降解的DNA的PCR分型—电洗脱回收大片断DNA法

本文分析了降解ＤＮＡ作ＰＣＲ分型易失败的原因，提出了因小片断ＤＮＡ过多，阻碍引物与膜板的复性及阻断引物延伸的新观点。将降解的ＤＮＡ电泳，切除小片断ＤＮＡ，电洗脱回收较大片断ＤＮＡ，成功地对降解ＤＮＡ进行了ＰＣＲ分型。本方法简便、快速、有效。     

HE染色涂片上精子DNA的提取与HLA-DQa基因扩增分型

本文对ＨＥ染色涂片上精子ＤＮＡ的提取、ＰＣＲ扩增分型的可行性与可靠性进行了研究。２０张ＨＥ染色涂片的实验结果表明：ＨＥ染色涂片上的精子是可以被回收并提取到ＤＮＡ进行ＨＬＡ－ＤＱａ基因的ＰＣＲ扩增分型的，其分型结果准确可靠。这说明精子的ＨＥ染色涂片也是进行ＤＮＡ分析的一种很好的检材来源。     

第三讲 法医DNA分型技术与方法

法医ＤＮＡ分型利用分子生物学技术检测、分析人类遗传标记 ,进行个体识别和亲子鉴定。 80年代以来 ,法医ＤＮＡ分型技术跨越了两大步 ,第一代分型是以ＤＮＡ分子杂交为核心的限制性片段长度多态性分析技术 ;第二代是以聚合酶链反应 (ＰＣＲ)为基础衍生出的各种ＤＮＡ标记分析技术。1　限制性片段长度多态性分析技术早年Ｊｅｆｆｒｅｙｓ建立的ＤＮＡ指纹技术本质是ＤＮＡ限制性片段长度多态性 (ＲＦＬＰ)分析 ,技术核心是ＤＮＡ分子杂交。ＤＮＡ指纹图谱的特异性取决于 2个因素 :一是限制性核酸内切酶特异性 ;二是探针特异性。ＲＦＬＰ分析…     

从聚丙烯酰胺凝胶中回收DNA片段直接PCR的简易方法

将聚丙烯酸胺凝胶中ＤＮＡ片段于ＰＣＲ缓冲液中电洗脱，加入ＰＣＲ反应试剂，直接扩增目的ＤＮＡ作进一步分析，ＤＮＡ片段回收效率为０．８９±０．０５，本方法简便、快速。     

DNA分析技术在法科学中的应用及展望

本综述对ＤＮＡ指纹、ＰＣＲ－ＶＮＴＲ、ＰＣＲ－ＳＴＲ、ＰＣＲ－ｍｔＤＮＡ测序等技术的发展,以及其在法科学中的应用领域和发展前景作了系统的阐述。认为由于ＤＮＡ分析技术所具有特点,使之已成为责令法科学生物检材检验的主要手段之一。阐述了现阶段ＤＮＡ分析技术已向标准化、自动化和高鉴别机率方向发展,以及建立ＤＮＡ罪犯数据库的必要性和应用价值。     

线粒体DNA测序分析及其法医学应用

本文建立一种以γ－３２ＰＡＴＰ直接标记ＰＣＲ扩增引物为测序引物、纯化的ＰＣＲ扩增产物为模板、Ｔａｑ酶直接测序的方法、对１００倒无血缘关系的中国汉族人线粒体ＤＮＡ主要高变区Ｄ环区的３２８个碱基（第１６０９１－１６４１８位）进行了核酸序列分析，检出８６种基因型，出现率最高者为７％；检出８１个可变碱基位点。两个非血缘关系的中国汉族人出现完全相同序列的概率为０．００６８。通过对２个四代家系．５个三代家系和１０个两代家系分析扯实线粒体ＤＮＡ是母系遗传。     

从聚丙烯酰凝胶中回收DNA片段直接PCR的简易方法

将聚丙烯酰胺凝胶中ＤＮＡＳ片段于ＰＣＲ缓冲液中洗脱，加入ＰＣＲ反应试剂直接扩增目的ＤＮＡ作进一步分析，ＤＮＡ片段回收效率为０．８９±０．０５，本方法简便、快速。     

直接同步扩增5种基因位点

来用直接同步ＰＣＲ扩增５种基因位点及反向打点杂交技术，使扩增产物ＤＮＡ与膜探针条杂交。再通过酶联底物显色反应对ＬＤＬＲ、ＧＹＰＡ、Ｄ７Ｓ８、Ｇｃ和ＨＢＧＧ进行联合分型。该方法特别适用于微量、陈旧或降解的ＤＮＡ样本，其操作简便、迅速，应用在亲子鉴定案例，亲子关系概率高达９９．７５％。该技术在法医学、遗传学及临床诊断中具有重要价值，并将改变ＤＮＡ分型技术难于标准化的局面．     

