激光显微捕获技术用于尿液脱落细胞DNA检测

目的采用激光显微捕获技术（LCM）捕获尿液脱落细胞,并进行STR分型。方法收集10份健康成人尿液样本,根据储存时间分组,其中新鲜尿液组（≤24h）分别采用Chelex-100及LCM联合DNA IQTM提取法提取DNA,储存尿液组（〉24h）再分为4℃组和室温组,分别在4～30d内不同时间点采用LCM联合DNA IQTM提取法提取DNA;各组提取的模板DNA进行扩增及SRT分型检验。结果新鲜尿液组采用LCM联合DNA IQTM提取法提取DNA,所有样本均可检出全部基因座（16个）,采用Chelex-100法则在部分基因座上出现等位基因丢失、非特异性扩增、峰值低等现象;4℃储存10d和室温储存4d以内的尿液经检验可明确判读12个以上基因座,4℃20～30d及室温7d,可检出7个以上基因座。结论 LCM技术可用于尿液检材的DNA分型检验,且检材应尽可能4℃保存并尽快检验。     

激光捕获显微分离技术及其法医学研究进展

激光捕获显微分离技术(laser capture m icrod issection,LCM)是一项显微捕获分离单个细胞和多个细胞的自动化新技术,本文综述了其在法医学领域的研究新进展。经过研究和实践,它将在法医学实践中解决混合和微量物证的DNA检验难题中发挥重要作用。     

FISH联用激光捕获显微分离技术对混合斑进行分离检验

目的建立荧光原位杂交(FISH)联用激光捕获显微分离技术(LCM)精确分离男女混合斑中精子的检验方法。方法收集健康志愿者精子和女性阴道上皮细胞制备模拟混合斑,经过预处理后用Vysis CEPX SpectrumOrangeTMY SpectrumGreenTM试剂盒进行荧光原位杂交,并用PALM激光捕获显微分离系统分离男、女性细胞,使用Identifiler试剂盒结合低体积扩增技术分别对男女成分进行STR扩增。结果荧光原位杂交后,可清晰分辨混合斑中的男女细胞。捕获20个精子细胞可以得到完整的STR分型,检出率为80%。随着精子数目增多,检出率提高而等位基因丢失率降低。30个精子检出率最高,为95%。结论激光捕获显微分离联用FISH技术可用于混合斑中男女细胞DNA的分离检验。     

Exclusion of a man charged with murder by DNA fingerprinting

DNA fingerprinting was used to demonstrate that two murder-rapes committed in 1983 and 1986, respectively, were connected. The probability of chance association of the fingerprint was calculated as 5.8 x 10(-8). The man who had been charged with the murder was excluded because his DNA fingerprint did not match sperm DNA fingerprints obtained from swabs and clothing attributed to the two victims.     

Development of a one-tube extraction and amplification method for DNA analysis of sperm and epithelial cells recovered from forensic samples by laser microdissection

Laser microdissection can be used in forensic casework to isolate specific cell types from mixtures of biological samples. Extraction of DNA from selected cells is still required prior to STR amplification. Because of the relatively pristine nature of the recovered cells, laser microdissection is more sensitive than more traditional methods of DNA analysis, theoretically resulting in DNA profiles from less cellular material. A one-tube extraction and amplification method minimises loss of DNA through liquid transfers and reduces the potential for contamination events occurring. In this paper, the development of a one-tube method for the effective extraction of DNA from laser microdissected sperm and epithelial cells is described. The performance of the in-house method was compared to that of a commercial DNA extraction kit for extraction of DNA from sperm and the downstream compatibility with STR amplification was determined for both sperm and epithelial samples. Full Identifiler™ profiles after 28 amplification cycles were obtained from as few as 15 epithelial cells and 30 sperm.     

