ABO基因分型及其在法医学中的应用

为建立一种ABO血型系统基因分型方法，采用PCR-RFLP技术，成功地将ABO系统区分为AA，AO，AB，OO，BB，BO六种基因型。对240名中国汉族无关个体血样的ABO（基因型频率调查结果表明，6种基因型的频率分布为0．0125～0．3834，符合Hardy－Weinbeng遗传平衡法则（P＞0．1），其DP值为0．8161。家系分析表明，亲代a、b、o基因传递遵守孟德尔遗传规律。对法医学中常见的血痕、混合斑、骨组织及毛发根部等生物样品进行检测，均能准确判定ABO基因型，并可在实际案件鉴定中应用。     

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     

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0.0250～0.4300,杂合度H值为0.5162,个体识别力DP值为0.7111。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。     

Evaluation of four deoxyribonucleic acid (DNA) extraction protocols for DNA yield and variation in restriction fragment length polymorphism (RFLP) sizes under varying gel conditions.

This study originated from discussions and recommendations of the Technical Working Group on DNA Analysis Methods (TWGDAM). Four bloodstain deoxyribonucleic acid (DNA) extraction protocols and five semen stain DNA extraction protocols were evaluated. Nine laboratories participated in the extraction of DNA from 20 bloodstains and 20 semen stains using each protocol. All blood and semen stains originated from a single donor and were prepared under uniform conditions to permit the direct comparison of DNA yields and restriction fragment lengths. The extracted DNA from approximately 600 bloodstains and 700 semen stains was quantified by yield gel analysis and a slot blot hybridization technique. The extracted DNA was digested and restriction fragment length polymorphism (RFLP) patterns were generated using three single-locus probes. The RFLP sizing data produced from the blood and semen stains were evaluated with respect to (1) DNA extraction method, (2) gel length, (3) agarose type, (4) presence or absence of ethidium bromide in the gel, and (5) fragment sizes obtained from DNA isolated directly from the donor's liquid blood. This study demonstrates conclusively that high-molecular-weight DNA can be isolated using either organic or nonorganic DNA extraction protocols and that the resulting RFLP sizes are highly reproducible regardless of gel length, agarose type, or presence/absence of ethidium bromide.     

用引物Y_3,Y_4和DNA扩增技术作血液(痕)和毛根性别鉴定的研究

作者用引物Y_3、Y_4和DNA聚合酶链式反应(PCR)作微量人类血液(痕)和毛根的性别鉴定。扩增的靶序列位于Y染色体DNA特异3.4kb重复序列中,扩增产物为460bp。检材用量为:新鲜血液0.5μl、血痕纱纤维1mm、毛根单个。20例保存4个月的血痕与2例保存6年半的血痕性别判定结果均正确,无性别记载的保存9～11年的3例血痕显现了清晰的460bpY特异DNA扩增带。15例保存20天的自然脱落毛根性别判定结果均正确。本法省略了检材处理中的酚-氯仿抽提DNA等纯化步骤,既简化了实验操作,又减少了检验过程中外源DNA的污染机会和样品DNA的损耗,使这一性别鉴定方法更符合法医学实践的需要。     

ABO genotyping by mutagenically separated polymerase chain reaction

ABO blood groups were determined by the mutagenically separated polymerase chain reaction (MS-PCR). The products from two sets of PCR reactions using the same program for the nucleotides at positions 261 and 703 from cDNA at the ABO locus were used to distinguish A, B and O alleles. Two forward mutagenic allele-specific primers of different lengths for the ABO polymorphic site were paired with the same reverse primer in each PCR reaction. The 216 bp fragment of the PCR products for the 261th nucleotide was A or B allele-specific and the 195 bp fragment was O allele-specific. The 126 bp fragment of the PCR products for the 703th nucleotide was B allele-specific and the 106 bp fragment was A or O allele-specific. The ABO genotypes were determined by the intersection of the predicted alleles from these two PCR reactions. The PCR products were obtained using 10 ng of DNA in 50 μL of PCR reaction mixture, and electrophoresed in 4% agarose gel. In this study, 265 ABO-phenotype known samples (A: 31, B: 48, AB: 6 and O: 180) in Chinese were used. The results of ABO genotypes were AA: 1, AO: 30, BB: 2, BO: 46, AB: 6 and OO: 180. These results were confirmed by the PCR-RFLP ABO genotyping method. This technique is a simple, rapid, and reliable method for ABO genotyping.     

