快速PCR仪与普通PCR仪扩增效果的比较研究

目的比较快速PCR仪与普通PCR仪的扩增效果。方法浓度为0.1、0.05、0.025、0.0125、0.0063ng/μL的9947A标准品各取样100例,采用Identifiler Plus试剂盒构建扩增体系,其中50例在普通PCR仪(9700型扩增仪)上进行扩增,50例在快速PCR仪(Speed cycler2扩增仪)上扩增,3500x L型遗传分析仪检测,对比每种浓度组两种扩增仪的检验结果。结果两种PCR仪的检出率均无差异(P0.05)。但当9947A的浓度为0.0125、0.0063ng/μL时,普通PCR仪检验样本的STR基因座检出数(13.7±1.0;11.3±1.5)均高于快速PCR仪(13.1±1.3;9.9±1.9),差异有统计学意义(P=0.029;P0.001);当9947A的浓度为0.1、0.05、0.025 ng/μL时,普通PCR仪检验样本的图谱峰高(18931±4625;13437±3165;5752±1344)均高于快速PCR仪(16929±4034;11815±4120;4865±1401),差异有统计学意义(P=0.023;P=0.030;P=0.002)。结论快速PCR仪可在较短的时间内达到与普通PCR仪相当的检出率,适合于实际案件中的应用;普通PCR仪检验获得的STR分型结果质量可能更高。     

生物检材中的PCR抑制物及其处理技术

DNA分型技术在刑事案件的侦破中已得到广泛应用,但不同来源的各种生物检材往往含有PCR抑制物,其对扩增反应的影响不容忽视。因此,DNA样本扩增前预处理就显得尤为重要。本文主要就PCR抑制物种类及其对PCR的抑制机制、PCR抑制物应对技术等相关研究进展进行简要综述,旨在为相关研究和应用提供参考。     

用DNA纯化试剂分离除去PCR抑制剂

作者对于影响PCR扩增成功的抑制剂进行了去除实验,发现使用Wizard~(?) DNA Clean-Up SystemDNA纯化试剂Wizard~(?) DNA Clean-Up System是一种去除色素和泥土PCR抑制剂的较好方法.经多起案件检材使用效果良好.     

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。     

部分腐败尸体的亲权鉴定——附案例报告

部分腐败尸体的亲权鉴定在法医实践中常会遇见,过去的血型测定法已无能为力。PCR 技术给这类案件的鉴定带来了希望。本文报告用 PCR 技术对部分腐败的女性尸体和胎儿进行基因分型并对此案的亲权进行鉴定,取得了令人满意的结果。同时讨论了一些注意事项。作者认为 PCR 技术在鉴定此类案件中有特殊价值。     

进行PCR时发生中断的补救方法

在进行PCR检验时被中断,常有发生,一般的补救办法就是重新建立反应体系,重加模板,重新扩增。本文在PCR的各个阶段人为地制造PCR中断,研究中断发生时所处阶段以及中断状态持续时间长短对继续PCR的影响。1材料与方法1.1试剂仪器与环境以硅珠法提取人血DNA作为扩增模板。AmpFl-STR PROFILER PLUS(以下简写为PP)和IDENTI-FILER(以下简写为ID)试剂盒(ABI)。2700、9700型PCR扩增仪(PE公司),3100基因分析仪(ABI)。环境室温在18℃~22℃间波动。1.2方法根据ABI AmpFlSTR PROFILER PLUS用户手册推荐,本文将热循环的条…     

AmpF(l)STR(R) Identifiler(R) Plus试剂盒快速直接PCR扩增初探

目的 探索可应用于法医实践的快速直接PCR扩增方法,以缩短扩增时间,提高STR分型速率.方法 联合运用AmpF(l)STR(R) Identifiler(R) Plus试剂盒与TaKaRa快速PCR检测试剂构建快速直接PCR体系,以FTA血卡为模板,采用优化后的扩增程序在快速PCR仪上进行扩增,分型结果与常规直接扩增方法相比较.结果 AmpF(l)fSTR(R) Identifiler(R) Plus试剂盒与TaKaRa的快速PCR检测试剂针对血卡的联合扩增,所得STR分型结果与常规方法一致,扩增用时由常规直接扩增所需的175 min缩短为55 min.结论 采用快速直接PCR方法进行扩增,可得到与常规直接扩增一致的DNA分型,扩增时间明显缩短,DNA分型速率大幅提高.     

