降解生物检材DNA分析研究新进展

在法医检案过程中,如何从降解生物检材中获得正确的DNA分型是法医学界的一个难题。本文对降解生物检材DNA分析研究取得的新进展进行综述,从检案的各个环节出发对近年来提高DNA分型成功率的方法和技术进行了详细介绍,例如新型遗传标记的开发和二代测序技术的应用等。希望为降解检材的分析提供新的思考和方法。     

二代测序技术在法医遗传学中的应用研究进展(2011～2016)

对生物检材进行DNA分型是解决法医遗传学实践中个体识别和亲权鉴定问题的重要步骤,法医学实践中复杂生物检材和复杂亲缘关系鉴定等一直是现有的检测分析技术的难点和挑战。随着DNA技术的发展,新的检测分析技术不断引入到法医遗传学领域,以期提高检测效能。二代测序技术具有测序通量高、成本低等特点,能够获得样本DNA详细序列和相对含量等信息,有助于生物检材的检测和案件的分析。二代测序技术在法医遗传学领域的应用受到广泛关注,相关的应用研究逐渐增多。本文就目前法医遗传学领域借助二代测序技术对遗传标记分析的研究进展进行总结,希望能为相关研究和应用提供参考。     

采用SNP分型方法对甲醛固定组织进行个体识别

目的通过对X染色体SNP遗传标记的检测,探讨甲醛固定组织块的DNA分型策略。方法提取甲醛固定组织块的DNA,在用SinofilerTM试剂盒、MiniFilerTM试剂盒检测未能获得分型结果的情形下,采用多重PCR和飞行质谱技术对X染色体上的51个SNP位点进行分型检测。结果对于常染色体STR、miniSTR分型失败的甲醛固定组织块,X-SNP分型获得成功。结论对于甲醛固定组织块等微量、降解的生物学检材,若常染色体STR基因座分型失败,可尝试进行SNP位点的分型,以获得更多的遗传信息。     

MiniSTR技术的研究进展

短串联重复序列(STR)是法医DNA鉴定中最常用和最重要的遗传标记,但是对于降解和微量的DNA样品,经常得不到完整的DNA分型甚至分型失败。MiniSTR技术通过设计更靠近重复序列的引物,得到更短一些的STR基因座,提高了降解和微量检材的DNA分型成功率。本文综述了miniSTR技术的研究进展,以服务于法医学实践。     

PCR-STR分型在刑事案件中的应用

应用PCR技术对多态DNA位点分型,使犯罪现场生物物证的个人识别鉴定取得了长足的进展,检测灵敏度明显提高,检验结论从否定到肯定.以往报告多为PCR-VNTR.PCR-ASO技术的应用.近几年PCR-STR分型成为法医学应用研究的热点.本文收集实际鉴定案件,着重对PCR-STR技术在刑事案件中的应用范围、热点及前景进行了讨论,现报告如下:1.材料我室自1995年4月至1997年12月受理的PCR检验案件中的7例典型案例,检材为血液(痕).混合斑、组织块等.     

二代测序技术在法医学中的应用

随着二代测序技术的快速发展,其高通量和低成本在生命科学领域应用广泛,测序的通量更高,测序时间和成本不断下降,使得其被广泛应用于微生物研究、古DNA研究、临床诊断、法医学研究等。本文阐述了二代测序技术平台及其遗传标记在法医学中的应用,包括STR分型、SNP分型、HLA基因型预测以及在降解检材中的应用等。     

降解生物检材DNA分析策略的研究进展

生物检材发生降解后的DNA分型是法医DNA领域的难题之一。本文对降解检材分析策略的研究进行综述。分析了目前常用的降解DNA分析方法与技术的优缺点,对近年来新出现的方法,如扩增前降解损伤DNA修复策略、扩增后纯化策略和核小体DNA策略做了介绍,希望能为降解生物检材DNA检验的研究和分析提供思路,为相关应用提供新方法的参考。     

