Report of the blind trial of the Cetus Amplitype HLA DQ alpha forensic deoxyribonucleic acid (DNA) amplification and typing kit

The AmpliType HLA DQ alpha forensic DNA amplification and typing kit is designed for the qualitative analysis of the human leukocyte antigen (HLA) DQ alpha alleles present in deoxyribonucleic acid (DNA) extracted from forensic samples. The AmpliType kit is the first forensic DNA typing product based on the GeneAmp polymerase chain reaction (PCR) process. The kit was evaluated by five forensic science laboratories (test sites) to assess their ability to perform DNA typing using PCR on sample types typically encountered by forensic laboratories. None of the DNA-containing samples was mistyped. Of the 180 DNA-containing samples analyzed, results were reported for 178 (98.9%). Of the 178 samples with results, all were correctly typed. Two sites did not report a result for one sample each. Four of the five laboratories experienced no significant levels of contamination in the DNA-containing samples. At the one site with the highest number of DNA-containing samples with contamination, the typing results were not compromised. This site was able to correct the contamination problem through simple procedural changes and stricter attention to sterile technique. Blank controls were important to monitor contamination. In conclusion, the trial demonstrated that forensic science laboratories are capable of setting up a PCR-based DNA typing laboratory and successfully using the AmpliType HLA DQ alpha forensic DNA amplification and typing kit to analyze forensic samples.     

Genetic markers in human bone: I. Deoxyribonucleic acid (DNA) analysis.

Deoxyribonucleic acid (DNA) was isolated from a number of spongy and compact human bone tissue specimens, and the yield was estimated on a "per milligram of starting tissue" basis. DNA was, in addition, isolated from a number of corresponding blood and bone tissue specimens. Spectrophotofluorometry and ethidium bromide visualization on minigels were used to estimate the quantity and degree of degradation of DNA. The DNA from several blood-bone pairs is shown to give concordant restriction fragment length polymorphism (RFLP) typing results by two different typing protocols with five different single-locus probes. DNA from several additional blood-bone pairs is shown to give concordant results for human leucocyte antigen (HLA)-DQ alpha phenotypes following polymerase chain reaction (PCR) amplification and hybridization to specific allele-specific oligonucleotide (ASO) probes, and for the variable numbers of tandem repeats (VNTR) length polymorphisms 3' to the human apolipoprotein B (APOB) gene following PCR amplification with specific primers and analysis of the products by electrophoresis and ethidium bromide visualization.     

PCR法对HLA-DQ_α基因的分型及其在性犯罪鉴定中的应用

本文应用 PCR 法及 ASO 探针斑点杂交技术,对100例无关个体血液 DNA 及10例性犯罪案件混合斑中精子 DNA 进行了 HLA-DQα基因的扩增及其 DNA 分型。结果正常人0.1～0.3μgDNA 就能满足 DQα基因扩增的需要。在100例个体中可以观察到由4种等位基因组成的10种 DQα基因型。10例不同条件的混合斑中精子 DNA 经30～60次扩增后与 ASO 探针杂交均能准确地判定 DQα基因型。HLA-DQα是个体识别能力较强的遗传标志。本文为性犯罪中精子来源的个体识别提供了一个新方法,具有一定的实用价值。     

Fixed human tissues: a resource for the identification of individuals

Polymorphic genetic loci of the deoxyribonucleic acid (DNA) present in formalin-fixed, paraffin-embedded tissues were successfully analyzed by utilizing the polymerase chain reaction. Using this analysis, with three different polymorphic loci [human leucocyte antigen (HLA) DQ alpha, low-density lipoprotein receptor, and parathyroid hormone], fixed tissues representing 14 different individuals were genotyped and could be distinguished from each other. The techniques were further applied to the fixed autopsy tissues of a man in which a question of paternity arose postmortem. Since many individuals have surgical procedures or autopsy, these readily available fixed tissues represent an additional resource for the identification of individuals.     

