多重置换扩增在含抑制物检材中的应用

目的研究多重置换扩增(multiple displacement amplification,MDA)在含抑制物检材中的抗抑制能力,与磁珠法纯化检材相比较,证明其在法医学中的应用及意义。方法将不同浓度血红素和腐殖酸与样本DNA进行混合,分为MDA处理组、磁珠法处理组和空白对照组,PCR-STR单基因座D3S1358扩增联合聚丙烯酰胺凝胶电泳检测,并应用Amp F詛STR誖IdentifilerTMPlus试剂盒联合毛细管电泳检测。结果血红素质量浓度大于1 ng/μL或腐殖酸质量浓度大于0.1 ng/μL时,空白对照组经单基因座STR检测不能得到扩增产物;磁珠法处理组血红素质量浓度大于100 ng/μL或腐殖酸浓度大于1 ng/μL时,不能够得到扩增产物;MDA处理组各浓度抑制物均能成功扩增,完全不受抑制物影响。结论 MDA技术可消除血红素及腐殖酸的抑制作用,其抗抑制能力优于磁珠法纯化DNA,具有一定的法医学应用意义。     

多重置换扩增技术及其法医学应用展望

多重置换扩增技术(multiple displacement amplification,MDA)是一种新型的全基因组扩增技术,具有操作简单、产物质量好、产物量稳定和扩增较均衡的优点,可以扩增低拷贝、混合、降解、含抑制物的DNA检材,为后续分析提供良好的高质量的扩增产物,在法医物证学领域具有很大的应用潜力,本文对该项技术的研究进展及应用前景进行综述。     

多重置换扩增技术用于法医学微量DNA检测效果

目的探讨多重置换扩增（MDA）技术对法医学微量DNA样品STR检测分型的效果。方法用MDA技术对不同模板量DNA进行全基因组扩增（WGA）．扩增产物用实时荧光定量PCR技术定量、用Prrfiler Plus^TM试剂盒检测基因型。结果该方法可对模板DNA增加10^4～10^6倍。1ng样品DNA的MDA产物可获得9个STR基因座和Amelogenin性别基因座的准确分型结果；低于0．1ng的样品DNA经MDA扩增后，基因座检出数增加。但可见等位基因不平衡或丢失现象。结论MDA技术可有效增加DNA模板量和提高微量DNA分型效果。但样品DNA量低于0．1ng时，MDA产物的STR分型结果判读须慎重。     

多重置换扩增技术用于法医学微量DNA检测效果

目的探讨多重置换扩增(MDA)技术对法医学微量DNA样品STR检测分型的效果。方法用MDA技术对不同模板量DNA进行全基因组扩增(WGA),扩增产物用实时荧光定量PCR技术定量、用Profiler PlusTM试剂盒检测基因型。结果该方法可对模板DNA增加104～106倍。1ng样品DNA的MDA产物可获得9个STR基因座和Amelogenin性别基因座的准确分型结果;低于0.1ng的样品DNA经MDA扩增后,基因座检出数增加,但可见等位基因不平衡或丢失现象。结论MDA技术可有效增加DNA模板量和提高微量DNA分型效果。但样品DNA量低于0.1ng时,MDA产物的STR分型结果判读须慎重。     

全基因组扩增在微量检材DNA分型中的应用

目的探索全基因组扩增技术对微量检材DNA分型的有效性。方法通过显微操作制备含1~20个细胞的模拟微量检材样本,在常规PCR-STR分型前加入全基因组扩增步骤,从等位基因不平衡、等位基因丢失、基因座丢失、伪等位基因(包含stutter峰)等方面探究PEP和MDA两种全基因组扩增方法对微量检材DNA分型的有效性。结果 MDA扩增效率高于PEP,但等位基因丢失和伪等位基因严重;PEP方法的正确分型率高于MDA,但小片段DNA优势扩增现象较严重。结论 MDA方法并不适合目前以STR分型为主导的法庭科学,当微量检材样本的绝对量相当少时,可以考虑使用PEP方法来扩大样本量,以满足重复检验的要求,但可能面临大片段DNA扩增失败的风险。     

荧光标记复合扩增毛细管电泳法在SNP分型中的应用

目的采用荧光标记复合扩增毛细管电泳法，对辽南地区汉族人群13个SNP进行等位基因频率调查，并评价其法医学应用价值。方法选择13个双等位基因SNP，应用荧光标记片段长度差异等位基因特异性复合扩增SNP分型方法，对辽南地区汉族人群进行群体调查。结果每个SNP纯合子为单一产物峰，杂合子则为长度不同的两个产物峰。不同位点扩增产物长度不同，根据产物长度和产物峰数量进行SNP分型，其结果与直接测序完全一致。同时获得辽南地区汉族人群13个SNP等位基因频率。结论采用荧光标记复合扩增毛细管电泳法进行SNP分型，方法简单实用，在法医学个人识别领域具有较高的应用。     

