荧光原位杂交技术在法庭科学DNA检验中的应用

目的运用荧光原位杂交技术结合激光显微切割技术,分离法医物证男女混合样本中的男性细胞和女性细胞,并进行DNA分型。方法通过双色荧光原位杂交,Y染色体标记上绿色信号,X染色体标记上红色信号,在荧光显微镜下识别男性细胞和女性细胞,并通过激光显微切割技术分别获得男性细胞和女性细胞进行DNA分型。结果运用荧光原位杂交技术,能够分别标记法医物证男女混合样本中的男性细胞和女性细胞,并通过激光显微切割技术获得各自的DNA分型。结论荧光原位杂交技术结合激光显微切割技术,可应用于法医物证男女混合样本的检验,提高个体识别能力。     

DNA recovery after sequential processing of latent fingerprints on copy paper

Forensic examiners must determine whether both latent fingerprint development and DNA profiling can be performed on the same area of an evidence item and, if only one is possible, which examination offers the best chance for identification. Latent fingerprints can be enhanced by targeting different components of fingerprint residues with sequential chemical treatments. This study investigated the effects of single-reagent and sequential latent fingerprint development processes on downstream DNA analysis to determine the point at which latent fingerprint development should be stopped to allow for DNA recovery. Latent fingerprints deposited on copy paper by one donor were developed using three sequential processes: 1,8-diazafluoren-9-one (DFO) → ninhydrin → physical developer (PD); 1,2-indanedione-zinc (IND-Zn) → ninhydrin → PD; and IND-Zn → ninhydrin → Oil Red O (ORO) → PD. Samples were examined after the addition of each chemical treatment. DNA was collected with cotton swabs, extracted, quantified, and amplified. DNA yields, peak heights, number of alleles obtained, and percentage of DNA profiles eligible for CODIS upload were examined. DNA profiles were obtained with varying degrees of success, depending on the number and type of treatments used for latent fingerprint development. The treatments that were found to be the least harmful to downstream DNA analysis were IND-Zn and IND-Zn/laser, and the most detrimental treatments were DFO, DFO/laser, and PD. In general, as the number of treatments increase, the opportunities for DNA loss or damage also increase, and it is preferable to use fewer treatments when developing latent fingerprints prior to downstream DNA processing.     

Rapid genomic DNA extraction (RGDE)

Hyperpolymorphic short tandem repetitive DNA sequences, STRs or microsatellites, have become widely used in human identification, particularly in criminal cases and in mass disasters. In such cases the substrates for the analyses may be decomposed as a biological material, a fact that has to be taken into account when choosing the appropriate casework methods. Nowadays expanded windows have been opened to the world especially in the area of genetic and biology science by performance of big projects such as human genome project. In this regard, one of the primary and important steps for all is DNA extraction with high quality and quantity in minimum time from biological material. By using RGDE method, genomic DNA with high quality and quantity can be acquired in the shortest time which has been presented in the world up to now. In this paper we report the evaluation of DNA extraction in this method.     

Extraction of high quality DNA from biological materials and calcified tissues

Calcified tissues, such as bone and tooth, and some other sample types, such as those containing adhesive, present a challenge to standard extraction protocols. We have developed a lysis reagent, BTA™ lysis buffer, which is designed for use with PrepFiler™ Kit reagents. The BTA™ lysis buffer disrupts calcified tissue matrices and achieves effective extraction of DNA from pulverized bone and tooth samples. In addition, the BTA™ lysis buffer mildly but efficiently extracts DNA from challenging substrates like tape, chewing gum, and cigarette butts and, as with bone and tooth, DNA from these lysates is purified using established PrepFiler™ reagent extraction protocols.We successfully extracted DNA from powdered human bone samples, chewed gum and smoked cigarettes using BTA™ lysis buffer. Extraction yields for bone, gum and cigarette samples tested were consistent and reproducible. This extraction method efficiently removed potential PCR inhibitors from all samples tested, and C_T values for the internal PCR control of Quantifiler^® Human DNA Quantification Kit were consistent and within the normal range. The DNA extracted from these samples also provided conclusive profiles that were free of PCR artifacts when amplified using the AmpF?STR^® Identifiler^® PCR Amplification Kit. The protocol is easily adapted for automation.     

