Alu家族在法医DNA分析中的应用

Alu家族是灵长类特有的短散在重复序列,在人类基因组内含量丰富,分布广泛,甲基化程度高,有种属特异性和插入变异,为法医DNA分析面临的许多问题提供了潜在的解决途径。目前,Alu元件在法医DNA分析中的应用包括:DNA定量、种属鉴定、种族鉴定、性别鉴定、个体识别和亲子鉴定,以及全基因组扩增等。本文总结各种基于Alu元件的法医DNA分析技术的原理和特点,探讨Alu元件的法医学研究和应用前景,供相关学者参考。     

An Alu-based, MGB Eclipse real-time PCR method for quantitation of human DNA in forensic samples

The forensic community needs quick, reliable methods to quantitate human DNA in crime scene samples to replace the laborious and imprecise slot blot method. A real-time PCR based method has the possibility of allowing development of a faster and more quantitative assay. Alu sequences are primate-specific and are found in many copies in the human genome, making these sequences an excellent target or marker for human DNA. This paper describes the development of a real-time Alu sequence-based assay using MGB Eclipse primers and probes. The advantages of this assay are simplicity, speed, less hands-on-time and automated quantitation, as well as a large dynamic range (128 ng/microL to 0.5 pg/microL).     

D17Z1探针点杂交DNA定量研究

本研究化学合成了高等灵长类特异性α卫星寡核苷酸片段(D17Z1),经辣根过氧化物酶标记、分子杂交、化学发光法检测,建立了高等灵长类特异性DNA精确定量的方法.制备了DNA浓度梯度标准对照.对人类DNA,猴、猪、牛、羊、鸡、兔、鱼、小鼠等常见动物DNA及JMl09大肠杆菌、入DNA、φ174DNA等微生物DNA进行了定量分析.结果表明,应用该方法对人类DNA定量不受非高等灵长类动物DNA与微生物DNA的影响,可实现组分定量;灵敏度测试,可对0.12ng的人类DNA进行定量,适用于法医DNA检验定量分析.     

Simultaneous determination of total human and male DNA using a duplex real-time PCR assay

A single duplex assay to determine both the amount of total human DNA and the amount of male DNA in a forensic sample has been developed. This assay is based on TaqMan technology and uses the multicopy Alu sequence to quantitate total human DNA and the multicopy DYZ5 sequence to quantitate Y chromosomal (male) DNA. The assay accepts a wide concentration range of input DNA (2 muL of 64 ng/microL to 0.5 pg/microL), and also allows detection of PCR failure. The PCR product sizes Alu (127 bp) and DYZ5 (137bp) approximate that of the smaller short tandem repeats (STRs) which should make the assay predictive of STR success with degraded DNA. The assay was optimized for probe/primer concentrations and BSA addition and validated on its reproducibility, on its human specificity, on its nonethnic variability, for artificial mixtures and adjudicated casework, for the effect of inhibitors and for state of DNA degradation. This assay should prove very usual in forensic analyses because knowing the relative amounts of male versus female DNA can allow the examiner to decide which samples may yield the most probative value in a case or direct the samples to methods that would yield the greatest information.     

Human genomic DNA quantitation system, H-Quant: development and validation for use in forensic casework

The human DNA quantification (H-Quant) system, developed for use in human identification, enables quantitation of human genomic DNA in biological samples. The assay is based on real-time amplification of AluYb8 insertions in hominoid primates. The relatively high copy number of subfamily-specific Alu repeats in the human genome enables quantification of very small amounts of human DNA. The oligonucleotide primers present in H-Quant are specific for human DNA and closely related great apes. During the real-time PCR, the SYBR Green I dye binds to the DNA that is synthesized by the human-specific AluYb8 oligonucleotide primers. The fluorescence of the bound SYBR Green I dye is measured at the end of each PCR cycle. The cycle at which the fluorescence crosses the chosen threshold correlates to the quantity of amplifiable DNA in that sample. The minimal sensitivity of the H-Quant system is 7.6 pg/microL of human DNA. The amplicon generated in the H-Quant assay is 216 bp, which is within the same range of the common amplifiable short tandem repeat (STR) amplicons. This size amplicon enables quantitation of amplifiable DNA as opposed to a quantitation of degraded or nonamplifiable DNA of smaller sizes. Development and validation studies were performed on the 7500 real-time PCR system following the Quality Assurance Standards for Forensic DNA Testing Laboratories.     

