Sex determination of blood stains with a recombinant DNA probe: comparison with radioactive and non-radioactive labeling methods

A recombinant DNA probe hybridizing specifically to human repeat DNA sequence (pHY10) of which about 3000 copies are present on the Y chromosome was used for sex determination of degraded DNA samples of blood stains. Human blood stains of male and female origin were readily differentiated with the pHY10 DNA probe. This radioactive technique enabled reliable and sensitive sex determination from blood or dried blood stains greater than 20 years old. Less than 1 microliter of blood or 1 piece of 0.5 cm length thread of blood stain from cotton fabric was sufficient for the test using dot blot hybridization. Compared with the radioactive labeling method, the photobiotin labeling method showed one thirtieth to one fiftieth lower sensitivity and presented some problems which are expected to be resolvable.     

Human identification and sex determination of dental pulp, bone marrow and blood stains with a recombinant DNA probe

Recombinant DNA hybridizing specifically to a 300 nucleotide repeat DNA sequence (BLUR8) of human specificity and to human repeat DNA sequence (pHY10) on the Y chromosome was used for human identification and sex determination of degraded DNA samples of blood stains, dental pulp, and bone marrow. This radioactive technique enabled reliable and sensitive human and sex determination from blood stains that were more than 80 years old. Less than 1 piece of 0.5 cm length thread of blood stain was enough for both tests. DNA from relatively fresh dental pulp and bone marrow was clearly identified. The human identification test, which could recognize up to 0.3 ng DNA correctly, was 3 to 5 times more sensitive than the sex determination test.     

RNA/DNA co-analysis from blood stains—Results of a second collaborative EDNAP exercise

A second collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Six human blood stains, two blood dilution series (5-0.001 μl blood) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by the participating laboratories using a RNA/DNA co-extraction or solely RNA extraction method. Two novel mRNA multiplexes were used for the identification of blood: a highly sensitive duplex (HBA, HBB) and a moderately sensitive pentaplex (ALAS2, CD3G, ANK1, SPTB and PBGD). The laboratories used different chemistries and instrumentation. All of the 18 participating laboratories were able to successfully isolate and detect mRNA in dried blood stains. Thirteen laboratories simultaneously extracted RNA and DNA from individual stains and were able to utilize mRNA profiling to confirm the presence of blood and to obtain autosomal STR profiles from the blood stain donors. The positive identification of blood and good quality DNA profiles were also obtained from old and compromised casework samples. The method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise involving a RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of blood in forensic casework that is compatible with current DNA analysis methodology.     

The analysis of biological samples from crime scene for a future human DNA profile confrontation. Effects of presumptive test reagents on the ability to obtain STR profiles for human identification

Because of the increase of evidence of blood stains, that have been washed or cleaned in an attempt to mask the analysis of DNA profiles, there is also an increase in the use of presumptive tests on samples sent to laboratories. Some of the presumptive tests, used to identify blood and semen stains, could potentially affect the recovery of high molecular weight DNA from the samples, or extinguish them, especially those already present in small quantities. After the presumptive tests, often these samples are discarded. This study aimed to examine the possibility of obtaining a DNA profile from samples submitted for presumptive testing and cleaned with bleaches with and without chlorine. Two different protocols were conducted: (a) A unique sample of human blood in natura (5 μL), already typed through the DNA techniques with the genetic profile previously known (control), was distributed onto cotton fabrics and dried at room temperature. Four samples of fabric were macerated in saline solution and Coombs serum and then stored for three months (room temperature and freezer −20 °C). (b) Another sample of human blood, type A, in natura, already typed through the techniques of DNA (control) was used. Aliquots of 200 μL were distributed in: cotton, denim and synthetic fabric. The samples were dried at room temperature for 24 h. The blood stains in those fabrics (cotton, denim and synthetic) were then divided into three groups: unwashed, cleaned with chlorine bleach and cleaned with chlorine bleach and soap powder. The samples were again dried at room temperature for 24 h, before the use of luminol. The DNA were extracted with Chelex 100 and amplified with the Identifiler Kit (Applied Biosystems). The blood stains exposed to saline and Coombs serum had DNA profiles consistent with untreated samples (controls). This result shows that the experts should keep and store the samples treated with saline and Coombs serum for future DNA confrontation when necessary. Also discussed in this paper the pattern of blood stains after washing with bleaching solutions, as well as the quantity of DNA obtained from these samples.     