Application of mtDNA sequence analysis in forensic casework for the identification of human remains

In four forensic cases of unidentified skeletal remains investigated in the last year, we were able to attach three to missing persons. In one case we could show that the discovered bone sample did not fit to a missing child. The method for mitochondrial DNA analysis for the routine identification of skeletal remains was established in our institute by typing bone samples of defined age obtained from Frankfurt's cemetery. Reproducible results were obtained for bones up to 75 years old. For analysis the bone samples were pulverised to fine powder, decalcified and DNA was extracted. From the DNA we amplified a 404-bp fragment from HV-1 and a 379-bp fragment from HV-2 of the mtDNA control region. After sequencing of the PCR products, the results were compared to the Anderson reference sequence and to putative maternal relatives.     

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.     

短串联重复位点ACTBP2(SE33)的扩增片段长度多态性研究

应用变性聚丙烯酸胺凝胶电泳（dn－PAGE）结合银染色技术对短串联重复（STR）位点ACTBP2（SE33）的扩增片段长度多态性（Amp－FLPs）进行了研究。在210名无关中国个体中观察到了25个等位基因，等位基因频率分布在0．007～0．093之间。基因型的分布符合Hardy－Weinberg定律，个体识别能力（DP）值为0．99，杂合度（H）为98．7％。七个家系分析的结果表明，该位点的遗传符合孟德尔遗传法则，未观察到变异。对几种常见的法医物证检材的分析表明，该分型系统对DNA降解放为严重的检村适用性强，而且灵敏度高（0．5ng），适合于法医学实际应用。     

Simple and Sensitive Method for Identification of Human DNA by Allele-Specific Polymerase Chain Reaction of FOXP2

Abstract:  The forkhead box P2 ( FOXP2 ) gene is specifically involved in speech and language development in humans. The sequence is well conserved among many vertebrate species but has accumulated amino acid changes in the human lineage. The aim of this study was to develop a simple method to discriminate between human and nonhuman vertebrate DNA in forensic specimens by amplification of a human-specific genomic region. In the present study, we designed an allele-specific polymerase chain reaction (PCR) using primers to amplify smaller than 70-bp regions of FOXP2 to identify DNA as being of human or nonhuman, including ape, origin. PCR amplification was also successfully performed using fluorescence-labeled primers, and this method allows a single PCR reaction with a genomic DNA sample as small as 0.01 ng. This system also identified the presence of human DNA in two blood stains stored for 20 and 38 years. The results suggested the potential usefulness of FOXP2 as an identifier of human DNA in forensic samples.     

HLA—A基因座分步PCR—SSP分型研究

目的使HLA基因分型能应用于法医常见检材的个人识别。方法 建立检测HLA—A基因座的分步PCR—SSP方法。先用一对HLA—A基因座特异的引物作第一次扩增,以所得产物为模板,分别用对HLA—A30、A31、A33特异的3对引物作第二次扩增,二次扩增的产物经电泳判型。结果 1130例血清分型为HLA—A30、A31、A33的血痕,其PCR—SSP分型和血清分型的不符合率为29％;室温保存2年的精斑、唾液斑,保存18年的血痕第一次扩增均获得满意的结果。结论法医亲子鉴定和个人识别宜用基因分型替代血清分型。HLA—A基因座分步PCR—SSP基因分型适用于法医检材。     

Transferrin (TF) typing from semen stains using isoelectric focusing and immunoblotting: correlation of TF types among blood, semen, urine, and vaginal secretion.

