15个STR基因座的突变观察和分析

目的调查15个STR基因座的突变情况。方法采集817例亲子鉴定的2722份血样本,采用Identi—filerTM系统扩增15个STR基因座分型,共有33060次等位基因传递,统计各基因座发生突变的频率。结果在15个基因座中发现涉及11个基因座共25次突变,平均突变率为0．8×101（95％C10．5—1．1×10-3）,其中一步突变20次,两步突变3次,三步突变2次;父、母来源突变比率为2．6：1,不能确定来源突变7次。结论STR基因座等位基因在IdentifilerTM复合扩增系统突变现象较为常见,亲子鉴定时应引起注意。     

Population data for a novel, highly discriminating tetra-local Y-chromosome short tandem repeat: DYS503

Population: Population samples for gene diversity studies were obtained from the Virginia Division of Forensic Science (bloodstains), Richmond, VA. Ninety-eight Caucasian and 100 African American samples were included in this study.     

亲权鉴定中常用STR基因座的基因组学和遗传学分析

美国联邦调查局建立的联合DNA索引系统即CODIS系统至今已有10年,该系统确定的13个STR基因座及近年来开发的PentaD、PentaE、D2S1338和D19S433基因座被全世界范围内的实验室广泛采用,在亲权鉴定和罪犯数据库建设等方面发挥了重要的作用。本文利用Web of Knowledge核心检索系统和Elsevier等全文数据库及网络资源,对近年来常用STR基因座的基因组学特征和遗传学特征进行了深入地分析,并对这些STR基因座在实践中的应用情况进行了综述。     

Laser microdissection separation of pure spermatozoa from epithelial cells for short tandem repeat analysis

Short tandem repeat (STR) analysis is a valuable tool in identifying the source of biological stains, particularly from the investigation of sexual assault crimes. Difficulties in analysis arise primarily in the interpretation of mixed genotypes when cell separation of the sexual assailant's sperm from the victim's cells is incomplete. The forensic community continues to seek improvements in cell separation methods from mixtures for DNA typing. The feasibility of applying laser microdissection (LMD) technology to precisely separate sexual assault cell mixtures by visual inspection coupled with laser dissection was assessed through three experiments. First, various histological stains were evaluated for use with LMD and DNA analysis. Second, different DNA isolation methods were evaluated on LMD-collected cells. Finally, STR analysis was performed on LMD-separated sperm cells from mixtures of semen and female buccal epithelial cells. The results indicated (a) hematoxylin/eosin staining performed best in its ability to differentiate sperm and epithelial cells while exhibiting the least negative effect on further downstream analysis; (b) both QIAamp and Lyse-N-Go methods were useful for recovery of DNA from LMD-collected sperm cells; and (c) LMD separation provided clear STR profiles of the male donor with the absence of any additional alleles from the female donor. This report describes an efficient, low-manipulation LMD method for the efficient separation of spermatozoa from two-donor sperm/epithelial cell mixtures.     

中国汉族人群41个STR基因座突变情况的观察分析

目的调查41个STR基因座在中国汉族人群中的突变情况。方法收集1 932个三联体家系4 546份血样本,采用AGCU_21+1、AGCU_EX22、Global Filer_Express~(TM)系统扩增41个STR基因座分型,统计各基因座发生突变的频率。结果 150个三联体在32个基因座共观察到154次突变,平均突变率为1.0×10~(-3)(95%CI:0.8~1.1×10~(-3)),突变率最高的是基因座SE33。其中一步突变152次(98.7%),两步突变2次(1.3%);146个三联体仅1个基因座发生突变(97.3%),4个三联体在2个基因座发生突变(2.7%);父、母来源突变比率约为4.7:1。结论 STR基因座等位基因突变现象较为常见,亲子鉴定时应引起注意。     

Development of a fast, simple profiling method for sample screening using high resolution melting (HRM) of STRs