多聚酶链反应(PCR)在法医生物学检材鉴定中的应用

法医物证学的鉴定客体主要是生物学检材，其形式是多种多样的：如在各种载体k的血痕，精液与阴道液的混合斑，毛发，骨组织，被害人指甲缝中残留的组织成份等．对这些检材进行检验的中要目的就是根据检验结果确定该生物学检材是谁所留．以往所采用的检验方法主要包括红细胞血型和蛋白质遗传标记分型等检测方法．现在我们可以在DNA水’【‘对有关生物学检材进行遗传多态性的检验，因为每一个个体（除非同卵双生）从遗传学上来看都是独一无二的，在DNA水平对生物学检材进行遗传标记的分型检测，可望达到接近个体绝对认定的结果．目前有两种…     

生物质谱技术在法医遗传学中的应用展望

随着基质辅助激光解吸电离质谱 (MALDI MS)和电喷雾离子化质谱 (ESI MS)两种技术的迅速发展 ,生物质谱技术已经成为生命科学研究中的重要工具。由于直接检测分子的质量 ,质谱技术同其他方法相比更加准确。生物质谱技术在核酸序列分析中具有快速性、微量化、和高通量的特点。利用MALDI TOF MS技术可以分析降解检材的SNP遗传标记 ,这在法医学中将有着重要的意义。本文对生物质谱技术的原理、进展、面临的困难以及在法医遗传学中的应用前景作了评述     

IDentifier DNA分型盒（炎黄34）的法医学验证及应用评估

目的 测试IDentifier DNA分型盒(炎黄34)的技术性能指标,评估其法医学应用价值。方法 根据《法庭科学人类荧光标记STR复合扩增检测试剂质量基本要求》(GB/T 37226—2018),从种属特异性、分型准确性、灵敏度、适应性、耐受性、一致性、均衡性、反应条件验证、混合样本、稳定性、批间差11个方面对IDentifier DNA分型盒(炎黄34)进行测试。比较IDentifier DNA分型盒(炎黄34)与Power Plex^?Fusion 6C系统、Versa Plex^?27PY系统、Veri Filer^TM Plus PCR扩增试剂盒的系统效能。使用IDentifier DNA分型盒(炎黄34)检测日常案件中的拭子类生物检材,观察其STR检验结果 。结果 IDentifier DNA分型盒(炎黄34)具有良好的种属特异性、分型准确性、适应性、耐受性和均衡性,灵敏度可达0.062 5 ng,能检测案件中不同类型的检材、降解检材及混有抑制剂的检材,对混合比例为4∶1以内的样本均能获得完整分型。该试剂盒的系统效...     

Preparation of degraded human DNA under controlled conditions

DNA typing through analysis of short tandem repeats (STRs) and mitochondrial DNA (mtDNA) by means of the polymerase chain reaction (PCR) and sequencing are the common methods for the forensic identification of persons and reconstruction of kinship, especially when skeletal human remains have to be analyzed. Furthermore, samples typically found at crime scenes may be both quantitatively and qualitatively inadequate since they may contain very scarce and often degraded DNA due to exposure to heat, light, humidity, and microorganisms. In order to improve the performance of STR typing technology in those cases where DNA availability is limited, it would be desirable to have a source of degraded DNA with known properties. For this purpose, we have developed a method to prepare artificially degraded DNA under controlled conditions. By treatment of genomic DNA with sonication and DNAse I we have produced DNA fragments within a defined range of lengths. STR typing of this degraded DNA with a commercially available multiplex kit could only produce partial profiles as indicated by the absence of STR alleles with sizes >200 bp. This artificially degraded DNA can be used for the improvement and standardization of STR typing protocols when only highly degraded DNA is available for analysis.     

A LDR‐PCR Approach for Multiplex Polymorphisms Genotyping of Severely Degraded DNA with Fragment Sizes <100 bp*

Abstract: Reducing amplicon sizes has become a major strategy for analyzing degraded DNA typical of forensic samples. However, amplicon sizes in current mini‐short tandem repeat‐polymerase chain reaction (PCR) and mini‐sequencing assays are still not suitable for analysis of severely degraded DNA. In this study, we present a multiplex typing method that couples ligase detection reaction with PCR that can be used to identify single nucleotide polymorphisms and small‐scale insertion/deletions in a sample of severely fragmented DNA. This method adopts thermostable ligation for allele discrimination and subsequent PCR for signal enhancement. In this study, four polymorphic loci were used to assess the ability of this technique to discriminate alleles in an artificially degraded sample of DNA with fragment sizes <100 bp. Our results showed clear allelic discrimination of single or multiple loci, suggesting that this method might aid in the analysis of extremely degraded samples in which allelic drop out of larger fragments is observed.     

