用酚 /氯仿有机溶剂方法提取的混合血样品中较少成份的最低检出量为 ,能够从10ng混合DNA中检出 1ng的较少成份 ;从 10∶90至 5 0∶5 0 5个不同比例组 ,较少成份所占比例多少与其对应基因型峰值的高低呈现一定的正相关趋势。应用荧光标记STRs复合扩增技术可能较好地解决混合血样品的个体识别问题     

Validation of the AmpF?STR SEfiler Plus™ PCR Amplification kit for forensic STR analysis

Validation of the AmpF?STR^® SEfiler Plus™ PCR Amplification kit with 29 and 30 PCR cycles for forensic STR analysis demonstrated that the kit had fewer artefacts than the AmpF?STR^® SGM Plus™ kit (28 PCR cycles). The SEfiler Plus kit was more sensitive and devoid of colour artefacts, but showed more stutters, drop-ins, drop-outs and allelic imbalances.     

Preliminary trials of low volume AmpFlSTR Profiler Plus amplification using AmpliGrid (AG480F) slides

Low volume PCR using the AmpliGrid (480F) slide system can potentially enhance the generation of more complete profiles from trace samples, in addition to providing a more cost-effective alternative for typing standard samples. Based on our preliminary results, implementation will require a reasonable investment in optimisation and validation for the intended purpose.     

Optimization and validation of a fast amplification protocol for AmpFlSTR Profiler Plus for rapid forensic human identification

The goal of this work was to optimize and validate a fast amplification protocol for the multiplex amplification of the STR loci included in AmpFlSTR^® Profiler Plus^® to expedite human DNA identification. By modifying the cycling conditions and by combining the use of a DNA polymerase optimized for high speed PCR (SpeedSTAR™ HS) and a more efficient thermal cycler instrument (Bio-RAD C1000™), we were able to reduce the amplification process from 4 h to 26 min. No modification to the commercial AmpFlSTR^® Profiler Plus^® primer mix was required. When compared to the current Royal Canadian Mounted Police (RCMP) amplification protocol, no differences with regards to specificity, sensitivity, heterozygote peak height ratios and overall profile balance were noted. Moreover, complete concordance was obtained with profiles previously generated with the standard amplification protocol and minor alleles in mixture samples were reliably typed. An increase in n − 4 stutter ratios (2.2% on average for all loci) was observed for profiles amplified with the fast protocol compared to the current procedure. Our results document the robustness of this rapid amplification protocol for STR profiling using the AmpFlSTR^® Profiler Plus^® primer set and demonstrate that comparable data can be obtained in substantially less time. This new approach could provide an alternative option to current multiplex STR typing amplification protocols in order to increase throughput or expedite time-sensitive cases.     

Population genetics for Y-chromosomal STRs haplotypes of Chinese Tujia ethnic group

Allele and haplotype frequencies of 11 Y-chromosomal short tandem repeats (STRs) included in the PowerPlex^® Y Systems (Promega) were determined in a sample of 215 unrelated healthy male individuals of Chinese Tujia ethnic group living in Chongqing (Southwest of China). The gene diversity values of the Y-STRs loci ranged from 0.3757(DYS391) to 0.9170 (DYS385a/b). A total of 195 haplotypes were identified in the Y-STR loci, among which 180 were unique, 11 were found in two individuals, 3 were shared in three individuals and 1 was shared in four individuals. The observed haplotype diversity value and discrimination capacity were 0.9942 and 0.9070, respectively.     

水浸和洗涤血样本的DNA检验

目的对经水作用的血样本DNA分型检验结果进行分析探讨。方法全血样本分为两组,水稀释组用水将全血样本稀释5、10、20、25、30倍后制作血斑;洗涤组分为纯水手洗、肥皂弱洗、肥皂强洗、84消毒液浸洗和洗衣粉机洗等5种洗涤方式。所有样本用IQ试剂盒提取DNA,Identifiler PlusTM试剂盒扩增,并进行分型检测。结果血液稀释组中心部位检材,均无等位基因丢失,除30倍稀释样本外,峰高均衡性均大于70%;外周部位检材出现2～10个等位基因丢失,峰高均衡性均小于50%。洗涤组中除84消毒液洗涤样本未检出DNA谱带外,其余均无等位基因丢失,而峰高及均衡性以手洗和肥皂弱洗样本更好。结论经水稀释或洗涤剂清洗的血样本,即使联苯胺预实验结果为阴性,选取合适的检验部位,仍可获得DNA分型。     

