6FAM-HEX-TMR-ROX四色荧光分析体系的构建

目的为在310基因分析仪上进行6FAM-HEX-TMR-ROX四色荧光STR复合扩增分型建立基础条件。方法以pGEM载体作为靶DNA,设计引物扩增获得500~80bp的13个长度不等DNA片段,以这些片段纯化物作为模板DNA,分别扩增获得6FAM、HEX、TMR和ROX标记的M atrix标准物,用4种M atrix标准物在310基因分析仪上构建可用于6FAM-HEX-TMR-ROX四色荧光分析的M atrix文件。结果扩增所得的13个非标记片段,长度分别为80、100、120、140、160、180、200、220、260、300、340、400、500bp,在非变性聚丙烯酰胺凝胶连续电泳-银染凝胶上均表现为单条带,6FAM、HEX、TMR、ROX分别标记的片段通过310基因分析仪检测均为单峰。混合ROX标记的80、120、180、220、260、300bp的6个片段获得的内标,显示出良好的线性关系。结论建立了有效的6FAM-HEX-TMR-ROX四色荧光分析M atrix,为进行多个STR基因座荧光标记复合扩增检测奠定了基础。     

基于分子克隆方法制备标准分子量片段混合物

目的制备标准分子量DNA片段混合物。方法选取pMD18-T载体部分序列作为插入片段,设计引物,制备包含不同大小的标准分子量片段的克隆。大量培养细菌提取质粒,用双酶切、连接荧光接头的方法制备不同大小的带有荧光的标准分子量片段,混合,纯化,最终获得标准分子量片段混合物(内标)。结果用分子克隆方法制备出具有实用性的标准分子量片段混合物,其单个标准分子量片段大小分别为80bp、124bp、194bp、224bp、254bp、304bp、349bp、399bp、424bp、454bp。并且这些标准分子量片段混合物可以准确地对DNATyper15试剂盒的扩增产物进行检测。结论应用该方法制备了能满足研究和实验室要求的标准分子量片段混合物,为DNA分子量标准物标准品的制作提供了一种新的方法。     

Biostatistical basis of individualization and segregation analysis using the multilocus DNA probe MZ 1.3: results of a collaborative study.

A collaborative study using the multilocus minisatellite DNA probe MZ 1.3 was carried out to investigate segregation information, mutation rate, DNA fragment frequencies as well as band sharing characteristics. The fingerprint patterns of 393 children as well as 694 unrelated individuals were analysed after digestion of DNA with the restriction enzyme HinfI. A mutation rate of 1% per meiosis or 0.04% per band was found with a mean number of 26 bands/individual. It was shown that maternal and paternal fragments are inherited in equal proportions. Population frequencies of restriction fragments demonstrated a distribution with increasing frequencies in the small fragment size range below 10 kb as well as the absence of very common or very rare fragments. Our data can be used to calculate simple exclusion probabilities based on the number of non-maternal bands in the child.     

Report of a European collaborative exercise comparing DNA typing results using a single locus VNTR probe

A collaborative exercise was carried out in 1989 among 12 European forensic laboratories using the single locus VNTR probe pYNH24, the restriction enzyme HinfI, the same set of human genomic DNA samples, and a standardized DNA size marker. The objectives of the exercise were: (1) to study the degree of variation within and between laboratories, (2) to obtain information on requirements for technical standardization allowing the exchange of typing results and (3) to compare different approaches for the identification of allelic DNA fragments of unknown size. Each laboratory carried out up to 10 independent typing experiments using the same DNA samples. The results were analysed independently by two laboratories using three different methods. The results of the exercise demonstrate the correlation of typing that can be achieved within and between laboratories under conditions of minimal standardization.     

RAPD和ISSR分子标记检测大麻的遗传多样性初探

目的利用随机扩增多态性DNA和简单序列重复区间扩增分子标记检测大麻遗传多样性,并探讨其在法医学中的应用价值。方法收集中国4省6个地区的100株大麻叶子样品,采用CTAB法提取基因组DNA,设计选择11个RAPD引物和13个ISSR引物,采用6%中性聚丙烯酰胺凝胶电泳-硝酸银染色法进行检测,根据出现的条带数目和片段大小等分析大麻的多样性。结果 11条RAPD引物扩增出的片段在200bp以上共52条,其中具有多态性的27条;ISSR引物扩增出126条,其中具有多态性的73条;多态性条带比率分别为51.9%和57.9%,其差异不具有统计学意义(P>0.05)。结论 RAPD和ISSR两种方法均可用于大麻遗传多样性分析,对检测毒品原植物的种类和来源地具有一定的应用前景。     

