A novel method for ABO genotyping using a DNA chip

ABO genotyping is often performed to identify the blood type of decomposed samples, which is difficult to be determined by a serological test. In this study, we developed a simple method for ABO genotyping using a DNA chip. In this method, polymerase chain reaction-amplified and fluorescent-labeled fragments in the ABO gene and primate-specific D17Z1 were hybridized with DNA probes on a chip designed to detect single nucleotide polymorphisms (SNPs) in the ABO gene and part of the D17Z1 sequence. Using blood samples from 42 volunteers and 10 animal species, we investigated whether the chip could be used to detect SNPs in the ABO gene and the D17Z1 sequence. This method was then applied to various forensic samples, and it was confirmed that this method was suitable for the simultaneous analyses of ABO genotyping and species identification. This method fulfills the recent need for the development of rapid and convenient methods for criminal investigations.     

Multicolor Real-Time PCR Genotyping of ABO System Using Displacing Probes

Abstract:  Rapid and informative ABO genotyping has become increasingly popular in forensic use. We developed a multiplex real-time polymerase chain reaction (PCR) approach to genotype ABO major groups and subgroups. Seven differently fluorophor-labeled displacing probes for O¹(261delG), A(261G), A(796C/803C), B(796A/803C), O² (802G>A), A² (1059delC), and A² (1009A>G) were combined in one or two PCRs to determine either ABO major groups or subgroups. The method correctly detected 13 reference DNA samples. A blind test of 237 samples resulted in complete agreement with their phenotypes, and 110 of these 237 samples as well as with PCR-SSP method. The whole analysis could be finished in less than 100 min at substantially low material cost and the template DNA ranging from 0.16 to 500 ng per reaction could be quantitatively detected. Despite the limited informativeness of ABO genotyping, the developed methods could find application in rapid and inexpensive screening of forensic settings.     

Simultaneous detection of ABO and Secretor-nonsecretor blood groups from forensic biological samples by fragment analysis

This paper describes the simultaneous detection of ABO and Secretor-nonsecretor (SE) blood groups from forensic biological samples by fragment analysis using the ABI PRISM^® 3130 genetic analyzer. The method allows the assay of well-known base changes at three nucleotide positions 261, 796 and 803 on cDNA of the ABO gene, and at 385 and 428 on cDNA of SE gene and a SE pseudo gene, so that reliable group prediction is established by the presence of representative alleles. As a result, simultaneous detection of ABO and SE blood groupings from biological samples was correctly determined by our methods.     

运用二重PCR和DNA芯片技术检测ABO基因型

目的以玻片为载体,用寡核苷酸探针杂交技术检测ABO基因型。方法根据ABO基因座外显子6和外显子7的3个SNP点的序列分布特征设计4条寡核苷酸探针,制成分型芯片。将待测样品DNA用末端标记了Cy5的引物进行二重PCR扩增,产物与芯片上的探针进行杂交,根据杂交产生的荧光信号确定样品的ABO基因型。结果利用ABO芯片,可对血斑、毛发等微量检材进行ABO基因型检测。对115名汉族无关个体的调查表明,ABO基因型的分布符合Hardy-Weinberg平衡,等位基因杂合度观察值和期望值分别为0.591、0.616,多态信息含量为0.544,二联体和三联体非父排除率分别为0.188、0.334,个体识别能力为0.777。结论通过DNA芯片检测ABO基因型的技术适用于法医学样本,可满足高通量的检测需求。     

A New Method for Human ABO Genotyping Using a Universal Reporter Primer System

Abstract: We developed a new method for forensic ABO genotyping based on a universal reporter primer (URP) system. This allows for the simultaneous detection of six single nucleotide polymorphism (SNP) sites in the ABO gene (nucleotide positions 261, 297, 526, 703, 796, and 803). This URP system provides obvious peaks, ranging from 82 to 151 bp in length. ABO genotypes were classified and successfully genotyped by our method, including minor alleles that may cause a discrepancy between the genetic data and serological phenotypes. Full profiles were identified using as little as 0.1 ng (0.05 ng/reaction) of standard K562 and 9947A DNA. Moreover, the success rate of genotyping from a URP system was much higher than that from a conventional primer extension method in degraded DNA. This method enables simple and rapid detection of multiple SNP sites on human ABO genes and is highly specific and sensitive when using limited and degraded DNA.     

