焦磷酸测序分析短片段牙釉质蛋白基因进行性别鉴定

目的采用焦磷酸测序技术分析短片段牙釉质蛋白基因进行性别鉴定并用于骨骼及腐败生物检材的检测。方法应用blast软件,确定牙釉质蛋白基因(Amel)上1段含有3个SNP位点及1个插入/缺失(indel)位点的序列作为待测靶序列,设计引物,扩增该段序列,应用焦磷酸测序技术分析扩增序列,进行性别鉴定。对方法进行准确性、灵敏度、种属特异性的测试,并用于对骨骼和高度降解DNA的检测。结果 PCR产物分别为44bp(Amel X)和45bp(Amel Y),女性测序结果为:G/G,T/T,…/…,C/C,男性测序结果为:G/T,T/A,…/C,C/A,分型图谱清晰。应用本文方法检测100份已知性别的DNA样本,结果均正确无误,方法最低DNA模板量为0.5ng,具有较好的人类种属特异性。用于高度降解DNA分析,较IdentifilerTM试剂盒具有更高的成功率且骨骼样本也得到清晰的分型结果。结论本文采用焦磷酸测序技术分析Amel的方法在法医学性别鉴定中有较好的应用价值。     

Intentional mixed buccal cell reference sample in a paternity case

We report a case where an alleged father (AF) attempted to substitute someone else's saliva sample for his reference sample in a paternity analysis. Buccal cells were collected from the AF and the child, and DNA analysis was performed using an autosomal STR loci (Identifiler). The profile from the AF showed extra peaks in some loci, as well as a much higher "X" allele peak relative to the "Y" allele peak at the amelogenin locus. After conducting reanalysis by another technician with another set of positive and negative controls, it was concluded that the only source of the mixed profile was by intentional introduction by the AF, at the time of sampling, of some foreign human biological material, most likely saliva from a woman. Owing to the inconclusive results, when the AF was called back to the lab and the peculiar results were explained to him, he admitted that he had introduced into his mouth saliva from another person in an attempt to be excluded as the father of the child. Although tampering with DNA reference samples is not common, some individuals may attempt to contaminate or otherwise adulterate specimens before DNA tests. Personnel responsible for sampling should be aware of this possibility and should try to establish procedures to avoid the problem.     

Validation of SRY Marker for Forensic Casework Analysis

Abstract:  Determining the gender of the source of forensic DNA evidence is based on the amelogenin test. However, at times the assay may not be indicative of gender assignment, because of deletions at the amelogenin site. Previously, we described successful coamplification of a marker residing within the SRY gene with the short tandem repeat markers from two commercially available human identification kits. The study herein addresses the validation of primers for the target SRY gene regarding specificity, sensitivity, and robustness. Among 115 unrelated male Slovenians no null allele was observed. Repeatable and reliable results were obtained from as little as 25 pg of template DNA, indicating a high sensitivity of detection for the assay. No polymerase chain reaction product was observed even at a concentration of 10 ng/μL of template female DNA. Additionally, the male specific marker could be detected in mixed male and female samples down to a ratio of 1:16.     

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.     

Concordance study between the AmpFlSTR MiniFiler PCR amplification kit and conventional STR typing kits

The AmpFlSTR MiniFiler polymerase chain reaction amplification kit developed by Applied Biosystems enables size reduction on eight of the larger STR loci amplified in the Identifiler kit, which will aid recovery of information from highly degraded DNA samples. The MiniFiler Kit amplifies CSF1PO, FGA, D2S1338, D7S820, D13S317, D16S539, D18S51, and D21S11 as well as the sex-typing locus amelogenin. A total of 1308 samples were evaluated with both the MiniFiler and Identifiler STR kits: 449 African American, 445 Caucasian, 207 Hispanic, and 207 Asian individuals. Full concordance between Identifiler and MiniFiler Kits was observed in 99.7% (10,437 out of 10,464) STR allele calls compared. The 27 differences seen are listed in Table 1 and encompass the loci D13S317 (n = 14) and D16S539 (n = 10) as well as D18S51 (n = 1), D7S820 (n = 1), and CSF1PO (n = 1). Genotyping discrepancies between the Identifiler and MiniFiler kits were confirmed by reamplification of the samples and further testing using the PowerPlex 16 kit in many cases. DNA sequence analysis was also performed in order to understand the nature of the genetic variations causing the allele dropout or apparent repeat unit shift.     