癌组织的基因突变对PCR反应及结果分析的影响

在实际检案中,办案人员有时送检石蜡包埋人体病理组织(癌)或切片。这类检材由于存在基因突变的可能,往往影响正确的结果分析,使给出鉴定结论造成困难。某地发生一起保险欺诈案件中,送检子宫癌病理组织蜡块,要求与当事人的血液进行同一认定,笔者通过该案例检验探讨癌组织的基因突变对PCR反应及结果分析的影响。1材料与方法采用Chelex-100法提取当事人的血液样本DNA,经PCR扩增,获得STR分型(图1)。采用文献[1]的方法提取DNA,经PCR扩增,获得STR分型(图2)。2讨论(1)本例检验结果表明,癌组织的基因突变会影响PCR扩增,使STR分型结果发…     

荧光定量PCR技术研究线粒体DNA nt16519单核苷酸多态性

目的应用荧光定量PCR技术,建立mtDNA nt16519位点的PCR分型方法。方法应用Primer Express3．0软件,设计1对Taqman MGB探针和1埘扩增引物,在探针的5’端分别标记FAM和VIC报告荧光,应用7500荧光定量PCR和直接测序两种方法,分别榆测62份血样和18份毛发样本的mtDNA nt16519单核苷酸多态性,比较二种方法结果的一致性。结果62份血样和18份毛发样本均得到了可靠的mtDNA nt16519分型结果。结论建立的荧光定量PCR的分型方法操作简单、结果准确,适用于法医DNA检案。     

PCR扩增3因素对低拷贝模板STR分型的影响研究

目的探讨低拷贝模板(low copy number,LCN)STR扩增方法,提高LCN检材的检验成功率。方法采用Profiler P lusTM试剂盒与9947A对照DNA,改变Taq酶量、体系、循环次数3个因素进行扩增检验,了解各变量对扩增检测的影响。结果对低拷贝模板DNA,单纯增加Taq酶量或反应体系,扩增效率改善不明显;增加循环数,显著提高检验灵敏度;低于0.01ng的模板DNA,同时增加扩增体系、Taq酶量、循环数在一定程度上提高扩增效率。结论对于影响扩增的Taq酶量、体系、循环次数3个因素中,循环数影响最大,但应慎用34次及以上循环数;三者同时增加,对于低于0.01ng模板DNA的扩增可有效改善。     

An Investigation of PCR Inhibition Using Plexor®‐Based Quantitative PCR and Short Tandem Repeat Amplification

A common problem in forensic DNA typing is PCR inhibition resulting in allele dropout and peak imbalance. In this paper, we have utilized the Plexor^® real‐time PCR quantification kit to evaluate PCR inhibition. This is performed by adding increasing concentrations of various inhibitors and evaluating changes in melt curves and PCR amplification efficiencies. Inhibitors examined included calcium, humic acid, collagen, phenol, tannic acid, hematin, melanin, urea, bile salts, EDTA, and guanidinium thiocyanate. Results were plotted and modeled using mathematical simulations. In general, we found that PCR inhibitors that bind DNA affect melt curves and C_T takeoff points while those that affect the Taq polymerase tend to affect the slope of the amplification curve. Mixed mode effects were also visible. Quantitative PCR results were then compared with subsequent STR amplification using the PowerPlex^® 16 HS System. The overall results demonstrate that real‐time PCR can be an effective method to evaluate PCR inhibition and predict its effects on subsequent STR amplifications.     

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.     

12个mini-X-STR和Amel基因座荧光标记复合扩增体系的初步研究

目的研究建立12个mini-X-STR和Amel基因座复合扩增体系。方法选择DXS101、DXS10159、DXS10162、DXS10164、DXS6789、DXS7133、DXS7423、DXS7424、DXS8378、DXS981、GATA165B12和GA-TA31E08共12个X-STR与Amelogenin基因座,自行设计引物,分别采用FAM、HEX、Tamra、ROX四色荧光标记5′端,并进行必要的修饰,按照合适的引物浓度进行混合。对97个血痕样本进行复合扩增,并用ABI3130XL进行电泳和分型。结果所建立的荧光标记复合扩增体系能够得到清晰、明确的分型图谱,灵敏度达50pg,稳定性、重复性与平衡性较佳。结论本研究建立的12个mini-X-STR和Amel基因座荧光标记复合扩增体系统检验方法简单,灵敏度高,适合实际办案的需要。     

6个五核苷酸STR基因座荧光复合扩增体系的建立

目的 构建6个五核苷酸STR基因座荧光复合扩增体系。方法筛选6个多态性程度较高的五核苷酸STR基因座D10S2325、Penta B、Penta W、PentaX、Penta D和PentaE,按照复合扩增引物设计要求,重新设计引物并标记荧光染料,经反复调整和优化,构建6基因座荧光复合扩增体系,并用该复合扩增体系对239名武汉汉族无关个体进行分型。结果6个五核苷酸STR基因座荧光复合扩增体系分型稳定,可重复性好,与各自相应单基因座分型结果完全一致;累积个人识别率达0．999999988,累积非父排除率达0．998063807。结论本文构建的6个五核苷酸STR基因座荧光复合扩增体系具有很高的法医学实用价值,可作为商品化试剂盒的有效补充。     