Trace element fingerprinting of Australian ocher using laser ablation inductively coupled plasma-mass spectrometry (LA-ICP-MS) for the provenance establishment and authentication of indigenous art

The expansion of indigenous art and the interest it has generated both at a domestic and international level means large monetary transactions are taking place between art galleries or centers and purchasers. As such, an accurate and conclusive method for provenance determination of traditional indigenous artistic materials must be established that can, if necessary, be used to assist in authentication of artworks. Laser ablation inductively coupled plasma mass spectrometry was utilized for elemental differentiation and provenance establishment of ocher samples. This research was used to develop a robust scientific protocol which facilitates definitive and accurate determination of provenance of Australian ochers and the artworks created using them. Analysis of the results obtained through this study show that the trace metal distribution patterns alone appear to be sufficient evidence to establish provenance of specific ochers, although additional differentiation between ocher samples, using major element distribution patterns, was achieved through the utilization of X-ray analytical techniques.     

Oligonucleotide fingerprinting using (GTG)5 and (GACA)4 probes for the differentiation of body fragments

For the detection of postmortem stability of DNA and for the identification of parts of dead bodies of unknown origin the oligonucleotide probes (GTG)5 and (GACA)4 can be used. (GTG)5 is a highly discriminating probe which allows to differentiate in the 4 to 25 kilobase range of DNA fragments. DNA fingerprints obtained by (GACA)4 show useful results especially in the short fragment range. The (GACA)4 probe can therefore be used to analyze partially degraded DNA.     

Sex identification in fresh blood and dried bloodstains by a nonisotopic deoxyribonucleic acid (DNA) analyzing technique

Deoxyribonucleic acid (DNA) specimens were prepared from blood or bloodstain extracts, and the content of a Y-chromosome specific DNA fragment was investigated by the Southern hybridization method using a nonisotopic staining technique. Thus obtained patterns of male DNA showed a clear band, whereas broad stains with some faint bands appeared on the patterns of DNA from both sexes. This method is expected to be a new powerful mean of forensic medical examination.     

Identification of decomposed human remains by deoxyribonucleic acid (DNA) profiling

After routine methods failed to establish positive identification of a decomposed homicide victim, deoxyribonucleic acid (DNA) typing techniques using blood from the victim and putative parents of the victim were used. This is the first report in the literature of a case using DNA fingerprinting in a "parentage" context to establish identity of unidentified, decomposed human remains.     

The transfer of human DNA by Lucilia cuprina (Meigen) (Diptera: Calliphoridae)

Blowflies leave deposits, termed artefacts, through the processes of excretion and regurgitation. To date, little consideration has been given to the possibility of adult blowflies consuming biological material and subsequently acting as vectors of human DNA through these artefacts. In this study, Lucilia cuprina (Meigen) (Diptera: Calliphoridae) were fed either human blood or human semen ad libitum and their artefacts were analysed for human DNA content. Samples containing 1, 10, 30 and 50 artefacts were tested. Quantifiable and typeable levels of human DNA were found in samples derived from both food sources, and even in samples containing a single artefact. Semen-derived artefacts contained significantly more human DNA than artefacts produced after a blood meal. Consequently a smaller number of artefacts was required to collect sufficient DNA for genotyping. These findings are forensically important as it provides investigators with another potential source of DNA at a crime scene where a body has been moved, or an attempt has been made to clean up biological material. They also highlight how fly artefacts could potentially contaminate and compromise evidence.     

Elemental analysis of glass by laser ablation inductively coupled plasma optical emission spectrometry (LA-ICP-OES)

