24个基因座复合扩增系统的应用

目的建立24个基因座的复合扩增系统,并对其性能指标进行评价。方法选择24个基因座（包含D8S1179、D5S818、D2S1338、D18S51、D6S1043、D2S441、D3S1358、vWA、D19S433、D16S539、CSF1PO、Penta D、D22S1045、D13S317、D1S1656、D7S820、TPOX、Penta E、D10S1248、TH01、D12S391、D21S11、FGA等23个STR基因座及1个Amelogenin基因座）;合成引物,上游端标记荧光染料;收集DNA数据库建库血痕及口腔拭子样本及相关11种动物样本,采用本文方法进行复合扩增;并对方法的准确性、灵敏度、稳定性、种属特异性、检材适用性、混合样本的检验等系统性能指标进行验证。结果本文复合扩增系统对最长保存9年的各种血痕检材完整分型成功率为100%,且均衡性良好,无非特异性扩增,口腔拭子的成功率为97.8%,灵敏度达125pg,对含有抑制剂样本,在血红素浓度≤600μmol/L,腐殖酸浓度≤50ng/μL时检出效果稳定,种属特异性好。结论本文24个基因座复合扩增系统在DNA数据库建设中具有较好的应用价值。     

16个线粒体DNASNP复合检测体系的建立

目的建立一种复合检测线粒体DNA（mtDNA）单核苷酸多态性（SNP）分型的方法。方法通过设计等位基因特异性引物并结合毛细管电泳分型技术平台,建立包含16个mtDNASNP位点的复合扩增检测体系。对50名汉族无关个体血样进行mtDNASNP检测,并通过直接测序法对其分型结果进行验证。结果50个样本经本检测体系复合扩增后,均得到清晰的SNP分型结果,当模板量在0．5～10pg时能得到较理想的分型图谱。样本的复合检测结果与直接测序法结果完全一致。结论建立的复合检测体系检测mtDNASNP方法灵敏度高、操作简便、分型准确,为针对mtDNA进行简便、有效的中高通量多态性分型提供了一种新方法。     

The calculation of DNA match probabilities in mixed race populations

A coherent method is offered to estimate likelihood ratios for DNA match probabilities from mixed racial populations that avoids the approach of reporting separate estimates for each race. The method is demonstrated for some cases involving profiles derived from several individuals and incorporates a correction for 'subpopulation' effects.     

Modified Midi‐ and Mini‐Multiplex PCR Systems for Mitochondrial DNA Control Region Sequence Analysis in Degraded Samples

Two multiplex polymerase chain reaction (PCR) systems (Midiplex and Miniplex) were developed for the amplification of the mitochondrial DNA (mtDNA) control region, and the efficiencies of the multiplexes for amplifying degraded DNA were validated using old skeletal remains. The Midiplex system consisted of two multiplex PCRs to amplify six overlapping amplicons ranging in length from 227 to 267 bp. The Miniplex system consisted of three multiplex PCRs to amplify 10 overlapping short amplicons ranging in length from 142 to 185 bp. Most mtDNA control region sequences of several 60‐year‐old and 400–500‐year‐old skeletal remains were successfully obtained using both PCR systems and consistent with those previously obtained by monoplex amplification. The multiplex system consisting of smaller amplicons is effective for mtDNA sequence analyses of ancient and forensic degraded samples, saving time, cost, and the amount of DNA sample consumed during analysis.     

Analytical Thresholds and Sensitivity: Establishing RFU Thresholds for Forensic DNA Analysis,

Determining appropriate analytical thresholds (ATs) for forensic DNA analysis is critical to maximize allele detection. In this study, six methods to determine ATs for forensic DNA purposes were examined and compared. Four of the methods rely on analysis of the baseline noise of a number of negatives, while two utilize the relationship between relative fluorescence unit signal and DNA input in the polymerase chain reaction (PCR) derived from a dilution series ranging from 1 to 0.06 ng. Results showed that when a substantial mass of DNA (i.e., >1 ng) was amplified, the baseline noise increased, suggesting the application of an AT derived from negatives should only be applied to samples with low levels of DNA. Further, the number and intensity of these noise peaks increased with increasing injection times, indicating that to maximize the ability to detect alleles, ATs should be validated for each post‐PCR procedure employed.     