Discrimination between human and animal DNA: application of a duplex polymerase chain reaction to forensic identification

Identification of a report's species is one of the basic analyses in forensic laboratories. The authors report the case of 6 bone fragments recovered in a wooded area, which were not attributable to 1 animal species on the basis of morphologic examination. The aim of this study was to develop a duplex polymerase chain reaction (PCR) to discriminate human and animal origin of bone fragments. The method is based on the PCR amplification of cytochrome b and a 16S ribosomal mitochondrial DNA fragment, which has never been tested up to now. Our protocol combines a single-round PCR with direct visualization of amplicons in agarose gel, without sequencing analysis of the PCR products. The presence of a single band (359 bp) indicates a nonhuman origin of the sample, whereas 2 bands (157 and 359 bp) indicate a human biologic sample.This method revealed to be useful for forensic purposes because the 16S ribosomal mitochondrial DNA is a small human-specific fragment that is easily amplifiable even with degraded DNA from biologic materials such as old bones.     

A molecular genetic approach for forensic animal species identification

This study investigated potential markers within chromosomal, mitochondrial DNA (mtDNA) and ribosomal RNA (rRNA) with the aim of developing a DNA based method to allow differentiation between animal species. Such discrimination tests may have important applications in the forensic science, agriculture, quarantine and customs fields. DNA samples from five different animal individuals within the same species for 10 species of animal (including human) were analysed. DNA extraction and quantitation followed by PCR amplification and GeneScan visualisation formed the basis of the experimental analysis. Five gene markers from three different types of genes were investigated. These included genomic markers for the beta-actin and TP53 tumor suppressor gene. Mitochondrial DNA markers, designed by Bataille et al. [Forensic Sci. Int. 99 (1999) 165], examined the Cytochrome b gene and Hypervariable Displacement Loop (D-Loop) region. Finally, a ribosomal RNA marker for the 28S rRNA gene optimised by Naito et al. [J. Forensic Sci. 37 (1992) 396] was used as a possible marker for speciation. Results showed a difference of only several base pairs between all species for the beta-actin and 28S markers, with the exception of Sus scrofa (pig) beta-actin fragment length, which produced a significantly smaller fragment. Multiplexing of Cytochrome b and D-Loop markers gave limited species information, although positive discrimination of human DNA was evident. The most specific and discriminatory results were shown using the TP53 gene since this marker produced greatest fragment size differences between animal species studied. Sample differentiation for all species was possible following TP53 amplification, suggesting that this gene could be used as a potential animal species identifier.     

考古样品中Amelogenin同源基因的提取和检测

目的利用人类性染色体Amelogenin同源基因在X、Y染色体上序列长度的差异,选择设计引物,对考古样本进行古DNA性别信息研究。方法采用苯酚/氯仿-二氧化硅-超滤离心方法提取东岭墓葬群殉人骨骼、牙齿古DNA,PCR扩增,非变性聚丙烯酰胺凝胶电泳(PAGE)分离和检测古DNA扩增片段。结果8个墓葬16个样品中有7个样品出现阳性扩增检测,目标基因片段清楚,男性为二条带(X、Y),女性为一条带(X),牙齿样品检测成功率优于骨骼样品。结论改进的苯酚/氯仿—二氧化硅—超滤分离法是较好的古DNA提取方法,具有降低PCR抑制剂、消耗成本低和提取成功率高等特点。基于人类X、Y染色体Amelogenin同源基因的古DNA性别分析方法可成为分子考古重要的技术方法。     

Internal validation of the 7500 Real Time PCR System for use in forensic casework in Hellenic Police

According to guidelines of the quality assurance standards for the forensic DNA testing laboratories, prior to introducing an existing DNA analysis procedure, reliability of the procedure has to be demonstrated by carrying out internal validation. In order to introduce a sensitive and accurate DNA quantification method in our laboratory, a validation study of the 7500 Real Time PCR System (Applied Biosystems) using the QUANTIFILER Human DNA Quantification Kit (Applied Biosystems) was performed. Here we report the results and the experience we have gained during this internal validation study.     

