Microchip-based cell lysis and DNA extraction from sperm cells for application to forensic analysis

The current backlog of casework is among the most significant challenges facing crime laboratories at this time. While the development of next-generation microchip-based technology for expedited forensic casework analysis offers one solution to this problem, this will require the adaptation of manual, large-volume, benchtop chemistry to small volume microfluidic devices. Analysis of evidentiary materials from rape kits where semen or sperm cells are commonly found represents a unique set of challenges for on-chip cell lysis and DNA extraction that must be addressed for successful application. The work presented here details the development of a microdevice capable of DNA extraction directly from sperm cells for application to the analysis of sexual assault evidence. A variety of chemical lysing agents are assessed for inclusion in the extraction protocol and a method for DNA purification from sperm cells is described. Suitability of the extracted DNA for short tandem repeat (STR) analysis is assessed and genetic profiles shown. Finally, on-chip cell lysis methods are evaluated, with results from fluorescence visualization of cell rupture and DNA extraction from an integrated cell lysis and purification with subsequent STR amplification presented. A method for on-chip cell lysis and DNA purification is described, with considerations toward inclusion in an integrated microdevice capable of both differential cell sorting and DNA extraction. The results of this work demonstrate the feasibility of incorporating microchip-based cell lysis and DNA extraction into forensic casework analysis.     

国产磁珠Wawasye试剂盒在法医DNA中应用

目的探讨国产磁珠Wawasye试剂盒在法医DNA检案中的应用效果。方法利用475份各类常见生物检材测试其效果,并与M48磁珠试剂盒作比较。结果国产磁珠Wawasye试剂盒与M48磁珠试剂盒在检验各类常见生物检材的结果无显著性差异。结论国产磁珠Wawasye试剂盒适用于公安机关DNA实验室。     

Automated DNA extraction using the QIAsymphony platform: Estimation of DNA recovery from simulated forensic stains

The aim of this study was to evaluate the forensic protocol recently developed by Qiagen for the QIAsymphony automated DNA extraction platform. Samples containing low amounts of DNA were specifically considered, since they represent the majority of samples processed in our laboratory. The analysis of simulated blood and saliva traces showed that the highest DNA yields were obtained with the maximal elution volume available for the forensic protocol, that is 200 μl. Resulting DNA extracts were too diluted for successful DNA profiling and required a concentration. This additional step is time consuming and potentially increases inversion and contamination risks. The 200 μl DNA extracts were concentrated to 25 μl, and the DNA recovery estimated with real-time PCR as well as with the percentage of SGM Plus alleles detected. Results using our manual protocol, based on the QIAamp DNA mini kit, and the automated protocol were comparable. Further tests will be conducted to determine more precisely DNA recovery, contamination risk and PCR inhibitors removal, once a definitive procedure, allowing the concentration of DNA extracts from low yield samples, will be available for the QIAsymphony.     

DNase-I纯化结合碱性裂解法提取混合斑精子DNA

目的研究脱氧核糖核酸酶I(DNase-I)纯化结合碱性裂解法提取混合斑精子DNA的方法在法医学中的应用。方法收集79份性犯罪案件混合斑检材,分别用DNase-I纯化结合碱性裂解法和差异裂解法提取精子DNA,采用STR荧光标记复合扩增体系进行16个STR基因座分型,并比较检验结果。结果应用DNase-I纯化结合碱性裂解法提取精子DNA,64例检材分型成功;应用差异裂解法提取精子DNA,57例检材分型成功;两种方法比较结果存在显著性差异(P=0.039),DNase-1纯化结合碱性裂解法提取精子DNA的STR分型成功率更高,成本低廉。结论DNase-I纯化结合碱性裂解法提取混合斑精子DNA可提高检验成功率,操作简便,快速,易于自动化,适于法医学个体识别鉴定。     