Divergent genetic strata in five Bahamian islands

Based on historical records, the genetic landscape of the Bahamian archipelago is presumed to be complex and to exhibit island-specific characteristics, yet the genetic composition of the island chain, which could corroborate or refute these past accounts, remains poorly defined. As such, the current investigation was undertaken to genetically characterize 5 Bahamian populations representing the Northwest (Grand Bahama and Abaco) and Central (Eleuthera, Exuma and Long Island) Bahamas across the 15 autosomal Identifiler loci routinely employed in forensic analyses. Altogether, our findings suggest that Bahamians are a genetically heterogeneous group, with each island sampled receiving differential contributions from African, European, East Asian and Native American sources. Even though the strongest genetic signal in all 5 collections emanates from continental Africa, inter-island differentiation is noted in both the Structure and admixture analyses. The presence of alleles not in common among the 5 insular populations also signals genetic heterogeneity among the islands of the archipelago. This is especially the case when considering the Long Island population, which exhibits statistically significant genetic differences in relation to the other Bahamian collections and the New World groups of African descent (Afro-American and Afro-Caribbean) in the G-test pair-wise comparisons, even after application of the Bonferroni adjustment.     

AGCU免提取STR荧光检测试剂盒的验证

目的考察AGCU免提取STR荧光检测试剂盒．对保存在滤纸片或FTA卡上血液样本的直接扩增检测情况。方法使用人GCU免提取STR荧光检测试剂盒,对未经提取的滤纸片血液样本、FTA卡血液样本675份进行直接扩增和18个基因座的DNA分型,并对结果的可靠性进行研究。结果18个基因座检测结果与PP16和ID试剂盒分型结果一致,2000年数据库样本成功率92．3％,2001年数据库样本成功率92．6％,2004以后年数据样本及案件样本、亲子鉴定样本成功率在99％以上。结论AGCU试剂盒可以成功地对滤纸片、FTA卡样本的18个STR基因座进行直接扩增检测,检验结果稳定,分型准确。     

Population genetic data of 15 tetrameric short tandem repeats (STRs) in Berbers from Morocco

The allele frequency distribution of 15 short tandem repeats (STR) loci contained in the AmpFlSTR Identifiler PCR Amplification Kit (Applied Biosystems), was determined in two Berber populations from Asni and Bouhria, in Central and Eastern Morocco, respectively. A total of 209 individuals were typed. No deviations from the Hardy-Weinberg equilibrium were observed for Asni at the 15 STRs loci whereas for the Bouhria samples, two loci (D5S818 and TH01) showed significant departures from Hardy-Weinberg expectations (after Bonferroni's correction). All loci are highly polymorphic and population differentiation tests showed that the Moroccan samples from Asni and Bouhria have significant differences in 4 out of 15 loci (D21S11, D7S820, D16S539 and TPOX). The aim of the study was to obtain accurate allele frequencies relevant for forensic applications. Comparative analyses between our population data and other population samples gathered from the literature are also presented.     

A comparison of three automated DNA purification methods in Forensic casework

Manual Chelex^®-100 and organic extractions (phenol/chloroform) are used as routine methods at the Swedish National Laboratory of Forensic Science, SKL. The aim of this study was to find an automated DNA purification system to replace the organic method. The following methods were evaluated and compared to each other and to the organic method used routinely; BioRobot^® EZ1 with EZ1 DNA Investigator Kit and Card (Qiagen), iPrep™ Purification Instrument with iPrep™ ChargeSwitch^® Forensic Kit and Card (Invitrogen), Magnatrix™ 1200 Workstation with the Magnatrix™ gDNA Blood Kit Forensic and two different protocols; Forensic protocol A and B (Magnetic Biosolutions). Blood on fats, cotton swabs, moist snuff, paper towels and leather, post-mortem blood and muscle tissue were extracted with the different methods. DNA concentration and quality of the electropherograms were examined. Individual comparisons between the four extraction methods showed that iPrep™ and Magnatrix™ 1200 gave significantly lower mean quantities compared to BioRobot^® EZ1 and the organic extraction method (p < 0.05). There were no significant differences between the latter two. BioRobot^® EZ1 generated the best results and is in the process of being validated for routine analysis at SKL.     