PEP-PCR法及其在法医学中应用的可行性研究

提高微量检材的利用率 ,建立PEP方法并对其法医学应用进行可行性研究。用 15个随机寡核苷酸的序列作为引物 ,低特异性下扩增出微量DNA ,以此扩增物作为模扳 ,进行特异基因座扩增。经PMCT118,GABAR ,DYS19,D18S849,D3S1744,D12S10 90 ,D8S1179,D2 1S11,D18S5 1,D5S818,D13S3 17,D7S82 0 ,D3S13 5 8,vWA ,FGA ,TH0 1,CSF1PO ,TPOX ,D16S5 3 9及Amelogene等 2 0个基因座的PEP PCR与直接的PCR分析比较 ,二者分型结果一致 ,即使 1ngDNA也能得到正确分型。PEP方法可用于法医学检验 ,有利于微量物证的利用 ,使物证检材提供尽可能多的信息     

Analysis of human specificity in AFLP systems APOB,PAH, and D1S80

We have previously characterized and databased three human amplified fragment length polymorphism (AFLP) loci: the hypervariable regions 3′ to apolipoprotein B (APOB), phenylalanine hydroxylase (PAH) and at locus D1S80. The analysis utilized polymerase chain reaction (PCR) technology for human identification in forensic and paternity testing. This study extended that work by assessment of specificity of amplicons produced with non-human and human control DMAs for APOB, PAH and D1S80 under high and low stringency PCR conditions. It was seen that primate and other animal templates (with the exception of chimpanzee) yielded products below the human allele range under high stringency PCR parameters. Under reduced stringency PCR with animal and primate samples, reproducible genetic fingerprints were generated spanning the human allele range. The patterns were produced with defined human AFLP primer pairs under specifically relaxed PCR reaction and thermalcycling parameters. They showed genetic relationships between species at the DNA level. Amplicon patterns were compared for band size and intensity matches within the PCR synthesis range defined by the conditions used. This technique could become a useful tool in species identification and molecular evolutionary studies.     

Quantitative analysis of amplifiable DNA in tissues exposed to various environments using competitive PCR assays

Competitive PCR assays were established for the mitochondrial DNA hypervariable region I and the human amelogenin locus. Using these assays, the copy numbers of DNA participating in PCR (amplifiable DNA) were quantified in tissues exposed to different environments. Human ribs, skin and nails were left in three exposure conditions (in the open air, in soil and in water). The amounts of amplifiable DNA in these tissues were quantified during a time period of up to two months. The amount of amplifiable DNA was well preserved in hard tissues (ribs and nails) regardless of the exposure conditions, whereas the soft tissues immersed in water showed a rapid decrease in amplifiable DNA. Strong PCR inhibition was observed in the DNA extracts obtained from buried bones. This phenomenon was clearly identified from an amplification failure of the internal standards in the competitive PCR. A preliminary examination to identify the PCR inhibitor suggested that the soil itself contributed to the inhibition. In addition, the amounts of amplifiable DNA in case samples were also investigated.     

6个新的STR基因座复合扩增体系的建立及应用

目的构建6个常染色体STR基因座的荧光复合扩增体系,应用于法医学DNA检验。方法筛选6个STR基因座D4S2366、D3S3045、D18S1002、D20S481、D22S689、D4S2639,根据复合扩增要求设计引物并采用不同荧光染料进行标记,经过反复调整和优化,建立6基因座荧光复合扩增体系,并用该复合扩增体系对224名华东汉族无关个体进行分型,计算出常用法医遗传学参数。结果使用该荧光复合扩增体系,在224名华东汉族无关个体中,6个STR基因座D4S2366、D3S3045、D18S1002、D20S481、D22S689、D4S2639分别检出7、8、9、10、9、9个等位基因和21、26、21、23、28、32种基因型,基因型分布符合Hardy-Weinberg平衡。杂合度（H）分布为0.714～0.808,个体识别率（DP）为0.874～0.934,二联体非父排除率（PED）为0.310～0.453,三联体非父排除率（PET）为0.485～0.628,多态信息含量（PIC）为0.672～0.784,二联体累积非父排除率（CPED）为0.947689,三联体累积非父排除率（CPET）为0.993345,累积个人识别能力（CDP）为0.999999543。结论构建的荧光复合扩增体系具有较高的法医学应用价值,D4S2366等6个常染色体STR基因座在华东汉族群体中均具有高度多态性,可作为常规商品化试剂盒的有效补充,用于突变情形的亲权鉴定以及依据亲权指数值不能明确鉴定意见的亲权鉴定。