How many DNA analyses are performed on adult sexual assault victims in Milan (Italy): A ten-year review

The aim of this study is to evaluate the actual use of serological/DNA analyses in the investigations carried out on adult sexual violence victims in Italy during the years 2006–2015.The victims were assisted in the largest Italian rape center, in Milan (Soccorso Violenza Sessuale e Domestica – SVSeD - Service for Sexual and Domestic Violence).The total number of sexual violence victims examined during the years 2006–2015 (adults and minors) was 3521, in 1697 of cases, biological evidence had been collected, while the number of adult victims (>18 y.o.) examined was 2300, in 1211 of cases biological evidence had been collected.Biological evidence was collected from the victims’ bodies using two swabs in five anogenital areas (labia maiora, labia minora, perineum, perianal and anal/rectum regions) and two swabs in all other skin areas suggested by the victims as areas of possible contact (double swab technique). Clothes were also collected on a case by case basis for the search of biological stains. Despite the proper collection, handling and chain of custody for all the swabs/items collected, serological/DNA analyses were requested in 86 cases out of 1211 only (710%). This percentage dropped to 190% when considering adolescent victims (13–19 y.o.).The reason why Italian Magistrates make little use of the powerful tool of DNA analyses in sexual assault cases, still remains unclear. Legal and procedural aspects are therefore also discussed.     

Busting the myths: DNA typeability after 48 hours of boil

The aim of our study was to monitor the quality and quantity of DNA in bone samples that were boiled for 48 h. Bos taurus bone disks were sampled every hour for 48 h. The subsequent DNA analysis used multiple mitochondrial DNA (mtDNA) targets (100–700 bp) to evaluate the quality and quantity of the DNA extracted. The DNA extracted from bone disks remained typeable after boiling for 48 h. We have proven that DNA typing results can be obtained even after long-term boiling.     

Quantifiler Human DNA Quantification Kit (Applied Biosystems) as a screening kit for DNA profiling

This study investigates the connection between the results of DNA quantification with Quantifiler Human DNA Quantification Kit (AB) and DNA profiling. For this purpose the DNA concentration of 3.068 routine casework samples was determined and DNA profiling was carried out. For discussion, depending on the specific DNA concentration, the samples were divided into four groups (0–5, 5–10, 10–30 and more then 30 pg/μl DNA) and the obtained number of full or partial profiles and the negative typing results was listed. Moreover group 1 (0–5 pg/μl DNA) results were subdivided and analysed more precisely. Based on the amount of 4% positive typing results in group 1 we decided to analyse every sample with quantification results >0 pg/μl DNA. A real cut-off no longer exists. Only samples showing 0 pg/μl on each result were sorted out.     

Single primer extension (SPEX) amplification to accurately genotype highly damaged DNA templates

High levels of non-authentic sequence data can be generated by traditional PCR-based methodologies when DNA is damaged, template numbers are small and/or the target amplification size too large. We therefore present an alternate methodology based on single primer extension (SPEX) amplification; that places no pre-defined size constraints on amplification and interacts with only one of the DNA strands at the target locus.     

Validation of PrepFiler™ forensic DNA extraction kit (Applied Biosystems)

The PrepFiler™ is a new kit recently introduced by Applied Biosystems for DNA extraction from a wide range of forensic samples. In the present study we tested the performance of PrepFiler™ kit against other commonly used commercially available kits on a variety of real forensic casework samples: bloodstains on different substrates, washed bloodstains, semen stains, saliva stains, hairs, bones, tissues, nails, prints after chemical treatments, skin swabs.     