Usefulness of dura mater in providing DNA samples for identifying cadavers

We examined the usefulness of the dura mater in identifying human remains. Dura mater was collected from 50 cadavers, including drowned, charred, and mummified remains. The STR genotype using the AmpFlSTR Identifiler Kit could be typed at 15 STR and amelogenin loci in 30 samples of 33 cases. Furthermore, the ABO genotype and amelogenin using gel-based methods could be typed in 44 samples of 50 cases. In cases with successful typing of STR, ABO-DNA, and amelogenin, the longest time after death was from 12 to 26 days in a drowned body. The minimum quantity of dura mater required for DNA extraction was about 2.5 mg, dried and fixed by ethanol, in a cadaver 15 h after death. The state of the DNA from the dura mater from the calvaria may be better than that from the basis cranii interna. We found that DNA from dura mater is one of the most useful samples for forensic identification.     

Male amelogenin dropouts: phylogenetic context, origins and implications

Several commercial PCR multiplex kits incorporate the amelogenin locus for the purpose of human gender identification. Consequently, erroneous results in the electropherogram profile of this locus can carry important forensic implications. In this study, dropout of the amelogenin Y allele was detected in 5 out of 77 phenotypically normal Kathmandu males using the AmpFlSTR Identifiler kit. A battery of male-specific markers including SNPs, STRs, STSs, and a minisatellite were amplified for the five amelogenin null samples in order to delineate the breakpoints of the deletions as well as assess the overall integrity of the Y-chromosome. This study represents the first to examine the haplogroup affiliation of the AMGY deletions. The analyses performed suggest a single origin for the five deletions as indicated by their allocation to a specific Y-haplogroup (J2b2-M241), related Y-STR haplotypes and identical regional localization of breakpoints. The age estimated from the microsatellite variation for the amelogenin deletions (if they are associated by descent) is approximately 6.5+/-3.3 ky, younger than the previously reported related age of the M241 haplogroup representatives (13-14 ky). Our data in combination with previous publications suggest a concentration of afflicted individuals in the Indian subcontinent, possibly as a result of common ancestry. The elevated incidence of the amelogenin dropout in these populations accentuates the need to utilize other loci for gender determination in order to obtain an accurate set of inclusion criteria in forensic casework.     

肿瘤组织中部分杂合性丢失判定标准的适用性评估

目的 对肿瘤组织中部分杂合性丢失(partial loss of heterozygous,pLOH)的判定标准在Identifiler系统中的适用性进行评估.方法 将696例正常无关个体Identifiler数据中共8428个杂合子基因座,对两个体同一杂合子基因座随机配对,构建两等位基因峰高或峰面积比值比.77对肿瘤-正常组织对Identi-filer分型数据中共896对杂合子基因座,将肿瘤组织与其身源正常组织配对构建两等位基因峰高或峰面积比值比.对上述比值比频率分布进行曲线拟合,按比值rt<0.5和>2.0的标准判断正常无关个体对杂合子不均匀扩增误判为pLOH的比例,并比较依据峰高比值比和峰面积比值比对pLOH检出率的差异.结果 正常无关个体对中共4214对杂合子基因座对的比值比和肿瘤-正常组织对中共896对杂合子基因座对的比值比均呈正态分布.按照峰高或峰面积比值比<0.5和>2.0的标准,正常无关个体对中有0.12%误判为pLOH.依据峰高比和依据峰面积比对肿瘤组织中pLOH检出率的差异无统计学意义(P=0.5632).结论 以峰高比值比或峰面积比值比(<0.5或>2.0)作为肿瘤组织中pLOH判断方法和标准适用于Identifiler系统.     

A Y-chromosome STR marker should be added to commercial multiplex STR kits

Abstract:  Autosomal short tandem repeat (STR) analysis has become highly relevant in the identification of victims from mass disasters and terrorist attacks. In such events, gender misidentification can be of grave consequences, yet the list reporting amelogenin amplification failure using STR multiplex kits continues to grow. Presented here are three such examples. In the first case, we present two male suspects who demonstrated amelogenin Y-deficient results using two commercial kit procedures. The presence of their Y chromosomes was proven by obtaining a Y-haplotype. The second case demonstrated a profile from a third male suspect where only the Y homolog of the XY pair was amplified. In events such as mass disasters or terrorist attacks, timely and reliable high throughput DNA typing results are essential. As the number of reported cases of amplification failure at the amelogenin gene continues to grow, we suggest that the incorporation of a better gender identification tool in commercial kits is crucial.     