Analysis of postmortem DNA degradation by single-cell gel electrophoresis

One of the most important longstanding problems in the field of forensic medicine is the determination of the time of death upon the discovery of a possible homicide victim. With a majority of homicide victims discovered within the first 48h, it is critically important to be able to determine time of death quickly, and with accuracy and precision. Current methods of determining postmortem interval (PMI) vary, but none can provide better than an 8-h window time estimate. In this paper, the potential application of single-cell gel electrophoresis (SCGE), also known as the comet assay, to evaluate postmortem cell death processes, specifically nuclear DNA fragmentation, is assessed. Upon the death of an organism, internal nucleases contained within the cells should cause chromosomal DNA to degrade into increasingly smaller fragments over time, and if these fragments can be isolated and visualized, the fragmentation should prove to be measurable and quantifiable. An original study providing proof of the concept of postmortem DNA fragmentation between early and late time periods was conducted using human leukocytes. With an established trend seen in the leukocyte results, this study was then expanded using a porcine animal model, over a longer time period, with more frequent time-points evaluated. DNA degradation in all samples was revealed by SCGE and quantified by the use of DNA-specific quantitative stains, as measured by digital camera affixed to a microscope. The comet 'tail-moment' gave a measure of the proportion of fragmented to non-fragmented DNA, while the 'tail-length' provided the relative size of degraded DNA fragments. In both models, an increase in DNA fragmentation was found to correlate with an increased PMI from 0 to 56h postmortem, as evaluated by comet-tail-moment and by comet-tail-length, with tail-length providing the strongest statistical correlation, based upon regression analysis. The postmortem DNA fragmentation observed in this study, reveals a sequential, time-dependent process with the potential for use as a predictor of PMI in homicide cases.     

Characterization of Protein Alterations in Damaged Axons in the Brainstem Following Traumatic Brain Injury Using Fourier Transform Infrared Microspectroscopy: A Preliminary Study

Axonal injury contributes greatly to neurological dysfunction following traumatic brain injury (TBI), but current histological diagnostic methods are limited in identifying the pathological profiles of injured axons and unable to provide an objective and accurate quantification. Fourier transform infrared microspectroscopy (FTIRM) has the ability to offer macromolecular bioinformatics of the tissues including biochemical composition and structure by calculating band absorption intensity. In this study, axonal injury in the brainstem of rats with traumatic brain injury at 72 h post‐trauma, which was confirmed with beta‐amyloid precursor protein (β‐APP) immunostaining, was detected with FTIRM technique. The lower intensity of infrared absorbance under the amide I band corresponds strongly to the area of axonal injury, and further analysis of amide I band shows significant differences in protein conformation between injured and normal axons. The findings indicate that using FTIRM technique, the amide I band has potentials to be a infrared spectral marker of axonal injury.     

A novel method for forensic DNA investigations: repeat-type sequence analysis of tandemly repeated mtDNA in domestic dogs

A highly variable and heteroplasmic tandem repeat region situated in the mitochondrial mt DNA control region (CR) in domestic dogs and wolves was studied to evaluate its suitability as a forensic genetic marker for analysis of single hairs. The tandem repeat array is composed of three 10-bp repeat types that are distributed so that a secondary DNA sequence is formed. Thus, the region presents two levels of variation: variation in the number of repeats and variation in the secondary DNA sequence of repeat types. Two analysis methods were therefore tested; fragment length analysis and analysis of the sequence of repeat types. Fragment analysis produced unique profiles that could be used to discriminate between blood samples from maternally closely related individuals. However, different hairs from one individual did not have the same fragment profile, and the method is, therefore, not suitable for analysis of single hairs. In contrast, analysis of the repeat type sequences (array types) is highly informative. When different hairs from one individual were studied, identical array types were found. The repeat-type sequence variation was studied among individuals having identical nonrepetitive CR mtDNA sequence variants. Seven, six, and two individuals, representing three different sequence variants, respectively, were analyzed. All these individuals had different array types, which implies a very high genetic variation between individuals in this region. The analysis method considerably improves the exclusion capacity of mtDNA analysis of domestic dogs compared with sequence analysis of non-repetitive DNA.     

