Optimization of the biological microchip for genotyping the AB0 locus: analytical aspects

The present work continues the search for methodological options facilitating the improvement and optimization of the biological microchip designed for genotyping the AB0 locus. It was shown in an earlier study designed to test a prototype biological microchip using a reference set of preparations with the known group specificity that under certain conditions some cells of the biochip appear to generate artifact hybridization signals that tend to make the results of genotyping either incorrect or difficult to interpret. We performed the correction of the molecular structure of DNA probes of the prototype biochip for the purpose of optimization of their hybridization potency. In addition, we developed and synthesized new DNA probes and designed new variants of the biochip. The experimental analysis of hybridization properties of all DNA probes thus obtained was carried out for the final choice of the most promising options suitable for the creation of the optimized biochip.     

Typing of ABO locus with biological microchip as a new level in solution of problems in forensic-medical biological expertise of material evidences

There are cases in practice when during expertise of material evidences, discrepancies between results of typing of ABO antigens and molecular-genetic typing of DNA occur. In this work, as a radical approach to objective solution of similar conflict situations, for some contradictory case of expertise, all examinations were performed on the unified methodological base--DNA level. Instead of biological (isoserological) typing of ABO antigen, molecular-genetic typing of ABO locus with biological microchip was performed. In all cases the results, received with the use of biological microchip, do not contradict but completely conform to the results of others molecular-genetic examinations performed in the case. Given results indicate irrationality of further use of traditional methods of isoserological typing of ABO antigen for primary differentiation of biological material. These analyses, if necessary, have to be performed on DNA level with molecular-genetic expertise.     

三个单位点DNA杂交联合应用的研究

本文从一些多态位点中筛选出在中国人群中，对于同一种限制酶HaeⅢ酶解都能检出良好多态性的三个单位点探针（PMLJ14、PYNH24、α－globin－3’HVR）。对这三个位点的等位基因频率进行了调查．用DNA指纹自动识别系统进行了数据处理，各位点的数据如下：PMLJ14、杂合度94％，等位基因频率分布0．002～0．051；PYNH24：杂合度89％，等位基因频率分布0．003～0．152；α－globin-3’HVR：杂合度78％，等位基因频率分布0．003～0．077。分析15个家系，未见到变异发生，符合孟德尔遗传规律。这三个位点个人识别中的累加机率是：1．7×10-5～2．1×10-14。     

寡核苷酸芯片技术在HLA-A抗原基因分型中的应用研究

目的建立一种HLA-A位点的基因芯片分型方法,为HLA-A位点的基因分型提供一个较新的思路。方法利用基因芯片技术,根据HLA-A位点不同基因亚型的独特序列设计探针,制成分型芯片;待检测样品经PCR反应标记上荧光之后,与芯片进行杂交,根据杂交产生的荧光信号值分析确定样品HLA-A位点的基因亚型。将这一方法应用于100份标准DNA和200份无关个体的HLA-A位点基因分型并将部分样品进行测序。结果检测结果表明HLA-A基因分型芯片可准确分辨出A位点等位基因20大类,耗时2.5h。结论寡核苷酸芯片技术用于HLA-A基因分型,分辨率高、特异性强、重复性好、操作简便、结果直观,适合于法医学实践和临床应用。     

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。     

Novel paternity testing by distinguishing parental alleles at a VNTR locus in the differentially methylated region upstream of the human H19 gene

Conventional PCR-based genotyping is useful for forensic testing but cannot be used to determine parental origins of alleles in DNA specimens. Here we describe a novel method of combined conventional genotyping and PIA typing (parentally imprinted allele typing) at a minisatellite region upstream from the H19 locus. The PIA typing uses two sets of primers and DNA digested with methylation-sensitive Hha I enzyme. The first amplification produces only the methylated fragment of paternal H19 allele, and the second detects polymorphism in the minisatellite. Hence, this distinguishes paternal and maternal alleles by difference in the DNA methylation. Furthermore, the polymorphism in this polymorphic locus was examined using 199 unrelated Japanese and 171 unrelated Germans, their polymorphism information content being 0.671 and 0.705, respectively. Feasibility of this typing is demonstrated for six families, and the usefulness is shown by application to paternity testing.     