Assessment of PowerPlex® Fusion 5C’s ability to type degraded DNA

DNA samples collected at crime scenes are often degraded so this research focused on the ability of the Promega PowerPlex® Fusion 5C amplification kit to type both naturally and artificially degraded DNA.DNA was degraded naturally by placing equal volumes of blood on white fabric that was stored either inside, outside in a shaded area, or outside in direct sunlight. Samples were then collected every 10 days for 60 days and the DNA extracted (QIAamp® DNA Investigator). Artificially degraded samples were created by exposing extracted DNA to either UV light or 95 °C heat for varying times. DNA was also degraded artificially by placing blood samples into a 50% bleach solution for varying times prior to extraction.Following sample treatment, standard forensic DNA analysis was performed including quantification (Investigator® Quantiplex) and amplification (PowerPlex® Fusion 5C). Separation and detection were performed on an ABI 3130xl capillary electrophoresis unit and analysis was performed using GeneMapper ID v3.2.1.While the time and shade samples showed similar amounts of degradation, the samples exposed to direct sun showed more degradation. The artificially degraded samples showed more signs of degradation such as reduced overall peak height and peak height imbalance at heterozygous loci. There were also some cases where an allele that was known to be in the profile exhibited total dropout. Although there were some instances of both allelic dropout and heterozygote peak imbalance, PowerPlex® Fusion was able to reliably type degraded DNA as all alleles detected were consistent with the known donor profile. The results show that PowerPlex® Fusion is a robust kit capable of handling forensically challenged samples.     

Comparison of Catalytic and Immunological Amylase Tests for Identifying of Saliva from Degraded Samples

The stability of salivary α‐amylase is a critical factor in both catalytic and immunological method‐based forensic saliva identification. This study aimed to assess the sensitivity of catalytic and immunological tests on degraded saliva samples. Degraded saliva stains were prepared by microbial decomposition using humid soil. Salivary α‐amylase activity was catalytically detected both qualitatively and quantitatively using the Phadebas^® amylase test. As immunological methods, we conducted qualitative and quantitative tests using the RSID?‐saliva test and ELISA, respectively. Salivary α‐amylase activity of degraded samples (incubated at 37°C for 12 h) was significantly lower than that of controls in the quantitative tests. All the degraded samples obtained by the humid soil produced negative results in the Phadebas^® tests, but showed positive results in the RSID?‐saliva test and ELISA. These results suggest that immunological tests are effective for testing degraded saliva samples that have lost their enzymatic activity.     

STR analysis of degraded DNA using a miniplex

Analysis of short tandem repeat (STR) markers currently represents the most useful instrument in the field of forensic genetics. The problem with forensic material is the degradation of the sample material. In recent years, several papers have demonstrated that short amplicon STR (miniSTR) represents one of the most useful tools for analyzing degraded DNA samples.In the present study, we attempted to develop a short amplicon STR multiplex system (autosomal and y-chromosomal) for analyzing degraded DNA using some newly designed primer sets for a multiplex polymerase chain reaction (PCR) systems for typing.An assay of degraded DNA samples using the designed multiplex systems, including artificially degraded samples and degraded forensic casework samples, proved remarkably effective. Comparing the multiplex with commercial kits, first results show a well success rate.     

Mini-SGM multiplex in degraded samples

A five Mini-SGM multiplex which encompasses TH01, FGA, D18S51, D16S539 and D2S1338, common STR markers in human identity testing, have been performed. Two cases with different biological tissues were selected to illustrate the usefulness of this technique in forensic casework. The use of routine methodology can sometimes give only a partial genetic profile or no profile at all. However, using the Mini-STR technique, a full profile was obtained for the majority of the degraded samples. We conclude that the Mini-SGM methodology is more sensible than routine methodology for degraded samples, although a full genetic profile is not obtained in all cases as results are still very much sample-dependent. This Mini-SGM multiplex can be considered a useful tool to complement conventional STR analysis in degraded samples.     

