New optimized DNA extraction protocol for fingerprints deposited on a special self-adhesive security seal and other latent samples used for human identification

Abstract: Obtaining complete short tandem repeat (STR) profiles from fingerprints containing minimal amounts of DNA, using standard extraction techniques, can be difficult. The aim of this study was to evaluate a new kit, Fingerprint DNA Finder (FDF Kit), recently launched for the extraction of DNA and STR profiling from fingerprints placed on a special device known as Self‐Adhesive Security Seal Sticker^® and other latent fingerprints on forensic evidentiary material like metallic guns. The DNA extraction system is based on a reversal of the silica principle, and all the potential inhibiting substances are retained on the surface of a special adsorbent, while nucleic acids are not bound and remain in solution dramatically improving DNA recovery. DNA yield was quite variable among the samples tested, rendering in most of the cases (>90%) complete STR profiles, free of PCR inhibitors, and devoid of artifacts. Even samples with DNA amount below 100 pg could be successfully analyzed.     

PE‐Swab Direct STR Amplification of Forensic Touch DNA Samples

The PE‐Swab direct STR amplification workflow was developed to process low‐level “touch DNA” samples. In this workflow, a forensic sample is first collected on a 4‐mm PE‐Swab (a novel sample collection device); two 2‐mm punches containing collected samples are then generated from the PE‐Swab and directly amplified for STR typing. Compared to the conventional STR workflow, which involves DNA extraction, purification, and elution volume reduction, the PE‐Swab direct STR amplification workflow does not require sample preparation and takes <60 sec before a touch sample is ready for STR amplification. Because there is no DNA loss due to sample preparation, the PE‐Swab workflow is more sensitive than the conventional STR workflow. The average peak height per sample obtained by the PE‐swab workflow is 3 times higher than that from the conventional workflow with both low‐level single source and two‐contributor mixture samples tested in this study.     

Recovery and STR amplification of DNA from RFLP membranes

Restriction fragment length polymorphism (RFLP) techniques were utilized in the forensic DNA community until the mid 1990s when less labor-intensive polymerase chain reaction short tandem repeat (PCR STR) techniques became available. During the transition from RFLP technology to PCR-based STR platforms, a method for comparing RFLP profiles to STR profiles was not developed. While the preferred approach for applying new technology to old cases would be to analyze the original biological stain, this is not always possible. For unsolved cases that previously underwent RFLP analysis, the only DNA remaining may be restriction cut and bound to nylon membranes. These studies investigate several methods for obtaining STR profiles from membrane bound DNA, including removal of bound DNA with bases, acids, detergents, various chemicals, and conventional cell extraction solutions. Direct multiplex STR amplification of template in the membrane-bound state was also explored. A partial STR profile was obtained from DNA that was recovered from an archived membrane using conventional extraction buffer components, indicating promise for recovering useful STR information from RFLP membranes that have been maintained in long-term frozen storage.     

Recovery of DNA from Latent Fingerprint Tape Lifts Archived Against Matte Acetate

This study was driven by court order to examine methods to remove, extract, and STR‐type potential DNA entrapped between latent fingerprint lifting tape and matte acetate that was collected from a 1977 crime scene. Results indicate that recovery of appreciable quantities of DNA is more challenging once adhesive is attached to matte acetate cards and even more difficult when fixed following black powder enhancement. STR amplification of extracts from entrapped fingermarks collected following the dusting/lifting procedure did not produce robust profiles, and extraneous peaks not expressed by print donors were detected for some samples. A hearing was set to argue whether there was DNA remaining to be tested, and if so, whether that DNA could be exculpatory in this postconviction matter. The studies herein provided the basis for the court's decision to not require the testing.     

