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.     

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。     

Development and validation of the AmpFℓSTR® Identifiler® Direct PCR Amplification Kit: a multiplex assay for the direct amplification of single-source samples

Abstract: The AmpF?STR^® Identifiler^® Direct PCR Amplification Kit is a new short tandem repeat multiplex assay optimized to allow the direct amplification of single‐source blood and buccal samples on FTA^® card without the need for sample purification and quantification. This multiplex assay has been validated according to the FBI/National Standards and SWGDAM guidelines. Validation results revealed that slight variations in primer concentration, master mix component concentration, and thermal cycling parameters did not affect the performance of the chemistry. The assay’s sensitivity was demonstrated by amplifying known amounts of white blood cells spotted onto FTA^® cards, and the assay’s specificity was verified by establishing minimal cross‐reactivity with nonhuman DNA. No effect on the age of the sample stored on the FTA^® substrate was observed and full concordance was established in the population study. These findings of the validation study support the use of the Identifiler^® Direct Kit for forensic standards and database samples genotyping.     

A 27‐Locus STR Assay to Meet All United States and European Law Enforcement Agency Standards,

Different national and international agencies have selected specific STR sets for forensic database use. To enhance database comparison across national and international borders, a 27‐locus multiplex system was developed comprising all 15 STR loci of the European standard set, the current 13 STR loci of the CODIS core, the proposed 22 STR loci of the expanded CODIS core, 4 additional commonly used STR loci, and the amelogenin locus. Development required iterative primer design to resolve primer‐related artifacts, amplicon sizing, and locus‐to‐locus balance issues. The 19.5‐min assay incorporated newly developed six‐dye chemistry analyzed using a novel microfluidic electrophoresis instrument capable of simultaneous detection and discrimination of 8 or more fluorescent dyes. The 27‐locus multiplex offers the potential for a new international STR standard permitting laboratories in any jurisdiction to use a single reaction to determine profiles for loci they typically generate plus an expanded common STR profiling set of global interest.     

Developmental validation of the AmpFℓSTR® Identifiler® Plus PCR Amplification Kit: an established multiplex assay with improved performance

Abstract: Analysis of length polymorphism at short tandem repeat (STR) loci utilizing multiplex polymerase chain reaction (PCR) remains the primary method for genotyping forensic samples. The AmpF?STR^® Identifiler^® Plus PCR Amplification Kit is an improved version of the AmpF?STR^® Identifiler^® PCR Amplification Kit and amplifies the core CODIS loci: D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, TH01, TPOX, and vWA. Additional loci amplified in the multiplex reaction are the sex‐determinant, amelogenin, and two internationally accepted loci, D2S1338 and D19S433. While the primer sequences and dye configurations were unchanged, the AmpF?STR^® Identifiler^® Plus PCR Amplification Kit features an enhanced buffer formulation and an optimized PCR cycling protocol that increases sensitivity, provides better tolerance to PCR inhibitors, and improves performance on mixture samples. The AmpF?STR^® Identifiler^® Plus PCR Amplification Kit has been validated according to the FBI/National Standards and Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. The validation results support the use of the AmpF?STR^® Identifiler^® Plus PCR Amplification Kit for human identity and parentage testing.     

Development and validation of the AmpFlSTR MiniFiler PCR Amplification Kit: a MiniSTR multiplex for the analysis of degraded and/or PCR inhibited DNA

DNA typing of degraded DNA samples can be a challenging task when using the current commercially available multiplex short tandem repeat (STR) analysis kits. However, the ability to type degraded DNA specimens improves by redesigning current STR marker amplicons such that smaller sized polymerase chain reaction (PCR) products are generated. In an effort to increase the amount of information derived from these types of DNA samples, the AmpFlSTR MiniFiler PCR Amplification Kit has been developed. The kit contains reagents for the amplification of eight miniSTRs which are the largest sized loci in the AmpFlSTR Identifiler PCR Amplification Kit (D7S820, D13S317, D16S539, D21S11, D2S1338, D18S51, CSF1PO, and FGA). Five of these STR loci (D16S539, D21S11, D2S1338, D18S51, and FGA) also are some of the largest loci in the AmpFlSTR SGM Plus kit. This informative nine-locus multiplex, which includes the gender-identification locus Amelogenin, has been validated according to the FBI/National Standards and SWGDAM guidelines. Our results demonstrate significant performance improvements in models of DNA degradation, PCR inhibition, and nonprobative samples when compared to the AmpFlSTR Identifiler and SGM Plus kits. These data support that the MiniFiler kit will increase the likelihood of obtaining additional STR information from forensic samples in situations in which standard STR chemistries fail to produce complete profiles.     

Uses of the NIST 26plex STR assay for human identity testing

Ongoing work at the U.S. National Institute of Standards and Technology has focused on the characterization of 26 autosomal STR loci for human identity testing. These 26 loci are in addition to the existing 13 U.S. core loci and those found in PowerPlex16 and Identifiler commercial STR typing kits. The amplification of the 26 loci has been optimized for degraded extracts in unique miniplex panels and also for reference samples as a single reaction 26plex assay. A study has been performed comparing genotypes obtained with the 26plex primers to those with miniplex panels for allele drop out and concordance. The forensic utility of the 26plex assay was evaluated for situations where additional loci are beneficial. The utility of this large multiplex was also tested in a case involving DNA extracted from degraded bone samples. The 26plex can serve as a low-cost assay (compared to commercially available kits) useful for both sorting comingled remains and providing additional markers for increased statistical support for samples that require “non-trio” family references for human identification.     