Development of Multiple Assays using 46 SNPs for Comprehensive mtDNA Haplogrouping and Application to Highly Degraded DNA

Six multiplex PCR systems using single‐base extension reactions to analyze 46 mitochondrial DNA (mtDNA)‐coding region single nucleotide polymorphisms (SNPs) that define 42 haplogroups, that is, 24 major mtDNA haplogroups and 18 subclades, were devised. To improve the usefulness of the established systems for the analysis of degraded DNA samples, novel primers to render amplicons with sizes <150 bp were designed. By applying these systems to 214 Japanese individuals, 24 different haplogroups (power of discrimination = 93.4%) were found. To assess the effectiveness of our systems in grouping degraded DNA, an ancient bone sample of a Jomon skeleton was analyzed and then classified as haplogroup N9b. We conclude that the present systems are powerful screening tools for major haplogroups of mtDNA in addition to the prevalent subhaplogroups in the Japanese population and that these systems are capable of analyzing highly degraded DNA samples in forensic studies.     

Developmental validation of reduced-size STR Miniplex primer sets

This paper describes a developmental validation study of three Miniplex sets covering 12 of the 13 CODIS loci. As these new sets will be used for the analysis of degraded and low level DNA, the validation studies were performed using 100-125 pg of DNA, the lowest input level at which peak balance, peak intensity, and allele consistency were stable. To demonstrate the applicability of the Miniplex sets to forensic casework, these validation studies were completed in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM). A range of tests were performed including studies of concordance with standard multiplex kits, sensitivity and reproducibility, and PCR amplification conditions. Additionally, studies of mixtures, nonhuman and environmentally degraded DNA, and simulated forensic samples were performed. Our results demonstrate that Miniplex STR amplification procedures are a robust and sensitive tool for the analysis of degraded DNA.     

Tri-allelic SNP markers enable analysis of mixed and degraded DNA samples

For the analysis of degraded DNA in disaster victim identification (DVI) and criminal investigations, single nucleotide polymorphisms (SNPs) have been recognized as promising markers mainly because they can be analyzed in short sized amplicons. Most SNPs are bi-allelic and are thereby ineffective to detect mixtures, which may lead to incorrect genotyping. We developed an algorithm to find non-binary (i.e. tri-allelic or tetra-allelic) SNPs in the NCBI dbSNP database. We selected 31 potential tri-allelic SNPs with a minor allele frequency of at least 10%. The tri-allelic nature was confirmed for 15 SNPs residing on 14 different chromosomes. Multiplex SNaPshot™ assays were developed, and the allele frequencies of 16 SNPs were determined among 153 Dutch and 111 Netherlands Antilles reference samples. Using these multiplex SNP assays, the presence of a mixture of two DNA samples in a ratio up to 1:8 could be recognized reliably. Furthermore, we compared the genotyping efficiency of the tri-allelic SNP markers and short tandem repeat (STR) markers by analyzing artificially degraded DNA and DNA from 30 approximately 500-year-old bone and molar samples. In both types of degraded DNA samples, the larger sized STR amplicons failed to amplify whereas the tri-allelic SNP markers still provided valuable information. In conclusion, tri-allelic SNP markers are suited for the analysis of degraded DNA and enable the detection of a second DNA source in a sample.     

Validation of the Short Amplicon Multiplex Q8 Including the German DNA Database Systems*

Abstract: We have developed a concept to enable the analyzing of degraded stains with limited DNA template quantity. Therefore we have constructed a short tandem repeat (STR) multiplex including the German DNA database systems (Q8). The amplicon lengths are smaller than 280 bp. For the validation of Q8 over 50 degraded samples were investigated. Amplifications were performed with “low copy number” PCR, the number of PCR cycles was increased to 33 and the reaction volume was decreased to 12.5 μL. Compared with the MPX2 and Nonaplex kit, the average success rate was increased using the Q8 kit by approximately 20% and 30%, respectively. The efficiency of a sensitive STR multiplex with reduced amplicon lengths was confirmed in comparing the success rates of Q8 for typing degraded samples and samples with limited amount of DNA template while partial profiles were observed with the majority of the samples using commercially available kits.