Characterization of 26 miniSTR loci for improved analysis of degraded DNA samples

An additional 20 novel mini-short tandem repeat (miniSTR) loci have been developed and characterized beyond the six previously developed by our laboratory for a total of 26 non-CODIS miniSTR markers. These new markers produce short PCR products in the target range of 50-150 base pairs (bp) by moving the primer sequences as close as possible-often directly next to the identified repeat region. These candidate loci were initially screened based on their small amplicon sizes and locations on chromosomes currently unoccupied by the 13 CODIS STR loci or at least 50 Mb away from them on the same chromosome. They were sequenced and evaluated across more than 600 samples, and their population statistics were determined. The heterozygosities of the new loci were compared with those of the 13 CODIS loci and all were found to be comparable. Only five of the new loci had lower values than the CODIS loci; however, all of these were much smaller in size. This data suggests that these 26 miniSTR loci will serve as useful complements to the CODIS loci to aid in the forensic analysis of degraded DNA, as well as missing persons work and parentage testing with limited next-of-kin reference samples.     

3个miniSTR基因座在高度降解检材中的应用及其遗传多态性

目的研究D1S1677、D4S2364、D10S1248 3个m in iSTR基因座在DNA高度降解检材中的法医学应用价值并调查河北地区汉族人群3个基因座的遗传多态性。方法对所有样本的3个基因座进行PCR扩增;PCR产物在AB I3100 Avant基因分析仪上电泳分离;GeneScan 3.7及Genotyper3.7软件分析结果。结果3个m in iSTR基因座均获得了清晰的基因型分型结果,扩增片段均小于125bp,分别检出7,5,8个等位基因和12,10,16种基因型,基因型分布均符合Hardy-W e inberg平衡。3个基因座在河北汉族人群的非父排除率和个人识别力分别为0.3530、0.3637、0.4901和0.7954、0.8113、0.8913。结论3个m in iSTR基因座在DNA高度降解检材的法医学分析中具有较高应用价值,并且在河北地区汉族人群中具有较好的遗传多态性。     

Development of a novel miniSTR multiplex assay for typing degraded DNA samples

A multiplex PCR was developed for the analysis of the sex-determining gene Amelogenin, four conventional STR (short tandem repeat; THO1, D18S51, D21S11 and FGA) loci with a reduced amplicon size and four miniSTR loci (D1S1677, D2S441, D10S1248 and D22S1045). A concordance study in a population of 198 Belgians revealed no differences for the conventional STR loci while a sensitivity study showed a reproducible DNA profile with as low as 30 pg of input DNA.     

8个miniSTR基因座复合扩增系统的建立及其应用

目的建立扩增片段〈200bp,包含CSF1PO,TH01,TPOX,D3S1358,FGA,D7S820,D21S11,PentaD 8个miniSTR基因座的复合扩增体系。方法采用四色荧光染料标记引物,PCR扩增后,应用ABI 3130遗传分析仪进行片段长度分析,对280例无关个体,137例疑难生物物证进行了检验。结果280例无关个体的调查结果为除1例在CSF1PO基因座外,其余样本的各基因座分型结果与AmpFLSTR Identifiler试剂盒完全相同。与应用ID试剂盒比较,明显提高了137例疑难生物物证的检出率。结论该检测技术方法稳定,结果准确,重复性好,且其判型结果可进行DNA数据库查询、比对,为刑事案件及灾难事故中疑难生物物证的检验提供了一条新的途径。     

MiniSTR multiplex systems based on non-CODIS loci for analysis of degraded DNA samples

We describe two short amplicon autosomal short tandem repeat (miniSTR) quadruplex systems for eight loci D1S1171, D2S1242, D3S1545, D4S2366, D12S391, D16S3253, D20S161, and D21S1437, unlinked from the combined DNA index system (non-CODIS) loci, using newly designed primer sets. The results of an assay of 411 Japanese individuals showed that polymerase chain reaction (PCR) products within the eight loci were less than 150bp in size, without the seven additional bases for adenylation. The frequency distributions in the loci showed no deviations from Hardy-Weinberg equilibrium expectations. The accumulated power of discrimination and power of exclusion for the eight loci were 0.9999999991 and 0.998, respectively. For assay of highly degraded DNA, including artificially degraded samples and the degraded forensic casework samples assessed with the present miniSTR quadruplex systems, the systems proved quite effective in analyzing degraded DNA.     