The Influence of Selected Fingerprint Enhancement Techniques on Forensic DNA Typing of Epithelial Cells Deposited on Porous Surfaces

Fingerprints deposited at crime scene can be a source of DNA. Previous reports on the effects of fingerprint enhancement methods have focused mainly on fingermarks deposited in blood or saliva. Here, we evaluate the effects of fingerprint enhancement methods on fingerprints deposited on porous surfaces. We performed real‐time quantification and STR typing, the results of which indicated that two methods (iodine fuming and 1,2‐indanedione in ethyl acetate enhancement) had no effect on the quantity of DNA isolated and resultant STR alleles when compared to control samples. DNA quantities and allele numbers were lower for samples enhanced with silver nitrate and 1,2‐indanedione in acetic acid when compared to control samples. Based on DNA quantity, quality, and observable stochastic effects, our data indicated that iodine fuming and 1,2‐indanedione in ethyl acetate were the preferred options for the enhancement of fingerprints on porous surfaces.     

The Influence of Swabbing Solutions on DNA Recovery from Touch Samples,

There has been minimal research into how to best obtain DNA from touch samples. Many forensic laboratories simply moisten a swab with water and use it for collecting cells/DNA from evidentiary samples. However, this and other methods have not been objectively studied in order to maximize DNA yields. In this study, fingerprints were collected using swabs moistened with water or laboratory or commercially available detergents, including sodium dodecyl sulfate (SDS), Triton X‐100, Tween 20, Formula 409^®, and Simple Green^®. Prints were swabbed, DNA isolated using an organic extraction, yields quantified, and relative yields compared. In all cases, the detergent‐based swabbing solutions outperformed water, with SDS and Triton X‐100 producing significant increases in yield. Short tandem repeat profiles were consistent with the individuals that placed them. Subsequent analysis of SDS concentrations for collecting touch DNA demonstrated an increase in DNA yield with increasing SDS concentration, with an optimal concentration of approximately 2%.     

Analysis of Mitochondrial DNA from a Burned,Ninhydrin‐Treated Paper Towel

Contact‐based evidence is likely to have limited quantities of DNA and may yield mixed profiles due to preexisting or contaminating DNA. In a recent arson investigation, a paper towel was collected and used as circumstantial evidence. The paper towel was partially burned and was likely set on fire with flammable liquid. As part of the investigation, the paper towel was treated with ninhydrin to visualize fingerprint evidence. Initial DNA analysis of two swabs was negative for short tandem repeat (STR) markers and revealed a mixture of mitochondrial DNA (mtDNA). Analysis of 13 additional cuttings yielded four more mixed profiles, but also two samples with a common single‐source profile. The single‐source mtDNA profile matched that of the primary suspect in the case. Thus, even if initial mtDNA analysis yields a mixed profile, a sampling strategy involving multiple locations can improve the chance of obtaining valuable single‐source mtDNA profiles from compromised evidence in criminal casework.     

An already archived latent fingerprint as a DNA source for STR typing in a murder case

Former studies have shown that even a single skin contact, resulting in a latent fingerprint, can transfer enough DNA for genetic analysis. However, up to now latent fingerprints have usually not been used for DNA typing. In the present case the smeared trace of a hand was found in the suspect's car and archived. As it could not be evaluated in a classical manner, the evidence had to be examined by molecular genetic methods. DNA was extracted and typed in five different STR loci. Based on the yielded results, the significance of the findings is discussed.     

STR Profiles from DNA Samples with "Undetected" or Low Quantifiler™ Results

Abstract:  Screening methods capable of identifying DNA samples that will not yield short tandem repeat (STR) profiles are desired. In the past, quantitation methods have not been sensitive enough for this purpose. In this study, low level DNA samples were used to assess whether Quantifiler™ has a minimum quantitation value below which STR profiles would consistently fail to be detected. Buccal swabs were obtained and the DNA extracted, quantified, and serially diluted to concentrations ranging from 0.002 to 0.250 ng/μL. Samples were analyzed once with Quantifiler™, followed by Profiler Plus™ amplification and capillary electrophoresis analysis. An absolute minimum value below which STR results were unobtainable could not be defined. From the 96 low level samples tested, STR loci (including one full profile) were successfully amplified and detected from 27% of the samples "undetected" by Quantifiler™. However, no STR alleles were detected in 73% of these "undetected" samples, indicating that Quantifiler™ data may be useful for predicting STR typing success.     