基于重组质粒制备STR分型阳性参照物

目的基于重组质粒制备可用于校准法医STR分型的阳性参照物。方法以常用阳性参照物9948人类基因组DNA STR分型为依据,基于重组质粒构建包含CSF1PO、D7S820、TH01等40个常染色体位点,DYS391、DYS522、DYS385a/b等22个Y染色体位点以及性别判定基因座Amelogenin的STR分型阳性参照物。将重组质粒定量、稀释后等比例混合,分别应用于DNATyper~?19、DNATyper~?24、DNATyper~?Y、Amp F?STR~?Identifiler~?Plus以及Power Plex~?18D System五种扩增试剂盒。结果阳性参照物中各重组质粒浓度为0.01pg/μL~0.001pg/μL;应用于Amp F?STR~?Identifiler~?Plus PCR扩增试剂盒,基于重组质粒制备的阳性参照物与人类基因组DNA扩增检测结果差异较小;将此阳性参照物分别应用于不同公司、不同STR基因座的四种STR扩增试剂盒,电泳检测图谱显示各基因座基因型完整,分型正确,峰高相当,基因座间均衡性良好。结论基于重组质粒制备STR分型阳性参照物,是一种可以替代细胞系制备阳性参照物的方法,具有一定的参考价值。基于此方法制备的阳性参照物可适用于市面上常用的STR检验试剂盒,普适性较强,对法医DNA分型检测有一定的实用价值。     

3种常用PCR扩增试剂盒检验血样DNA的结果比较

目的探讨常用的ProfilerPlusTM、IdentifilerTM与Powerplex(16试剂盒检验血样DNA的差异。方法510名无关中国汉族个体血样,分别用ProfilerPlusTM与Powerplex(16试剂盒进行DNA检验,然后对有不同检验结果的同一样本,再用ProfilerPlusTM、IdentifilerTM与Powerplex(16等三种试剂盒进行检验,并比较其结果。结果在510名个体血样的DNA检测结果中,发现同一样本有不同结果的有7例,其差异率为1.3725%;ProfilerPlusTM、IdentifilerTM与Powerplex(16各有1例在D13S317或FGA基因座上出现等位基因缺失现象,缺失率为0.1961%;ProfilerPlusTM有5例在D8S1179基因座上出现扩增严重不平衡现象,相应的IdentifilerTM与Powerplex(16试剂盒的检验结果为正常杂合子。结论ProfilerPlusTM、IdentifilerTM与Powerplex(16试剂盒检验血样DNA均会出现扩增不平衡和/或基因丢失现象,其发生几率IdentifilerTM与Powerplex(16试剂盒较ProfilerPlusTM少。     

Typing of XY (male) genotype from malignant neoplastic tissue by the amelogenin-based sex test

DNA profiling of a cancer tissue can be problematic because of genomic instability. Here we have analyzed gastrointestinal cancer specimens from 46 males, of which seven (15%) showed aberrations in determination of gender by the widely used amelogenin test. The X-type amelogenin allele in all cases remained intact. All male tumor samples showing frequent autosomal loss of heterozygosity had a decreased signal of the Y allele from the amelogenin marker. When tested with an alternate set of primers for the amelogenin locus, the Y-type allele showed loss of heterozygosity in the same seven cases. However, when amplified with 15 Y-specific STR primers, all the cancerous tissue Y chromosomes seemed to be intact. These results indicate when malignant neoplastic tissue specimens are used, that amelogenin-based gender determination should be carefully interpreted.     

AMELY无效扩增个体Yp11.2区域STS位点检测

目的通过Y-STR复合扩增技术检测AMELY缺失的男性个体Y染色体的完整性,并对数据库中的STS位点进行筛选并设计引物,检测Y染色体AMEL区域的STS位点缺失情况,为进一步研究中国人口中AMELY的缺失提供数据和理论依据。方法应用Goldeneye 20A PCR扩增试剂盒、华夏TM白金PCR扩增试剂、Y filer plus试剂盒和Goldeneye 27YB试剂盒进行PCR扩增、通过ABI 3500遗传分析仪进行毛细管电泳以及STR分型。通过UniSTS database和UCSC对Y染色体p11.2区域的多个STS位点进行筛选,部分STS位点自行设计引物,梯度PCR和常规PCR扩增结合琼脂糖凝胶电泳进行STS位点的分析。结果 Goldeneye 20A和华夏TM白金两种试剂盒分型结果均显示两父子AMELY等位基因无效扩增。Y filer plus和27YB试剂盒分型结果显示两父子均在DYS570和DYS576位点无效扩增。STS位点扩增显示两父子的Y染色体缺失的STS位点有BV703904、BV703923、SY2137、SY716、DYS256、DYS267、SY2232、SY2233、DYS261和DYS260。结论 AMELY无效扩增的父子Yp11.2缺失片段包含了BV703904-DYS260之间的区域,长度为2.63~2.74 Mb。     

Higher failures of amelogenin sex test in an Indian population group

The human sex test in forensic multiplexes is based on the amelogenin gene on both the X and Y chromosomes commonly used in sex genotyping. In this study of 338 male individuals in a Malaysian population comprising Malays, Chinese and Indians, using the AmpFlSTR Profiler Plus kit, the amelogenin test gave a significant proportion of null alleles in the Indian ethnic group (3.6% frequency) and 0.88% frequency in the Malay ethnic group due to a deletion of the gene on the Y chromosome. This sex test also failed in a forensic casework sample. Failure of the amelogenin test highlights the need for more reliable sex determination than is offered by the amelogenin locus in the Malay and Indian populations. The gender of the Indian-Malay amelogenin nulls was confirmed by the presence of three Y-STR alleles (DYS438, DYS390 and DYS439). For the Indian ethnic group, one of the Y-STR forms a stable haplotype with the amelogenin null. The amelogenin-deletion individuals also showed a null with a male-specific minisatellite MSY1, indicating that a very large deletion was involved that included the amelogenin and the MSY1 loci on the short arm of the Y chromosomes (Yp).     

