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.     

循环次数与体系对DNA检测灵敏度的影响

目的探讨通过增加PCR循环次数和缩小扩增体系改善DNA检测灵敏度的可行性。方法将10例无血缘关系健康志愿者精液DNA样本分别定量为50、40、30、25、20、15、10 pg/μL,分别用10、5、3μL的体系和28、30、32、34、36次循环进行扩增,用3130遗传分析仪检测15个常染色体STR基因座。结果 28次循环下,3μL体系可对40pg/μL及以上样本正确分型。20pg/μL及以上样本在10、5、3μL体系中,34次循环均可正确分型,增加到36次循环,出现非特异性谱带,无法正确分型。结论增加循环次数和缩小扩增体系在一定程度上可以提高DNA检测的灵敏度。     

Evaluation and quantification of nuclear DNA from human telogen hairs

Nuclear DNA was extracted from human telogen hairs from 60 individuals. Six to nine hairs from each individual were individually extracted. The amount of DNA recovered from each individual varied greatly, and most samples yielded a quantity of 550 pg or less per hair. A selective extraction buffer was used to remove epithelial cell DNA and the amount of exogenous DNA was determined. DNA was also quantified by real time PCR using three different sized amplicons targeting an Alu sequence. The results were used to determine the state of degradation of the extracted DNA. Different quantities of sample (<100 pg, 100-500 pg, >500 pg) were amplified with the Miniplex kits to determine the minimum DNA template required for successful amplification. DNA recovered from hair showed degradation; however, partial profiles were obtained for those samples containing at least 60 pg using MiniSTRs.     

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.     

Multiplexing of Y chromosome specific STRs and performance for mixed samples

A combination of four Y-specific polymorphic STR loci was amplified simultaneously using fluorescently labeled primers. Multiplex conditions required optimization to eliminate constant bands and amplification products for female DNA. A series of experiments was carried out for mixtures of DNA from two males, and from male and female individuals for the Y-specific STRs and an autosomal locus. For the male/male mixtures amplified with the Y specific system, and amplified for an autosomal locus, the minor component in the mixture could only be identified up to a ratio of 1:10, 1:50 respectively. In male/female DNA mixtures the Y STR alleles could be identified for the highest ratio tested, 400 pg male in DNA in 800 ng female DNA which amounts to a ratio of 1:2000.     

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.     

Optimization of a duplex amplification and sequencing strategy for the HVI/HVII regions of human mitochondrial DNA for forensic casework

A duplex primer set for the amplification of mitochondrial DNA HVI and HVII control regions was evaluated for the optimization of a DNA sequencing protocol suitable for forensic casework. HVI and HVII products, with the absence of non-specific products, could be detected by agarose gel electrophoresis when as little as 0.5 and 0.1pg of DNA were amplified for 34 and 38 cycles, respectively. Because HVI and HVII amplicons are co-synthesized in the duplex PCR, fewer steps are required (lessening the risk of cross contamination events) and more frugal use of precious extracted DNA samples is possible, both desirable features for forensic casework. The ABI Prism BigDyetrade mark version 1.1 chemistry provided high quality sequencing data, with little or no background noise and uniform peak heights, outcomes that favored reliable detection of heteroplasmy, particularly at early sequence reads (<40 bases). Optimal compromise between sensitivity and sequence accuracy in the absence of noise was achieved starting at 150 mitochondrial genome copies. The protocol is effective (no sequence errors) with highly degraded DNA (average detectable template size of 200bp). Dual artificial template mixtures with the minor component at 15% suggests that heteroplasmy should be detected at this level with confidence.     

An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA

By increasing the PCR amplification regime to 34 cycles, we have demonstrated that it is possible routinely to analyse <100 pg DNA. The success rate was not improved (without impairing quality) by increasing cycle number further. Compared to amplification of 1 ng DNA at 28 cycles, it was shown that increased imbalance of heterozygotes occurred, along with an increase in the size (peak area) of stutters. The analysis of mixtures by peak area measurement becomes increasingly difficult as the sample size is reduced. Laboratory-based contamination cannot be completely avoided, even when analysis is carried out under stringent conditions of cleanliness. A set of guidelines that utilises duplication of results to interpret profiles originating from picogram levels of DNA is introduced. We demonstrate that the duplication guideline is robust by applying a statistical theory that models three key parameters - namely the incidence of allele drop-out, laboratory contamination and stutter. The advantage of the model is that the critical levels for each parameter can be calculated. This information may be used (for example) to determine levels of contamination that can be tolerated within the strategy employed. In addition we demonstrate that interpreting one banded loci, where allele dropout could have occurred, using LR=1/2f(a) was conservative provided that the band was low in peak area. Furthermore, we demonstrate that an apparent mis-match between crime-stain and a suspect DNA profile does not necessarily result in an exclusion. The method used is complex, yet can be converted into an expert system. We envisage this to be the next step.     