3个miniSTR基因座荧光标记复合扩增体系的建立及应用

目的建立扩增片段小于115 bp,包括D1S1676、D6S1274和D17S1299 3个非CODIS系统的miniSTR基因座复合扩增系统,用于高度降解DNA样本的基因分型。方法采用不同荧光染料标记引物,通过PCR扩增,利用310遗传分析仪对100份成都汉族健康无关个体血样以及2份高度降解检材进行检测。结果荧光标记复合扩增D1S1676、D6S1274和D17S1299 3个miniSTR基因座,每个基因座均获得了清晰的基因型分型结果。100份样本,3个miniSTR基因座分别检出个9、9、7个等位基因和27、23、18种基因型,基因型分布均符合Hardy-Weinberg平衡。3个基因座在成都汉族人群的累积非父排除率、累积个体识别能力分别为0.9991和0.9160。结论本系统可以应用于个体识别和亲权鉴定,为DNA高度降解样本分型提供了新的方法。     

2个miniSTR等位基因分型标准物的制备

目的采用分子克隆技术制备miniSTR D3S4529和D12ATA63基因座等位基因分型标准物,并评价其应用价值。方法用荧光引物对835份无关个体血卡样本进行扩增并分型检测,筛选2个基因座的等位基因片段,用分子克隆方法制备等位基因分型标准物,并对中国汉族群体进行遗传学调查。结果根据筛选出的等位基因片段制备出分型标准物,各等位基因峰形尖锐,无双肩峰,峰高基本一致,荧光值在4 000 RFU左右。中国汉族人群D3S4529、D12ATA63基因座分别检出7个、11个等位基因,杂合度分别为0.752、0.723,多态信息含量均为0.71。结论通过分子克隆法制备的miniSTR D3S4529和D12ATA63等位基因分型标准物,在法医学研究中具有较高的应用价值。     

Population data of nine miniSTR loci in Koreans

For highly degraded DNA samples of forensic casework, new miniSTR systems have been developed to supplement the current STR systems. In the present study, nine miniSTR loci were analyzed in 300 unrelated Koreans using three multiplex PCR systems (multiplex I: D10S1248, D14S1434 and D22S1045; multiplex II: D1S1677, D2S441 and D4S2364; and multiplex III: D3S3053, D6S474 and D20S482), and allele frequencies and forensic parameters were calculated. These data demonstrated that D10S1248, D2S441, D22S1045, D14S1434, and D6S474 are as highly informative as the CODIS STRs suggesting that the miniSTRs could be useful for forensic analysis of degraded DNA.     

荧光标记3个miniSTR基因座复合扩增系统的建立

目的建立扩增片段<135bp,包括D5S818,D8S1179,D16S539 3个miniSTR基因座复合扩增系统。方法采用不同荧光染料标记引物,通过PCR扩增,利用ABI 3100遗传分析仪进行片段长度分析,对100份无关个体血样,10个家系样本以及30份高度降解检材进行检测。结果本系统DNA分型结果与AmpFLSTR Identifiler试剂盒完全一致,且灵敏度高于AmpFLSTR Identifiler试剂盒。结论本系统可以应用于个人识别和亲权鉴定,为降解DNA样本分型提供了新的方法。     

9个miniSTR基因座在烧骨中的检测与分型

目的对300℃焚烧后成人股骨样本进行9个miniSTR（D20S1082、D6S474、D12ATA63、D9S1122、D2S1776、D1S1627、D3S4529、D2S441、Amelogenin）基因座的检测与分型。方法样本为8根经300℃焚烧后的成人股骨,用改良酚-氯仿法提取烧骨DNA,在Mastercylcerpro梯度PCR仪上对9个miniSTR基因座分别进行扩增,3130基因分型仪检测并收集电泳结果,GeneMarkerV2.2.0软件计算扩增产物片段相对大小以及进行样本基因型分型。结果8根烧骨样本均能够提取到DNA,浓度平均值为25ng/μL,D260/D280值在1.7~1.9之间。9个miniSTR基因座在样本中的检出率在78%~100%之间,分型图谱较清晰,个别样本出现额外带。结论本文9个miniSTR基因座分型检测的方法,可用于对烧骨捡材的DNA分型检验。     

4个miniSTR基因座复合扩增体系及应用

目的构建D6S474、D20S482、D4S2408、D6S1017等4个miniSTR基因座复合扩增体系,评价其对腐败检材的应用价值,调查4个基因座在汉族人群中的遗传多态性。方法采用不同荧光标记4个miniSTR基因座上游引物,构建复合扩增体系。用分子克隆方法制备等位基因分型标准物。采用上述体系对135份汉族无关个体血样进行检测,并计算群体遗传学参数。比较该体系与ID试剂盒在降解检材分析中的成功率。结果采用本文复合扩增体系检测,汉族人群中4个基因座基因型频率分布均符合Hardy-Weinberg平衡定律,累积个人识别能力为0.999 666,累积非父排除率为0.914 902。本文体系较ID试剂盒对自然腐败检材的分型成功率更高。结论 4个miniSTR基因座复合扩增体系对法庭科学实践,特别是对腐败检材的检测有应用价值。     

Allele frequencies of six miniSTR loci in the population of Northern Portugal

A possible approach to try to recover information from degraded DNA is to reduce the size of the PCR products by designing primers that bind as close as possible to the STR repeat region, known as miniSTRs. Allele frequencies and forensic parameters for the six miniSTRs loci D1S1677, D2S441, D4S2364, D10S1248, D14S1434 and D22S1045 were investigated in a sample group consisting of 228 anonymous apparently healthy unrelated individuals living in North of Portugal. The results show that all loci were in Hardy–Weinberg equilibrium. The combined power of discrimination and power of exclusion for the six loci were 0.99999 and 0.9789, respectively. All but one (D4S2364) loci showed a moderate degree of polymorphism (observed heterozygosity >0.6). The allele sizes ranged between 66 and 118 bp in our population, which is beneficial for typing degraded samples than those of a commercial STR kit.     