The elemental analysis of glass evidence has been established as a powerful discrimination tool for forensic analysts. Laser ablation inductively coupled plasma optical emission spectrometry (LA-ICP-OES) has been compared to laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and energy dispersive micro X-ray fluorescence spectroscopy (μXRF/EDS) as competing instrumentation for the elemental analysis of glass. The development of a method for the forensic analysis of glass coupling laser ablation to ICP-OES is presented for the first time. LA-ICP-OES has demonstrated comparable analytical performance to LA-ICP-MS based on the use of the element menu, Al (Al I 396.15 nm), Ba (Ba II 455.40 nm), Ca (Ca II 315.88 nm), Fe (Fe II 238.20 nm), Li (Li I 670.78 nm), Mg (Mg I 285.21 nm), Sr (Sr II 407.77 nm), Ti (Ti II 368.51 nm), and Zr (Zr II 343.82 nm). The relevant figures of merit, such as precision, accuracy and sensitivity, are presented and compared to LA-ICP-MS. A set of 41 glass samples was used to assess the discrimination power of the LA-ICP-OES method in comparison to other elemental analysis techniques. This sample set consisted of several vehicle glass samples that originated from the same source (inside and outside windshield panes) and several glass samples that originated from different vehicles. Different match criteria were used and compared to determine the potential for Type I and Type II errors. It was determined that broader match criteria is more applicable to the forensic comparison of glass analysis because it can reduce the affect that micro-heterogeneity inherent in the glass fragments and a less than ideal sampling strategy can have on the interpretation of the results. Based on the test set reported here, a plus or minus four standard deviation (± 4s) match criterion yielded the lowest possibility of Type I and Type II errors. The developed LA-ICP-OES method has been shown to perform similarly to LA-ICP-MS in the discrimination among different sources of glass while offering the advantages of a lower cost of acquisition and operation of analytical instrumentation making ICP-OES a possible alternative elemental analysis method for the forensic laboratory.     

Demonstration of incompatible mother-child pairs in PGM1 and Duffy systems in a three-generation family from the upper Silesia (Poland)

A silent allele PGM1(0) in three generations of a Polish family was indicated by an abnormal segregation pattern and by reduced enzyme activity on electrophoresis and densitometric assay. There was also found in this family the silent allele Fy0 in the Duffy system (the incompatibility in the mother-child pair).     

Analysis of artificially degraded DNA using STRs and SNPs--results of a collaborative European (EDNAP) exercise

Recently, there has been much debate about what kinds of genetic markers should be implemented as new core loci that constitute national DNA databases. The choices lie between conventional STRs, ranging in size from 100 to 450 bp; mini-STRs, with amplicon sizes less than 200 bp; and single nucleotide polymorphisms (SNPs). There is general agreement by the European DNA Profiling Group (EDNAP) and the European Network of Forensic Science Institutes (ENFSI) that the reason to implement new markers is to increase the chance of amplifying highly degraded DNA rather than to increase the discriminating power of the current techniques. A collaborative study between nine European and US laboratories was organised under the auspices of EDNAP. Each laboratory was supplied with a SNP multiplex kit (Foren-SNPs) provided by the Forensic Science Service, two mini-STR kits provided by the National Institute of Standards and Technology (NIST) and a set of degraded DNA stains (blood and saliva). Laboratories tested all three multiplex kits, along with their own existing DNA profiling technique, on the same sets of degraded samples. Results were collated and analysed and, in general, mini-STR systems were shown to be the most effective. Accordingly, the EDNAP and ENFSI working groups have recommended that existing STR loci are reengineered to provide smaller amplicons, and the adoption of three new European core loci has been agreed.     

Lewis typing of human bloodstains by enzyme-linked immunosorbent assay (ELISA) using monoclonal anti-Le(a) and anti-Le(b)

The Lewis blood grouping of human dried bloodstains could be determined by an enzyme-linked immunosorbent assay (ELISA) using monoclonal anti-Le(a) and anti-Le(b) antibodies with an avidin-biotin complex (ABC). The bloodstains aged 1 year were used as samples, and approximately 1 mg of the stains was enough to type each Lewis antigen reliably by this method. The Lewis substances of 106 individual stains were correctly typed regardless of their ABO blood group system.     