A screening assay has been developed to provide preliminary individualization of crime scene samples thus eliminating expensive, time-consuming short tandem repeat (STR) profiling of nonprobative samples. High resolution melting performed in a real-time PCR instrument is used to detect the slight melting differences between the length and sequence variations of 22 forensic STRs. Three STRs (vWA, D18S51, THO1) were chosen to develop an assay which was optimized for Mg++ concentration, annealing/extension time/temperature, assay volume, and bovine serum albumin addition. The assay was tested for reproducibility, uniformity for genotype, melting profile consistency, effects of inhibitors, and mixture effects. The assay could be used to determine DNA concentration when a standard curve is run simultaneously. Calculations of costs show that the assay can save significant time and money for a crime with many samples or suspects.     

Evaluation of 19 short tandem repeat markers for individualization of Papaver somniferum

Papaver somniferum, commonly known as opium poppy, is the source of natural opiates, which are used as analgesics or as precursors in the creation of semi-synthetic opioids such as heroin. An increase in opioid addiction in the United States has resulted in high rates of illicit opioid use and overdoses. It has recently been shown that P. somniferum DNA suitable for genetic analysis can be recovered from heroin samples. The development of a comprehensive genetic individualization tool for opium poppy could serve to link cases and strengthen programs such as the Drug Enforcement Administration’s (DEA) Heroin Signature Program, which seeks to combat rising opioid use.The purpose of this study was to develop a quantitative real-time PCR (qPCR) method for the quantification of opium poppy DNA, compare three commercial DNA extraction kits for their ability to isolate DNA from poppy seeds, and evaluate nineteen opium poppy short tandem repeat (STR) markers for their use in a forensic identification panel. Such a panel could be used for individualizing samples and determining the geographic origin in heroin or poppy seed tea cases. The qPCR method was proven to be reproducible and reliable, specific for P. somniferum, and sensitive enough for forensic case-type samples. Of the three kits tested, the nexttec™ one-step DNA Isolation Kit for Plants was the optimal method and facilitated rapid extraction of DNA from poppy seeds. The majority of evaluated STR primer sets were unreliable or had low discriminatory power, limiting their use for individualization of poppy samples. A six-locus STR multiplex was developed and evaluated according to Scientific Working Group on DNA Analysis Methods (SWGDAM) and International Society of Forensic Genetics (ISFG) guidelines, including the use of a sequenced allelic ladder. The multiplex was found to have low discriminatory power, with greater than two-thirds of samples analyzed having just two different genotypes. The multiplex was determined to be unsuitable for individualization; however, a genotype map was developed as a proof of concept that these markers may be useful for determining the biogeographical origin of samples. Searching the poppy genome for new STR markers and developing new primer sets may be necessary for the creation of a powerful genetic tool for the individualization of P. somniferum.     

Multiplex short tandem repeat amplification of low template DNA samples with the addition of proofreading enzymes

Abstract:  With <100 pg of template DNA, routine short tandem repeat (STR) analysis often fails, resulting in no or partial profiles and increased stochastic effects. To overcome this, some have investigated preamplification methods that include the addition of proofreading enzymes to the PCR cocktail. This project sought to determine whether adding proofreading polymerases directly in the STR amplification mixture would improve the reaction when little template DNA is available. Platinum Taq High Fidelity and GeneAmp High Fidelity were tested in Profiler Plus? STR reactions alone and in combination with AmpliTaq^® Gold. All reactions included the additional step of a post‐PCR purification step. With both pristine low template DNA and casework samples, the addition of these polymerases resulted in comparable or no improvement in the STR amplification signal. Further, stochastic effects and artifacts were observed equally across all enzyme conditions. Based on these studies, the addition of these proofreading enzymes to a multiplex STR amplification is not recommended for low template DNA work.     