浓缩DNA法结合miniSTR分型技术检验微量DNA

目的优化低拷贝数DNA STR分型方法。方法对采用磁珠法或Chelex-100法提取DNA,Identi-filer试剂盒扩增,未获得分型结果的日常检案检材,采用物理浓缩法或过柱浓缩法浓缩DNA,采用miniFilerTM试剂盒再次扩增分型。结果 127例检材中,47例磁珠法提取DNA未获得分型的样品,分型成功率为36%;80例Chelex-100法提取DNA未获分型的样品,分型成功率为30%。结论采用浓缩法和miniFilerTM试剂盒,可以提高日常检案中低拷贝数检材的STR检验分型成功率。     

STR analysis of degraded DNA using a miniplex

Analysis of short tandem repeat (STR) markers currently represents the most useful instrument in the field of forensic genetics. The problem with forensic material is the degradation of the sample material. In recent years, several papers have demonstrated that short amplicon STR (miniSTR) represents one of the most useful tools for analyzing degraded DNA samples.In the present study, we attempted to develop a short amplicon STR multiplex system (autosomal and y-chromosomal) for analyzing degraded DNA using some newly designed primer sets for a multiplex polymerase chain reaction (PCR) systems for typing.An assay of degraded DNA samples using the designed multiplex systems, including artificially degraded samples and degraded forensic casework samples, proved remarkably effective. Comparing the multiplex with commercial kits, first results show a well success rate.     

Allele frequencies of six miniSTR loci of three ethnic populations in Singapore

MiniSTR loci has demonstrated to be an effective approach to recover genetic information from degraded sample, due to the improved PCR efficiency of their reduced PCR product sizes. This study investigated the allele frequency of six miniSTR loci, D1S1677, D2S441, D4S2364, D10S1248, D14S1434 and D22S1045, in three Singapore populations. All loci showed a moderate degree of polymorphism with observed heterozygosity >0.6 for all three populations. The allele frequencies, forensic parameters and heterozygosity comparison with other CODIS STR in similar populations are presented.     

Allele frequencies for six miniSTR loci of two ethnic populations in China

Allele frequencies and forensic parameters for the six miniSTR loci D1S1677, D2S441, D4S2364, D10S1248, D14S1434, and D22S1045 were investigated in two ethnic China populations. Allele frequencies for each locus are reported along with nomenclature based on sequence analysis. The polymerase chain reaction (PCR) products contained within the six loci were less than 125 bp in size. All loci showed a moderate degree of polymorphism with observed heterozygosity >0.7 except for D4S2364 and did not show departures from Hardy-Weinberg equilibrium for all two populations. The accumulated powers of discrimination for the six loci were 0.999994, and 0.999996 for Han ethnic, and Korean ethnic minority group, respectively.     

Construction and application of four fluorescence labeled multiplex typing system for 3 miniSTR loci

We constructed a multiplex PCR system for 3 miniSTR loci D20S482, D3S3053, D6S474. This typing system showed high stability and sensitivity (0.05 ng). Population data investigated in 120 healthy unrelated Chinese Han individuals showed higher genetic polymorphism, with the combined power of discrimination and power of exclusion being 0.998 and 0.84. The amplification product length ranged from 88 bp to 127 bp for all three loci. The successful rate of typing highly degraded samples using this miniSTR multiplex PCR system was significantly higher than using identifiler kit, indicating the multiplex set represents a useful tool in Chinese forensic practice, especially for the highly degraded DNA sample.     

一种用于降解DNA样本的性别鉴定方法

目的 建立一种采用PCR技术对降解DNA样本进行性别鉴定的新方法。方法 采用针对amelogenin基因X染色体外显子3bp缺失设计的引物AMELU1及AMELD1,对在室温环境下放置5－15年的男、女血痕标本各50例、毛发各20例、骨骼各20例以及现场提取5－－20天的男、女腐败肌肉各10例标本中提取的降解DNA样本进行扩增。用PGA（9％T,3％C）电泳、银染显带检测扩增产物。结果 所有样本均得到正确结果,男性检材表现为83bp的Y特异性及80bp的X特异性2条谱带,而女性检材仅有1条80bp的X特异性谱带。结论 用针对amelogenin基因X染色体外显子3bp缺失设计的引物AMELU1及AMELD1鉴定性别的方法灵敏、可靠、方便,是降解DNA检材性别鉴定十分理想的方法。     

Resistance of degraded hair shafts to contaminant DNA

We have investigated the susceptibility of degraded human hair shaft samples to contamination by exogenous sources of DNA, including blood, saliva, skin cells, and purified DNA. The results indicate that on the whole hair shafts are either largely resistant to penetration by contaminant DNA, or extremely easy to successfully decontaminate. This pertains to samples that are both morphologically and biochemically degraded. We suggest that this resistance to the incorporation of contaminant DNA relates to the hydrophobic and impermeable nature of the keratin structures forming the hair shaft. Therefore, hair samples represent an important and underestimated source of DNA in both forensic and ancient DNA studies.