Chelex法和两种磁珠法提取接触DNA效果的比较

目的比较Chelex法、DNA IQ磁珠法、EQ国产磁珠法对接触DNA的提取效果。方法将稀释为10ng、100ng的标准品DNA,分别采用Chelex法、DNA IQ磁珠法、EQ国产磁珠法处理;对30例烟蒂和30例牙刷分别采用Chelex法、DNA IQ磁珠法和EQ国产磁珠法提取DNA,然后进行PCR定量和STR检测。结果Chelex法对DNA的提取无损失,DNA IQ磁珠法、EQ国产磁珠法对DNA的提取均有不同程度的损失;烟蒂、牙刷等检材采用Chelex法提取的接触DNA量和IPC CT值显著高于IQ磁珠法、EQ国产磁珠法,但STR检验成功率却低于IQ磁珠法、EQ国产磁珠法。2种磁珠法提取的DNA量、IPC CT值和STR检验成功率无显著性差异。结论污染轻、杂质少的接触DNA检材,用Chelex法提取最为方便快捷;IQ磁珠法、EQ国产磁珠法更适合污染接触DNA检材的提取及自动化操作。     

Efficiency of Casework Direct Kit for extraction of touch DNA samples obtained from cars steering wheels

Analysis of STR profiles obtained from touch DNA has been very useful to the elucidation of crimes. Extraction method may be determinant for the recovery of genetic material collected from different surfaces. Vehicle theft is one of the most common crimes in São Paulo city, Brazil, but collection of biological traces in car steering wheels is not considered, because of the belief that profiles generated won’t be able to identify the thief, only the owner. This study aimed to analyze the efficacy of extraction methods for obtaining DNA profiles in samples collected from steering wheels. Eight criminal acts were simulated with 2 different individuals each (mixture of victim and thief), in duplicate, in order to compare two extraction methods: DNA IQ™ and Casework Direct Kit (both Promega Corporation). Genetic material was collected by double swab method and quantified by Quantifiler™Trio (ThermoFisher Scientific). Amplification was conducted with PowerPlex® Fusion System (Promega). It was possible to obtain STR profiles for all experiments. The mixtures were compared with reference profiles to evaluated how many alleles of each donor were observed. Samples extracted with Casework Direct Kit obtained STR profiles with higher averages of alleles for primary and secondary donors (88.7% and 59.9%, respectively) than those extracted with DNA IQ™ (60.4% and 38.1%, respectively). This could be explained by the differences established in the protocols of both methods, since DNA IQ™ is based on successive washes and can result in loss of DNA, whereas Casework Direct Kit minimizes this problem. We concluded that Casework Direct Kit was more efficient for processing touch DNA samples than DNA IQ™.     

AutoMate Express™ forensic DNA extraction system for the extraction of genomic DNA from biological samples

Abstract: The AutoMate Express? Forensic DNA Extraction System was developed for automatic isolation of DNA from a variety of forensic biological samples. The performance of the system was investigated using a wide range of biological samples. Depending on the sample type, either PrepFiler? lysis buffer or PrepFiler BTA? lysis buffer was used to lyse the samples. After lysis and removal of the substrate using LySep? column, the lysate in the sample tubes were loaded onto AutoMate Express? instrument and DNA was extracted using one of the two instrument extraction protocols. Our study showed that DNA was recovered from as little as 0.025 μL of blood. DNA extracted from casework‐type samples was free of detectable PCR inhibitors and the short tandem repeat profiles were complete, conclusive, and devoid of any PCR artifacts. The system also showed consistent performance from day‐to‐day operation.     

DNA Profiles from Fingerprint Lifts—Enhancing the Evidential Value of Fingermarks Through Successful DNA Typing

This study evaluated the compatibility of the most common enhancement methods and lifting techniques with DNA profiling. Emphasis is placed on modern lifting techniques (i.e., gelatin lifters and Isomark?) and historical fingerprint lifts for which limited research has been previously conducted. A total of 180 fingerprints were deposited on a glass surface, enhanced, lifted, and processed for DNA typing. DNA could be extracted and profiled for all the powders and lifts tested and from both groomed fingerprints and natural prints with no significant difference in the percentage of profile recovered. DNA profiles could also be obtained from historical fingerprint lifts (79.2% of 72 lifts) with one or more alleles detected. These results demonstrate the compatibility between different powder/lift combinations and DNA profiling therefore augmenting the evidential value of fingerprints in forensic casework.     