pYNZ22位点扩增片段长度多态性法医应用的研究

对120名中国人进行pYNZ22位点扩增、银染检测，已找到11个等位基因，频率分布于0.4～30.4％，片段长170bp～870bp，相邻等位基因长度相差70bp，杂合度为73％，DP值为0．938。分析了五个家系均符合孟德尔定律。同一个体的血液、血斑、精液、混合斑中分离出的精子、毛囊、唾液及其它有核细胞组织的DNA多次进行pYNZ22位点扩增，得到一致的结果。灵敏度达到0．5ng核DNA。     

应用DNA工作站进行批量血斑STR分型的研究

目的建立对大批量血斑样品PCR-STR基因分型检测的自动化新方法。方法应用自动化DNA工作站改良优化Chelex-100法和DNAIQ磁珠法的实验条件,建立两种批量血斑的自动化DNA提取方法;筛选确定PCR-STR反应体系的构建和PCR-STR产物测序电泳分析前处理程序。结果1104份血斑样品经Chelex-100法批量提取、Profiler Plus试剂盒扩增均一次检出9个STR基因座,定量PCR测定模板浓度均值为0.43ng/ml,荧光检测信号在200~800RFU之间;对其中114份血斑样品用DNAIQ磁珠法批量提取、同试剂盒扩增均一次检出9个STR基因座,定量PCR测定模板浓度均值为0.7ng/ml,荧光检测信号在1000~2000RFU之间;对其中50份血斑进行自动和手动Chel-ex-100检验法比较,成功率分别为100%和98%,且前者等位基因峰高信号更均衡。结论本文建立的自动化DNA工作站批量检测方法,在成功率、稳定性、均一性等方面具有优势。     

车辆内接触DNA检测方法的分析比较

目的探讨汽车内接触DNA的分离方法及遗传标记分型效率。方法收集单人长期驾驶的11辆小型轿车,采用粘取法和擦拭法富集方向盘、变速杆和手刹三个部位的脱落细胞,采用磁珠法和硅胶膜法提取基因组DNA,采用GoldenEye^TM 20A和PowerPlex■Fusion进行扩增,并对检验结果进行比较分析。结果方向盘在基因座分型正确率、等位基因drop-in和drop-out基因座比率、单基因座正确率以及单基因座等位基因drop-in和drop-out率六个方面均表现最好,其次为变速杆,最差为手刹;擦拭法和粘取法之间DNA提取在获得的DNA总量和STR检测正确成功率方面无统计学差异;PPFusion与20A的总体基因座分型比较正确率无差异,但单基因座正确率优于20A,drop-out发生率低于20A,drop-in发生率高于20A。结论汽车内脱落细胞的检测可优先采集方向盘部位,根据载体质地选择擦拭法或粘取法采集脱落细胞,选用硅胶膜法或磁珠法提取DNA,PPFusion和20A两个试剂盒均可,分析结果时需特别注意drop-in和drop-out。     

联合应用常染色体STR和X-STR标记鉴定单亲疑难案例

目的通过对常染色体和X染色体遗传标记的检测,探讨单亲疑难案例的鉴定策略。方法提取3个单亲鉴定案例的6份血样,采用Goldeneye 20A试剂盒和AGCU21＋1试剂盒检测常染色体上39个STR基因座,采用自主研制的16重X-STR扩增系统检测X染色体上16个STR基因座。结果用Goldeneye 20A试剂盒检测后发现每个单亲案例均有一个基因座不符合遗传规律,当常染色体STR基因座增加到39个时,案例1累计出现3个矛盾基因座;案例2和案例3均没有出现新的矛盾基因座。X染色体STR分型结果显示案例1有8个矛盾基因座,案例2和案例3无矛盾基因座,与常染色体分型结论相符。结论对于出现单基因座不符合遗传规律的母女、母子、父女单亲案例鉴定,不仅可以增加新的常染色体STR检测,也可以增加X染色体STR的检测,这样在相互验证的同时也能获得更加可靠的鉴定意见。     

