Simplified low-copy-number DNA analysis by post-PCR purification

Frequently, evidentiary items contain an insufficient quantity of DNA to obtain complete or even partial DNA profiles using standard forensic gentotyping techniques. Such low-copy-number (LCN) samples are usually subjected to increased amplification cylces to obtain genetic data. In this study, a 28-cycle polymerase chain reaction (PCR) was used to evaluate various methods of post-PCR purification for their effects on the sensitivity of fluorophore-based allelic detection subsequent to capillary electrophoretic separation. The amplified product was purified using filtration, silica gel membrane, and enzyme mediated hydrolysis purification techniques and evaluated for their effect on fluorescent allelic signal intensity. A purification method was selected and its effect on fluorescent allelic signal intensity was compared with that of the unpurified PCR product. A method of post-PCR purification is described which increases the sensitivity of standard 28-cycle PCR such that profiles from LCN DNA templates (<100 pg DNA) can be obtained. Full DNA profiles were consistently obtained with as little as 20 pg template DNA without increased cycle number. In mock case type samples with dermal ridge fingerprints, genetic profiles were obtained by amplification with 28 cycles followed by post-PCR purification whereas no profiles were obtained without purification of the PCR product. Allele dropout, increased stutter, and sporadic contamination typical of LCN analysis were observed; however, no contamination was observed in negative amplification controls. Post-PCR purification of the PCR product can increase the sensitivity of capillary electrophoresis to such an extent that DNA profiles can be obtained from <100 pg of DNA using 28-cycle amplification.     

Mini-STRs: A powerful tool to identify genetic profiles in samples with small amounts of DNA

Degraded human remains and crime scene evidences with small amounts of DNA typically reveal incomplete or null genetic profiles when using standard (large) STR amplicons. The technology of mini-STRs, using reduced-size STR amplicons, can help to recover information from these samples. In our Forensic Genetic Service several genetic profiles were obtained or completed using MiniFiler kit (Applied Biosystems) increasing the success rate in sample typing. In all studied cases no inconsistencies were found between profiles obtained with MiniFiler and Identifiler, suggesting that this mini-STR kit can be used to include low copy number (LCN) evidence profiles in STR databases.     

Short tandem repeat (STR) genotyping of keratinised hair. Part 2. An optimised genomic DNA extraction procedure reveals donor dependence of STR profiles

A feasibility study of short tandem repeat (STR) genotyping of telogen phase hairs in particular, and hair shaft in general, is presented. A number of extraction procedures in common use were investigated and the quantities of nuclear DNA (nuDNA) delivered were quantified via a real-time PCR assay. The extracts were subjected to two variations on AmpFlSTR Profiler Plus PCR amplification strategies (extended cycles, two rounds of PCR) and the genotypes compared. Nuclear DNA was found to persist in human hair shafts, albeit at very low levels. Full Profiler Plus profiles consistent with the hair donor were obtained from 100 mg hair shaft samples (bleached and unbleached). These were, however, mixed profiles, indicating low copy number (LCN) contamination in the extracts. Single telogen hair clubs and single hair shafts delivered partial profiles with usually only one allele of heterozygous loci. Telogen phase hairs yielded the same amount of nuDNA (and no more) as hair shafts (either anagen or telogen). Whether hair shafts dissolved or not in lysis buffer had no effect on either the quantitated yield of DNA or on the chance of obtaining a correct genotype. These results provide evidence that genomic DNA resides on the exterior of the hair shaft and we use this information to suggest an optimal procedure for nuDNA extraction from keratinised hair samples: soaking hairs in simple digestion buffers containing Tris-HCl, a salt and a chelating agent without prior cleaning of the hair shafts. It is proposed that cleaning removes most of the recoverable DNA. This procedure was applied to obtain genotypes from 3 cm hair shafts which matched reference profiles from the donors at up to 9 out of 10 AmpFlSTR Profiler Plus STR loci. When the genotyping success was measured by counting the number of matches between the two dominant alleles at each locus for each extract with the reference DNA profile of the hair donor, the success was found to be highly dependent on the donor. The number of matching alleles varied between not less than 10 for one donor to no more than two for another donor. These results may well be linked to the environmental experience of the hairs from each donor prior to removal.     