甲醛固定及石蜡包埋组织的DNA分型及其应用

目的：建立甲醛固定及石蜡包埋组织ＤＮＡ的基因分型方法并使之应用于实际检案。方法：用Ｃｈｅｌｅｘ和有机溶剂提取法提取ＤＮＡ,以ＰＣＲ与反向探针杂交技术作ＰＭ和ＨＬＡ－ＤＱＡ１位点分型。结果：２６份包埋组织块有２４份能得到理想的分型结果。结论：本法可用于法医学个人识别和亲子鉴定     

血清学方法结合PCR—DNA分型方法进行个人识别1例

血清学方法结合ＰＣＲ－ＤＮＡ分型方法进行个人识别１例方建新，李莉，阙庭志，林源，柳燕（司法部司法鉴定科学技术研究所法医物证室；上海２０００６３）１案情摘要１９９５年８月７日．浙江省嘉兴市郊区一信用社遭到抢劫，在值班人员的围堵过程中．一名歹徒从三楼跳下...     

Discrimination between human and animal DNA: application of a duplex polymerase chain reaction to forensic identification

Identification of a report's species is one of the basic analyses in forensic laboratories. The authors report the case of 6 bone fragments recovered in a wooded area, which were not attributable to 1 animal species on the basis of morphologic examination. The aim of this study was to develop a duplex polymerase chain reaction (PCR) to discriminate human and animal origin of bone fragments. The method is based on the PCR amplification of cytochrome b and a 16S ribosomal mitochondrial DNA fragment, which has never been tested up to now. Our protocol combines a single-round PCR with direct visualization of amplicons in agarose gel, without sequencing analysis of the PCR products. The presence of a single band (359 bp) indicates a nonhuman origin of the sample, whereas 2 bands (157 and 359 bp) indicate a human biologic sample.This method revealed to be useful for forensic purposes because the 16S ribosomal mitochondrial DNA is a small human-specific fragment that is easily amplifiable even with degraded DNA from biologic materials such as old bones.     

利用荧光AFLP技术检测罂粟DNA多态性

目的 利用荧光AFLP检测技术检测罂粟植物DNA多态性。方法 用Axygen公司的AxyPrep DNA试剂盒提取了12株产于缅甸和中国云南省昆明市宜良县罂粟植株的DNA，用Eco RI和Mse I对总DNA进行酶切。连接人工接头，预扩增和选择性扩增，其产物在CEQ8000遗传分析系统上检测。结果 64对选择性扩增引物中8对引物能得到20条以上的扩增片断，具有高度多态性。结论 荧光AFLP技术可用于罂粟DNA多态性的检测。     

DNA Analysis of Natural Fiber Rope*

Abstract: When rope is found at a crime scene, the type of fiber is currently identified through its microscopic characteristics. However, these characteristics may not always unambiguously distinguish some types of rope from others. If rope samples contain cells from the plants of origin, then DNA analysis may prove to be a better way to identify the type of rope obtained from a crime scene. The objective of this project was to develop techniques of DNA analysis that can be used to differentiate between ropes made from Cannabis sativa L. (hemp), Agave sisalana Perrine (sisal), Musa textilis Née (abaca, “Manila hemp”), Linum usitatissimum L. (flax), and Corchorus olitorus L. (jute). The procedures included extracting the DNA from the rope, performing polymerase chain reaction (PCR) using the extracted DNA as a template, and analyzing the DNA products. A primer pair for PCR, chosen from within a chloroplast gene for the large subunit of ribulose bisphosphate carboxylase/oxygenase, was designed to be specific for plant DNA and complementary to the genes from all five plants. The resulting PCR fragments were approximately 771 base pairs long. The PCR fragments, distinguished through base sequence analysis or restriction enzyme analysis, could be used to identify the five different rope types. The procedure provides a useful addition to visual methods of comparing rope samples.     

DNA polymorphism detection of Papaver somniferum L using fluorescent amplified fragment length polymorphism

OBJECTIVE: To detect DNA polymorphism of Papaver somniferum L using fluorescent Amplified Fragment Length Polymorphism. METHODS: Genomic DNA was isolated using the AxyPrep DNA Kit, double-digested by two restrictional endonucleases (Eco RI and Mse I) and ligated to oligonucleotide adapters. After Pre-amplification and selective amplification, the DNA fragments were separated by capillary electrophoresis using the CEQ8000 DNA Fragment Analyzer. RESULTS: More than 20 fragments of highly polymorphic products were obtained by 8 pairs of primer from 64 selective amplifying primer pairs. CONCLUSION: The fluorescent AFLP technique can be used to detect the DNA polymorphism of Papaver somniferum.     

