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。     

改良IPEP法用于痕量DNA检测的实验研究

目的探讨改良扩增前引物延伸(IPEP)法对痕量DNA样本STR检测分型的效果。方法用改良IPEP法对痕量样品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AmpFLSTR~ Indentifiler~试剂盒作基因型检测。结果该方法可增加模板DNA约200~1100倍。基因组DNA不低于0.025ng时,可获得15个STR基因座和Amelogenin性别基因座的分型结果。基因组DNA0.01~0.025ng时,可获得9个以上基因座的分型结果。结论改良IPEP法可有效提高痕量DNA样本STR分型检验的灵敏度,有较好的实用价值。     

Chelex-100提取生物检材DNA实时PCR定量研究

目的研究Chelex-100法提取的生物检材DNA用量与复合STR分型成功率的关系。方法113份各种生物检材采用Chelex-100法提取DNA，应用Quantifiler人类DNA定量试剂盒在ABI 7500荧光定量PCR仪上进行实时PCR定量，同时用Identifiler复合扩增系统在ABI 3100遗传分析仪上对这些DNA样品进行STR分型。结果各种生物检材提取的DNA浓度分别为：37份滤纸、纱布血痕0．042～5．28ng／μl，16份口腔拭子1．15—4．21ng／μl，18份烟头0．016～1．46ng／μl，10份肋软骨0．531—14．40ng／μl，8份肌肉5．75—24．80ng／μl，7份指甲0．788—11．50ng／μl，17份精斑0．79～99．50ng／μl。在建立的8μl扩增体系中，根据上述结果，调整用于复合STR扩增的DNA模板量在0．5—3ng之间，大部分样品可获得完全的STR分型。结论Chelex-100法提取的检材DNA模板用量在0．5—3ng之间可得到有效STR扩增，浓度为0．5ng／μl以上的DNA样品，用小体积模板（1μl）比大体积（3μl）模板扩增效果好。     

19个常染色体与4个Y染色体STR复合扩增体系的建立

目的建立19个常染色体STR及Amelogenin和4个Y染色体STR基因座复合扩增体系,并对其效能进行评估。方法用五色荧光标记20＋4Y—STR基因座,建立同步扩增检测体系,用ABI3130XL遗传分析仪对扩增产物进行电泳,GeneMapperID3．2软件进行基因分型;检测体系的灵敏度、均衡性、稳定性、特异性、同一性和稳定性,并观察混合、降解及微量检材的分型情况。结果采用本文体系,DNA模板量在0．05～1．00ng时,分型准确,均衡性、特异性好;混合、降解及微量检材分型正确。该19个常染色体STR基因座的累计个人识别率大于0．999999999,三联体累计非父排除率达0．999999985,Y—STR单倍型多态性为0．592。结论本文建立的复合扩增体系分型准确,稳定,在法医学案件检验及数据库建设等方面有良好的应用前景。     

19个常染色体与4个Y染色体STR复合扩增体系的建立

目的建立19个常染色体STR及Amelogenin和4个Y染色体STR基因座复合扩增体系,并对其效能进行评估。方法用五色荧光标记20+4Y-STR基因座,建立同步扩增检测体系,用ABI 3130XL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;检测体系的灵敏度、均衡性、稳定性、特异性、同一性和稳定性,并观察混合、降解及微量检材的分型情况。结果采用本文体系,DNA模板量在0.05~1.00ng时,分型准确,均衡性、特异性好;混合、降解及微量检材分型正确。该19个常染色体STR基因座的累计个人识别率大于0.999 999 999,三联体累计非父排除率达0.999 999 985,Y-STR单倍型多态性为0.592。结论本文建立的复合扩增体系分型准确,稳定,在法医学案件检验及数据库建设等方面有良好的应用前景。     

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。     

磁珠法自动化纯化现场检材DNA方法研究

目的利用TE-MAGS在TECAN工作站上结合磁珠试剂盒,建立自动化工作站批量纯化现场检材DNA的方法,并探讨其在法医物证检案中的应用。方法灵敏度测试:标准品使用0.1ng/μL 9947A,用200μL TES稀释制备DNA总量0.1ng~1ng共10种的标准样品,采用本文方法提取纯化,使用IdentifilerTM试剂盒扩增,用3130XL型测序仪检测,Gene Mapper ID-X分析,分析STR图谱质量;纯化能力测试:在1ng总量的标准样品中加入腐殖酸、血红素,采用本文方法提取纯化、扩增检测,分析STR图谱质量;实际案件应用对比:收集304份现场检材,分别采用本方法和硅珠法进行提取纯化,经扩增检测,统计对比两种提取纯化方法 STR分型成功率。结果灵敏度测试:0.1ng~0.2ng总量标准样品提取的DNA模板,扩增后可检测到部分基因座STR图谱,0.3ng~1ng总量标准样品提取的DNA模板,扩增后可以得到完整的STR图谱;纯化能力测试:对混合有一定浓度的腐殖酸、血红素的标准样品的提取产物检测图谱未见明显抑制;实际案件应用对比测试:304份现场检材工作站磁珠法检出成功率(50%)高于硅珠法(40.8%)。结论本文所建立的方法缓冲范围较大,回收率高,纯化能力强,提取产物STR分型成功率高,适合现场检材批量化DNA检验。     