Persisting fetal microchimerism does not interfere with forensic Y-chromosome typing

Forensic Y-chromosome typing applies Y-chromosomal polymorphisms to the analysis of male/female mixed stains such as vaginal swabs in rape cases. The sensitivity of this approach exceeds that of cytological techniques combined with autosomal DNA typing. Y-chromosome typing is based on the assumption that Y-chromosomal DNA found in tissue or secretions of women must originate from a male individual, usually the perpetrator. Nevertheless, it was shown recently that fetal cells can migrate into the female body during pregnancy and can persist for decades ("persisting fetal microchimerism"). The body of a woman after a pregnancy with a male embryo can thus display a small fraction of fetal cells with Y-chromosomes. Using high sensitivity PCR protocols (reamplification with nested primers and up to 60 PCR cycles) fetal cells were previously identified in a number of maternal tissues including skin, blood, muscle and solid organs. It is, however, not clear at present, whether these cells can occur in vaginal secretions, and whether they are capable of producing false positive results in forensic Y-chromosome typing. To evaluate these questions, 66 blood samples of women with at least one son and nine vaginal swabs of women without sexual intercourse in the last 2 weeks were amplified for a stretch of the SRY gene. Eight thyroid gland tissues with already established male fetal microchimerism were used as positive control samples. Blood samples of 10 young girls without history of pregnancy were used as negative controls. Using a PCR with 10 ng of extracted DNA and 30 PCR cycles ("routine sensitivity assay") none of the samples yielded positive results. However, in a PCR with 200 ng of extracted DNA and 45 PCR cycles ("high sensibility assay"), 14% of the blood samples of mothers and 33% of the vaginal swabs amplified for SRY. Our results thus show that increasing the sensitivity of the PCR method and the amount of template DNA produce positive results while protocols used for routine Y-chromosomal typing with small amounts of DNA (approximately 10 ng of DNA) and with a limited number of PCR cycles (approximately 30) can clearly eliminate this peril.     

Extended PCR conditions to reduce drop-out frequencies in low template STR typing including unequal mixtures

Forensic laboratories employ various approaches to obtain short tandem repeat (STR) profiles from minimal traces (<100 pg DNA input). Most approaches aim to sensitize DNA profiling by increasing the amplification level by a higher cycle number or enlarging the amount of PCR products analyzed during capillary electrophoresis. These methods have limitations when unequal mixtures are genotyped, since the major component will be over-amplified or over-loaded. This study explores an alternative strategy for improved detection of the minor components in low template (LT) DNA typing that may be better suited for the detection of the minor component in mixtures. The strategy increases the PCR amplification efficiency by extending the primer annealing time several folds. When the AmpF?STR^® Identifiler^® amplification parameters are changed to an annealing time of 20 min during all 28 cycles, the drop-out frequency is reduced for both pristine DNA and single or multiple donor mock case work samples. In addition, increased peak heights and slightly more drop-ins are observed while the heterozygous peak balance remains similar as with the conventional Identifiler protocol. By this extended protocol, full DNA profiles were obtained from only 12 sperm heads (which corresponds to 36 pg of DNA) that were collected by laser micro dissection. Notwithstanding the improved detection, allele drop-outs do persist, albeit in lower frequencies. Thus a LT interpretation strategy such as deducing consensus profiles from multiple independent amplifications is appropriate. The use of extended PCR conditions represents a general approach to improve detection of unequal mixtures as shown using four commercially available kits (AmpF?STR^® Identifiler, SEfiler Plus, NGM and Yfiler). The extended PCR protocol seems to amplify more of the molecules in LT samples during PCR, which results in a lower drop-out frequency.     