Reliable and Robust Molecular Sexing of the Hen Harrier (Circus cyaneus) Using PCR‐RFLP Analysis of the CHD 1 Gene

The hen harrier (Circus cyaneus) is a bird of prey that is persecuted in the United Kingdom, and there is a need for a DNA‐based individual identification and sexing system for the use in forensic investigations. This study reports a new set of PCR primers for the chromo‐helicase‐DNA‐binding protein 1 gene, which allows sexing using PCR‐RFLP. Instead of exonic primers that amplify across a large intron, this set consists of a primer within the intron, enabling reduction in amplicon sizes from 356 to 212 bp and 565 to 219 bp in W and Z chromosomes. DNA degradation and dilution experiments demonstrate that this set is significantly more robust than one that amplifies across the intron, and sequencing of the intronic primer‐binding region across several individuals shows that it is highly conserved. While our objective is to incorporate this primer set into an STR‐based individualization kit, it may in the meantime prove useful in forensic or conservation studies.     

DNA Barcoding Identifies all Immature Life Stages of a Forensically Important Flesh Fly (Diptera: Sarcophagidae)

Carrion‐breeding insects, such as flesh flies (Diptera: Sarcophagidae), can be used as evidence in forensic investigations. Despite their considerable forensic potential, their use has been limited because morphological species identification, at any life stage, is very challenging. This study investigated whether DNA could be extracted and cytochrome oxidase subunit I (COI) barcode sequences obtained for molecular identification of each immature life stage of the forensically important Australian flesh fly, Sarcophaga (Sarcorohdendorfia) impatiens (Walker). Genomic DNA extracts were prepared from all larval instars and puparia. Amplification of the barcoding region was successful from all extracts, but puparia amplicons were weak. All sequences were identified as S. impatiens with 99.95% confidence using the Barcoding of Life Database (BOLD). Importantly, crop removal was necessary to eliminate PCR inhibition for specimens from late second and early third instars. Similar results are expected for immatures of other carrion‐breeding species, enhancing the use of evidence from immature flies in forensic investigations.     

New York State TrueAllele® Casework Validation Study

DNA evidence can pose interpretation challenges, particularly with low‐level or mixed samples. It would be desirable to make full use of the quantitative data, consider every genotype possibility, and objectively produce accurate and reproducible DNA match results. Probabilistic genotype computing is designed to achieve these goals. This validation study assessed TrueAllele^® probabilistic computer interpretation on 368 evidence items in 41 test cases and compared the results with human review of the same data. Whenever there was a human result, the computer's genotype was concordant. Further, the computer produced a match statistic on 81 mixture items (for 87 inferred matching genotypes) in the test cases, while human review reported a statistic on 25 of these items (30.9%). Using match statistics to quantify information, probabilistic genotyping was shown to be sensitive, specific, and reproducible. These results demonstrate that objective probabilistic genotyping of biological evidence can reliably preserve DNA identification information.     

Identification of Human Remains by DNA Analysis of the Gastrointestinal Contents of Fly Larvae

Dipterous fly larvae (maggots) are frequently collected from a corpse during a criminal investigation. Previous studies showed that DNA analysis of the gastrointestinal contents of maggots might be used to reveal the identity of a victim. However, this approach has not been used to date in legal investigations, and thus its practical usefulness is unknown. A badly burned body was discovered with its face and neck colonized by fly larvae. Given the condition of the body, identification was not possible. Short tandem repeat (STR) typing was performed using the gastrointestinal contents of maggots collected from the victim and was compared to STR profiles obtained from the alleged father. The probability of paternity was 99.685%. Thus, this comparative DNA test enabled the conclusive identification of the remains. This is the first reported case of analysis of human DNA isolated from the gastrointestinal tract of maggots used to identify a victim in a criminal case.     

福尔马林固定石蜡包埋组织3种DNA提取方法比较

目的探讨经福尔马林固定1d石蜡包埋组织(FFPET)提取DNA的简易有效方法。方法比较水浴加热、微波加热和二甲苯脱蜡的效果。组织脱蜡后分别采用Chelex-100+层析柱纯化法、DNA IQTM试剂盒磁珠提取法和Chelex-100+磁珠纯化法提取DNA;实时荧光定量PCR技术定量DNA;荧光标记毛细管电泳技术进行STR分型。结果二甲苯脱蜡的效果好于其他两种加热的脱蜡方法(P<0.05)。Chelex-100+层析柱纯化所获得的DNA量显著高于其他两种方法(P<0.05)。结论二甲苯脱蜡、Chelex-100+层析柱纯化法是一种简单、有效的FFPET处理方法。