Optimization of the biological microchip for genotyping the AB0 locus: analytical aspects

The present work continues the search for methodological options facilitating the improvement and optimization of the biological microchip designed for genotyping the AB0 locus. It was shown in an earlier study designed to test a prototype biological microchip using a reference set of preparations with the known group specificity that under certain conditions some cells of the biochip appear to generate artifact hybridization signals that tend to make the results of genotyping either incorrect or difficult to interpret. We performed the correction of the molecular structure of DNA probes of the prototype biochip for the purpose of optimization of their hybridization potency. In addition, we developed and synthesized new DNA probes and designed new variants of the biochip. The experimental analysis of hybridization properties of all DNA probes thus obtained was carried out for the final choice of the most promising options suitable for the creation of the optimized biochip.     

Typing of ABO locus with biological microchip as a new level in solution of problems in forensic-medical biological expertise of material evidences

There are cases in practice when during expertise of material evidences, discrepancies between results of typing of ABO antigens and molecular-genetic typing of DNA occur. In this work, as a radical approach to objective solution of similar conflict situations, for some contradictory case of expertise, all examinations were performed on the unified methodological base--DNA level. Instead of biological (isoserological) typing of ABO antigen, molecular-genetic typing of ABO locus with biological microchip was performed. In all cases the results, received with the use of biological microchip, do not contradict but completely conform to the results of others molecular-genetic examinations performed in the case. Given results indicate irrationality of further use of traditional methods of isoserological typing of ABO antigen for primary differentiation of biological material. These analyses, if necessary, have to be performed on DNA level with molecular-genetic expertise.     

Optimization of recovery of human DNA from envelope flaps using DNA IQ™ System for STR genotyping

An optimized protocol based on the DNA IQ™ System has been tested for the extraction of DNA from envelope flaps. DNA is extracted directly without the need for opening and swabbing the flaps. The method is repeatable with <10% R.S.D. (relative standard deviation). The results of a systematic study show that it is an equilibrium extraction, and a small sample volume as well as high lysis buffer content in sample contribute to high extraction efficiency. The extracted DNA requires no further purification steps following its extraction with the DNA IQ™ System. Complete but skewed 15-locus short tandem repeat (STR) profiles, which is typical of degraded of DNA, have been generated from the DNA extracted from 6 to 9 years old casework envelope samples.     

Diagnostics of the AB group of AB0(H) system in saliva traces

On the basis of the mixed agglutination reaction of the saliva spots, obtained from persons with groups A, B, and AB (extractors) as well as mixtures of saliva obtained from the above subjects in various combinations, were experimentally studied. It was shown to be possible to diagnose the AB group both in the "pure" saliva traces and as an admixture of the AB group saliva to the groups A and B saliva obtained by using the anti-A and anti-B hetero-immune gemagglutinating sera. Our results confirm the previously published opinion on that antigens A and B of the ABse group saliva are positioned in the cis-acting mode, i.e. they are located on one molecule of surface structure of the epithelial (buccal) cell.     

Investigation of the between-gel and within-gel variation in fragment size determinations found when using single locus DNA probes.

Measurement variation in the sizing of DNA fragments has been assessed, examining within-gel and between-gel variability. Also, measurement variation detected between two different laboratories, using both manual and automated measurement techniques, has been investigated and discussed.     

Challenging DNA: Assessment of a range of genotyping approaches for highly degraded forensic samples

It is common in forensic casework to encounter highly degraded DNA samples from a variety of sources. In this category bone and teeth samples are often the principal source of evidential material for criminal investigations or identification of long-deceased individuals. In these circumstances standard STRs are prone to fail due to their long amplicon sizes (since DNA becomes progressively more fragmented as it degrades). To successfully resolve such cases alternative markers can be used and until recently the only other tool available was mitochondrial DNA, which despite being more resistant to degradation, is much less informative. A rapidly developing approach to analyzing degraded DNA is the typing of loci from short-amplicon PCR products based on markers such as mini-STRs and autosomal SNPs. We have performed an analysis of several cases with naturally degraded DNA using established STRs plus mini-STRs and autosomal SNPs in order to make an objective comparison of the performance of each method using challenging DNA. The main aim was to establish the benefits and drawbacks of each marker set to help the practitioner choose the DNA analysis method most suited to the circumstances of each case.     