DNA指纹图在法医学鉴定中应用的研究(Ⅲ)——应用“Myo”DNA探针进行混合斑中精液的个体认定

本文利用蛋白酶K、SDS对精液和阴道液、精液和血液的混合斑进行前处理,除去女性阴道脱落上皮细胞和血液细胞成份获得精子。提取精子DNA,用“Myo”小卫星DNA探针杂交进行DNA指纹检验,获得了高度多态性的精子DNA指纹图谱,与同一个体血液DNA指纹图谱比较完全一致,实现了混合斑中精液来源的个体认定。在对20多起强奸案例混合斑的实际应用中,成功地认定了强奸罪犯。     

Evaluation of a Visualization Assay for Blood on Forensic Evidence

In forensics, bloodstains on dark fabrics might be invisible for the naked eye. Although several visualization, presumptive, and confirmatory blood tests have been developed, all have one or more disadvantages, especially on DNA analysis. We report here the use of a visualization assay that can visually detect blood drops up to 1/20 dilution. In this assay, the fabric is placed between two wet filter papers and covered by glass surfaces on both sides. Pressure is applied on the glass surfaces in which bloodstains transfer onto the filter papers through capillary forces. Detected stains can be tested with other more sensitive presumptive blood tests performed on the filter paper. Even more, DNA analysis can be performed on the transferred bloodstains. The presented visualization assay is easy to perform, extremely cheap, requires little hands on time, and does not affect bloodstain pattern analysis.     

Developmental Validation of the PrepFiler™ Forensic DNA Extraction Kit for Extraction of Genomic DNA from Biological Samples*

Abstract: The PrepFiler? Forensic DNA Extraction Kit enables isolation of genomic DNA from a variety of biological samples. The kit facilitates reversible binding of DNA with magnetic particles resulting in high DNA recovery from samples with very low and high quantities of biological materials: 0.1 and 40 μL of human blood (donor 2) provided 14 and 2883 ng of DNA, respectively. Following the revised SWGDAM guidelines, performance of the developed method was investigated using different sample types including saliva on swabs, semen stains on cotton fabric, samples exposed to environment, samples with polymerase chain reaction (PCR) inhibitors, blood stains (on denim, cotton cloth, and FTA^® paper), and touch evidence‐type samples. DNA yields for all samples tested were equal or better than those obtained by both phenol–chloroform extraction and commercial kits tested. DNA obtained from these samples was free of detectable PCR inhibitors. Short tandem repeat profiles were complete, conclusive, and devoid of PCR artifacts.     

The application of visible wavelength reflectance hyperspectral imaging for the detection and identification of blood stains

Current methods of detection and identification of blood stains rely largely on visual examination followed by presumptive tests such as Kastle–Meyer, Leuco-malachite green or luminol. Although these tests are useful, they can produce false positives and can also have a negative impact on subsequent DNA tests. A novel application of visible wavelength reflectance hyperspectral imaging has been used for the detection and positive identification of blood stains in a non contact and non destructive manner on a range of coloured substrates. The identification of blood staining was based on the unique visible absorption spectrum of haemoglobin between 400 and 500 nm. Images illustrating successful discrimination of blood stains from nine red substances are included. It has also been possible to distinguish between blood and approximately 40 other reddish stains. The technique was also successfully used to detect latent blood stains deposited on white filter paper at dilutions of up to 1 in 512 folds and on red tissue at dilutions of up to 1 in 32 folds. Finally, in a blind trial, the method successfully detected and identified a total of 9 blood stains on a red T-shirt.     