Quantitation of human genomic DNA through amplification of the amelogenin locus

An alternate method for quantitation of human genomic DNA is presented. Quantitative template amplification technology (abbreviated "Q-TAT") estimates the quantity of human DNA present in an extract by comparing fluorescence in X and Y amplicons produced from unknowns with fluorescence in a standard curve amplified from known quantities of reference DNA. Q-TAT utilizes PCR and electrophoresis with fluorescent detection/quantitation, precluding the need for new instrumentation, methodology, or quality assurance associated with slot-blot or real-time PCR. In a comparison study incorporating shared samples, Q-TAT was found to be more sensitive than widely used slot-blot methods but somewhat less sensitive than real-time PCR. Among samples containing DNA concentrations ranging from 100 pg/microL to 2-4 ng/microL, Q-TAT produced DNA concentration estimates that agreed reasonably well with either Quantiblot or real-time PCR. Q-TAT was reproducible with a typical coincidence of variation of about 35%. Quantitation of human DNA in this study involved summing fluorescence in X and Y amplicons in unknowns and quantitation standards. However, analyzing fluorescence in X and Y amplicons individually could allow estimates of male and female DNA present in mixtures to be made. Moreover, since X and Y amplicons exhibit sizes of 210 and 216 bp, respectively, the integrity as well as the concentration of the genomic DNA template can be assessed. Q-TAT represents an alternate method useful for the quantitation of human genomic DNA prior to amplification of STR loci used for identity testing purposes. The method uses existing equipment and procedures in conjunction with a well-characterized DNA standard to produce concentration estimates for unknowns that reliably produce STR profiles suitable for analysis.     

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 successful DNA typing of samples following a thermal cycler power loss

An approach for generating DNA profiles when critical samples have been consumed and a power outage occurs during the polymerase chain reaction (PCR) amplification reaction is described. This study demonstrates that a complete and accurate DNA short tandem repeat profile can be obtained: (1) when single source DNA samples are amplified for 26, 27, or 28 cycles using the Profiler Plus and COfiler Amplification Kits after an interruption in amplification, (2) from mock samples when PCR amplification has been interrupted early (after five cycles) or late (after 18 cycles) and the sample is subjected to an additional round of amplification, even after incubation of the sample at room temperature overnight, and (3) from nonprobative casework samples interrupted after approximately 18 cycles of amplification, an overnight incubation at room temperature and subjected to one or two additional rounds of PCR amplification for approximately 26 total cycles. Samples interrupted before five completed cycles and subjected to additional PCR cycles yielded variable results.     

A 26plex Autosomal STR Assay to Aid Human Identity Testing*†

Abstract: A short tandem repeat multiplex assay has been successfully developed with 25 autosomal loci plus the sex‐typing locus amelogenin for a total of 26 amplified products in a single reaction. Primers for the loci were designed so that all of the amplicons present were distributed from 65 base pairs (bp) to less than 400 bp within a five‐dye chemistry design with the fifth dye reserved for the sizing standard. A multiplex design strategy was developed to overcome challenges encountered in creating this assay. The limits of the multiplex were tested, resulting in the successful amplification of a wide range of genomic DNA sample concentrations from 2 ng to as low as 100 pg with 30 cycles of PCR. The 26plex has the potential to benefit the forensic community for reference sample testing and complex relationship evaluation.     

DYS385检测方法的改进

目的建立检测DYS385的新方法。方法比较基因数据库(GDB)中推荐的引物和Schneider所设计的引物扩增DYS385的效果;在此基础上,以GDB推荐的引物作为外引物,Schneider所设计的引物作为内引物,通过调整内、外引物的浓度,优化扩增条件,建立DYS385的半巢式PCR体系。结果与常规方法相比,采用Schneider所设计的引物扩增DYS385,扩增片段缩短112bp,电泳分离的效果好,灵敏度提高2倍,达500pg;建立的半巢式扩增方法,能特异性扩增短片段,灵敏度提高20倍,达50pg。结论建立的DYS385半巢式扩增方法具有更高的特异性和灵敏度,适合法医学应用。     