3种DNA提取法在污染严重混合斑分型中的应用比较

目的比较Chelex-100法、酚/氯仿法和二氧化硅膜法3种DNA提取法在污染严重混合斑分型中的应用效果。方法从日常案例中收集污染严重的混合斑25份,差异消化法分离精子后同时用Chelex-100法、酚/氯仿法和二氧化硅膜技术3种方法提取DNA,采用PCR-STR技术对D19S253、FGA和CSF1PO 3个基因座进行分型,Gel-Pro软件处理电泳图谱,SPSS软件分析比较不同方法之间的差异。结果采用Chelex-100法提取DNA,25份检材分型结果均未成功;采用酚/氯仿法,25份检材中10份分型成功,3份检材FGA和CSF1PO基因座可分型,4份检材CSF1PO基因座可分型;采二氧化硅膜纯化法,25份检材均成功分型;酚/氯仿法和二氧化硅膜法两种方法比较,结果存在显著性差异(P<0.05)。结论二氧化硅膜纯化技术可以有效去除PCR抑制物,提取的DNA扩增效果明显优于Chelex-100法和酚/氯仿法,具有较高的应用价值。     

Typing of STR locus in biological objects with pronounced degeneration: analysis of DNA in exhumed remains of victims of war actions

The authors describe some specific features of enzymatic amplification typing of DNA preparations obtained from degraded tissues of remains of humans, which were brought from regions of war actions in the Chechen Republic. Special attention is paid to the specific artefact of polymerase chain reaction, for the first time detected by the authors in examinations of the above-mentioned objects. This so-called "ladder" effect manifesting by simultaneous nonspecific amplification of many variants of allele fragments of the STR locus on the individual DNA matrix, which can be erroneously interpreted as an evidence of mixed DNA preparation, is apparently characteristic of individual objects with pronounced degradation of biological material. Such phenomena were observed in typing of STR locuses in biological tissues subjected to biological, thermal, and physicohemical degradation. The phenomenon was studied in detail.     

法医DNA标准物质备选细胞STR等位基因片段长度的定值

目的 研究建立法医DNA标准物质备选细胞基因组STR基因座等位基因片段长度标准定值的方法.方法 利用有机法提取HPF和HSSM细胞基因组DNA并进行STR复合扩增,将产物进行电泳检测.利用Gene Mapper软件分析电泳结果,记录STR基因座等位基因的数值,并对目前我国公安系统应用较为广泛的DNATyperTM15、IdentifilerTM两种试剂盒扩增产物进行相应的DNA片段长度(bp)统计以及定值.结果 HPF为男性个体细胞,HSSM为女性个体细胞.HPF和HSSM细胞DNATyperTM15系统等位基因片段长度范围分别为126.26±0.05～367.53±0.20bp和125.33±0.07～370.08±0.17bp,IdentifilerTM系统等位基因片段长度范围分别为117.22±0.04～340.02±0.08bp和117.21±0.03～323.86±0.09bp.结论 对STR基因座等位基因片段长度进行标准定值,可为法医DNA标准物质提供有效的溯源途径.     

Establishing paternity using minisatellite DNA probes when the putative father is unavailable for testing

A paternity case involving a putative father who had died a few years earlier in an automobile accident was referred to the laboratory for testing. The child and his mother, the deceased's parents, and nine of the deceased's siblings were available for analysis. As previously reported, paternity testing using red blood cell groups, human leukocyte antigens (HLA), red blood cell enzymes, serum proteins, and immunoglobulin allotypes gave a cumulative paternity index of 43,300 and a combined probability of paternity equal to 99.998%. RFLP analysis using Hinf I and Sau 3A single digests and the minisatellite deoxyribonucleic acid (DNA) probes 15.1.11.4 and 6.3 showed no exclusion of paternity and gave nearly conclusive evidence that the putative father was the biological father of the child.     