The application of ultraviolet irradiation to exogenous sources of DNA in plasticware and water for the amplification of low copy number DNA

Using high sensitivity forensic STR polymerase chain reaction (PCR) typing procedures, we have found low concentrations of DNA contamination in plasticware and water assumed to be sterile, which is not detected by standard DNA procedures. One technique commonly used to eliminate the presence of DNA is ultraviolet (UV) irradiation; we optimized such a protocol used in the treatment of water, tubes, plates, and tips for low copy number DNA (LCN) amplification. UV light from a Stratalinker((R)) 2400 was administered to 0.2, 1.5 mL tubes, and PCR plates contaminated with up to 500 pg of DNA. They were subsequently quantified with an ALU-based real-time PCR method using the Rotorgene 3000. Overall, there was a decrease in concentration of DNA recovered as the duration of treatment increased. Nonetheless, following 45 min of irradiating a PCR plate with 500 pg of DNA, nearly 6 pg were still detected. However, when the plate was raised within an inch of the UV source, less than 0.2 pg of DNA was detected. Additionally, lining the area around the samples with aluminum foil further reduced the amount of time necessary for irradiation, as only 30 min eliminated the presence DNA in the raised PCR plate. Similar experiments were conducted using tubes filled with a solution of DNA and water in equivalent concentrations for 50, 15, and 1.5 mL tubes with comparative results. It is plausible that the aluminum foil increased the amount of reflection in the area thereby enhancing penetration of UV rays through the walls of the plasticware. This protocol was tested for the possibility of inhibitors produced from irradiation of plastic tubes. As our protocols require less irradiation time than previous studies, PCR sensitivity was not affected. Moreover, the lifespan of the UV lamps was extended. Our findings demonstrate that this method is useful as an additional precautionary measure to prevent amplification of extraneous DNA from plasticware and water without compromising the sensitivity of LCN DNA amplifications.     

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.     

A systematic analysis of PCR contamination.

In light of the strict legal scrutiny surrounding DNA typing at this time, it has become necessary to systematically address the issue of PCR contamination. To precisely define the parameters affecting PCR contamination under casework analysis conditions, PCR amplification reactions were intentionally compromised by employing sub-standard laboratory technique and by introducing secondary sources of DNA. The PCR parameters considered for potential sources of contamination include amplification set-up, amplification product handling, aerosol DNA and storage. In addition, analyst technique was evaluated by modifying or eliminating standard safeguards. Under the circumstances normally encountered during casework analysis, PCR contamination was never noted. Significantly, using the dot blot detection method, contamination was never observed when nanogram quantities of genomic DNA were mishandled or aerosolized. Contamination occurred only when amplification product was carelessly manipulated or purposefully sprayed near or directly into open tubes containing water or genomic DNA. Although standard precautions should be employed during PCR-based DNA typing, our data indicates that contamination during amplification procedures is not prevalent when detected by dot blot analysis.     

STR genotyping and mtDNA sequencing of latent fingerprint on paper

A systematic study was conducted to investigate whether DNA can be successfully extracted from latent fingerprints deposited on ordinary paper and analysed using short tandem repeat profiling and mitochondrial DNA sequencing. In order to evaluate the performance of latent fingerprint analysis in a criminal case, experiments with varying conditions were carried out to improve our understanding of low copy number (LCN) DNA typing. After optimising the extraction methods to achieve increased sensitivity, the examination of touched paper can routinely yield the STR profile of the individual who has touched it. A fingerprint can therefore be considered as a potential source of DNA for genetic identification. Nevertheless, the findings of our "after enhancement experiment" (using chemically or physically pre-treated fingerprints), and our "mixture experiment" (using fingerprints from three to four people on the same sheet of paper) help to define the limitations of the low copy number PCR technique in forensic casework.     

The probability of achieving full allelic representation for LCN-STR profiling of haploid cells

Modern forensic techniques allow DNA to be extracted from ever decreasing amounts of cellular material. Low copy number (LCN) profiling enables the production of STR profiles from small numbers of cells. Moreover, methods such as laser micro-dissection enables forensic scientists to potentially isolate individual cells for PCR. The DNA derived from haploid cells (semen) is a common source of forensic evidence in sexual assault cases. Haploid cells contain only half the DNA complement of diploid cells (3 pg compared to 6 pg). The smaller the number of cells sampled, the smaller the probability that there is a full representation of the alleles comprising the donor profile. This paper investigates the relationship between the number of cells sampled and the probability of full representation of all alleles in the donor sample. It also considers the effect of typing several loci as opposed to just a single locus.     