全基因组扩增法应用于低拷贝数DNA检测

目的建立基于多重置换扩增(MDA)技术的全基因组扩增(WGA)方法,实现对低拷贝数(LCN)DNA样品进行分析。方法采用REPLI-g试剂盒对样本进行等温全基因组扩增,扩增产物采用ProfilerPlus试剂盒确定样本的十个STR基因座的等位基因型。结果10pgDNA模板经全基因组扩增后,能够进行DNA分型。结论全基因组扩增可以用于LCN的DNA分析,帮助提高微量物证的检出成功率。     

23个STR基因座荧光复合扩增体系的法医学应用评估

目的评估新建立的23个STR复合扩增体系EX23的法医学应用价值。方法使用磁珠法提取样本DNA,应用23个STR复合扩增体系进行扩增,ABI3130XL遗传分析仪对扩增产物进行电泳,GeneMapperID 3.2软件进行基因分型,对法医学应用参数、灵敏度、种属、脱落细胞检材及降级检材分型效果进行观察,并与Sinofiler试剂盒比较。结果 DNA模板量在0.05~1.00ng时,各基因座分型结果清晰准确,均衡性好、特异性强。应用该复合扩增体系检验混合样本、降解检材及脱落细胞检材,均能获得正确的分型结果。统计结果显示该23个STR基因座累计个人识别（TDP）率达0.999999999,三联体累计非父排除率（CPE）达0.999999997。结论新建立的23个STR复合扩增体系分型效果良好,在广东地区汉族人群中具有高度多态性,可满足日常法医鉴定的需要。     

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。     

低拷贝模板DNA分型及其在法医学中的应用

近年来,低拷贝模板类生物物证在法庭科学领域中的应用越来越广泛.然而由于其自身存在的一些限制性因素,始终是法庭科学工作者关注的一个焦点.本文从低拷贝模板DNA的定义及其在法庭科学应用中的有效性、应用范围、分型策略、质量控制、重复性原则、随机阈值等方面对低拷贝数分型及其在法庭科学应用中的最新研究进展进行了综述.     

线粒体16SrRNA和Cytb基因复合扩增进行种属鉴定

目的建立一种用于种属鉴定的线粒体DNA16SrRNA基因和细胞色素b基因荧光标记复合扩增检测体系。方法利用引物设计软件Primer5.0对mtDNA序列的16SrRNA基因和细胞色素b基因各设计一对引物,建立复合扩增体系,分别扩增人和牛、猪、狗、鸡、草鱼5种常见动物,用310遗传分析仪对产物进行分析。结果人和5种动物DNA扩增产物均出现两个峰,Cytb通用引物的扩增产物为人与动物的共有峰,为358bp;16SrRNA基因的扩增产物为人与动物间存在位置差异的特异峰,位于231～256bp之间。结论该复合扩增体系可以明确区分人和5种动物样本,可用于种属鉴定。     

Typing the 1.1 kb control region of human mitochondrial DNA in Japanese individuals

This study presents a reliable method that uses high-fidelity long-range PCR and optimized primers to assess polymorphism and to genotype human mitochondrial DNA (mtDNA). This method was used to analyze polymorphic sites in the human mtDNA control region, including hypervariable regions I, II, and III (HVI, HVII, and HVIII), from 124 unrelated Japanese individuals. In HVI, HVII, and HVIII, 80, 37, and 14 polymorphic sites were identified, respectively, excluding those in the homopolymeric cytosine stretch (C-stretch) regions. The region between HVI and HVII also contained 15 polymorphic sites. On the other hand, C-stretch length heteroplasmy in HVI or HVII was observed in 66 of 124 Japanese individuals (53%), which is much higher than in Caucasian populations. The variants in the C-stretch regions were characterized by counting the number of heteroplasmic peaks split from the single peak in homoplasmic sequences (i.e., 16244G and 16255G in HVI and 285G in HVII). Including the C-stretch length heteroplasmy, the 124 Japanese mtDNA samples were classified into 116 distinct haplotypes. The random match probability and the genetic diversity were estimated to be 0.95% and 0.998581, respectively, indicating that the method presented here has higher discrimination than the conventional method for mtDNA typing using HVI and HVII. [Correction added after publication 30 January 2007: in the preceding sentence random match probability and genetic diversity estimates were corrected from 0.95 and 0.998581%, respectively, to 0.95% and 0.998581, respectively.] The haplogroups and their frequencies observed in this study (i.e., D4; 13.7%, M7a1; 11.3%, D4a; 9.7% and M7b2; 8.9%) were similar to those observed in other studies of Japanese mtDNA polymorphism. The method described here is suitable for forensic applications, as shown by successful analysis of tissues from highly putrefied remains of an infant, which allowed maternal relationship to be determined via mtDNA haplotyping.     