3种提取胶带粘面汗潜指印中DNA的方法比较

目的比较胶带粘面汗潜指印中DNA提取的方法。方法分别采用硅珠法、QIAMicrokit法、硅珠-QIAMicrokit法提取胶带粘面的汗潜指印中DNA,STR复合扩增,荧光电泳检测。结果用QIAMicrokit法、硅珠-QIAMicrokit法提取胶带粘面汗潜指印中DNA,检测成功率分别为21%和36%。硅珠法检测未获成功。结论硅珠-QIAMicrokit法提取胶带粘面汗潜指印中的DNA比QIAMicrokit法,检验时间更短,检测成功率更高。     

刑事照相常用紫外光源照射对DNA检验的影响研究

目的探究刑事照相常用紫外光源照射不同客体上的血指印和汗潜指印后对DNA检验产生的影响。方法分别使用254nm和365nm紫外光源在不同照射距离,不同照射时间下对不同客体表面制备的原血指印、微量血指印和汗潜指印检材进行照射,随后进行DNA定量分析。结果原血指印经紫外照射后对DNA检验结果无显著影响,微量血指印和汗潜指印经紫外照射对后续DNA检验结果认定影响较大。结论紫外光波长越短、功率越大、照射距离越短、照射时间越长,对DNA检验结果的影响越大。     

Threat mail and forensic science: DNA profiling from items of evidence after treatment with DFO

Two cases of threatening letters with their accompanying envelopes were received to the Division of Forensic Identification unit of the Israel Police. The envelopes, including the stamps, and the letters were initially examined for latent fingerprints by the DFO reagent, known to cause degradation of DNA. Although no latent fingerprints could be visualized on any of the items, the biology laboratory using organic DNA extraction, was successful in defining genetic profiles from all the items employing six STR loci, even after treatment with DFO. In a controlled experiment, a known donor attached a stamp, by licking, to an envelope. This item was treated with DFO and then profiled using STR loci. The results showed that previous DFO treatment on the control stamp before DNA analysis had no negative effects on obtaining the DNA profile of the known donor using STR loci.     

Trace DNA Sampling Success from Evidence Items Commonly Encountered in Forensic Casework

Trace DNA analysis is a significant part of a forensic laboratory's workload. Knowing optimal sampling strategies and item success rates for particular item types can assist in evidence selection and examination processes and shorten turnaround times. In this study, forensic short tandem repeat (STR) casework results were reviewed to determine how often STR profiles suitable for comparison were obtained from “handler” and “wearer” areas of 764 items commonly submitted for examination. One hundred and fifty‐five (155) items obtained from volunteers were also sampled. Items were analyzed for best sampling location and strategy. For casework items, headwear and gloves provided the highest success rates. Experimentally, eyeglasses and earphones, T‐shirts, fabric gloves and watches provided the highest success rates. Eyeglasses and latex gloves provided optimal results if the entire surfaces were swabbed. In general, at least 10%, and up to 88% of all trace DNA analyses resulted in suitable STR profiles for comparison.     

DNA Typing for the Identification of Old Skeletal Remains from Korean War Victims*

Abstract: The identification of missing casualties of the Korean War (1950–1953) has been performed using mitochondrial DNA (mtDNA) profiles, but recent advances in DNA extraction techniques and approaches using smaller amplicons have significantly increased the possibility of obtaining DNA profiles from highly degraded skeletal remains. Therefore, 21 skeletal remains of Korean War victims and 24 samples from biological relatives of the supposed victims were selected based on circumstantial evidence and/or mtDNA‐matching results and were analyzed to confirm the alleged relationship. Cumulative likelihood ratios were obtained from autosomal short tandem repeat, Y‐chromosomal STR, and mtDNA‐genotyping results, and mainly confirmed the alleged relationship with values over 10⁵. The present analysis emphasizes the value of mini‐ and Y‐STR systems as well as an efficient DNA extraction method in DNA testing for the identification of old skeletal remains.     