DNA提取方法和PCR循环次数对STR扩增成功率的影响

目的探讨常见载体上的微量血痕DNA的提取方法、PCR循环次数对STR扩增成功率的影响。方法分别应用Chelex-100法及Chelex-100结合纯化法对8种载体上不同大小的血痕样本进行DNA提取,并采用28次、30次及34次PCR循环进行STR扩增,分别观察其扩增成功率。结果经28次、30次及34次PCR循环,Chelex-100法提取DNA后的STR扩增成功率分别为0.2917,0.3333,0.4583,Chelex-100结合纯化法的STR扩增成功率分别为0.3750,0.4583,0.8750。结论用Chelex-100结合纯化法提取DNA,用34次循环扩增可提高STR基因座的检测成功率。     

牙齿的DNA提取及STR分型研究

目的建立有效的牙齿DNA提取方法。方法使用物理及化学方法去除牙齿表面污染物,经脱钙、裂解、纯化从牙粉中提取DNA进行STR分型。结果对96例牙齿检材进行DNA检验,获得STR分型的有94例,在查找尸源的案件中发挥了重要作用。结论本方法操作简单快速,能够显著提高牙齿DNA检验的成功率。     

Direct STR amplification from whole blood and blood- or saliva-spotted FTA without DNA purification

The DNA purification step has been thought to be essential for typing of STR DNA. However, this process is time-consuming, and there is a risk of unexpected cross-contamination during purification. We report a new method for direct short tandem repeat (STR) amplification using a newly developed direct PCR buffer, AnyDirect, which can amplify STR loci from whole blood and blood- or saliva-spotted FTA cards without DNA purification. The autosomal and Y chromosomal STR loci were analyzed for whole blood and blood or saliva spots of random individuals, followed by comparison of the results with those of corresponding purified DNA. The results from whole blood and blood spots showed perfect concordance with those from purified DNA without allele or locus drop-out. However, in the case of saliva spots, no amplification or locus drop-out was observed in some of the samples, which offers a topic for further study. Additionally, some commercial hot-start DNA polymerases other than AmpliTaq Gold DNA polymerase were also found to be compatible with this buffer system. Therefore, this direct PCR buffer was demonstrated to be useful for fast forensic DNA analysis or criminal DNA databases for which there is no need to store DNA samples.     

人骨骼及牙齿DNA提取方法的比较和优化

目的人骨骼和牙齿DNA提取方法的比较和优化。方法收集18份不同个体的长骨、30颗磨牙和同一个体2根股骨、8颗磨牙。利用TissueLyser-Ⅱ组织破碎仪和PreFiler Express BTA^TM法医DNA提取试剂盒（BTA法）,应用Automate Express^TM自动化法医DNA提取系统提取DNA,进行STR分型,与脱钙法进行比较,并进行实验条件优化。结果用TissueLyser-Ⅱ结合BTA法,约2.5h即可完成骨骼和牙齿的DNA提取,分型成功率分别为94.4%和96.7%。与脱钙法比较,两种方法获得DNA质量浓度和检出率比较接近（P〈0.05）,但BTA法在操作过程方面更具优势。最佳样本量为100mg,消化时间为2h。结论采用TissueLyser-Ⅱ组织破碎仪结合BTA法对骨骼和牙齿进行DNA提取和分型检验,能满足实际检案的要求,可在法医学实践中选择使用。     

A simple method of DNA extraction and STR typing from urine samples using a commercially available DNA/RNA extraction kit

We devised a simple DNA extraction procedure suitable for STR typing of urine sample. Use of a commercially available DNA/RNA extraction kit equipped with a silica-gel-based membrane made it possible to omit the recovery of urinary nucleated cells by sedimentation before the extraction. Successful genotyping of the TH01, HumTPO and multiplex STRs was achieved using aliquots of urine as small as 100 microL. Furthermore, application of this DNA extraction procedure to frozen urine samples provided STR allele results comparable to results obtained from fresh samples. Therefore, this extraction procedure is considered to be effective for STR typing of urine samples in both the frozen and aqueous state. Furthermore, addition of sodium azide to fresh urine samples prolonged their storage duration even at room temperature.     