A new sensitive short pentaplex (ShoP) PCR for typing of degraded DNA

Analysis of short tandem repeat makers has become the most powerful tool for DNA typing in forensic casework analysis. Unfortunately, typing of DNA extracted from telogen shed hairs, bones buried in the soil or from paraffin-embedded, formalin-fixed tissue often reveals no results due to the degradation of DNA. The reduction in size of the target fragments by development of new primers and their combination in multiplex approaches open a new field of DNA analysis. Here we present a new sensitive short pentaplex PCR including the loci amelogenin, TH01, VWA, D3S1358 and D8S1179. Validation tests of our new method included sensitivity, mixtures, human specificity, artificial degradation of DNA by DNase I and case work analysis on a panel of different forensic samples. The detection limit was 12.5 pg of human DNA, and mixtures of 50 pg in a total of 1000 pg were clearly detectable and revealed complete profiles. Only DNA extracts of human primates displayed a few signals, whereas other animal, fungal or bacterial DNA showed no signals. Our method proved extremely valuable in the analysis of artificially degraded DNA and in forensic cases, where only poorly preserved DNA was available. This approach and other similar methods can aid in the analysis of samples where allelic drop out of larger fragments is observed. It is highly recommended to develop more of these multiplexes to improve poor quality DNA typing.     

两个体混合样本比例的PCR-STR检测研究

目的探讨PCR-STR检测两个体混合样本比例的灵敏度。方法按不同比例将两个体全血、单个核细胞及纯DNA 3种不同的样本进行混合,应用AB I公司生产的profile p lus商品化试剂盒多重PCR扩增后,用AB I3100基因分析仪进行毛细管电泳,检测基因座及其峰面积,并就混合样本中两单个核细胞样本DNA含量百分比与扩增前混合样本比例的关系进行相关分析。结果当样本混合比例为4%+96%时,可明确区分混合样本中两个体的基因型,且扩增前混合细胞百分率与扩增后两者峰面积比值呈直线相关。结论PCR-STR检测两个体混合样本比例的灵敏度有一定范围,并可进行半定量。     

Immunohistochemical analysis of forensic evidence from a double homicide

We report the use of immunohistochemical staining for analysis of forensic evidence from a double homicide. A 38-year-old woman and her 7-year-old daughter were murdered by multiple blows to the head and face with a tomahawk, resulting in multiple fragments of brain tissue scattered about the murder scene. The victims' husband and father was the main suspect, who maintained that he was out of town on business during the evening of the murders. However, a shirt taken from the suspect's car on the morning after the murders (secured by the police before the suspect visited the murder scene) was found to have two small stains. DNA analysis on the stains showed the presence of the deceased wife's DNA, and immunohistochemical stains on shirt fragments conclusively documented the presence of deep central nervous system tissue, providing the critical piece of evidence needed to arrest and prosecute the suspect. This report demonstrates that shirt or similar cloth fragments can be processed into paraffin blocks and subsequently immunostained to search for and classify types of tissue fragments that may be present on the fabric.     

降解DNA的PCR分型-电洗脱回收大片断DNA法

本文分析了降解ＤＮＡ作ＰＣＲ分型易失败的原因，提出了因小片断ＤＮＡ过多，阻碍引物与膜机的复性及阻断引物延伸的新现点。将降解的ＤＮＡ电泳，切除小片断ＤＮＡ，电洗脱回收较大片断ＤＮＡ，成功地对降解ＤＮＡ进行了ＰＣＲ分型．本方法简便、快速、有效。     

Possibilities of DNA sex determination in hair roots

Species- and sex-determination on hair roots were simultaneously performed using extracted DNA and a human Y chromosomespecific probe. (pH Y 2.1) After Southern hybridization, Hae III-digested DNA fragments were detected by non-radioactive digoxigenin detection system. DNA was extracted from one to five freshly plucked hair roots. The specific 2.12 kb fragment was successfully detected in male DNA samples from a single hair root. A positive identification of female DNA was more difficult. The hair root DNA was revealed to be stable at room temperature for at least 2 weeks (examination time) and produced the same specific band pattern as the DNA of fresh hair roots. In the blind tests with DNA samples from randomly plucked one to four hair roots, the rate of successful sex-determination was 95.8% on male samples (23 out of 24 samples) and 25% on female samples (4 out of 16 samples).     