A comparative study of two extraction methods routinely used for DNA recovery from simulated post coital samples

In sexual assault cases DNA profiling of spermatozoa can be of critical importance. Most methods use differential extraction of the spermatozoa to separate it from the female component. Here we have compared two commercially available differential extraction methods, the QIAamp^® DNA mini kit (Qiagen) and Differex™ with the DNA IQ^® System (Promega). Simulated postcoital samples were prepared using buccal cells from a female donor and spermatozoa from three male donors. A dilution series ranging from neat semen to a 1:1500 dilution (semen:dH₂O) was prepared and mixed with an equal volume of saliva from a female donor. Extraction efficiency was assessed using DNA concentration measured with NanoDrop 2000 and Quantifiler^® Human DNA Quantification Kit and the profile count of full, partial and mixed DNA profiles generated using SGM Plus and PowerPlex^® ESI 17. Statistical analysis was carried out using Randomisation in R, which is a robust model making no assumption of the distribution of data. Based on the amount of DNA extracted and the types of profiles no significant difference in the performance of the two extraction kits was seen. However, the processing time taken with the Differex™ System was about half than that of the QIAamp^® DNA mini kit and involved fewer liquid transfers.     

Implementation of Prep-n-Go™ Buffer for DNA extraction from buccal swabs

The efficacy of two extraction methods; room temperature and heat protocols was assessed for buccal swabs using the Prep-n-Go™ Buffer. DNA was extracted from buccal swabs using both extraction methods and their effectiveness to produce good quality DNA profiles was evaluated. Heat protocol was found to yield more DNA, however room temperature protocol produced better quality DNA profiles with fewer artefacts when the samples from both extraction methods were amplified directly without any normalisation with the VeriFiler™ Express PCR Amplification Kit.     

The fate of the chemical warfare agent during DNA extraction

Forensic laboratories do not have the infrastructure to process or store contaminated DNA samples that have been recovered from a crime scene contaminated with chemical or biological warfare agents. Previous research has shown that DNA profiles can be recovered from blood exposed to several chemical warfare agents after the agent has been removed. The fate of four toxic agents, sulfur mustard, sodium 2-fluoroacetate, sarin, and diazinon, in a lysis buffer used in Promega DNA IQ extraction protocol was studied to determine if extraction would render the samples safe. Two independent analytical methods were used per agent, selected from GC-MS, 1H NMR, 19F NMR, (31)P NMR, or LC-ES MS. The methods were validated before use. Determinations were carried out in a semi-quantitative way, by direct comparison to standards. Agent levels in the elution buffer were found to be below the detectable limits for mustard, sarin, sodium 2-fluoroacetate or low (<0.02 mg/mL) for diazinon. Therefore, once extracted these DNA samples could be safely processed in a forensic laboratory.     

Recovery of Latent Fingerprints and DNA on Human Skin*

Abstract: The project “Latent Fingerprints and DNA on Human Skin” was the first systematic research in Europe dealing with detection of fingerprints and DNA left by offenders on the skin of corpses. One thousand samples gave results that allow general statements on the materials and methods used. The tests were carried out according to a uniform trial structure. Fingerprints were deposited by natural donors on corpses. The latent fingerprints were treated with magnetic powder or black fingerprint powder. Afterward, they were lifted with silicone casting material (Isomark^®) or gelatine foil. All lifts were swabbed to recover DNA. It was possible to visualize comparable and identifiable fingerprints on the skin of corpses (16%). In the same categories, magnetic powder (18.4%) yielded better results than black fingerprint powder (13.6%). The number of comparable and identifiable fingerprints decreased on the lifts (12.7%). Isomark^® (14.9%) was the better lifting material in comparison with gelatine foil (10.1%). In one‐third of the samples, DNA could be extracted from the powdered and lifted latents. Black fingerprint powder delivered the better result with a rate of 2.2% for full DNA profiles and profiles useful for exclusion in comparison with 1.8% for the magnetic powder traces. Isomark^® (3.1%) yielded better results than gelatine foil (0.6%).     