Developmental validation of a multiplex qPCR assay for assessing the quantity and quality of nuclear DNA in forensic samples

Forensic scientists are constantly searching for better, faster, and less expensive ways to increase the first-pass success rate of forensic sample analysis. Technological advances continue to increase the sensitivity of analysis methods to enable genotyping of samples containing minimal amounts of DNA, yet few tools are available that can simultaneously alert the analyst to both the presence of inhibition and level of degradation in samples prior to genotyping to allow analysts the opportunity to make appropriate modifications to their protocols and, consequently, to use less sample. Our laboratory developed a multiplex quantitative PCR assay that amplifies two human nuclear DNA target sequences of different length to assess DNA degradation and a third amplification target, a synthetic oligonucleotide internal PCR control (IPC), to allow for the assessment of PCR inhibition. We chose the two nuclear targets to provide quantity and fragment-length information relevant to the STR amplification targets commonly used for forensic genotyping. The long target (nuTH01, 170-190 bp) spans the TH01 STR locus and uses a FAM-labeled TaqMan probe for detection. The short nuclear target (nuCSF, 67 bp) is directed at the upstream flanking region of the CSF1PO STR locus and is detected using a VIC-labeled TaqManMGB probe. The IPC target sequence is detected using a NED-labeled TaqManMGB probe. The assay was validated on the Applied Biosystems 7500 Real-Time PCR system, which is optimized for NED detection. We report the results of a developmental validation in which the assay was rigorously tested, in accordance with the current SWGDAM guidelines, for precision, sensitivity, accuracy, reproducibility, species specificity, and stability.     

Development and application of hydrogel oligonucleotide microchips for forensic-medical personal identification as illustrated by ABO locus

The article describes the method defining 5 alleles of ABO blood group typing system by molecular hybridization in hydrogel oligonucleotide microchip. The testing points were SNP variants in positions 261 and 297of exon 6 and in positions 646 and 657 of exon 7. Therefore, 5 ABO blood groups can be easily revealed: A, B, 0(1), 0(1v), 0(2). The method was tested on 10 DNA samples isolated from blood and saliva of unrelated individuals. The results were confirmed by sequencing of the identified allelic fragments. Estimation sensitivity was 25 pg of total DNA input. This technique is cost-effective and easy for use and, therefore, promising for forensic-medical personal identification.     

Results of applying molecular-and-genetic markers of chromosomal DNA in identification of unrecognizable remains of servicemen lost in military actions in the Northern Caucasus

The results of applying molecular-and-genetic markers of chromosomal DNA in identifying unrecognizable remains of servicemen lost as dead in 1994-1996 and 1999-2001 military campaigns in the Northern Caucuses are summarized in the paper. Some of the specific features related with enzymatic amplification typing of DNA preparations sampled from degraded biological tissues of strongly deformed or decayed cadavers were analyzed. The typing results were analyzed by the AB0 system of degraded expertise samples in order to check the reliability of routine forensic-biological examinations as applicable to cadaver tissues with pronounced putrefactive changes. It was established that group-specific antigens of the AB0 system were correctly determined only in 67.5% of cases. False results were obtained in the other 32.5% of cases. Most of them were related with determination of groups 0(I) and A(II).     

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.     