Identification of DNA of human origin based on amplification of human-specific mitochondrial cytochrome b region

Species-specific differences in a non-polymorphic region of the mitochondrial cytochrome b gene appear to be large enough to allow human-specific amplification of forensic DNA samples. We therefore developed a PCR-based method using newly designed primers to amplify a 157-bp portion of the human mitochondrial cytochrome b gene. The forward and reverse primers were designed to hybridize to regions of the human mitochondrial cytochrome b gene with sequences differing from those of chimpanzee by 26% (7 bp/27 bp) and 26% (6 bp/23 bp), respectively. Using this primer pair, we successfully amplified DNA extracted from blood samples of 48 healthy adults. All these human samples produced a single band of the expected size on agarose gel electrophoresis, and the sequence of the single band was shown to be identical to that of the target region (157 bp) by sequence analysis. On the other hand, no visible bands were amplified from DNA extracted from blood samples of animals including non-human primates (chimpanzee, gorilla, Japanese monkey, crab-eating monkey) and other species (cow, pig, dog, goat, rat, chicken and tuna). Thus, DNA producing a single band following PCR amplification using this primer pair can be reasonably interpreted as being of human origin. In addition, aged biological specimens comprising bloodstains, hair shafts and bones were successfully identified as being of human origin, illustrating the applicability of the present method to forensic specimens.     

Response of short tandem repeat systems to temperature and sizing methods

In capillary gel electrophoresis (CE), changing run conditions such as temperature can result in minor variations in the size determination of an allele. These effects are caused by secondary structure differences that can occur between the amplified sample and the internal standard. The type of method chosen to generate the sizing curve in STR analysis can influence the relationship between estimated allele size and temperature. To better understand the effects of temperature and sizing method on the reproducibility of DNA migration, two fluorescently labeled allelic ladders, CTTv and Y-PLEX 6 were analyzed using the ABI Prism 310 Genetic Analyzer. The default method on the Genetic Analyzer utilizes an electrophoretic temperature of 60 degrees C and a Local Southern method to generate a sizing curve from the fragment migration times of the internal lane standard. In this work, electrophoresis was conducted at 35-70 degrees C using the commercially available POP 4 buffer at pH 8 and two sizing methods, Global Southern and Local Southern, were compared. The slopes of the regression line between estimated allele size and temperature, using either sizing method, were measured in order to demonstrate the temperature sensitivity of migration time and the importance of the operator-chosen method. Our results indicate that the Global Southern method is a better choice in situations where temperature fluctuations can occur. In addition, the temperature dependence of the DNA size estimates using the POP 4 system were compared to results obtained using an experimental buffer consisting of 3% hydroxyethylcellulose at pH 11. These results demonstrate that secondary structure effects are minimized at an elevated pH, increasing the precision of size estimates obtained.     

Determining the human origin of fragments of burnt bone: a comparative study of histological, immunological and DNA techniques.

In situations where badly burnt fragments of bone are found, identification of their human or non-human origin may be impossible by gross morphology alone and other techniques have to be employed. In order to determine whether histological methods were redundant and should be superseded by biomolecular analyses, small fragments of artificially burnt bone (human and non-human) were examined by quantitative and standard light microscopy, and the findings compared with newer biomolecular analyses based on identifying specific human albumin by ELISA and amplifying human mitochondrial DNA by PCR. For quantitative microscopy, reference data were first created using burnt bones from 15 human and 20 common domestic and farm animals. Measured osteon and Haversian canal parameters were analysed using multivariate statistical methods. Highly significant differences were found between values for human and non-human bone, and a canonical discriminant function equation was derived, giving a predicted correct classification of 79%. For the main study, samples of cortical bone were taken from three fresh cadavers, six human skeletons and ten freshly slaughtered animals and burnt by exposure to temperatures ranging from 800 to 1200 degrees C; charred fragments of human cortical bone from two forensic cases were also tested. Quantitative microscopy and canonical discriminant function gave the correct origin of every sample. Standard microscopy falsely assigned burnt bone from one human skeleton and one forensic case to a non-human source, but otherwise gave correct results. Human albumin was identified in five individuals, including one of the forensic cases, but mitochondrial DNA could not be amplified from any of the human bone. No false positive test results were seen with either biomolecular method; and human albumin and mitochondrial DNA were correctly identified in all unburnt control specimens. It was concluded that histological methods were not redundant and that quantitative microscopy provided an accurate and consistent means of determining the human or non-human origin of burnt bone and was more reliable than standard microscopy or the newer immunological and DNA techniques tested here.