PCR扩增循环数与低拷贝模板DNA的STR分型

目的探讨PCR扩增循环数对低拷贝模板DNA的STR分型的影响。方法模板DNA(9947A)的不同扩增用量(含低拷贝模板量),采用ProfilerPlus试剂盒,扩增循环数分别为28、30、32、34、38次,3100型基因分析仪(ABI,美国)检测结果。结果循环数从28次增至38次,模板DNA量最低检出量可从0.25ng减少至0.0312ng;先循环28次后,每反应加0.3μlAmpliTaqGoldDNA聚合酶,再循环6次较1次34个循环的检测灵敏度高,相当于1次38个循环的效果。结论增加PCR扩增循环数,可能影响低拷贝模板DNA的STR分型。     

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。     

PEP-PCR法及其在法医学中应用的可行性研究

提高微量检材的利用率 ,建立PEP方法并对其法医学应用进行可行性研究。用 15个随机寡核苷酸的序列作为引物 ,低特异性下扩增出微量DNA ,以此扩增物作为模扳 ,进行特异基因座扩增。经PMCT118,GABAR ,DYS19,D18S849,D3S1744,D12S10 90 ,D8S1179,D2 1S11,D18S5 1,D5S818,D13S3 17,D7S82 0 ,D3S13 5 8,vWA ,FGA ,TH0 1,CSF1PO ,TPOX ,D16S5 3 9及Amelogene等 2 0个基因座的PEP PCR与直接的PCR分析比较 ,二者分型结果一致 ,即使 1ngDNA也能得到正确分型。PEP方法可用于法医学检验 ,有利于微量物证的利用 ,使物证检材提供尽可能多的信息     

A systematic approach to improve downstream single-cell analysis for the DEPArray™ technology

Most commercially available STR amplification kits have never been fully validated for low template DNA analysis, highlighting the need for testing different PCR kits and conditions for improving single-cell profiling. Here, current strategies rely mainly on adjusting PCR cycle number and analytical threshold settings, with a strong preference for using 30 amplification cycles and thresholds at 30–150 RFU for allele detection. This study aimed to (1) determine appropriate conditions for obtaining informative profiles utilizing a dilution series, and (2) test the outcome on single cells using the DEPArray™ technology. Four routinely applied forensic STR kits were compared by using three different amplification volumes and DNA dilutions down to 3.0 pg, while two well-performing kits were used for single/pooled leucocyte and sperm cell genotyping. Besides reduced costs, the results demonstrate that a 50%–75% PCR volume reduction was beneficial for peak height evaluation. However, this was counteracted by an increased artifact generation in diluted DNA volumes. Regarding profile completeness, the advantage of volume reduction was only prominent in samples processed with Fusion 6C. For single and pooled cells, ESIFast and NGMDetect provided a solid basis for consensus profiling regarding locus failure, although locus dropouts were generally observed as stochastic events. Amplification volume of 12.5 μL was confirmed as appropriate in terms of peak heights and stutter frequencies, with increased stutter peaks being the main artifact in single-cell profiles. Limitations associated with these analyses are discussed, providing a solid foundation for further studies on low template DNA.     

Typing of deoxyribonucleic acid (DNA) extracted from compact bone from human remains.

The application of deoxyribonucleic acid (DNA) typing methods for the potential identification of unknown human remains was investigated. DNA was isolated from compact bone tissue from badly decomposed bodies and from known and unknown human remains, using a decalcification and ion wash procedure. Restriction fragment length polymorphism (RFLP) analysis of variable number of tandem repeats (VNTR) loci yielded results in some cases, but more often the DNA was too degraded to produce RFLP patterns. No RFLP profiles could be obtained from putrefied soft tissues. However, DNA extracted from compact bone tissue of human remains up to eleven years old was successfully amplified using the polymerase chain reaction (PCR) for the VNTR loci D1S80, D17S5, COL2A1, and APO B, as well as the HLA-DQ alpha locus. This is especially significant, since PCR results were obtained from those samples whose DNA had been degraded substantially and had yielded no RFLP patterns. All DNA types determined from the compact bone tissue from decomposed bodies whose identification had been established first by other means (and whose parents or offspring were available for typing) demonstrated mendelian inheritance of the alleles of the loci analyzed. These results suggest that amplification and typing of DNA extracted from compact bone of human remains could be useful in establishing the identity of a person, as well as in excluding possible false identifications.