用引物Y_3,Y_4和DNA扩增技术作血液(痕)和毛根性别鉴定的研究

作者用引物Y_3、Y_4和DNA聚合酶链式反应(PCR)作微量人类血液(痕)和毛根的性别鉴定。扩增的靶序列位于Y染色体DNA特异3.4kb重复序列中,扩增产物为460bp。检材用量为:新鲜血液0.5μl、血痕纱纤维1mm、毛根单个。20例保存4个月的血痕与2例保存6年半的血痕性别判定结果均正确,无性别记载的保存9～11年的3例血痕显现了清晰的460bpY特异DNA扩增带。15例保存20天的自然脱落毛根性别判定结果均正确。本法省略了检材处理中的酚-氯仿抽提DNA等纯化步骤,既简化了实验操作,又减少了检验过程中外源DNA的污染机会和样品DNA的损耗,使这一性别鉴定方法更符合法医学实践的需要。     

PCR扩增ZFY/ZFX基因鉴定人类干血痕性别

应用 PCR 技术同时扩增人 ZFY 和 ZFX 基因特异的 DNA 序列,在男性血痕中可检测到两种扩增产物,即340bp 长的 ZFY 基因及488bp 长的 ZFX 基因特异 DNA 片段;在女性血痕中仅可检测到488bp 长的 ZFX 基因特异 DNA 片段,据此判定干血痕性别。干血痕的最小检出需要量为0.125μl 血液量的血痕。室温保存10年的血痕可以准确判定性别。ZFY 基因位于 Y 染色体短臂。本方法同时检测两条性染色体,可以避免由于扩增失败或 Y 染色体长臂变异出现的假阴性或假阳性。扩增产物经琼脂糖凝胶电泳即可区分。     

Comparison of DNA typing using AmpFlSTR Yfiler and PowerPlex Y System,for specimens subject to very long storage

We performed DNA typing from blood stains stored at room temperature for 22–30 years, using AmpFlSTR Yfiler PCR amplification Kit and PowerPlex Y System to extract and amplify the DNA and performing electrophoresis with an ABI 310 Genetic Analyzer. We identified alleles using GeneMapper ID v3.2.1 software. We found that long-term storage tended to reduce the number of detectable loci in the blood stains and that it was easier to detect the loci of smaller amplicons than larger amplicons.     

Human blood stain identification and sex determination in dried blood stains using recombinant DNA techniques

DNA was extracted from human and non-primate dried blood stains. Human male and female specimens were readily distinguished by analysis with a Y-chromosome specific DNA probe. Human and non-primate blood stains were also readily differentiated using a repeat sequence (Alu) DNA probe. The potential power of recombinant DNA analysis in forensic science is discussed.     

Evaluation of four deoxyribonucleic acid (DNA) extraction protocols for DNA yield and variation in restriction fragment length polymorphism (RFLP) sizes under varying gel conditions.

This study originated from discussions and recommendations of the Technical Working Group on DNA Analysis Methods (TWGDAM). Four bloodstain deoxyribonucleic acid (DNA) extraction protocols and five semen stain DNA extraction protocols were evaluated. Nine laboratories participated in the extraction of DNA from 20 bloodstains and 20 semen stains using each protocol. All blood and semen stains originated from a single donor and were prepared under uniform conditions to permit the direct comparison of DNA yields and restriction fragment lengths. The extracted DNA from approximately 600 bloodstains and 700 semen stains was quantified by yield gel analysis and a slot blot hybridization technique. The extracted DNA was digested and restriction fragment length polymorphism (RFLP) patterns were generated using three single-locus probes. The RFLP sizing data produced from the blood and semen stains were evaluated with respect to (1) DNA extraction method, (2) gel length, (3) agarose type, (4) presence or absence of ethidium bromide in the gel, and (5) fragment sizes obtained from DNA isolated directly from the donor's liquid blood. This study demonstrates conclusively that high-molecular-weight DNA can be isolated using either organic or nonorganic DNA extraction protocols and that the resulting RFLP sizes are highly reproducible regardless of gel length, agarose type, or presence/absence of ethidium bromide.     

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.     