压力循环技术用于人指甲DNA提取及方法学评价

目的将压力循环技术（PCT）用于指甲DNA提取,并对方法学进行评价。方法收集10份人指甲样本,剪碎约为1mm×1mm大小,采用10%漂白粉水,10%SDS,10%漂白粉水,无菌水清洗样本。10份样本各分成两组,1组用压力循环技术处理,另1组不作处理,提取DNA经复合扩增并进行STR分型检测,用于评价压力循环技术的作用。取5份指甲样本用血浸泡,5份用去离子水浸泡,之后采用上述清洗方法各清洗1-3次,收集各次清洗用的无菌水提取DNA,经STR分型检测,用于评价清洗对去除外源性DNA的效果。结果 10份经压力循环技术处理的样本中有7例比相应未经处理样本DNA提取量更高,但两组进行统计学处理,差异不具有统计学意义（P〉0.05）;两组样本中提取DNA含量在0.026 ng以上的样本均得到完整的STR分型,与相应口腔拭子样本对照准确无误。血污染和非血污染样本清洗二次以上,均可避免外源性DNA的污染。结论使用压力循环技术并配合本文清洗方法,可有效提高人指甲DNA的提取效率,并避免外源性生物DNA的干扰,保证DNA分型结果的准确。     

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.     

法医物证DNA自动化检验技术体系的研究

目的建立自动化工作站同步提取不同种类涉案法医生物检材DNA的新方法。方法选用TECAN Freedom EVO100．4、75—2型自动化提取、加样工作站,采用磁珠法及Chelex-100法对各类涉案生物检材进行DNA提取、PCR扩增、毛细管电泳检测其STR分型,进行比较测试。在“全国公安机关DNA数据库应用系统”中建立并应用实验室信息管理系统（LIMS）模拟实施规范化DNA检案。结果1552份各类检材,采用工作站-磁珠法提取DNA效果最佳,STR检测成功率为95％,工作站-Chelex法为88％;二者分别与其手工提取法比较,成功率无明显差异。92个样本同期检测,自动化工作站较手工操作DNA检案时间可缩减1．25倍。结论工作站域珠法提取涉案检材DNA,可获得满意的STR分型结果。应用LIMS管控,可有效防控污染,明显提高检案效率及鉴定质量。     

Comparison of two whole genome amplification methods for STR genotyping of LCN and degraded DNA samples

The analysis of LCN or highly degraded DNA samples presents a challenge for forensic science. Improving the quantity and/or quality of samples would greatly increase the profiling success rate from LCN and degraded samples. Whole genome amplification (WGA) is one method that has such potential. Two commercially available WGA kits, GenomePlex and GenomiPhi, were investigated for use on LCN and degraded DNA samples. Both kits amplified genomic DNA, producing microgram quantities from sub-nanogram templates. Profiling success of LCN DNA samples was increased, with improvements of over 700% from 10pg template DNA compared to non-WGA-amplified control samples. The amplification success with degraded DNA was also improved by WGA. Degraded DNA was simulated using restriction enzymes to demonstrate that the application of WGA can result in the typing of STR loci that could not previously be amplified. An increase in artefacts, such as stutter alleles and amplification biases, were observed in many samples. Results show that WGA is capable of increasing both the quality and quantity of DNA, and has the potential to improve profiling success from difficult samples in forensic casework.     

两种DNA提取法对签字笔上脱落细胞STR分型比较

目的比较Chelex-100法和硅珠法两种DNA提取法,在签字笔上附着微量脱落上皮细胞分型中的应用效果。方法 17名志愿者每人使用14支签字笔,每支笔每天使用20min,为期1个月,平均分为两组,分别保存1、3、5、7、14、21和28d,同时运用Chelex-100法和硅珠法两种方法提取签字笔上遗留微量脱落细胞中的DNA,用Identifiler复合扩增系统在AB I 3100遗传分析仪上对这些DNA样品进行STR分型,同时采集上述17名志愿者口腔拭子作为对照。结果以基因座检出个数为指标,使用后签字笔保存1、3、5、7、14、21和28d后,采用Chelex-100法和硅珠法两种方法提取DNA并进行分型检出的基因座个数相比差异均有统计学意义(P0.01);口腔拭子保存1、3、5、7、14、21和28d后,采用Chelex-100法和硅珠法两种方法提取DNA并进行分型检出的基因座个数相比差异均无统计学意义(P0.05)。结论对于微量检材,应用硅珠法提取的DNA分型效果明显优于Chelex-100法,具有较高的应用价值,而在检材量比较多时区别不明显。     