Development and usage of a NIST standard reference material for real time PCR quantitation of human DNA

National Institute of Standards and Technology SRM 2372 human DNA quantitation standard has been produced to support the need for a human-specific DNA quantitation standard in forensic casework and calibration of new quantitative polymerase chain reaction (qPCR) assays. The conventional DNA concentration has been assigned with one of the U.S. National Reference UV/Visible Spectrophotometers, assuming an absorbance of 1.0 at 260 nm equals 50 ng/μL of double stranded DNA. In addition, an interlaboratory study has been conducted, to verify that the SRM 2372 materials perform well in currently used DNA quantitation assays by the forensic DNA community. Each unit of SRM 2372 consists of three well-characterized DNA extracts. Component A is a single-source human male material derived from blood. Component B is a multiple-source human female material derived from blood. Component C was purchased as a purified unsheared genomic human DNA (Sigma-Aldrich Co., St. Louis, MO) obtained as a lyophilized human genomic extract and has both male and female donors. SRM 2372 is intended to enable the comparison of DNA concentration measurements across time and place. Manufacturers can use SRM 2372 to validate the values assigned to their own reference materials. Individual forensic laboratories can use SRM 2372 to validate DNA quantitation methods and to verify the assigned concentration of in-house or commercial DNA calibration standards.     

Development of an Alu-based,QSY 7-labeled primer PCR method for quantitation of human DNA in forensic samples

Determining the amount of human DNA extracted from a crime scene sample is an important step in DNA profiling. The forensic community relies almost entirely upon a technique (slot blot) to quantitate human DNA that is imprecise, time consuming, and labor intensive. This paper describes the development of a new technique based on PCR amplification of a repetitive Alu sequence. Specific primers were used to amplify a 124-bp fragment of Alu sequence; amplification was detected by SYBR Green I staining in a fluorescent plate reader. To reduce background in the plate reader assay, QSY-7 labeled primers were utilized. The assay was tested on animal DNAs, human blood spots, mock crime samples, and degraded DNA in comparison with the slot blot technique. The QSY Alu assay has a dynamic range of 10 ng to 10 pg, and is sensitive, specific, fast, quantitative, and comparable in cost to the slot blot assay.     

Molecular Assay for Screening and Quantifying DNA in Biological Evidence: The Modified Q‐TAT Assay*

Abstract: A method is described for the quantitation of total human and male DNA. Q‐TAT utilizes end‐point, multiplex polymerase chain reaction (PCR) amplification of the amelogenin and SRY loci to quantify DNA and incorporates a cloned nonhuman template to detect PCR inhibition. Standard curves of fluorescence from amelogenin or SRY amplicons were generated from amplification of known amounts of NIST traceable SRM‐female or SRM‐male DNA. Curves showed good linearity up to 500 pg of SRM‐template (R² > 0.99) and reliably estimated total and male DNA content in casework samples. The nonhuman pRL_null template included in each PCR was a sensitive indicator of known PCR inhibitors including EDTA, hemin, blue denim dye, and humic acid. Finally, the SRY amplicon was a sensitive indicator of male DNA and, in mixtures, could reliably estimate male DNA present in an excess of female DNA. The Q‐TAT multiplex is a reliable quantitation method for forensic DNA typing.     

Run-specific limits of detection and quantitation for STR-based DNA testing

STR-based DNA profiling is an exceptionally sensitive analytical technique that is often used to obtain results at the very limits of its sensitivity. The challenge of reliably distinguishing between signal and noise in such situations is one that has been rigorously addressed in numerous other analytical disciplines. However, an inability to determine accurately the height of electropherogram baselines has caused forensic DNA profiling laboratories to utilize alternative approaches. Minimum thresholds established during laboratory validation studies have become the de facto standard for distinguishing between reliable signal and noise/technical artifacts. These minimum peak height thresholds generally fail to consider variability in the sensitivity of instruments, reagents, and the skill of human analysts involved in the DNA profiling process over the course of time. Software (BatchExtract) made publicly available by the National Center for Biotechnology Information now provides an alternative means of establishing limits of detection and quantitation that is more consistent with those used in other analytical disciplines. We have used that software to determine the height of each data collection point for each dye along a control sample's electropherogram trace. These values were then used to determine a limit of detection (the average amount of background noise plus three standard deviations) and a limit of quantitation (the average amount of background noise plus 10 standard deviations) for each control sample. Analyses of the electropherogram data associated with the positive, negative, and reagent blank controls included in 50 different capillary electrophoresis runs validate that this approach could be used to determine run-specific thresholds objectively for use in forensic DNA casework.     

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.     

Development of a human-specific real-time PCR assay for the simultaneous quantitation of total genomic and male DNA

A duplex real-time quantitative PCR assay was developed for forensic DNA analysis, which provides simultaneous quantitation of total genomic human DNA and human male DNA. The assay utilizes two spectrally resolved fluorogenic probes in a 5' nuclease (TaqMantrade mark) assay. Within the range of organisms empirically tested and based upon theoretical specificity using National Center for Biotechnology Information GenBank sequences, primer and probe sequences were shown to be human specific, and the Y-chromosome probe, male-specific. A mixture-challenge study resulted in accurate quantitation of 25 pg male DNA in a mixture of up to 1:5000 (male:female DNA). Additional experimental results include comparisons with the slot blot method and commercial real-time PCR kits. The assay developed addresses the shortcomings of the traditional slot blot method as well as the commercial real-time PCR kits. This method is shown to be specific, relatively simple, rapid, has low limits of detection, and consumes limited sample in addition to reporting both the male and total genomic DNA concentrations present.     