ABO基因型检验及其法医学应用研究

目的采用ＰＣＲ技术对ＡＢＯ血型系统进行基因型检验。方法选择最佳扩增条件进行四引物复合扩增,用限制性内切酶ＫｐｎＩ和ＡｌｕＩ分别酶解扩增产物,电泳分离、银染显色法检验ＡＢＯ基因型。结果对２７０例血斑、２０例混合斑、２０根毛发（有毛囊）、１２份唾液斑等不同的生物检材进行了分型,与血清学方法检验结果相符。结论该方法能够应用于法医学的检验     

DNA芯片分析单核苷酸多态性及其法医学应用

DNA芯片技术作为一门新兴的高科技生物技术,显示了它旺盛的生命力和迅猛的发展势头。单核苷酸多态性(SingleNucleotidePolymorphisms,SNPs)是最常见的人类基因组变异类型。它作为一种有效的人类遗传标记,在疾病相关性研究、药物基因组学、法医学、人类进化和迁移等研究中发挥了重要作用。它同DNA芯片技术结合运用也将在法医检验,尤其是亲子鉴定和个人识别中发挥重要作用。本文主要讨论了DNA芯片和SNPs的特点,以及二者联合运用于法医学的价值。     

Rapid direct PCR for ABO blood typing

Many different molecular typing methods have been reported to complement routine serological ABO blood typing in forensics. However, these ABO genotyping methods are often time-consuming and call for an initial DNA isolation step that requires the use of expensive kits or reagents. We report here a rapid direct ABO genotyping method that eliminates the need for DNA extraction from fresh blood, hair, and body fluid stains before PCR. Using a fast PCR instrument and an optimized polymerase, the genotyping method-which employs a multiplex allele-specific primer set for the simultaneous detection of three single-nucleotide polymorphism (SNP) sites (nucleotides 261, 526, and 803)-identifies A, B, O01/O02, O03, and cis-AB01 alleles in around 70 min from sample collection to electropherogram. Not only will this ABO genotyping method be efficiently used in forensic practice for rapid screening of samples before full-blown multilocus short tandem repeat profiling, but it will also demonstrate an example of rapid direct genotyping of SNPs that offers the advantages of time- and cost-efficiency, convenience, and reduced contamination during DNA analysis.     

PCR-RFLP技术鉴定ABO基因型的法医学应用研究

目的 建立 PCR－ RFLP、非变性 PAG胶垂直电泳和银染技术进行 ABO基因分型的方法体系,并对 200名广东汉族人群 ABO基因型频率进行了调查。方法 用 Chelex－ 100和酚、氯仿抽提法处理样本, PCR扩增后用非变性聚丙烯酰胺凝胶垂直电泳和银染技术检测分型。结果 ABO位点特异性扩增片段长度为 175bp～ 210bp, 6种基因型频率分布为 0.025 0～ 0.430 0,杂合度 H值为 0.516 2,个体识别力 DP值为 0.711 1。结论 该方法可成功运用于血液、血痕、精斑、毛发、骨组织和混合斑等检材的个体识别及亲权鉴定的检验。     

Detection and classification of composite resins in incinerated teeth for forensic purposes

The great demand for esthetic restorations has resulted in placement of large numbers of composite resin fillings. The popularity of these materials is reflected in the quantity and variety of resin brands currently on the market. The ability to distinguish resin brands can aid in positive identification of burn victims, assuming that appropriate dental records exist. Scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDS) was used to characterize the structure and composition of 10 modern resins. The structure of each resin was unique to manufacturer, and elemental analysis allowed separation into distinct groups. These 10 resins were also placed in extracted teeth and incinerated at 900 degrees C for 30 min, simulating near cremation conditions. The resins were identifiable by SEM/EDS after incineration, and the elemental composition remained almost unchanged. The data produced are immediately useful for resin identification in forensics, and comparative analysis can be readily performed using standard equipment. This work represents the initial stage of database generation.     