降解DNA的PCR分型-电洗脱回收大片断DNA法

本文分析了降解ＤＮＡ作ＰＣＲ分型易失败的原因，提出了因小片断ＤＮＡ过多，阻碍引物与膜机的复性及阻断引物延伸的新现点。将降解的ＤＮＡ电泳，切除小片断ＤＮＡ，电洗脱回收较大片断ＤＮＡ，成功地对降解ＤＮＡ进行了ＰＣＲ分型．本方法简便、快速、有效。     

Application of DNA Forensic Techniques for Identifying Poached Guanacos (Lama guanicoe) in Chilean Patagonia*

Abstract: Guanaco (Lama guanicoe) is a protected and widely distributed ungulate in South America. A poacher, after killing guanacos in Valle Chacabuco, Chilean Patagonia, transported and stored the meat. Samples were retrieved by local police but the suspect argued that the meat was from a horse. Mitochondrial cytochrome b gene (774 pb), 15 loci microsatellites, and SRY gene were used to identify the species, number of animals and their population origin, and the sex of the animals, respectively. Analysis revealed that the samples came from a female (absence of SRY gene) Patagonian guanaco (assignment probability between 0.0075 and 0.0282), and clearly distinguishing it from sympatric ungulates (E‐value = 0). Based on the evidence obtained in the field in addition to forensic data, the suspect was convicted of poaching and illegally carrying fire arms. This is the first report of molecular tools being used in forensic investigations of Chilean wildlife indicating its promising future application in guanaco management and conservation.     

The efficiency of Y-chromosome markers in forensic trace analysis and their inclusion in the Austrian National DNA Database

Y-STR markers are a valuable tool in the analysis of biological traces in which a mixture of male and female trace material is to be expected. It is possible to generate a Y-chromosome DNA profile, even if all the prior sperm tests are negative and no sign of any male component is found in amelogenin. In 38 of a total of 239 sexual offences a perpetrator trace was identified solely using Y-STR analysis. Based on these findings, the Austrian National DNA Database was expanded to include Y-STRs in 2012 with the primary objective to identify serial sexual offences.     

MiniFiler试剂盒运用于法医微量物证的检验

目的应用MiniFiler试剂盒对法医微量物证进行DNA分析。方法采用MiniFiler试剂盒对样本进行DNA分型,确定样本9个STR基因座的等位基因型。结果10pg DNA模板经MiniFiler试剂盒扩增后,能够进行DNA分型,但40pg以上的DNA方可得到稳定可靠的分型结果。结论MiniFiler试剂盒可以用于法医微量物证的STR分析。     

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.     

Implementation and Validation of the Teleshake Unit for DNA IQ™ Robotic Extraction and Development of a Large Volume DNA IQ™ Method

Abstract: Automated platforms used for forensic casework sample DNA extraction need to be versatile to accommodate a wide variety of sample types, thus protocols frequently need modification. In this study, DNA IQ? methods previously developed for the Biomek^® 2000 Automation Workstation were adapted for the Teleshake Unit using normal volumes and all deepwell extraction, and a large volume DNA IQ? method developed. DNA purification without detectable contamination of adjacent reagent blanks is reported in the extraction of tissue samples containing several micrograms of DNA. Sensitivity and contamination studies demonstrated similar performance with the manual organic extraction method for bloodstain dilution samples. Mock casework samples demonstrated the effectiveness of the Teleshake and Teleshake large volume methods. Because of the performance and increased versatility of the DNA IQ? extraction with these modifications, the Teleshake Unit has been implemented in both normal and large volume automated DNA extractions at the Virginia Department of Forensic Science.     

大鼠死后骨骼肌细胞核DNA降解的彗星电泳检测

目的探讨彗星电泳检测大鼠死后骨骼肌细胞核DNA降解碎片与死后时间的关系。方法建立SD大鼠死亡模型,18只雌性大鼠断颈处死,在25.1℃分别于死后6、12、24、36、48、60提取大鼠骨骼肌组织进行彗星电泳,荧光显微成像系统采集图像,同时获取多个分析参数(Comet4.0),并进行统计学处理。结果在死后6～48h,彗星尾长(TL)在死后各时间点的均数依次为11.56±0.10、17.76±0.18、18.82±0.21、21.68±0.18和23.33±0.07;Oliver尾矩(TM)在各时间点的均数依次为1.63±0.46;2.12±0.90;2.15±1.03;2.22±0.76;3.35±0.80;尾DNA(TDNA)在各时间点的均数依次为29.57±8.42;32.36±10.92;30.11±12.55;37.81±12.03;54.76±8.60。结论细胞DNA降解随着死后时间的延长而增加;彗星尾长用于推断死后时间优于Oliver尾矩和尾DNA。     