人类反转座核元件甲基化研究进展

人类反转座核元件LINE-1序列和Alu序列甲基化水平的变化与多种疾病的发生、发展有密切的联系,因此是相关疾病,尤其肿瘤和癌症预测、诊断、治疗及预后的新标记。近年来,二者在法医学方面的研究也逐渐展开,主要集中在同卵双生子的鉴别、年龄的鉴定、组织的鉴定等方面,有望成为法医DNA分析领域新一代的遗传标记。本文综述LINE-1序列和Alu序列甲基化在医学、法医学等领域的研究进展,希望能为相关研究和应用提供参考。     

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.     

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.     

Human blood stain identification and sex determination in dried blood stains using recombinant DNA techniques

DNA was extracted from human and non-primate dried blood stains. Human male and female specimens were readily distinguished by analysis with a Y-chromosome specific DNA probe. Human and non-primate blood stains were also readily differentiated using a repeat sequence (Alu) DNA probe. The potential power of recombinant DNA analysis in forensic science is discussed.     

Quantifiler observations of relevance to forensic casework

The Quantifiler (QF) kit is regularly used by forensic scientists for DNA quantitation. We performed in-house validation studies which revealed some interesting observations. The QF standard displayed a two-fold difference between two different lot numbers which suggests that every standard should be tested prior to use. The Promega K562 DNA standard works well with the QF kit. c. 41% of samples that inhibited the internal PCR control (IPC) system within the QF kit still produced good Profiler Plus reactions. QIAquick was effective at removing inhibitors. The presence of dyes within casework samples were observed not to inhibit QF amplifications. Template DNA greater than 100 ng/muL appeared to inhibit the IPC. Close to identical concentration results were obtained when alternative analysis settings were used. These validation findings will assist DNA processes involved in forensic casework.     

Effects of processing techniques on the forensic DNA analysis of human skeletal remains

Human remains processed by forensic anthropologists may potentially be used for genetic analysis. Therefore, the condition of the deoxyribonucleic acid (DNA) in processed remains may become an issue for future analysis. Processing techniques employed by anthropologists are highly variable and scanning electron microscopy reveals significant alterations to the bone surface depending upon the technique used. Such damage to the bone indicates differences may exist in quality and quantity of DNA extracted. This study assessed how five processing procedures used by major forensic anthropology laboratories around the country affects the amounts of DNA extracted from human rib bones and the subsequent DNA analysis. The DNA was analyzed using the short tandem repeat (STR) locus CSF1PO and amelogenin. The findings indicate processing procedures used by forensic anthropologists do not adversely affect DNA analysis but prolonged exposure to heat during processing may decrease the yield of information from the DNA.     

Identifying background microbiomes in an evidence recovery laboratory: A preliminary study

16S rRNA profiling of bacterial communities may have forensic utility in the identification or association of individuals involved with criminal activities. Microbial profiling of evidence may, in the future, be performed within environments currently utilised for human DNA recovery, such as a forensic biology laboratory. It would be important to establish the background microbiome of such an environment to determine the potential presence of human or environmental microbial signatures to assist forensic scientists in the appropriate interpretation of target microbial communities. This study sampled various surfaces of an Evidence Recovery Laboratory (ERL) on three occasions including (a) before a monthly deep-clean, (b) immediately following the deep-clean, and (c) immediately after the laboratory’s use by a single participant for the purposes of routine item examinations. Microbial profiles were also generated for the involved participant and researcher for comparison purposes. Additionally, human nuclear DNA was profiled for each of the samples collected, using standard forensic profiling techniques, to provide a prospective link to the presence or absence of a background microbial signature within the ERL after its use. Taxonomic distributions across ERL samples revealed no consistent signature of any of the items sampled over time, however, major phyla noted within all ERL samples across the three timepoints were consistent with those found in human skin microbiomes. PCoA plots based on the Unweighted Unifrac metric revealed some clustering between participant microbial reference samples and surfaces of the ERL after use, suggesting that despite a lack of direct contact, and adherence to standard operating procedures (SOPs) suitable for human DNA recovery, microbiomes may be deposited into a forensic setting over time. The reference samples collected from the involved participant and researcher generated full STR profiles. Human DNA was observed to varying degrees in samples taken from the ERL across each of the sampling timepoints. There was no correlation observed between samples that contained or did not contain detectable quantities of human nuclear DNA and microbial profile outputs.     

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.