DYS385检测方法的改进

目的建立检测DYS385的新方法。方法比较基因数据库(GDB)中推荐的引物和Schneider所设计的引物扩增DYS385的效果;在此基础上,以GDB推荐的引物作为外引物,Schneider所设计的引物作为内引物,通过调整内、外引物的浓度,优化扩增条件,建立DYS385的半巢式PCR体系。结果与常规方法相比,采用Schneider所设计的引物扩增DYS385,扩增片段缩短112bp,电泳分离的效果好,灵敏度提高2倍,达500pg;建立的半巢式扩增方法,能特异性扩增短片段,灵敏度提高20倍,达50pg。结论建立的DYS385半巢式扩增方法具有更高的特异性和灵敏度,适合法医学应用。     

体外DNA扩增技术鉴定血痕性别三例报告

本文报道用体外DNA扩增技术对3例刑事案中的人血痕标本进行性别鉴定。其方法是从血痕标本中微量抽提DNA,用蛋白酶K进行消化。然后用两组引物Y1.1与Y1.2和Alu9.1与Alu9.2及国产FD耐热DNA聚合酶进行聚合酶链反(PCR)扩增DNA,电泳分析Y及Alu重复序列,从而判断血痕的性别。     

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.     

PCR typing of DNA fragments of the two short tandem repeat (STR) systems upstream of the human myelin basic protein (MBP) gene in Danes and Greenland Eskimos

DNA from the double short tandem repeat (STR) system MBP (locus 18q23-pter) was amplified by the polymerase chain reaction (PCR) and the two polymorphic repeat systems were separated by cutting with the restriction enzyme NlaIII. The lengths of the DNA fragments of the two MBP STR systems MBP-A and MBP-B were analyzed by vertical electrophoresis in polyacrylamide gels followed by silver staining. DNA samples from 112 unrelated Danes, 140 unrelated Greenland Eskimos, and 88 Danish mother/child pairs were analyzed. The distributions of MBP phenotypes were in Hardy-Weinberg equilibrium in both the Eskimo and Danish populations. Significant differences were observed between the distribution of fragments (‘alleles’) in Greenland Eskimos and in Danes. The allele MBP-A7 was considerably more frequent in Eskimos (0.2214) than in Danes (0.0775) and also the allele MBP-B9 was considerably more frequent in Eskimos (0.225) than in Danes (0.06). Strong gametic associations were found between fragments from the MBP-A and MBP-B series in both Danes and Eskimos. Some of the associations were different in Danes and Eskimos. In the 88 Danish mother/child pairs, the segregation of the MBP genotypes were in accordance with a genetic model of co-dominant inheritance and no mutation was found. Two MBP STR regions with irregular structures were sequenced. One fragment had a single base G to A transition at position 124 in the primer binding region between the MBP-A and MBP-B regions. In the other fragment, a deletion starting at position 117 and including the primer binding region between the MBP-A and MBP-B regions was found.     

微腔型PCR芯片用于法医DNA分析的初步研究

目的为了克服传统PCR热循环仪体积大,运行电压高,耗时长,只能在实验室中应用的缺点,研究了一种微腔型PCR芯片,以期实现现场对STR片段的复合扩增。方法采用在PCR反应缓冲液中加入不同浓度的BSA溶液对芯片进行表面优化处理的方法及不同酶量优化实现对STR片段的有效扩增。结果使用浓度为0．5mg／mL的BSA可得到清晰完整的STR分型结果;加大酶量有益于扩增效率的提高。结论该种微腔型PCR芯片经初步优化后可有效地对STR片段进行复合扩增,经进一步优化可真正实现法医DNA分析的更加微量化和快速化。     

Simplified low-copy-number DNA analysis by post-PCR purification

Frequently, evidentiary items contain an insufficient quantity of DNA to obtain complete or even partial DNA profiles using standard forensic gentotyping techniques. Such low-copy-number (LCN) samples are usually subjected to increased amplification cylces to obtain genetic data. In this study, a 28-cycle polymerase chain reaction (PCR) was used to evaluate various methods of post-PCR purification for their effects on the sensitivity of fluorophore-based allelic detection subsequent to capillary electrophoretic separation. The amplified product was purified using filtration, silica gel membrane, and enzyme mediated hydrolysis purification techniques and evaluated for their effect on fluorescent allelic signal intensity. A purification method was selected and its effect on fluorescent allelic signal intensity was compared with that of the unpurified PCR product. A method of post-PCR purification is described which increases the sensitivity of standard 28-cycle PCR such that profiles from LCN DNA templates (<100 pg DNA) can be obtained. Full DNA profiles were consistently obtained with as little as 20 pg template DNA without increased cycle number. In mock case type samples with dermal ridge fingerprints, genetic profiles were obtained by amplification with 28 cycles followed by post-PCR purification whereas no profiles were obtained without purification of the PCR product. Allele dropout, increased stutter, and sporadic contamination typical of LCN analysis were observed; however, no contamination was observed in negative amplification controls. Post-PCR purification of the PCR product can increase the sensitivity of capillary electrophoresis to such an extent that DNA profiles can be obtained from <100 pg of DNA using 28-cycle amplification.