Getting Ahead: Extraction of DNA from Skeletonized Cranial Material and Teeth

Between 1990 and 2018, the Defense POW/MIA Accounting Agency submitted 2177 cranial elements and 1565 teeth to the Armed Forces Medical Examiner System—Armed Forces DNA Identification Laboratory for DNA testing. In an effort to identify missing United States service members, materials were recovered from wartime losses inclusive of World War II, the Korean War, and Southeast Asia. Using four different DNA extraction protocols, DNA testing was performed using mitochondrial DNA Sanger sequencing, modified AmpFlSTR^® Yfiler?, AmpFlSTR^® MiniFiler?, PowerPlex^® Fusion, or Next Generation Sequencing. This paper aims to provide optimal strategies for the DNA testing of skeletonized cranial materials. Cranial elements produced the most consistent results in Sanger sequencing using an organic purification; however, teeth were most successful for the same platform with an inorganic purification. The inverse is true for STR testing of cranial bones. Of the cranial elements, the temporal provided the most consistent results.     

Duplex-specific nuclease (DSN): A method for the rehabilitation of low-copy number DNA profiles

A continual challenge in the field of forensic DNA analysis is the amplification and interpretation of degraded and low-copy number (LCN) DNA obtained from amounts of limited biological evidence. It has been well established that DNA profiles obtained from the amplification of low quality, degraded, and/or LCN DNA samples are often of limited value due to the frequent occurrence of preferential amplification during polymerase chain reaction (PCR). The by-products of preferential PCR amplification are often observed as inter- and intra-locus peak imbalance, allelic dropout, and/or locus dropout. These are all artifacts that are identified during the interpretation phase of analysis rather than by improving the quality of the DNA present. While it is theoretically possible to obtain a complete DNA profile from a single cell, in reality, profiles obtained from suboptimal amounts of DNA are difficult to interpret and frequently inconsistent when replicated. Inspired by advances in next-generation sequencing techniques, we propose a methodology for simultaneously normalizing the abundance of PCR products across all short tandem repeat (STR) loci using the DNA exonuclease, duplex-specific nuclease (DSN). DSN is an enzyme isolated from the hepatopancreas of Red King (Kamchatka) crab that possesses a strong affinity for digesting double stranded DNA (dsDNA) and has limited activity toward single stranded DNA (ssDNA). Degraded DNA known to display peak imbalance and allele dropout was amplified using AmpFlSTR^® Identifiler^® Plus for 28 cycles. Following amplification, samples were denatured at 99.9 °C for 5 min and incubated with one unit of DSN at 62 °C in a 28 μl volume for 1 min. Nuclease activity was terminated through the addition of equal volume of 10 mM EDTA and 95 °C incubation for 2 min. Following DSN treatment, 21 of 30 alleles within the known profile exhibited some improvement in peak height balance. The findings obtained support the potential use of DSN treatment as a method for normalizing STR profiles and improving the quality of data from degraded and low quantity DNA samples.     

国际刑警组织DNA技术的应用和数据交换

国际刑警组织为了对各成员国的DNA检验工作提供技术支持 ,促进DNA技术的广泛应用 ,设立了DNA组。DNA专家组是DNA组的主要咨询机构 ,负责推荐DNA采样和证据采集、DNA数据库、质量控制、DNA技术培训等方面的指导原则。国际刑警组织还定期召开国际DNA用户大会 ,开展地区性DNA技术培训 ,以促进DNA技术的普及、应用和发展。本文主要介绍国际刑警组织在DNA技术方面开展的工作及推荐的一些指导原则。