Evaluation of a semi-automated, magnetic bead-based DNA extraction method for genetic fingerprinting of forensic casework samples

In order to cope with the demanding workload for DNA profiling of forensic casework samples a concept for a semi-automated processing system was developed at the Landeskriminalamt (Office of Criminal Investigation) Baden-Württemberg, Germany [K. Vollack, et al., Implementation of a semi-automated processing system for DNA profiling of forensic casework samples, this issue]. The applied magnetic bead extraction method is based on ChargeSwitch^® Technology (CST) from Invitrogen and was established on a liquid handling workstation Freedom EVO^® 150 from Tecan.     

Immunological analysis of human placental lactogen in blood stains and its application to forensic science

Sex determination of blood stains from women who were in the late stages of pregnancy was possible by detecting human placental lactogen (HPL) in them. However, the agglutination time for positive reactions was prolonged as the stains aged.     

An alternative for the extraction and storage of DNA from insects in forensic entomology

An important area of recent research in forensic entomology has been the use of insect DNA to provide identification of insects for fast and accurate estimation of time since death. This requires DNA to be extracted efficiently and in a state suitable for use in molecular procedures, and then stored on a long-term basis. In this study, Whatman FTA cards were tested for use with the Calliphoridae (Diptera). In particular, testing examined their ability to effectively extract DNA from specimens, and store and provide DNA template in a suitable condition for amplification using the polymerase chain reaction (PCR). The cards provided DNA that was able to be amplified from a variety of life stages, and thus appears to be of sufficient quality and quantity for use in subsequent procedures. FTA cards therefore appear suitable for use with calliphorids, and provide a new method of extraction that is simple and efficient and allows for storage and transportation without refrigeration, consequently simplifying the handling of DNA in forensic entomological cases.     

A dedicated automated system for extraction, quantification and STR amplification of forensic evidence samples

Forensic DNA analysis is a multi-step process involving extraction of DNA, quantification of human DNA in the extract, amplification using multiplex STR systems, separation of products, and data analysis. The backlog of forensic casework is increasing worldwide. Automation is one significant way to alleviate the bottleneck of sample processing in forensic labs. The HID EVOlution™ Combination System described here is a robust, reliable sample processing platform, easily adapted to forensic laboratory workflows. Using a variety of forensic sample types including: blood stained FTA paper, cotton fabric and denim, dried blood spiked with known PCR inhibitors, saliva on cotton swabs, and semen stains, we found that yields of human DNA and STR profiles obtained with AmpFlSTR^® Idenitfiler^® kits were complete, highly reproducible, and equivalent to results obtained using the manual PrepFiler™ reagent extraction method. Automated operation was clean, and no cross-contamination was detected between extraction blanks and interspersed high DNA content samples.     

Development and validation of a sensitive UPLC-MS/MS method for the analysis of narcotic analgesics in urine and whole blood in forensic context

Narcotic analgesics are widely (ab) used and sometimes only occur in low concentrations in biological samples. Therefore, a highly sensitive liquid chromatography tandem mass spectrometry method was developed for simultaneous analysis of 9 narcotic analgesics and metabolites (buprenorphine, O-desmethyltramadol, fentanyl, norbuprenorphine, norfentanyl, pethidine, piritramide, tilidine and tramadol) in urine and whole blood. Sample preparation was performed on a mixed-mode cation exchange solid phase extraction cartridge with an additional alkaline wash step to decrease matrix effects and thus increase sensitivity. Ionization with electrospray ionization was found to be more efficient than atmospheric pressure chemical ionization. The use of a mobile phase of high pH resulted in higher electrospray ionization signals than the conventional low pH mobile phases. In the final method, gradient elution with 10mM ammonium bicarbonate (pH 9) and methanol was performed on a small particle column (Acquity C18, 1.7 μm, 2.1 mm × 50 mm). Selectivity, matrix effects, recovery, linearity, sensitivity, precision, accuracy and stability were validated in urine and whole blood. All parameters were successfully evaluated and the method showed very high sensitivity, which was the major aim of this study. The applicability of the method was demonstrated by analysis of several forensic cases involving narcotic analgesics.     