Development of a deoxyribonucleic acid (DNA) restriction fragment length polymorphism (RFLP) database for Punjabis in East Punjab,India

In response to continuing interest in obtaining reference deoxyribonucleic acid (DNA) analysis data for previously unstudied population groups, blood samples were collected from Punjabi individuals living in East Punjab, India. This first segment of our research is focused on restriction fragment length polymorphism (RFLP) analysis, with future segments anticipated for various polymerase chain reaction (PCR) based techniques. In this study, the samples were subjected to RFLP analysis using HaeIII, followed by hybridization with variable number tandem repeat (VNTR) probes for loci D2S44, D1S7, D10S28, D4S139, D17S79 and D5S110. The band sizes of the resulting patterns were estimated using an FBI imaging system. The resulting data were subjected to statistical analysis for conformity with Hardy-Weinberg expectations, first for the total population of Punjabis, and additionally for the subgroups of Sikhs and Hindus. The loci are highly polymorphic in all sample populations studied. Except for D5S110, there is no evidence for departure from Hardy-Weinberg equilibrium (HWE) for the VNTR loci in the population groups. In addition, there is little evidence of correlation between the alleles at any of the pairs of loci and no evidence of association across the six loci. Finally, the data suggest that a multiple locus VNTR profile would be rare in the Punjabi or either of its subgroups.     

Identification of male bloodstains by dot hybridization of human Y chromosome-specific deoxyribonucleic acid (DNA) probe

The sex determination of bloodstains was performed using a human Y chromosome-specific (DNA) fragment of 1.9-kb length as a hybridization probe. The DNA samples were taken from 1- and 4-week-old bloodstains of males and females, respectively. Strong signals with male DNA were observed by Y-probe, while faint signals with female DNA were detected. In addition, clear signals were observed in the extract samples from male bloodstains (16-week-old) on paper. Dot hybridization of the Y-probe would be widely applicable to studies on sex determination of medicolegal materials such as blood, bloodstains, teeth, and cadaverous parts.     

Analysis of rosin and modified rosin esters in adhesives by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)

The optimum method for analyzing glyceryl rosinate by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was studied. The performances of dithranol, anthracene, and 2,5-dihydroxybenzoic acid, pyrene, trans,trans-1,4-diphenyl-1,3-butadiene, 9-nitroanthracene, indole-3-acrylic acid and retinoic acid as a matrix agent, and those of sodium trifluoroacetate (NaTFA) and silver trifluoroacetate (AgTFA) as a cationization agent were measured. Dithranol showed higher signal to noise ratio and lower base-line, and AgTFA showed better performance than NaTFA. In this study, however, NaTFA was chosen since the ester peaks with high signal to noise ratios were observed, and the spectra of sodium-cationized molecules are simpler than those of silver-cationized molecules. Rosin esters and modified rosin esters in 22 rubber-based pressure sensitive adhesives and a paper-cement were analyzed. Glyceryl rosinate, glyceryl disproportionated rosinate, pentaerythrityl rosinate and pentaerythrityl hydrogenated rosinate were easily detected despite the fact that rubber-based adhesives are complex mixtures of elastomers, tackifiers, antioxidants and other additives. The detection limit of this method was studied and 2% of glyceryl rosinate in a rubber-based pressure sensitive adhesive was detected. It has been proved that MALDI-TOF-MS is a useful analytical method to analyze rosin and modified rosin esters in adhesives.     

Lewis typing of human saliva stains by enzyme-linked immunosorbent assay (ELISA) using monoclonal anti-Le(a) and anti-Le(b) antibodies

The Lewis blood grouping of human saliva stains could be detected by an enzyme-linked immunosorbent assay (ELISA) using anti-Le(a) and anti-Le(b) monoclonal antibodies with an avidin-biotin complex (ABC). The saliva stains (1.0 by 1.0 cm in size) were used as samples and not only could the Lewis substances of 57 individual stains be correctly typed by this method, but also it was clarified that there are several different secretion patterns of amounts of Le(a) and Le(b) substances in 3 individual Lewis types.