Effect of blood stained soils and time period on DNA and allele drop out using Promega 16 Powerplex kit

Blood stained soils may be of great interest in forensic incidents. Amplification of DNA from soil is often inhibited by co-purified contaminants. Different soils types from Pakistan and Turkey were stained with blood and samples were collected systematically after specified intervals. Rapid, inexpensive, large-scale DNA extraction method involving minimal purification was developed. DNA was quantized using Spectrophotometer and Fluorometer and was confirmed by Agarose Gel Electrophoresis. DNA extracted from different soils in different periods showed a remarkable decrease in yield as well as degradation in every extraction. PCR amplification was performed using various DNA targets present in Promega 16 Powerplex^® System kit. Amplification could not carry out in all loci especially in degraded samples taken after 20 days. Allele n locus drop out was noticed which shows that DNA was degraded. For some loci more than 2 alleles were also noticed showing contamination while working with the blood stained soils.     

Direct STR amplification from whole blood and blood- or saliva-spotted FTA without DNA purification

The DNA purification step has been thought to be essential for typing of STR DNA. However, this process is time-consuming, and there is a risk of unexpected cross-contamination during purification. We report a new method for direct short tandem repeat (STR) amplification using a newly developed direct PCR buffer, AnyDirect, which can amplify STR loci from whole blood and blood- or saliva-spotted FTA cards without DNA purification. The autosomal and Y chromosomal STR loci were analyzed for whole blood and blood or saliva spots of random individuals, followed by comparison of the results with those of corresponding purified DNA. The results from whole blood and blood spots showed perfect concordance with those from purified DNA without allele or locus drop-out. However, in the case of saliva spots, no amplification or locus drop-out was observed in some of the samples, which offers a topic for further study. Additionally, some commercial hot-start DNA polymerases other than AmpliTaq Gold DNA polymerase were also found to be compatible with this buffer system. Therefore, this direct PCR buffer was demonstrated to be useful for fast forensic DNA analysis or criminal DNA databases for which there is no need to store DNA samples.     

Allele frequency distribution for 21 autosomal STR loci in Bhutan

We studied the allele frequency distribution of 21 autosomal STR loci contained in the AmpFlSTR^® Identifiler™ (Applied Biosystems), the Powerplex^®16 (Promega) and the FFFL (Promega) multiplex PCR kits among 936 individuals from the Royal Kingdom of Bhutan. As such these are the first published autosomal DNA results from this country.     

Y-SNPs analysis using two different approaches in a Northern Portugal male population sample

The non-recombining portion of the human Y (NRY) chromosome has various types of variation, including single nucleotide polymorphism (SNP). In spite of their low discrimination power, they provide a powerful and simple exclusion tool for forensic purposes. A special advantage of SNPs is that it can potentially detect smaller DNA fragments (analysis of degraded DNA).The aim of this work consisted in the analysis of a group of SNP polymorphisms (M2, M9, M35, M89, M45, M170, M172, M173, M207 and P25) in a Northern Portugal male population sample, which allows the determination of the most common European haplogroups, including the Northern Portugal ones.The method used for typing these polymorphisms was the real-time PCR with TaqMan probes on the ABI 7000 platform (Applied Biosystems).We had some difficulties in typing some of the markers using this approach. However, the preliminary results obtained for the defined haplogroups are in accordance with those described in close European populations. To confirm the typing and solve the doubts that emerged from the real-time approach, the samples were also typed using SNapShot.     

单细胞检验技术用于混合上皮细胞的分离和检验

目的建立单细胞显微捕获联合低体积扩增技术,用于混合上皮细胞检材分离检验。方法取5名男性口腔上皮细胞拭子浸泡液30μL,分别滴加到5份含同一女性皮肤表皮细胞拭子上,制成5份混合上皮细胞样本为实验组,同时制备同样的5份样本为对照组。实验组样本采用显微捕获单个口腔上皮细胞,并使用低体积扩增技术进行扩增;对照组用M48纯化试剂盒提取DNA,Identifiler试剂盒复合扩增,扩增体系为10μL。所有产物均用ABI 3130遗传分析仪进行STR分型。结果 5份实验组样本均得到男性STR分型结果,5份对照组样本则均仅得到混合分型结果。将该方法应用于1例强奸杀人案例检验,取得了满意效果。结论单细胞显微捕获联合低体积扩增技术可用于混合上皮细胞样本的分离检验。     

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.