Analysis of DNA in minute volumes of blood from stains and crusts

As the first step, the locus D1S80 was amplified by the polymerase chain reaction technique from genomic DNA extracted from artificial bloodstains and crusts with different amount of blood (32 microl, 16 microl, 8 microl, 4 microl, 2 microl, and 1 microl). In all samples of bloodstains and crusts, identification by DNA analysis was possible. As the second step, the locus HLA-DQA1 was amplified from genomic DNA extracted from diluted blood samples (640, 320, 160, 80, 40, 20, 10, and 5 leukocytes). DNA amplification was possible in diluted blood samples with at least 10 leukocytes. Considering the conditions in which the present study was carried out, it was possible to conclude that 1 microl of bloodstains or crusts was enough for identification. It was also concluded that five leukocytes are not enough material to render consistent DNA identification.     

Recovery of DNA for forensic analysis from lip cosmetics.

To obtain a reference DNA profile from a missing person, we analyzed a variety of personal effects, including two lip cosmetics, both of which gave full DNA profiles. Further investigations were undertaken to explore this previously unreported source of DNA. We have tested a range of brands and types of lip cosmetics. Our studies have revealed that lip cosmetics are an excellent source of DNA, with almost 80% of samples giving a result. However, artifacts are frequently observed in the DNA profiles when Chelex is used for the DNA extraction and additional DNA purification procedures are required to ensure that an accurate DNA profile is obtained.     

一次性牙刷上脱落细胞的DNA提取及STR分型

目的研究一次性使用牙刷上脱落细胞的DNA提取和STR分型。方法对一次性使用牙刷的采集方法、采集部位、DNA提取方法、存放时间对STR分型的影响进行比对研究。结果割取法可获得较高浓度的DNA,30例中检出9个以上基因座达27例,与擦拭法存在统计学差异（P〈0.05）。Chelex-100法、DNA IQTM法检出9个以上基因座分别为26例、24例,STR分型结果无统计学意义（P〉0.05）。提取牙刷的前、后部三束刷毛检出9个以上基因座分别达27例、28例,STR分型结果无统计学意义（P〉0.05）;放置1天、1周、1个月、3个月、6个月的时间后检出9个以上基因座分别为28例、27例、22例、12例、7例。结论割取法提取一次性牙刷上的脱落细胞进行STR分型效果良好;放置时间越长的牙刷,检出率越低。     

A validation study for the extraction and analysis of DNA from human nail material and its application to forensic casework.

A validation study was conducted to demonstrate that deoxyribonucleic acid (DNA) could be successfully extracted from human nail material and analyzed using short tandem repeat (STR) profiling and/or mitochondrial DNA (mtDNA) sequencing. This study involved the development of a DNA extraction protocol that includes a cleaning procedure designed to remove external contaminants (e.g., biological, chemical). This protocol was used to test human nail material that had been soaked in whole blood from a second donor and coated with gold-palladium to simulate scanning electron microscopic analysis. The results showed no indication of a mixture and were consistent with that of the nail donor. Fresh human nail material usually yielded both STR profiles and mtDNA sequence information; however, aged human nail material (approximately eight years old) yielded only mtDNA sequence information. Upon completion of the validation study, the extraction protocol was used for the analysis of a torn fingernail fragment recovered from the scene of a violent homicide in 1983. A partial STR profile and mtDNA sequence information indicated that the fingernail fragment was excluded as originating from the suspect and was, in fact, consistent with originating from one of the victims.