Beware of the possibility of fingerprinting techniques transferring DNA

Fingerprinting brushes have the potential to collect and transfer DNA during powdering. Squirrel-hair fingerprint brushes exposed to specific sets of saliva stains and brushes used in routine casework were tested for their ability to collect and transfer DNA containing material using standard DNA extraction procedures and AmpFlSTR Profiler Plus amplification and typing procedures. The tests found that the risk of transferring DNA during powdering and having a detrimental impact on the analysis increases if the examiner powders over either biological stains (such as blood or saliva) or very fresh prints and uses more sensitive PCR amplification and typing procedures. We advocate caution when powdering prints from which DNA may also be collected and provide options for consideration to limit the risk of transferred DNA contamination while fingerprinting.     

Successful DNA typing of urine stains using a DNA purification kit following dialfiltration

To evaluate the utility of DNA polymorphism typing of urine stains in forensic investigations, the amplifiable amount of DNA was estimated in 20 urine specimens obtained from 10 male and 10 female volunteers using a DNA purification kit following dialfiltration. DNA obtained from both urine and urine stains was amplified with the AmpflSTR Profiler PCR Amplification Kit, and was analyzed by capillary electrophoresis using the Genetic Analyzer. The amount of male and female urine necessary for obtaining a complete DNA profile was 0.2 mL and 0.08 mL, respectively. When 0.2 mL of male urine were used to create urine stains, complete DNA profiles could be obtained from just some of the stains. However, when only 0.1 mL of female urine was used, complete profiles could be successfully obtained from all of the stains. DNA on bleached cotton remained amplifiable for 3-6 weeks. This method using a DNA purification kit following dialfiltration can be recommended for the genotyping of urine stains.     

体外DNA扩增技术鉴定血痕性别三例报告

本文报道用体外DNA扩增技术对3例刑事案中的人血痕标本进行性别鉴定。其方法是从血痕标本中微量抽提DNA,用蛋白酶K进行消化。然后用两组引物Y1.1与Y1.2和Alu9.1与Alu9.2及国产FD耐热DNA聚合酶进行聚合酶链反(PCR)扩增DNA,电泳分析Y及Alu重复序列,从而判断血痕的性别。     

DNA指纹图在法医学鉴定中应用的研究(Ⅱ)——应用‘Myo’小卫星探针进行血斑、精斑、个体组织的个体认定

应用‘Myo’小卫星 DNA 探针,Southern 印迹杂交技术,对血斑、精斑、同一个体不同组织进行 DNA 指纹图分析,均获得清晰的图谱。同一个体的血斑与血液、精斑与精液以及不同的组织其 DNA 指纹图谱完全相同。可以根据斑痕或组织与嫌疑个体的血液或某一组织 DNA 的指纹图谱比对以做出同一认定。50μl 血液量的血斑、5μl 精液量的精斑可以获得清晰易辨的指纹图谱。五年的精斑、两年的血斑亦可做出与同源个体新鲜精液、血液完全一致的 DNA 指纹图谱。对杀人、强奸杀人、碎尸等不同案件的血痕、精斑、不同组织碎块进行了 DNA 指纹图检验,均做出了正确的个体认定。本方法的应用为我国法医物证检验提供了新的分析手段,使个体认定得以实现。     

ABO genotyping by polymerase chain reaction.

ABO blood group system's genotyping by polymerase chain reaction in genomic DNA level is developed. The positions of nucleotide 258 and 700 of cDNA from A transferase were used to distinguish A, B, and O alleles by restriction enzyme digestion. To identify the 258th nucleotide, a 199- or 200-bp DNA fragment was amplified by PCR and digested with Kpn I. For the 700th nucleotide, a 128-bp PCR amplified fragment was designed and digested with Alu I. By examining the DNA fragment digested patterns, ABO genotypes were easily determined. Results obtained using this method on 20 ABO-known peripheral blood samples showed that this new technique could provide accurate ABO genotype. Biologic forensic samples, such as, blood stains, saliva stains, semen stains, hair, bone tissue, and semen contaminated with vaginal secretion were also successfully typed. This rapid, sensitive and reliable method should be applicable not only in forensic identification but also in medical examination.