We describe a method for obtaining nondistorted and reproducible transferrin (TF) typing from liquid semen and semen stains. Isoelectric focusing of TF isoproteins on polyacrylamide gel (IEF-PAGE, pH 4 to 6.5) was accomplished using a 0.5 mm thick gel. The separated isoproteins were then visualized by immunoblotting with TF-specific antibody. Pretreatment of semen samples with neuraminidase enhanced the TF band resolution. The method was reliable, sensitive and simple, with a high resolution. When maintained at room temperature, laboratory-prepared semen stains were TF-typable for up to at least 50 weeks. The TF types in semen stains were correlated with the types found in the corresponding blood and urine samples. TF typing could therefore provide an additional discriminant characteristic in the forensic examination of semen stains. An evaluation of TF typing by IEF-PAGE and immunoblotting was also performed on casework samples submitted to our laboratory.     

Evaluation of ABO and Lewis genotypes using primer extension preamplification

There are some difficulties with blood typing from ABO variant bloodstains and Lewis negative samples using serologic methods. In these samples, DNA analysis should be employed simultaneously to avoid errors in typing. Primer extension preamplification (PEP) produces copies of template DNA. The minimum quantity to examine nucleotide substitutions of ABO and Lewis genotypes by PCR ranged from 1 to 3 ng DNA. The PCR products with or without PEP treatment showed identical ABO and Lewis genotyping results. Performing both serologic and PCR testing served to crosscheck the ABO and Lewis grouping of such specimens. Errors in ABO and Lewis typing can be avoided as discrepancies are investigated further. The application of the PEP method to limited amounts of DNA samples for ABO and Lewis blood groupings is useful.     

An evaluation of the polymerase chain reaction method for forensic applications

This paper describes the use of polymerase chain reaction (PCR) for amplifying small amounts of DNA obtained from samples of interest to the forensic scientist. A region of the HLA DQalpha (DQa) locus was amplified in DNA prepared from the following: hair roots, liquid blood, blood-stains, semen and vaginal swabs (semen free and semen contaminated). A population study was conducted using DNA from 78 unrelated individuals. The observed distribution of HLA DQa alleles varied from that reported for an American population but obeyed the Hardy-Weinberg equilibrium. Interpretation problems associated with the PCR technique are discussed.     

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.     

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.     

PCR法对HLA-DQ_α基因的分型及其在性犯罪鉴定中的应用

本文应用 PCR 法及 ASO 探针斑点杂交技术,对100例无关个体血液 DNA 及10例性犯罪案件混合斑中精子 DNA 进行了 HLA-DQα基因的扩增及其 DNA 分型。结果正常人0.1～0.3μgDNA 就能满足 DQα基因扩增的需要。在100例个体中可以观察到由4种等位基因组成的10种 DQα基因型。10例不同条件的混合斑中精子 DNA 经30～60次扩增后与 ASO 探针杂交均能准确地判定 DQα基因型。HLA-DQα是个体识别能力较强的遗传标志。本文为性犯罪中精子来源的个体识别提供了一个新方法,具有一定的实用价值。     

用引物Y_3、Y_4和PCR方法鉴定性别的法医学应用

用Y3、Y4和Alu9．1、Alug．2两对引物和PCR方法检测陈旧血痕和毛根的性别获得成功。引物Y3、Y4扩增的靶序列位于Y染色体特异3．4Kb重复序列中，扩增产物为460bp；引物Alu9．1、Alu9.2用以扩增男女共有的Alu重复序列，扩增产物为130bP。室温保存13年之久的19例脐带血血痕（男性9冽，女性10例）和室温保存10～11个月的10例已知性别自然脱落毛根（男性6例，女性4例）的性别测定结果均正确；对一起凶杀案的血痕性别测定为定案提供了重要证据。本方法简化了样品的前处理过程。     

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。     

单管实时PCR快速检验ABO基因型

目的建立快捷特异的ABO基因分型检测方法。方法根据ABO基因结构特点,设计特异性引物和四色双链探针,采用单管实时PCR方法检测ABO基因,结果与传统免疫学方法相对比。结果该方法可检出常见的3个等位基因,区分常见的6种基因型,全部检测过程可在100min内完成。110例中国人的随机个体定型结果与传统免疫学方法一致。结论实时PCR法进行ABO基因分型,简便快捷,灵敏度高,可以有效地为侦查破案服务。