Direct comparison of post-28-cycle PCR purification and modified capillary electrophoresis methods with the 34-cycle “low copy number” (LCN) method for analysis of trace forensic DNA samples

The investigation of samples with low amounts of template DNA remains at the forefront of forensic DNA research and technology as it becomes increasingly important to gain DNA profile information from exceedingly trace levels of DNA. Previous studies have demonstrated that it is possible to obtain short tandem repeat (STR) profiles from <100 pg of template DNA by increasing the number of amplification cycles from 28 to 34, a modification often referred to as “low copy number” or LCN analysis. In this study, we have optimised post-PCR purification techniques applied after only 28 cycles of PCR, as well as using modified capillary electrophoresis injection conditions and have investigated the progressive application of these enhanced approaches. This paper reviews the characteristics of the profiles obtained by these methods compared with those obtained on the same samples after 34-cycle PCR. We observed comparable sensitivity to 34-cycle PCR in terms of the number of profiles with evidence of DNA and the number of allelic peaks per profile and we noted improved peak height and area magnitude with some sample types. Certain parameters reported to be adversely affected in 34-cycle LCN investigations, such as non-donor allele peaks and increased stutter peak ratio, were reduced by this approach. There are a number of advantages for trace samples in progressing from the standard 28-cycle process to the post-PCR processing method as compared to 34-cycle PCR method, including reduced sample consumption, reduced number of PCR amplifications required, and a staged approach to sample processing and profile interpretation.     

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.     

An Optimized DNA Analysis Workflow for the Sampling,Extraction, and Concentration of DNA obtained from Archived Latent Fingerprints

DNA profiles have been obtained from fingerprints, but there is limited knowledge regarding DNA analysis from archived latent fingerprints—touch DNA “sandwiched” between adhesive and paper. Thus, this study sought to comparatively analyze a variety of collection and analytical methods in an effort to seek an optimized workflow for this specific sample type. Untreated and treated archived latent fingerprints were utilized to compare different biological sampling techniques, swab diluents, DNA extraction systems, DNA concentration practices, and post‐amplification purification methods. Archived latent fingerprints disassembled and sampled via direct cutting, followed by DNA extracted using the QIAamp® DNA Investigator Kit, and concentration with Centri‐Sep? columns increased the odds of obtaining an STR profile. Using the recommended DNA workflow, 9 of the 10 samples provided STR profiles, which included 7–100% of the expected STR alleles and two full profiles. Thus, with carefully selected procedures, archived latent fingerprints can be a viable DNA source for criminal investigations including cold/postconviction cases.     

Effects of storage time on DNA profiling success from archived latent fingerprint samples using an optimised workflow

Due to recent improvements in forensic DNA testing kit sensitivity,there has been an increased demand in the criminal justice community to revisit past convictions or cold cases.Some of these cases have little biological evidence other than touch DNA in the form of archived latent fingerprint lift cards.In this study,a previously developed optimised workflow for this sample type was tested on aged fingerprints to determine if improved short tandem repeat(STR)profiles could be obtained.Two-year-old samples processed with the optimised workflow produced an average of approximately five more STR alleles per profile over the traditional method.The optimised workflow also produced detectable alleles in samples aged out to 28 years.Of the methods tested,the optimised workflow resulted in the most informative profiles from evidence samples more representative of the forensic need.This workflow is recommended for use with archived latent fingerprint samples,regardless of the archival time.     

全基因组扩增法应用于低拷贝数DNA检测

目的建立基于多重置换扩增(MDA)技术的全基因组扩增(WGA)方法,实现对低拷贝数(LCN)DNA样品进行分析。方法采用REPLI-g试剂盒对样本进行等温全基因组扩增,扩增产物采用ProfilerPlus试剂盒确定样本的十个STR基因座的等位基因型。结果10pgDNA模板经全基因组扩增后,能够进行DNA分型。结论全基因组扩增可以用于LCN的DNA分析,帮助提高微量物证的检出成功率。     

汗潜指印的STR分型检测在案件中的应用

目的探索案例中所涉及的汗潜指印DNA的提取和检验方法。方法采用Chlex-100法提取DNA,进行STR复合扩增,通过毛细管电泳检测荧光信号。结果案例中涉及的汗潜指印在ProfilerPlus试剂盒10个基因座的分型检测均获成功。结论含有汗潜指印的检材的发现和正确提取对最终检测成功至关重要。     

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。     

Comparisons of protocols for enhanced DNA profile quality from FTA®-deposited urine samples