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。     

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。     

17个STR基因座快速多重扩增体系的建立及应用

目的建立17个STR基因座多重PCR快速扩增体系。方法采用人血样本提取DNA并定量,用于多重快速扩增体系分型准确性检测;采用标准品9948,设定稀释度,检测体系灵敏度;采用定量男女DNA样本按11种比例混合,检测体系对混合样本的分型能力;在标准品中加入干扰物质血红素和腐植酸,检测体系的抗干扰能力;对5种非人样本进行检测,评价体系的特异性;对实际案例进行检验,评价体系实际应用价值。结果采用本文体系,在65min内,用0.5~2ng DNA模板量能获得较好的扩增效果,分型结果准确稳定,扩增均衡;种属特异性好;血红素≤50μmol/L,腐植酸≤25ng/μL时可不受干扰准确分型;男女混合样本中单一样本量不低于1/10即可进准确进行判断;对实际案例常见生物检材的检验结果良好。结论本文17个STR基因座快速多重扩增体系可显著缩短扩增时间,技术性能符合实际检案要求,可在实践中选用。     

混合斑中精子细胞分离及其DNA制备方法

目的尝试建立一种检测混合斑中精子细胞的方法。方法使用显微操作法捕获精子细胞,全基因组扩增(多重置换扩增)精子细胞DNA。结果对10管精斑检材的全基因组扩增,获得了高产、保真的产物。使用50μL体系对20个精子细胞直接进行全基因组扩增,省去了对起始模板的纯化过程,DNA扩增倍数达30000倍以上,片段长度大多在15 kb以上,其STRs复合扩增分型结果有可参照性。结论显微操作法可以有效捕获精子细胞,排除干扰,多重置换扩增可以提供足够量的产物用于法医DNA分析,该方法具有可行性。     

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。     

Comparison of two whole genome amplification methods for STR genotyping of LCN and degraded DNA samples

The analysis of LCN or highly degraded DNA samples presents a challenge for forensic science. Improving the quantity and/or quality of samples would greatly increase the profiling success rate from LCN and degraded samples. Whole genome amplification (WGA) is one method that has such potential. Two commercially available WGA kits, GenomePlex and GenomiPhi, were investigated for use on LCN and degraded DNA samples. Both kits amplified genomic DNA, producing microgram quantities from sub-nanogram templates. Profiling success of LCN DNA samples was increased, with improvements of over 700% from 10pg template DNA compared to non-WGA-amplified control samples. The amplification success with degraded DNA was also improved by WGA. Degraded DNA was simulated using restriction enzymes to demonstrate that the application of WGA can result in the typing of STR loci that could not previously be amplified. An increase in artefacts, such as stutter alleles and amplification biases, were observed in many samples. Results show that WGA is capable of increasing both the quality and quantity of DNA, and has the potential to improve profiling success from difficult samples in forensic casework.     

Whole-genome amplification by MDA to improve sensitivity of forensic expert examination of chromosomal DNA

This pilot project has the objective to evaluate the possibility of application of the multiple displacement amplification (MDA) technique to whole-genome amplification with a view to improving sensitivity of molecular-genetic test-systems. Preparations of total cellular DNA were amplified by MDA and analysed to assess conserved specificity of chromosomal DNA and its enhanced template activity in the standard polymerase chain reaction (PCR) for typing allele variants of polymorphous DNA loci. DNA samples before and after MDA showed virtually identical genotypic combinations of alleles. Allele fragments were stably detected at a level of DNA 4-5 times lower than in the standard test. The results of the study indicate that the MDA technique provides a promising tool to improve reliability of forensic- expert examination of chromosomal DNA and imply the necessity to further develop forensic-medical aspects of this method.     

Quantitation of human genomic DNA through amplification of the amelogenin locus

An alternate method for quantitation of human genomic DNA is presented. Quantitative template amplification technology (abbreviated "Q-TAT") estimates the quantity of human DNA present in an extract by comparing fluorescence in X and Y amplicons produced from unknowns with fluorescence in a standard curve amplified from known quantities of reference DNA. Q-TAT utilizes PCR and electrophoresis with fluorescent detection/quantitation, precluding the need for new instrumentation, methodology, or quality assurance associated with slot-blot or real-time PCR. In a comparison study incorporating shared samples, Q-TAT was found to be more sensitive than widely used slot-blot methods but somewhat less sensitive than real-time PCR. Among samples containing DNA concentrations ranging from 100 pg/microL to 2-4 ng/microL, Q-TAT produced DNA concentration estimates that agreed reasonably well with either Quantiblot or real-time PCR. Q-TAT was reproducible with a typical coincidence of variation of about 35%. Quantitation of human DNA in this study involved summing fluorescence in X and Y amplicons in unknowns and quantitation standards. However, analyzing fluorescence in X and Y amplicons individually could allow estimates of male and female DNA present in mixtures to be made. Moreover, since X and Y amplicons exhibit sizes of 210 and 216 bp, respectively, the integrity as well as the concentration of the genomic DNA template can be assessed. Q-TAT represents an alternate method useful for the quantitation of human genomic DNA prior to amplification of STR loci used for identity testing purposes. The method uses existing equipment and procedures in conjunction with a well-characterized DNA standard to produce concentration estimates for unknowns that reliably produce STR profiles suitable for analysis.     

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。