Multiplex short tandem repeat amplification of low template DNA samples with the addition of proofreading enzymes

Abstract:  With <100 pg of template DNA, routine short tandem repeat (STR) analysis often fails, resulting in no or partial profiles and increased stochastic effects. To overcome this, some have investigated preamplification methods that include the addition of proofreading enzymes to the PCR cocktail. This project sought to determine whether adding proofreading polymerases directly in the STR amplification mixture would improve the reaction when little template DNA is available. Platinum Taq High Fidelity and GeneAmp High Fidelity were tested in Profiler Plus? STR reactions alone and in combination with AmpliTaq^® Gold. All reactions included the additional step of a post‐PCR purification step. With both pristine low template DNA and casework samples, the addition of these polymerases resulted in comparable or no improvement in the STR amplification signal. Further, stochastic effects and artifacts were observed equally across all enzyme conditions. Based on these studies, the addition of these proofreading enzymes to a multiplex STR amplification is not recommended for low template DNA work.     

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.     

DNA IQ磁珠法结合Maxwell~（TM） 16自动仪提取接触DNA

目的研究DNA IQ磁珠法结合MaxwellTM 16自动仪对接触DNA提取的应用价值。方法 151份案件接触DNA检材95℃裂解后,采用DNA IQ磁珠法结合MaxwellTM 16自动仪提取DNA,然后进行DNA定量和STR分型检测,统计各种类型的接触DNA含量I、PC CT值和STR分型成功率。结果 151份案件接触DNA检材中,除果核平均DNA获得量为9.51ng以外,其它接触检材的平均DNA获得量均大于10ng,烟蒂检验成功率最高为93%,果核检验成功率较低,为60%。所有DNA样品的IPC CT值均在27左右,纯度高。结论大部分接触DNA检材采用DNA IQ磁珠法结合MaxwellTM 16自动仪可提取到足以进行STR分型的DNA。     

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。     

DNA profiling from heroin street dose packages

A large amount of heroin street doses are seized and examined for drug content by the Israel police. These are generally wrapped in heat-sealed plastic. Occasionally it is possible to visualize latent fingerprints on the plastic wrap itself, but the small size of the plastic item and the sealing process makes the success rate very low. In this study, the possibility of extracting and profiling DNA from the burnt edge of the plastic wrap was investigated. The idea was based on the assumption that epithelial cells might be trapped during the sealing process. The results show that there are sufficient quantities of DNA deposited at the "amorphic" burnt edges of sealed street doses for DNA profiling to be carried out. A controlled experiment using a known donor was performed. This subject carried out sealing of "street drug" packages and consequent DNA extractions were performed to show that known DNA profiles could be recovered from such packages, as a result of handling by the "packer." "Square-like" burnt edges did not yield DNA profiles, probably because of differences in the sealing process. It was also shown that DNA could be recovered from the plastic wrap itself and not only from the amorphic burnt edges. As heroin dealers and drug users are often involved in other crimes and run-ins with the law, the effective extraction and addition of their DNA profiles from such items of evidence to the newly established DNA database in Israel provides new avenues in the continued fight against crime and drug traffickers.     

Evaluation of a Freezer Mill for Bone Pulverization prior to DNA Extraction: An Improved Workflow for STR Analysis

Traditional methods for bone pulverization typically generate heat, risking stability of DNA sample. SPEX? has developed cryogenic grinders which introduce liquid nitrogen to cool the sample and aid in the grinding process. In this study, the Freezer Mill 6970 EFM was used with two DNA extraction methods and routine downstream STR analysis procedures. DNA from as little as 0.1 g of bone powder was used to develop full STR profiles after freezer mill pulverization, and the method was reproducible. Further, no contamination was detected upon cleaning/reuse of the sample vials. There were no significant differences in DNA yield, STR alleles detected, or peak heights using the freezer mill as compared to traditional grinding, and successful DNA profiles were achieved from as low as 0.1 g of bone powder with this method. Overall, this work indicates that this cryogenic mill method may be used as a viable alternative to traditional tissue grinders.