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.     

Recovery of DNA and fingerprints from touched documents

This study investigated the various factors affecting DNA profiling from DNA recovered from fingerprints deposited on paper before and after fingerprint enhancement treatments. The DNeasy^® plant mini kit (QIAGEN^®) was found to improve DNA recovery from paper by over 150% compared with the QIAamp^® mini kit. A significant decrease in the amount of DNA recovered was observed following treatment with DFO and/or Ninhydrin. This decrease in yield did not have a comparably significant effect on the quality of the SGM Plus™ profiles. Furthermore, this study found that whilst certain paper types, such as newspaper, magazine and filter paper allowed for the good recovery of DNA, common office paper and white card, strongly interfered with the recovery of DNA resulting in poor quality profiles.     

Comparison of five DNA quantification methods

Six commercial preparations of human genomic DNA were quantified using five quantification methods: UV spectrometry, SYBR-Green dye staining, slot blot hybridization with the probe D17Z1, Quantifiler™ Human DNA Quantification kit and RB1 rt-PCR. All methods measured higher DNA concentrations than expected based on the information by the manufacturers. UV spectrometry, SYBR-Green dye staining, slot blot and RB1 rt-PCR gave 39, 27, 11 and 12%, respectively, higher concentrations than expected based on the manufacturers’ information. The DNA preparations were quantified using the Quantifiler™ Human DNA Quantification kit in two experiments. The measured DNA concentrations with Quantifiler were 125 and 160% higher than expected based on the manufacturers’ information. When the Quantifiler™ human DNA standard (Raji cell line) was replaced by the commercial human DNA preparation G147A (Promega) to generate the DNA standard curve in the Quantifiler™ Human DNA Quantification kit, the DNA quantification results of the human DNA preparations were 31% higher than expected based on the manufacturers’ information. The results indicate a calibration problem with the Quantifiler™ human DNA standard for its use with the Quantifiler™ Human DNA Quantification kit. The possible reasons for the problem are discussed and a solution is suggested. The results emphasise the need for standard reference DNA material and standard methods for DNA quantification.     

The development of visual and chemical methods for predicting the likelihood of obtaining a DNA profile from degraded bone samples

Gross morphology, histology and nitrogen content were examined in bone samples from individuals that had been buried for approximately 12 years in Kuwait and Iraq. The results indicate that the gross morphology and histology are useful indicators of DNA survival. Nitrogen content did not show a significant correlation with DNA preservation.     

Single primer extension (SPEX) amplification to accurately genotype highly damaged DNA templates

High levels of non-authentic sequence data can be generated by traditional PCR-based methodologies when DNA is damaged, template numbers are small and/or the target amplification size too large. We therefore present an alternate methodology based on single primer extension (SPEX) amplification; that places no pre-defined size constraints on amplification and interacts with only one of the DNA strands at the target locus.     

法医线粒体DNA检测的系统效能及应用价值

线粒体DNA（mitochondrial DNA，mtDNA）遗传标记的检测在法医学实践中有着重要的价值。线粒体DNA异质性的出现，对检测体系的系统效能存在着影响，主要表现在对所检测样本的单倍型的认同及个体识别概率的计算上。通过建立mtDNA数据库，找出异质性在人群中的发生频率，并使检测异质性的方法标准化．对线粒体DNA的更有效检测具有重要意义；线粒体DNA编码区多态性位点在提高线粒体DNA系统效能中具有独到之处。因此，更进一步地进行mtDNA编码区多态性的探索．将进一步提高线粒体DNA检测的系统效能。     

Following the transfer of DNA: How far can it go?

The issue of DNA transfer is becoming increasingly important in crime scene situations, as DNA analytical techniques now detect tiny amounts. Whereas primary and secondary DNA transfers have been well studied, subsequent transfer steps have received much less focus. This study aimed to measure the detectability of a DNA source after multiple transfer events. Transfer of wet blood gave a full genetic profile well beyond the secondary transfer events on both cotton and glass substrates. Dry blood gave a full profile well beyond the secondary transfer events on glass only, but to a lesser extent than wet blood. Touch DNA only produced a full profile on the primary substrate on both cotton and glass, and detectable quantities beyond the secondary transfer event on glass only. Our results will contribute to a better understanding of the tertiary and subsequent transfer of DNA, which will allow for improved evaluation of the likelihood of alternative scenarios explaining why an individual's DNA was found at a crime scene.     

STR基因座与Amelogenin的峰面积比在DNA降解评估中的应用

目的研究DNA降解过程中STR基因座与性别基因座Amelogenin(AMEL)峰面积比(STR/AMEL)的变化规律,探讨STR/AMEL值在评估DNA降解程度中的应用。方法取人体髂腰肌组织抽提DNA,分析DNaseⅠ酶人工降解后STR/AMEL值(Penta E/AMEL、Penta D/AMEL、FGA/AMEL)的变化,并分析室外环境自然降解的髂腰肌组织中三者比值的变化。以降解时间为自变量(x),STR/AMEL值为因变量(y),进行回归曲线分析,建立两种条件下三组曲线方程。结果人工降解及自然降解条件下STR/AMEL值与降解时间均呈负相关,一元三次方程能够较好地模拟STR/AMEL值随降解时间的变化规律。人工降解条件下,R~2均大于0.99;自然降解条件下,R~2均大于0.86。结论 STR/AMEL值(Penta E/AMEL、Penta D/AMEL、FGA/AMEL)与DNA降解程度呈负相关,有望应用于DNA降解程度的评估。     

法医DNA鉴定结论的局限性及审查判断

DNA鉴定解决了困扰司法鉴定多年的、以生物检材进行人身同一认定的技术难题。但DNA鉴定技术本身存在着一定的局限性，主要是：不同个体的DNA结构有偶然重合的可能性、DNA鉴定的高灵敏度增加了污染的可能性、全国范围内DNA鉴定水平参差不齐。因此，DNA鉴定结论如同其他鉴定结论一样，只是普通的证据，并非“铁证”，盲目迷信DNA鉴定，具有危险性，DNA鉴定需要接受审查判断。文章就此些问题作了相关论述。     

3株减蛋综合征病毒毒株核酸酶切图谱分析

选用EcoRⅠ、BamHⅠ、HindⅢ、EcoRⅤ、PvuⅡ、BglⅠ、SmaⅠ和PstⅠ等8种限制性内切酶,对不同地区分离的3株减蛋综合征(EDS)病毒进行酶切图谱比较.结果发现3株毒用前6种酶切割后产生的片段数及各片段的大小均相同,而用SmaⅠ和PstⅠ切割后,贵州分离毒HS-1与国际标准毒AV-127、南京分离毒GC2相比,多出1个片段.表明不同地区EDS分离毒的核酸结构基本相同而又略有差异.