Case study of dissolving allele dropout

Allele typing performed using the original components of the AmpFlSTR Identifiler kit led to unbalanced peak heights and allele dropouts. However, allele typing was completely successful when substituting the QIAGEN Multiplex PCR Master Mix for the AmpFlSTR PCR Reaction Mix of the Identifiler kit. The successful range for allele typing was 250 pg to 1.5 ng of DNA. The running times for PCR amplification were reduced by about 30 min from the original protocol.     

Use of amelogenin test for DNA sex appurtenance in analysis of mixed biological traces

Theoretical and experimental study of amelogenin tests system for identification of DNA sex appurtenance has been performed. In many cases amplification can fail to reflect the true quantitative ratio between the mixture components during typing sex-specific variants of amelogenin gene in DNA preparations of mixed sex appurtenance. In practical studies these properties of the amelogenin system can lead to difficulties and errors in interpretation of the results of forensic medical expert evaluations, and we therefore studied the use of amelogenin DNA test for identification of sex appurtenance of mixed biological traces and compared the characteristics of autosomal PDAF system and amelogenin system for sex identification in analysis of DNA mixtures.     

A multiplexed system for quantification of human DNA and human male DNA and detection of PCR inhibitors in biological samples

Forensic analysts routinely encounter samples containing DNA mixtures from male and female contributors. To obtain interpretable Short Tandem Repeat (STR) profiles and select the appropriate STR analysis methodology, it is desirable to determine relative quantities of male and female DNA, and detect PCR inhibitors. We describe a multiplex assay for simultaneous quantification of human and human male DNA using the ribonuclease P RNA component H1 (RPPH1) human target and the sex determining region Y (SRY) male-specific target. A synthetic oligonucleotide sequence was co-amplified as an internal PCR control. Standard curves were generated using human male genomic DNA. The SRY and RPPH1 assays demonstrated human specificity with minimal cross-reactivity to DNA from other species. Reproducible DNA concentrations were obtained within a range of 0.023-50 ng/μl. The assay was highly sensitive, detecting as little as 25 pg/μl of human male DNA in the presence of a thousand-fold excess of human female DNA. The ability of the assay to predict PCR inhibition was demonstrated by shifted IPC Ct values in the presence of increasing quantities of hematin and humic acid. We also demonstrate the correlation between the multiplex assay quantification results and the strength of STR profiles generated using the AmpF?STR^®PCR Amplification kits.     

Study of species specificity of the amelogenin system for genetic sex determination

The properties of amelogenin amplification system and, in particular, of its species specificity, were studied. DNA preparations extracted from cattle (cow/bull), pig, ram and from poultry (hen), as well as from dog and cat, were used as a matrix for polymerase chain reaction (PCR) involving a standard scheme of enzymatic amplification of the amelogenin gene. It was demonstrated that, unlike for the human DNA, the amelogenin test couldn't be used for the DNA of examined animals as a sex-specific marker. However, there is a danger of a false determination of the male sex in the female origin samples during a forensic-experts' typing of the amelogenin gene, if there is an admixture of an animal DNA to a human DNA preparation. As for the biological samples of the animal origin, there is a possibility of a false-female sex determination. It can be attributed to an incorrectly selected standard of the molecular weight or to its remote location on the gel as well as to the use of an inaccurately calculated algorithm designed for determining the sizes of analyzed fragments.     

“YFlag” – A Single‐base Extension Primer Based Method for Gender Determination

Assigning the gender of a DNA contributor in forensic analysis is typically achieved using the amelogenin test. Occasionally, this test produces false‐positive results due to deletions occurring on the Y chromosome. Here, a four‐marker “YFlag” method is presented to infer gender using single‐base extension primers to flag the presence (or absence) of Y‐chromosome DNA within a sample to supplement forensic STR profiling. This method offers built‐in redundancy, with a single marker being sufficient to detect the presence of male DNA. In a study using 30 male and 30 female individuals, detection of male DNA was achieved with c. 0.03 ng of male DNA. All four markers were present in male/female mixture samples despite the presence of excessive female DNA. In summary, the YFlag system offers a method that is reproducible, specific, and sensitive, making it suitable for forensic use to detect male DNA.     

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.