4种溶液对陈旧斑迹胶体金试剂条ABO检测结果比较

目的比较4种不同溶液用于陈旧斑迹ABO胶体金试剂条分型检验的结果。方法采集已知血型的静脉血127份,唾液73份制备斑迹,放置1~2年;分别用去离子水、PBS、PBS(含2%吐温20)、PBS(含2%吐温20、1%吐温80)4种溶液浸泡,再用ABO胶体金试剂条检测其血型,观察结果的清晰度及准确度。结果用不同溶液浸泡后进行ABO分型检测结果中,去离子水和PBS溶液分型检测线不清晰,难以判型;含吐温的PBS溶液分型检测线比较清晰,所测样本结果均准确,其中同时含吐温20和吐温80的溶液结果更佳。结论根据本文结果,在陈旧斑迹的ABO血型检验中可选择使用含有吐温的PBS溶液作为斑迹浸泡溶液。     

ABO genotyping by duplex amplification and oligonucleotide arrays assay

Objective

Research on the application feasibility of ABO genotyping for forensic identification by oligonucleotide arrays assay.

Methods

Oligonucleotide microarrays which detect three different SNPs in exon 6 and exon 7 for ABO genotyping were used. After hybridization wash, the arrays were scanned and fluorescence intensities were analyzed using microarray population studies on ABO was carried out in a sample of 115 unrelated Chinese Han individuals oligonucleotide arrays for genotype detection. The method was also applied to cases.

Results

Technique could identify six genotypes of ABO system and the results of GeneChip analyses confirmed by PCR–RFLP. According to the results of population studies, no significant deviations Hardy–Weinberg equilibrium could be found. The observed heterozygosity (H-obs) was 0.591. Expected heterozygosity (H-exp) was 0.616. The polymorphic information content (PIC) was the average exclusion probability in paternity testing for duos (PE (1)) was 0.188. The average exclusion probability in paternity testing for trios (PE(2)) was 0.344. The discrimination power 0.777.

Conclusion

The data and case application demonstrated that ABO typing by oligonucleotide probe arrays was a useful technique for paternity testing and individual identification.     

47个SNPs复合检测方法的建立及其法医学应用

目的建立47-plexSNPs复合检测方法，评价其在法医学中的应用价值。方法筛选46个常染色体SNPs和1个Y—SNPs，使用2个检测体系分别对47个SNPs进行单管内复合PCR扩增，采用荧光标记单碱基延伸法和毛细管电泳检测技术进行分型检测；并用建立的方法对260份广东地区无关个体血样进行47个SNPs分型。结果建立的47-plex SNPs的复合检测体系灵敏度高，种属特异性好；260名个体所有SNPs均能准确分型，群体内基因型频率分布均符合Hardy—Weinberg平衡，累积个人识别率大于0．9999，累积非父排除率为0．99982，累积偶合率为6．24×10一。结论本文47-plex SNPs复合检测方法能同时对47个SNPs进行快速、准确的检测，在法医学个体识别鉴定中具有良好的应用前景。     

微测序技术检测12个Y—SNP及其遗传多态性

目的应用SNaPshotKit对Y染色体上12个SNP位点进行快速而准确的检测,对四川地区78个汉族男性无关个体进行群体遗传学研究,并对陈旧骨骼和性犯罪案件的相关物证进行检验。方法对SRY2627、SRY1532、M13、M20、SRY8299、Tat、M69及M9、92R7、M17、M19、M112两组共12个Y-SNP位点进行复合扩增,PCR产物经纯化处理后,采用SNaPshotKit试剂结合毛细管电泳技术对单核苷酸多态性进行检测。结果建立了12个Y-SNP位点的微测序快速检测系统,在四川地区人群中发现M9、SRY8299二个位点存在变异。结论复合扩增结合微测序技术能够同时对多个Y-SNP的多态性进行快速而准确的检测,建立的检测系统在法医学个体识别中具有应用价值。     

ABO基因分型与多重STR联合检测在法医学中的应用

目的建立ABO基因型和Goldeneye16A试剂盒联合检测的方法,并评价其在法医学实践中的应用价值。方法将6种ABO基因型(A/A,A/O,B/B,B/O,A/B,O/O)的序列特异性引物(PCR-SSP)检测方法与Goldeneye16A试剂盒相整合进行同步分型。通过对460份男性个体血痕样本、9947A DNA及90份案件样本进行检测,考察方法的一致性、灵敏度及对法庭科学检材的适用性。结果应用本文方法可同时检出6种ABO基因型和15个常染色体STR基因座及性别决定基因座,检测灵敏度为125pg,其中ABO基因检测灵敏度达63pg。460份男性血痕和90份案件检材证实该联合分型方法用于各类检材结果准确、稳定。结论本文ABO基因分型与多重STR联合检测方法,适用于各类含有核细胞的生物检材,在法庭科学DNA鉴定中有较好的应用前景。     