Sequence analysis of a new short tandem repeat locus D17S2266E located in the human growth hormone gene cluster HumGH@

D17S2266E is a new, variable genetic marker exhibiting polymorphism of the number of repeats of four- and two-nucleotide motifs. This study, carried out on a group of 250 unrelated persons from various regions of Poland, revealed the presence of 24 different alleles ranging in size from 232 to 290 base pairs. Analysis of the sequenced fragments demonstrated that the alleles consisted of two flanking regions and two variable blocks that were separated by a consensus sequence. There were (AAAG)(5)(AG)(1)(AAAG)(3-4) repeats in the first block, and [(AAAG)(2)(AG)(1)](0-1)[(AAAG)(6)(AG)(1)](0-1)(AAAG)(n) repeats in the second block. On the basis of the allele frequencies in the population, we were able to do biostatistical calculations, which gave the following results: expected heterozygosity 0.8947 +/- 0.0137, power of discrimination 0.9793, polymorphism information content 0.8837, probability of exclusion (PE) 0.7859, PE for motherless cases 0.6473, and an average paternity index of 4.7470. These biostatistical parameters show that the marker D17S2266E can find a wide range of applications in forensic testing.     

A proposal for standardization in forensic canine DNA typing: allele nomenclature of six canine-specific STR loci

In this study a proposal for the allele nomenclature of six polymorphic short tandem repeat (STR) loci (PEZ3, PEZ6, PEZ8, PEZ10, FHC2161, and FHC2328) for canine genotyping (Canis lupus familiaris) is presented. The nomenclature is based on the sequence data of the polymorphic region of the microsatellite markers as recommended by the DNA commission of the International Society of Forensic Haemogenetics (ISFH) in 1994 for human DNA typing. To cover commonly and rarely occurring alleles, a selection of homozygous and heterozygous animals were analyzed and subjected to sequence studies. The alleles consisted of simple tri- and tetra-nucleotide repeat patterns as well as compound and highly complex repeat patterns. Several alleles revealing the same fragment size but different repeat structures were found. The allele designation described here was adopted to the number of repeats, including all variable regions within the amplified fragment. In a second step the most commonly occurring alleles were added to an allelic ladder for each marker allowing a reliable typing of all alleles differing in size. A total number of 142 unrelated dogs from surrounding municipal animal homes, private households, and canines in police duty were analyzed. The data were added to a population database providing allele frequencies for each marker.     

Rapid and high-throughput forensic short tandem repeat typing using a 96-lane microfabricated capillary array electrophoresis microdevice

A 96-channel microfabricated capillary array electrophoresis (muCAE) device was evaluated for forensic short tandem repeat (STR) typing using PowerPlex 16 and AmpFlSTR Profiler Plus multiplex PCR systems. The high-throughput muCAE system produced high-speed <30-min parallel sample separations with single-base resolution. Forty-eight previously analyzed single-source samples were accurately typed, as confirmed on an ABI Prism 310 and/or the Hitachi FMBIO II. Minor alleles in 3:1 mixture samples containing female and male DNA were reliably typed as well. The instrument produced full profiles from sample DNA down to 0.17 ng, a threshold similar to that found for the ABI 310. Seventeen nonprobative samples from various evidentiary biological stains were also correctly typed. The successful application of the muCAE device to actual forensic STR typing samples is a significant step toward the development of a completely integrated STR analysis microdevice.     

Combining autosomal and Y-chromosomal short tandem repeat data in paternity testing with male child: methods and application

Paternity testing is being increasingly requested with the aim of challenging presumptive fatherhood. The ability to establish the biological father is usually based on the genotyping of autosomal short tandem repeat (STR) in alleged father, mother and child, but the use of Y-chromosomal STR has gained interest in the last few years. In this work, we propose a new probabilistic approach that combines autosomal and Y-chromosomal STR data in paternity testing with father/son pairs taking into account mutation events. We also suggest a new two-stage approach where we first type Y-STRs and possibly autosomal STR for the putative father and son, conditional on Y-STR results. We applied this approach to 22 cases. Our results show that Y-STRs can identify nonpaternity cases with high accuracy but need to be validated with autosomal STR to establish paternity. Moreover, the two-stage approach is less costly than the standard approach and is very useful in motherless cases.