单位点DNA探针在早孕期亲子鉴定中的应用研究

本研究利用限制性片段长度多态性分析技术，从3O例早孕期绒毛组织及其父母血液中提取DNA，以两种单位点DNA探针PAC255及PAC256进行检测，根据所获等位基因的大小，查国人此二位点基因频率及每一家系三组合中该基因传递相对机会，计算出相应的亲子关系指数和亲子关系概率。结果．累积条子关系指数和累积亲子关系概率的范围分别是56．80～8333.50和98.27～99.99％、按照国际通用标准，这30个家系中，可以肯定亲手关系者20例（66.7％），极可能有亲子关系者8例（26.7％），非常可能有亲子关系者2例（6.7）。说明同时使用此二单位点探针，在早孕期亲子鉴定中有很高的应用价值。     

短串联重复基因座突变率的分析研究

分析亲子鉴定案例进行中的STR基因座的突变率。采用chelex 100快速抽提DNA,DNA扩增,聚丙烯酰胺凝胶电泳、银染色法,参照标准品进行DNA分型。在300例已确定亲生关系的案例中,有11例出现STR基因座变异,其中D11S554基因座6例,D19S253基因座2例,SE33、D12S391、D13S631基因座各1例。结果提示:用STR基因座进行亲子鉴定,必须考虑STR基因座突变因素。     

Study of Short- and Long-Term Storage of Teeth and Its Influence on DNA

Abstract:  DNA degradation can interfere with the resolution of forensic cases. Allelic dropout often reduces the opportunity for adequate comparisons between degraded and reference samples. This study analyzed DNA degradation in 24 extracted teeth after storage at room temperature for 0, 2, 5, and 10 years. DNA concentration, quantified by dot-blot hybridization, declined significantly for the first 2 years, but there was no significant further degradation from the second to the tenth year of storage. COfiler™ analysis was used and the allelic dropout ratio for the amelogenin locus relative to CSF1PO locus was also estimated. Statistically significant differences were found between fresh teeth and teeth from the 2- and 5-year groups but not from the 10-year group. Under our storage conditions most of the DNA degradation occurred during the first 2 years. Further research is needed to control for individual and external factors that could affect DNA.     

混合生物样品的组分分析及其STR基因型判定

目的应用荧光标记STR多基因座联合检测技术对混合生物样品较少组分最低比例检出限和STR基因型的判定进行研究。方法 将已知浓度的人标准细胞系DNA:9947A和K562分别按照1:1、1:4、1:9、1:19、1:39、1:59、1:79和1:99比例混合后作为扩增模板,应用Profiler plus试剂盒对D3S1358、VWA、FGA、D8S1179、D21S11、D18S51、D5S818、D13S317、D7S820和性别鉴定基因座检验。结果在比例为1:19时能明确判定两个体各基因座基因型,且两样品基因型结果峰高平均值之比与样品浓度之比正相关。结论应用荧光标记STR多基因座联合检测技术可以对一定比例的混合生物样品进行STR基因型判定,并对其混合比例状况进行大致推断。     

Hypervariable regions of DNA for parentage testing and individual identification

Four kinds of DNA probes that recognize hypervariable regions (HVR) were studied for parentage testing and individual identification. Allele frequencies and their confidence intervals among unrelated Japanese individuals were obtained. Codominant segregation of the polymorphism was confirmed in family studies. Two a priori probabilities were calculated for each HVR locus: the exclusion probabilities for an alleged father/mother/child trio and for an alleged parent/child duo, and the probabilities of matching of genotypes of two unrelated individuals or two siblings. The ease of availability of the probes and their highly discriminating polymorphic patterns mean they could be very useful for forensic purposes.     

强奸致孕形成部分性葡萄胎的检验分析

目的探讨强奸致孕案葡萄胎组织的DNA检验和STR结果分析。方法用DNA Typer TM15试剂盒对犯罪嫌疑人、受害人血样和流产胚胎组织进行荧光复合STR基因座扩增检测。结果检测的一例流产胚胎组织DNA的STR峰谱表现出部分3条带,且3条带中有两个等位基因来源于犯罪嫌疑人,另外一个来源于受害人,推断为单卵双精子受精的部分性葡萄胎。结论应用STR分型技术可以推断葡萄胎的DNA来源和受精类型,为亲缘关系鉴定提供依据。