焦磷酸测序技术分析单核苷酸多态性在法医学中的应用

单核苷酸多态性（SNP）是新一代的法医学遗传标记,有望成为法医实践中解决高度降解检材及特殊案件DNA鉴定的重要工具。近年来涌现出许多高通量的SNP分析方法,如引物延伸结合时间飞行质谱分析法、微测序法、SNP lex以及焦磷酸测序法等。本文重点对焦磷酸测序技术的原理、步骤及其在法医学中的应用进展进行简要的综述。     

SNP genotyping of forensic casework samples using the 52 SNPforID markers

The analysis of degraded DNA is one of the biggest challenges in forensic casework. SNPs, which can be amplified using small amplicons, have previously been successfully applied to the profiling of forensic evidence that could not be analyzed using conventional STRs. Here we selected the 52 SNPforID SNP markers, with amplicons that ranged in size from 59 bp to 115 bp, and used them to profile a range of casework samples from Malaysia. DNA degradation is a common problem in Malaysia due to the high temperatures and humidity. To carry out the study we modified the 52 SNPforID markers into four 13-plex SNaPshot assays to enable easier interpretation of profiles on the ABI PRISM^® 310 and 3500.Fifty-one crime samples comprising bloodstains on cloth, swabs, and a mat and 2 swabs of trace DNA from 10 crime scenes in Malaysia were profiled after DNA extraction using a phenol–chloroform method. The samples were also subjected to STR analysis using the Powerplex^® 16 system (Promega), which resulted in only 17 full profiles and 9 partial profiles; using SNPs, 36 full profiles and 5 partial profiles could be generated.     

东北地区4个民族ABO血型基因频率的调查分析

目的研究东北地区汉族、满族、蒙古族和朝鲜族人群中ABO基因型及等位基因频率分布特点。方法应用GoldeneyeTMABO基因分型系统共检测1588例4民族人群样本的ABO基因型,并绘制遗传系统发生树。结果 4民族A、B、O基因频率分别为:0.208 9、0.241 5、0.549 6(汉族),0.230 8、0.233 0、0.536 2(满族),0.213 0、0.237 7、0.549 3(蒙古族),0.223 0、0.230 0、0.547 0(朝鲜族)。辽宁满族与吉林满族ABO血型分布存在显著性差异,并在系统发生树中,吉林满族相对其他六个群体,自成一簇。辽宁满族与吉林朝鲜族,辽宁汉族与辽宁蒙古族遗传关系较近。结论东北地区汉、满、蒙、朝4民族人群ABO血型分布状况及基因频率均相对稳定。比较相关群体间遗传距离发现,同地区或近地域内的不同民族群体之间存在一定的基因交流,个别群体又相对独立。     

The analysis of illicit methaqualone containing preparations by gas chromatography-mass spectrometry for forensic purposes

A validated gas chromatographic-mass spectrometric method for quantitative analysis of methaqualone (MTQ) in illicit preparations is reported. The method proved to have a coefficient of variation of below 5%. Four batches of seized tablets, two pairs with similar imprints, were analyzed. It was found that the average MTQ concentration in all four batches of tablets differed significantly (p = 0.01) rendering it impossible to conclude that, on the basis of MTQ concentration alone, the batches with a similar logo originated from the same manufacturer or manufacturing batch. Conversely, it can be said that in this case, the four batches originated from either different clandestine laboratories or manufacturing batches.     

Analytical survey of restorative resins by SEM/EDS and XRF: databases for forensic purposes

Frequently in forensic cases, unknown substances must be identified. Automated databases can ease the burden of comparison as materials may be compared against many known standards in a relatively short period of time. It has been shown that dental resins can be named according to brand or brand group even in conditions as harsh as cremation. Databases are already in use for many materials, but no such database exists for dental resins. Thus, two databases were generated. One utilized a laboratory-based method, scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM/EDS), in conjunction with the Spectral Library Identification and Classification Explorer (SLICE) software. The other was based on portable X-ray fluorescence (XRF). The ability to perform database comparison with portable instrumentation can thus be brought directly to the field. Both the SLICE and XRF databases were evaluated by testing unknown resins. EDS is a well-established technique and the SLICE program was demonstrated to be a good tool for unknown resin identification. Portable XRF is a relatively new instrument in this regard and its databases have been constructed mostly for metal alloy comparison and environmental soil testing. However, by creation of a custom spectral library, it was possible to distinguish resin brand and bone and tooth from other substances.