Disputes over the identity of a urine sample donor have been reported, and urine authentication by genetic profiling has helped resolved the cases. However, since genotyping of urine is not always required, many drug-testing laboratories may face sample storage issues. Several studies have investigated the use of FTA® cards as a convenient tool for keeping specimen at room temperature for extended periods of time. However, generating complete STR profile from some FTA®-deposited urine samples remains challenging due to low levels of genetic material content, necessitating amendments to the laboratory’s standard protocols. This work therefore aims to evaluate the effects of two DNA template preparation methods, both employing FTA® cards as the storage medium, on the success rates of STR profiling from urine. Specimen from a female volunteer, representing a particularly low-yield sample, was employed. Aliquots of 1 and 2 mL were used as the starting material to evaluate DNA template preparation using the FTA® manufacturer’s protocol for disc purification against elution of DNA from the FTA® using Prepfiler™ Forensic DNA Extraction Kit. AmpFℓSTR™ Identifiler™ Plus PCR Amplification Kit was used to amplify the STR markers, and the PCR products were analysed using Applied Biosystems™ 3500xL Genetic Analyzer. The DNA profile qualities were examined in terms of number of loci detected and peak height balance. Comparisons with the profiles obtained from DNA isolated using QIAamp® DNA Micro Kit from 1 and 2 mL of the same batch of urine were also made. The optimised protocol was then tested on urine samples from three male volunteers. The results showed that the purification of FTA® punches according to the manufacturer’s protocol enabled full DNA profiles to be obtained from both 1 and 2 mL of urine from all samples tested, including male samples. In contrast, no DNA profile could be generated from the DNA eluted with the Prepfiler™ kit. When compared with the more conventional solid-phase DNA extraction method, the profiles generated from the FTA® punches exhibited similar reproducibility and quality to those from the template isolated by the QIAamp® Kit. This work further demonstrated the feasibility of FTA® cards as a tool for specimen storage and DNA template preparation from small volumes of urine for authentication by STR profiling. Full STR profiles could be generated from sample from both sexes without modification of the PCR conditions or injection time.     

8种方法显现的汗潜指印STR分型研究

目的研究常见指印显现方法对指印STR检验的影响。方法采用Invisorb spin forensic试剂盒提取纯化人汗潜指印DNA,低拷贝模板(LCN)STR复合扩增,荧光电泳检验。结果用铜粉、铝粉、荧光粉、黑磁粉、"502"胶、茚三酮、磺酸双三嗪荧光显色液显现的玻片、纸张和胶带纸粘面上的汗潜指印可成功进行STR分型。结论常见指印显现方法不影响指印STR检验。     

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.     

Optimising direct PCR from anagen hair samples

Anagen hairs are in the active growth phase, and when forcefully removed, may contain an intact root or sheathing. The hair root or sheathing is a source of nucleic DNA and can be amplified using direct PCR. Human identification STR kits are optimised to a small range of input DNA for PCR. Anagen hairs are unable to be quantified prior to amplification and can exhibit characteristics of an over-loaded DNA sample when analysed. The aim of this study was to optimise direct PCR for anagen hair sampling. Two separate modifications to the downstream processes were carried out in order to determine the most effective method at minimising PCR artefacts. Decreasing the cycle number from the standard 29 cycles to 27 cycles when using the NGM™ kit displayed the best results for this method. However, decreasing the cycle number may increase allelic drop-out and would be costly for laboratories to perform an in-house validation. Diluting the PCR product during electrophoresis analysis minimises the effects of PCR artefacts in the same way decreasing the cycle number does. Diluting the PCR product is the most cost-effective method and does not increase the chance of allelic drop-out.     

A comparison of the characteristics of profiles produced with the AMPFlSTR SGM Plus multiplex system for both standard and low copy number (LCN) STR DNA analysis.

DNA STR profiles have been generated from 1 ng and low copy number (LCN) templates using 28 and 34 cycles of amplification, respectively. Characteristics which facilitate the interpretation of profiles, such as heterozygous balance, allelic dropout and stutter proportions have been quantified. We demonstrate that a reduction in DNA template coupled with an increase in amplification cycle number produces an increased rate of allelic dropout out which can be correlated to the peak areas of those alleles observed. In addition, the LCN conditions increase the degree of peak area asymmetry observed from heterozygotes and the size range of stutters. Analysis of the data allows us to develop sets of guidelines appropriate for interpreting both single and mixed DNA profiles.