Recovery of DNA from bone without demineralization

This study assessed the performance of five different DNA extraction methods for the recovery of DNA from bone: ChargeSwitch® gDNA Plant Kit, DNA IQ™ System Kit, DNeasy® Blood & Tissue Kit, PrepFiler® BTA Forensic DNA Extraction Kit and phenol-chloroform-isoamyl alcohol. DNA was extracted from pig rib and femur bones that was fresh, had undergone surface decomposition for three months, and had undergone surface decomposition for one year. Extracted DNA was analyzed using real-time PCR and amplification of an in-house PCR multiplex that assessed the quality and quantity of DNA and for the presence of inhibitors. The phenol-chloroform-based method consistently yielded the highest amounts of DNA and DNA IQ the lowest; however, all methods produced relatively high yields of DNA from both pig rib and femur samples that could be amplified without any detected inhibition. The data demonstrate that with reasonable quality bone samples any of the tested methods can isolate DNA that can be successfully analyzed. The effective use of internal PCR controls is also demonstrated.     

汗潜指印DNA提取方法的初步研究

目的建立渗透性载体及非渗透性载体上汗潜指印DNA的提取和检验方法。方法采用C-有-柱法及SiO2法两种DNA提取方法,PCR扩增后310型遗传分析仪检测。结果载玻片上3枚汗潜指印采用2种方法均可扩增出Amel及9个STR位点;纸上3枚汗潜指印用SiO2法检见Amel及9个STR位点,而用C-有-柱法检验结果不稳定。渗透性及非渗透性载体上1及2枚汗潜指印,采用上述两种DNA提取方法,检验结果均不稳定。结论所建立的方法可以检见渗透性及非渗透性载体上汗潜指印DNA,并达到同一认定的程度。     

用Chelex100法及有机溶剂提取法联合提取DNA扩增STR基因座的比较研究

为了比较不同方法提取DNA扩增STR基因座的成功率 ,本文用Chelex10 0法及Chelex10 0法联合有机溶剂提取法分别提取性犯罪案件 6份血斑及 6份混合斑中精子DNA ,并用PE公司ProfilerPlus系统复合扩增 ,ABI 310型基因分型仪检测。结果表明 ,常规采用Chelex10 0法提取血斑及精斑等生物检材DNA作STR基因座检验失败或所检测的位点较少时 ,应对Chelex10 0法提取的DNA上清液再用有机溶剂提取法提取 ,可极大提高DNA检验成功率。采用本文建立的Chelex10 0法联合有机溶剂提取法 ,提高了PCR扩增阳性率 ,对实际检案很有帮助 ;在PCR扩增前应常规进行DNA定量检验 ,否则对于重要检材应进行梯度扩增。     

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.     

DNA typing from skeletal remains using GlobalFiler™ PCR amplification and Investigator® 24plex QS kits

Since the beginning of our work in 2003 our laboratory has focused exclusively on STR DNA from bone, a powerful tool in missing person cases. In cases such as mass disasters or missing persons, human remains are challenging to identify as they may be fragmented, burnt, recovered from water, degraded, and/or contain inhibitory substances. To address these challenges, this study has evaluated the performance of relatively new STR kits Investigator® 24plex QS kit (Qiagen) and GlobalFiler™ PCR Amplification kit (Thermo Fisher Scientific) by comparing it with current uses of the AmpFLSTR® Identifiler® Plus kit (Applied Biosystems) to obtain genetic information from skeletal remains. We analyzed 20 bone samples of skeletal remains from routine casework submitted for body identifications by law enforcement corresponding using Investigator® 24plex QS kit and GlobalFiler™ PCR Amplification kit, previously analysed AmpFLSTR® Identifiler® Plus kit (Thermo Fisher Scientific). The data indicates that the STR profiles obtained using the GlobalFiler™ and Investigator® 24plex QS kit for analysis of skeletal remains has shown results in an increased number of reportable genetic loci, and provide greater power of discrimination in comparison to the Identifiler® Plus Kit. Advanced extraction and purification techniques, together with more sensitive and robust new amplification kits allowed us to overcome the challenges associated with processing compromised skeletal remains and ultimately obtain full STR DNA profiles in 99% of the bones.     

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.     

Establishing an integrated pipeline for automatic and efficient detection of trace DNA encountered in forensic applications