DNA mixture genotyping by probabilistic computer interpretation of binomially-sampled laser captured cell populations: Combining quantitative data for greater identification information

Two person DNA admixtures are frequently encountered in criminal cases and their interpretation can be challenging, particularly if the amount of DNA contributed by both individuals is approximately equal. Due to an inevitable degree of uncertainty in the constituent genotypes, reduced statistical weight is given to the mixture evidence compared to that expected from the constituent single source contributors. The ultimate goal of mixture analysis, then, is to precisely discern the constituent genotypes and here we posit a novel strategy to accomplish this. We hypothesised that LCM-mediated isolation of multiple groups of cells (‘binomial sampling’) from the admixture would create separate cell sub-populations with differing constituent weight ratios. Furthermore we predicted that interpreting the resulting DNA profiling data by the quantitative computer-based TrueAllele® interpretation system would result in an efficient recovery of the constituent genotypes due to newfound abilities to compute a maximum LR from sub-samples with skewed weight ratios, and to jointly interpret all possible pairings of sub-samples using a joint likelihood function.As a proof of concept, 10 separate cell samplings of size 20 recovered by LCM from each of two 1:1 buccal cell mixtures were DNA-STR profiled using a specifically developed LCN methodology, with the data analyzed by the TrueAllele® Casework system. In accordance with the binomial sampling hypothesis, the sub-samples exhibited weight ratios that were well dispersed from the 50% center value (50 ± 35% at the 95% level). The maximum log(LR) information for a genotype inferred from a single 20 cell sample was 18.5 ban, with an average log(LR) information of 11.7 ban. Co-inferring genotypes using a joint likelihood function with two sub-samples essentially recovered the full genotype information. We demonstrate that a similar gain in genotype information can be obtained with standard (28-cycle) PCR conditions using the same joint interpretation methods. Finally, we discuss the implications of this work for routine forensic practice.     

Identification of the human Y-chromosomal microsatellite locus DYS19 from degraded DNA

STR DYS19 seems to be one of the most useful markers for population genetic, evolutionary, and forensic applications. However, the authors have noticed that the amplification of the DYS19 polymorphism fails when highly degraded DNA is used as a template. The authors designed a new pair of primers that reduce the DYS19 fragment sizes compared with those of the known protocol. Using these primers, an improved success rate can be achieved, particularly when putrefied samples are under investigation.     

生物检材中DNA损伤及修复研究进展

从犯罪现场遗留的生物斑迹中提取到的DNA样本可能因为长时间暴露于恶劣的环境中而遭受到各种类型的损伤,导致扩增失败不能获得分型结果。对这些受损伤的DNA样本进行修复并提高其检测成功率,是近年来法庭遗传学领域新的研究热点。本文通过对DNA损伤的原因和类型、DNA损伤修复机制、生物检材中DNA损伤修复研究和应用进行综述,希望能对相关研究和应用提供帮助。     

生物检材DNA碱性裂解提取法的优化与改良

目的通过对DNA碱性裂解提取法进行优化和改进,建立一种操作更简便、检验快速、结果更可靠的生物检材DNA提取方法。方法以抗凝血作为检验样本,通过改变提取试剂的浓度、pH值、保存时间及孵化样本的温度、时间等条件,观测对检验结果峰值的影响,以确定提取试剂最佳条件,DNA提取物最佳保存条件。通过用改良后的碱性裂解法及Chelex100法同时提取不同量的抗凝血样本、各类法医生物检材,用不同厂家的DNA试剂盒扩增,对碱性裂解法的灵敏度、适应性和适用性进行评估。结果改良后的碱性裂解法只需将生物检材用NaOH（0.25mol/L）99℃孵化8min,振荡后加入TrisHCl（0.05mol/L,pH=6.5）中和液,离心后直接扩增检测。DNA提取物于-20℃冰箱可长期保存。对于毛发及精斑类检材优于Chelex100法。DNA提取物适用于现有各种DNA试剂盒扩增检测,其灵敏度与Chelex100法相似。结论改良后的碱性裂解法,操作简便、检验快速、结果可靠,可适合于法庭科学的检案与建库。