Typing of mitochondrial DNA from isolated cells of the human buccal epithelium

The authors have developed a method for molecular-genetic analysis of DNA from isolated cells for the purpose of forensic medical diagnostics. The method is based on the use of the laser capture microdissection (LCM) technology in combination with typing of mitochondrial DNA. Optimization of the conditions for amplification of polymorphic mtDNA loci in preparations containing minimal amounts of the genetic material was accomplished at the initial stage of the work. To this effect, the two-round polymerase chain reaction was employed that allowed the amplified material to be accumulated in the amount sufficient for sequenation. At the next stages, the system thus obtained was tested on the cell model (using isolated cells of human buccal epithelium). It was shown that the proposed method is suitable for the analysis of specific mtDNA characteristics in a single human cell.     

运用PCR技术对人骨组织和牙齿进行性别鉴定的应用研究

为了解决刑事案件中利用人骨组织和牙齿进行性别鉴定的问题，我们通过骨骼和牙齿中ＤＮＡ的提取床用Ｙ染色体特异ＤＮＭ列引物扩增Ｙ染色体的ＤＹＺＩ和ＳＲＹ基因序列．结果：１．骨组织中ＤＮＡ的提取可在１小时内完成；牙齿中ＤＮＡ的提取可在４小时内完成；２．牙齿中的ＤＮＡ保存的完好性大于骨组织；４．煮沸过的骨组织中的ＤＮＡ降解严重，无法用于实验．     

DNA Profiles from Fingerprint Lifts—Enhancing the Evidential Value of Fingermarks Through Successful DNA Typing

This study evaluated the compatibility of the most common enhancement methods and lifting techniques with DNA profiling. Emphasis is placed on modern lifting techniques (i.e., gelatin lifters and Isomark?) and historical fingerprint lifts for which limited research has been previously conducted. A total of 180 fingerprints were deposited on a glass surface, enhanced, lifted, and processed for DNA typing. DNA could be extracted and profiled for all the powders and lifts tested and from both groomed fingerprints and natural prints with no significant difference in the percentage of profile recovered. DNA profiles could also be obtained from historical fingerprint lifts (79.2% of 72 lifts) with one or more alleles detected. These results demonstrate the compatibility between different powder/lift combinations and DNA profiling therefore augmenting the evidential value of fingerprints in forensic casework.     

Evaluation of the Reliability of DNA Typing in the Process of Identification of War Victims in Croatia

Abstract:  Aiming to estimate the frequency of various types of errors that can occur in the large-scale process of identification, we identified and compared genotypes of 911 parent–child pairs in the database of 3498 relatives of people that disappeared during the 1991/1992 war in Croatia. Genotypes of 891 pairs (97.8%) were matching, while 20 pairs did not match in one or more loci. Reanalysis of these samples revealed that out of 1822 analyzed genotypes, one genotype was completely wrong, and two genotypes had one wrong allele because of human errors. Five genotypes had a single wrong allele due to either polymerase chain reaction or electrophoresis errors. In five genotypes mutations were the cause of mismatch. Genetic inconsistencies with parentage were found in four "fathers" (4.2%) and three "mothers" (0.36%). As the majority of observed single-locus errors were caused by nonhuman errors, all databases produced with similar technology would probably have comparable level of errors.     

DNA Typing for the Identification of Old Skeletal Remains from Korean War Victims*

Abstract: The identification of missing casualties of the Korean War (1950–1953) has been performed using mitochondrial DNA (mtDNA) profiles, but recent advances in DNA extraction techniques and approaches using smaller amplicons have significantly increased the possibility of obtaining DNA profiles from highly degraded skeletal remains. Therefore, 21 skeletal remains of Korean War victims and 24 samples from biological relatives of the supposed victims were selected based on circumstantial evidence and/or mtDNA‐matching results and were analyzed to confirm the alleged relationship. Cumulative likelihood ratios were obtained from autosomal short tandem repeat, Y‐chromosomal STR, and mtDNA‐genotyping results, and mainly confirmed the alleged relationship with values over 10⁵. The present analysis emphasizes the value of mini‐ and Y‐STR systems as well as an efficient DNA extraction method in DNA testing for the identification of old skeletal remains.     

Alterations of Length Heteroplasmy in Mitochondrial DNA Under Various Amplification Conditions*

Abstract: There are several areas within mitochondrial DNA that show length heteroplasmy. If the heteroplasmy pattern is unique and consistent for each person, it may be used to support an interpretation of exclusion in identity testing. We investigated whether the length heteroplasmy pattern would be consistent under different amplification conditions. We also determined whether various amplification parameters would affect the homopolymeric cytosine stretches (C‐stretch) in HV1. Monoclonal samples tended to be heteroplasmic after amplification. After several repetitions, C‐stretch patterns of all samples were inconsistent even under the same amplification conditions. Increased PCR cycles and high template concentrations resulted in a more frequent heteroplasmic tendency. These amplification parameters seem to have little effect if samples are not long enough in C‐stretch or total length of the segment from nt 16180 to nt 16193. It is suggested that the pattern of length heteroplasmy cannot be used as an additional polymorphic marker.