The analysis of trace DNA is a crucial component in forensic applications. Biological materials containing low-level DNA collected at crime scenes, such as fingerprints, can be valuable as evidence. Automatic detection of biological samples has been largely embraced in forensic applications to meet the increasing throughput requirements. However, the amount of DNA automatically retrieved from trace evidence often tends to be small and unstable, ultimately resulting in poor detection of DNA profiles. Thus, in this work, we introduced a robust DNA extraction and purification platform named Bionewtech® BN3200 (Bionewtech®, Shanghai, China) with the goal of constructing a rapid automatic detection system for trace DNA. The establishment of automatic detection system for trace DNA mainly encompassed two parts: assessing the sensitivity of automatic extraction platform and screening the optimal short tandem repeat (STR) typing kit. The sensitivity of Bionewtech® BN3200 platform based on Ultra-sensitive DNA Extraction kit was initially estimated, demonstrating that this extraction platform might contain large potential in the trace DNA extraction. For the amplification part, three sets of commercial multiplex STR typing kits were selected as candidates, and the amplified products were further genotyped on the Applied Biosystems 3500xl Genetic Analyzer. After comparation, SiFa™ 23 Plex Kit was determined as the most suitable amplification system for trace DNA. Eventually, the newly exploited trace DNA detection system was successfully implemented in the detection of fingerprints derived from glass surfaces with the five-seconds contact time. As a result, the DNA recovered from the fingerprints fluctuated approximately from 57.60 pg to 18.05 ng, in addition, over 70% of the total STR loci were detected in 75% of the fingerprint samples.     

A Simple and Efficient Method of Extracting DNA from Aged Bones and Teeth

DNA is often difficult to extract from old bones and teeth due to low levels of DNA and high levels of degradation. This study established a simple yet efficient method for extracting DNA from 20 aged bones and teeth (approximately 60 years old). Based on the concentration and STR typing results, the new method of DNA extraction (OM) developed in this study was compared with the PrepFiler? BTA Forensic DNA Extraction Kit (BM). The total amount of DNA extracted using the OM method was not significantly different from that extracted using the commercial kit (p > 0.05). However, the number of STR loci detected was significantly higher in the samples processed using the OM method than using the BM method (p < 0.05). This study aimed to establish a DNA extraction method for aged bones and teeth to improve the detection rate of STR typing and reduce costs compared to the BM technique.     

PrepFiler Express BTATM Lysis Buffer Combined with Silicon Microbeads for Rapid DNA Extraction from BoneCSCD

Objective: To establish a convenient and rapid method for extracting DNA from bone. Methods: Fifteen long bone samples were washed and sterilized. The skeletal fragments were obtained by electric drill, and lysed by PrepFiler Express BTATM lysis buffer. DNA was then manually extracted by silicon microbeads for further analysis. Results: STR genotyping was successfully obtained in 14 out of the 15 samples, and the detection rate was 93.33%. Conclusion: The method for DNA extraction from bone established in present study is convenient, quick, effective, and with a strong applicability, which is worth spreading and applying. © 2017 by the Editorial Department of Journal of Forensic Medicine.     

A Powder-free DNA Extraction Workflow for Skeletal Samples

The processing of skeletal material poses several challenges for forensic laboratories. Current methods can be laborious, time-consuming, require dedicated equipment, and are vulnerable to contamination. In this study, various sample mass (1 × 50 mg, 3 × 50 mg, and 1 × 150 mg chip(s)) and incubation times (2, 4, and 16 h) were tested using the PrepFiler^® BTA™ Forensic DNA Extraction Kit to digest whole bone chips in lieu of powdering. The most effective method was then applied to bones and tooth fragments collected from contemporary human cadavers exposed to various environmental conditions using an automated platform. Over a third of the samples tested generated full DNA profiles without having to powder the bone/tooth fragment or further alter the manufacturer's protocol. However, for most samples resulting in incomplete STR profiles due to low amounts of DNA, slightly better results were achieved with powdered tissue. Overall, this work demonstrates the potential use of a faster, nonpowdering DNA extraction method for processing skeletal samples as an effective first-pass screening tool.     

DNA profiles from biological trace material contained in underwear and toothbrushes

There are special cases where it is extremely difficult to find a close relative for obtaining reference samples, such as in adoption cases or foreigners without relatives in the country. In these cases, it is possible to use personal objects from the missing person. We developed a protocol for obtaining DNA profiles from toothbrushes and underwear and evaluated two DNA extraction methods (Silica and magnetic particles), comparing the DNA quantity and the quality of the obtained profiles. Results showed that for underwear, silica in suspension recovered higher DNA quantity but magnetic particles (PrepFiler®) generated more complete profiles. For toothbrushes PrepFiler® recovered a higher DNA quantity and generated more complete DNA profiles. Statistical analyses showed there were no significant differences between both methods. Higher DNA quantities were recovered from underwear samples, however toothbrushes generated higher quality in DNA profiles, without obtaining mixed profiles, concluding that toothbrushes are a better item than underwear. Considering that silica in suspension method is three-times cheaper than PrepFiler®, we propose it is possible to successfully obtain DNA profiles from personal objects using a low-cost DNA extract method.