Detection of polymorphisms of human DNA after polymerase chain reaction by miniaturized SDS-PAGE.

PCR followed by SDS-PAGE in miniaturized non-denaturing gels permits in some cases the identification of single base pair substitutions in small DNA fragments and therefore, the study of human DNA polymorphisms. The usefulness of the system in forensic science is investigated by typing the HLA-DQA1 locus and the VNTR recognized with the probe pMCT118 (locus D1S80) and it shows to be advantageous over previously published methods for typing the MCT118 system, whereas in HLA-DQA1 typing for forensic casework, both dot-blot with ASO probes and this method could be complementary.     

HLA-A基因座多态性在亲子鉴定中的应用研究

首次将HLA A基因座的聚合酶链反应 寡核苷酸探针 (PCR SSOP)杂交分型技术应用于亲子鉴定案例分析 ,以获得HLA A基因座多态性用于法医学鉴定分析的基础数据。HLA A基因座基因分型的等位基因检出率 ( 2 4个 )和非父排除率 ( 73 3 % )均高于血清学检测方法 ;正常家系分析结果符合孟德尔遗传规律。对 3 9个亲子鉴定例的成员进行HLA A基因检测 ,9例排除 ( 2 3 1% ) ;未排除的 3 0例的亲子关系概率在 65 2 %～ 99 5 %之间。用PCR SSOP技术对HLA基因座基因分型不仅可以应用于亲子鉴定和法医学个人识别 ,还可以应用于器官移植配型及人类遗传学研究。     

HLA—A基因座分步PCR—SSP分型研究

目的使HLA基因分型能应用于法医常见检材的个人识别。方法 建立检测HLA—A基因座的分步PCR—SSP方法。先用一对HLA—A基因座特异的引物作第一次扩增,以所得产物为模板,分别用对HLA—A30、A31、A33特异的3对引物作第二次扩增,二次扩增的产物经电泳判型。结果 1130例血清分型为HLA—A30、A31、A33的血痕,其PCR—SSP分型和血清分型的不符合率为29％;室温保存2年的精斑、唾液斑,保存18年的血痕第一次扩增均获得满意的结果。结论法医亲子鉴定和个人识别宜用基因分型替代血清分型。HLA—A基因座分步PCR—SSP基因分型适用于法医检材。     

Polymerase chain reaction (PCR) amplification and human leukocyte antigen (HLA)-DQ alpha oligonucleotide typing on biological evidence samples: casework experience.

The polymerase chain reaction (PCR) method of specific gene amplification was used in casework to synthesize millions of copies of the polymorphic second exon of the human leukocyte antigen (HLA)-DQ alpha (or DQA1) locus from a variety of evidence samples. The HLA-DQ alpha allelic variants in the amplified deoxyribonucleic acid (DNA) were determined in a rapid non-radioactive test by hybridization to sequence-specific oligonucleotide probes in both the dot-blot and reverse dot-blot formats. This genetic typing system has been subjected to blind proficiency testing; the performance of this test in the analysis of experimentally mixed samples was also evaluated. As of August 1990, over 250 cases have been tested and more than 2000 individual evidence (bloodstains, semen stains, individual hairs, bone fragments, and tissue sections) and reference samples have been analyzed. The first 198 of these cases are summarized in this paper; in 65% of the cases with conclusive results a suspect was included, and in 35%, all suspects were excluded. Individual cases as well as some of the general issues relating to forensic science analysis and this genetic typing system are discussed. The high rate of exclusion reported here combined with the ability of PCR to type old evidence samples suggests the relevance of this genetic test for postconviction review; two cases in which the convicted suspect was excluded are discussed.     

Use of deoxyribonucleic acid (DNA) fingerprints for identity determination: comparison with traditional paternity testing methods--Part II

Six red blood cell (RBC) antigen systems, coupled with human lymphocyte antigen (HLA) phenotyping, were used to establish paternity on 28 mother/child/alleged-father trios. Samples were subsequently examined using the deoxyribonucleic acid (DNA) fingerprinting test with the multilocus Jeffreys DNA probes 33.6 and 33.15. In 27 of 28 paternity cases, the DNA fingerprinting test results supported and enhanced the results of RBC and HLA typing by resolving disputed paternity cases conclusively. One discrepancy between conventional serological methods and DNA analysis is discussed.     

HLA-DRB1位点的PCR-SSP分型在亲子鉴定中的应用研究

应用PCR－SSP（PCRamplificationwithsequencespecificprimer）方法将HLAⅡ类DRB1位点基因分型应用于亲权鉴定。对42例亲子鉴定案例进行分析研究的结果表明,本方法简单、快速、结果可靠,且具有较高的非父排除概率（66．3％）,不仅适用于法医学亲手鉴定和个人识别,亦可应用于移植配型、HLA相关疾病及人类遗传学研究。     

HLA基因分型技术的进展与展望

研究人的主要组织相容性复合物HLA(humanleucocyteantigen,人类白细胞抗原)的结构和功能有助于认识生命现象的本质以及疾病的致病机理,因此长期以来一直是生命科学研究的重点之一。随着分子生物学技术的迅速发展,HLA分型已经由传统的血清学水平向DNA水平过渡,并取得了很好的效果。本文对目前各种HLA基因分型方法的原理和优缺点结合自己的应用经验做了评价,并简要介绍了在HLA基因分型研究中出现的新的动态。     

Human Leukocyte Antigen alleles as an aid to STR in complex forensic DNA samples

Human biological samples with multiple contributors remain one of the most challenging aspects of DNA typing within a forensic science context. With the increasing sensitivity of commercially available kits allowing detection of low template DNA, complex mixtures are now a standard component of forensic DNA evidence. Over the years, various methods and techniques have been developed to try to resolve the issue of mixed profiles. However, forensic DNA analysis has relied on the same markers to generate DNA profiles for the past 30 years causing considerable challenges in the deconvolution of complex mixed samples. The future of resolving complicated DNA mixtures may rely on utilising markers that have been previously applied to gene typing of non-forensic relevance. With Massively Parallel Sequencing (MPS), techniques becoming more popular and accessible even epigenetic markers have become a source of interest for forensic scientists.The aim of this review is to consider the potential of alleles from the Human Leukocyte Antigen (HLA) complex as effective forensic markers. While Massively Parallel Sequencing of HLA is routinely used in clinical laboratories in fields such as transplantation, pharmacology or population studies, there have not been any studies testing its suitability for forensic casework samples.     

Distribution of HLA-DQA1 alleles in Arab and Pakistani individuals from Dubai,United Arab Emirates

PCR-based typing of the HLA-DQA1 locus, using allele specific oligonucleotide (ASO) probes and reverse dot blot methodology was used to determine allelic distributions and construct a database for Arab and Pakistani individuals living in Dubai. Genotype and allelic frequencies were calculated, and the data were tested for departures from Hardy-Weinberg (HWE) equilibrium. The most frequent HLA-DQA1 alleles among Dubaian Arabs are DQA1 4 and 1.2. Among Pakistanis, the most frequent allele is also DQA1 4. No significant deviations from HWE were detected.     

Highly sensitive HLA-DNA typing from formalin-fixed and paraffin-embedded tissue samples

Nucleotide sequences have been determined for more than 1700 different alleles at the core of the human leukocyte antigen (HLA) system. The highly polymorphic character of these genes affects adaptive immune response and is also useful for forensic applications. HLA typing from formalin-fixed and paraffin-embedded tissue provides abundant useful information for both clinical settings and forensic investigations. This study, which investigated the potential use of DNA from formalin-fixed and paraffin-embedded tissue samples in an HLA PCR sequence-specific primer and probe (SPP) system, showed that tissue fixed in formalin for less than 3 days and embedded in paraffin can serve as a useful source of DNA for PCR-SPP typing kits.     

Identifying the Source of Mystery Waterborne Oil Spills—A Case for Quantitative Chemical Fingerprinting

Oil spills of unknown origin, so-called “mystery” spills, occur routinely in rivers, open water, and navigable coastal waterways. The natural resources damage (NRD) liability associated with even a small volume of oil released into the environment warrants that a thorough chemical characterization of the spilled oil be conducted by agencies and potentially responsible parties (PRPs). Chemical fingerprinting methods have played an important role in the identification of mystery oil spills. These methods fall into two categories, viz., qualitative and quantitative. The qualitative approach relies upon visual comparison of various chromatographic fingerprints obtained by GC/FID and GC/MS analysis of spill and candidate source oils and are represented ASTM methods. The quantitative approach relies upon measurements of the concentrations (relative or absolute) of dozens of diagnostic chemicals, typically PAHs and biomarkers, and a subsequent statistical or numerical analysis of various diagnostic parameters calculated from these concentrations. The quantitative approach is represented by the revised Nordtest methodology. The quantitative approach is preferable for most oil spill investigations since the means of interpretation are objective, whereas the ASTM methods are subjective. Quantitative fingerprinting data are particularly important when the mystery spill and source oils are qualitatively similar and are required when mystery spills may include mixed sources or prespill oil signatures.     

DIP-STR在不平衡混合DNA样本中的分析研究

当前,DNA检验技术作为打击犯罪的利器,在法医鉴定中发挥着巨大作用。但对于性侵、暴力犯罪等案件中提取的混合DNA样本,尤其是从受害人或嫌疑人的接触物上采集的高度不平衡混合DNA样本,利用常染色体STR检验方法得到的结果通常不是很理想。由于PCR扩增偏倚,从混合样本中检测出痕量DNA分型是一个巨大的挑战,也是当前法医DNA检验的一个难点。近年来的研究显示,利用新型连锁遗传标记DIP-STR,即结合缺失或插入多态性片段DIP(deletion–insertion polymorphisms)和STR的连锁位点,可以用来检测出混合DNA样本中任一性别和细胞起源的微量DNA,甚至在DNA混合比例高达1:1000时,DIP-STR标记的灵敏度、特异性仍旧相对较为理想。因此,DIP-STR标记的分析可以作为常染色体STR检验的有效补充。本文将对DIP-STR在不平衡混合DNA样本分析中的研究背景、方法及其应用前景进行综述。     

Identification in blood stains through DNA typing with C4 and HLA-DR probes

A restriction fragment length polymorphism analysis using double digestion of DNA preparations with XbaI and BglII restriction enzymes and hybridization with C4 and HLA-DR probes is described. The typing conditions selected reveal extensive individual variation in both C4 and DR gene regions. In our panel of 46 unrelated individuals, 37 different phenotypic patterns were recognized when both probes were used, and preliminary discriminative power values of 0.865 and 0.914 were calculated for C4 and DR beta, respectively. The probability of a chance match using both systems is probably about 1.5.10(-2). The potential of this method for individual identification of blood stains was demonstrated on DNA prepared from 6-month-old dried blood stains from seven panel individuals. The seven individuals were all identified when comparing stain DNA patterns with panel control patterns. No RFLP pattern changes were observed following storage of blood stains. Based on these experiments with C4 and DR beta DNA typing under laboratory conditions, it is concluded that DNA typing with such probes may become a powerful tool in future stain identification analyses.     

Use of DNA polymorphism in forensic paternity evaluation

Problematical paternity cases were additionally subjected to DNA-polymorphism analysis. 5 cases are reported, focusing on problems due to, 'silent' allele transmittance, relatively low probability for paternity, obvious occurrence of the extremely rare Rh gene complex CWc, involvement of brothers as putative fathers, non-paternity of a man although his W-value was 99.975%. The aim of this paper is to demonstrate the need for DNA-polymorphism analysis, if conventional blood group tests do not lead to a clear-cut decision. Extended typing of conventional blood group polymorphisms (except HLA) cannot compete with highly polymorphic DNA loci.     

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.