武汉汉族人群D20S85和D6S477基因座遗传多态性调查

目的对武汉地区280名汉族无关个体的D20S85和D6S477位点遗传多态性进行调查,研究其在法医学检验中的应用价值。方法应用PCR和PAGE技术进行分型检验。结果分别检出10个、9个等位基因,获得各等位基因在该地区汉族人群分布频率,基因型频率分布符合Hardy-Weinberg平衡。两位点的DP值分别为0.9085、0.9127。结论D20S85和D6S477是法医学中重要的遗传标记。     

A quantitative PCR assay for the assessment of DNA degradation in forensic samples

A multiplex quantitative PCR assay has been designed to amplify target sequences of different length, which allows for the assessment of DNA degradation in samples of forensic interest. The targets were chosen to provide quantification and fragment length information relevant to the STR amplification targets commonly used for forensic genotyping. The longer target (nuTH01, 170-190 bp) spans the TH01 STR locus. Although not one of the longest loci used for STR genotyping, it was chosen as a good compromise given the target length limitations on qPCR efficiency with TaqMan detection. The shorter target (nuCSF, 67 bp) was designed in the upstream flanking region of the CSF1PO STR locus. In addition to these human nuclear targets, the assay includes an internal PCR control target sequence to allow for an assessment of PCR inhibition. The assay was rigorously tested on samples with varying amounts of degradation, and the ratio of nuCSF:nuTH01 quantifications was shown to provide a good estimation of the degree of degradation present in a sample. This estimate, along with the internal control for PCR inhibition, provides a valuable tool for post-extraction sample assessment.     

New autosomal STR loci

Additional STR loci can be beneficial for a number of human identity, forensic casework, and DNA database applications. The marker selection and characterization process applied at NIST in developing these new loci and assays are described along with concordance testing results from non-overlapping PCR primers. A 23plex for simultaneous amplification of 22 autosomal STR loci and an amelogenin sex-typing assay is also demonstrated.     

D3S1358等7个DNA位点的荧光法在法医学中的应用

报道了D3S1358、D16S539等7个DNA位点的复合扩增和四色荧光分析技术。经310自动测序仪检测7个位点 ,实现了STR位点同步扩增和自动检测目的。同一性实验表明 ,同一个体肌肉、血液、唾液和头发等组织STR位点基因型完全一致。对2个四代家系、3个三代家系的调查结果表明 ,这些位点符合孟德尔遗传定律。D3S1358、D16S539等7个位点对陈旧、微量的检材适用性很强,灵敏度达75pg ,在法医个人识别和亲子鉴定中有着重要意义     

Modified Midi‐ and Mini‐Multiplex PCR Systems for Mitochondrial DNA Control Region Sequence Analysis in Degraded Samples

Two multiplex polymerase chain reaction (PCR) systems (Midiplex and Miniplex) were developed for the amplification of the mitochondrial DNA (mtDNA) control region, and the efficiencies of the multiplexes for amplifying degraded DNA were validated using old skeletal remains. The Midiplex system consisted of two multiplex PCRs to amplify six overlapping amplicons ranging in length from 227 to 267 bp. The Miniplex system consisted of three multiplex PCRs to amplify 10 overlapping short amplicons ranging in length from 142 to 185 bp. Most mtDNA control region sequences of several 60‐year‐old and 400–500‐year‐old skeletal remains were successfully obtained using both PCR systems and consistent with those previously obtained by monoplex amplification. The multiplex system consisting of smaller amplicons is effective for mtDNA sequence analyses of ancient and forensic degraded samples, saving time, cost, and the amount of DNA sample consumed during analysis.     

VNTR遗传标记的亲权鉴定研究

采用Amp－FLP技术研究人类血液、组织VNTR位点D1S80（pMCT118）、D17S30（pCNZ－22）和ApoB3’位点的遗传多态性，并应用于亲权鉴定案件，获得满意结果。D1S80、D17S30和ApoB3’的DP值分别为0．962、0．956和0．960，累积父权排除率（EPP）为94．51％，远远高于传统血型的DP值和EPP值，是亲权鉴定和个体识别有效方法。     

A LDR‐PCR Approach for Multiplex Polymorphisms Genotyping of Severely Degraded DNA with Fragment Sizes <100 bp*

Abstract: Reducing amplicon sizes has become a major strategy for analyzing degraded DNA typical of forensic samples. However, amplicon sizes in current mini‐short tandem repeat‐polymerase chain reaction (PCR) and mini‐sequencing assays are still not suitable for analysis of severely degraded DNA. In this study, we present a multiplex typing method that couples ligase detection reaction with PCR that can be used to identify single nucleotide polymorphisms and small‐scale insertion/deletions in a sample of severely fragmented DNA. This method adopts thermostable ligation for allele discrimination and subsequent PCR for signal enhancement. In this study, four polymorphic loci were used to assess the ability of this technique to discriminate alleles in an artificially degraded sample of DNA with fragment sizes <100 bp. Our results showed clear allelic discrimination of single or multiple loci, suggesting that this method might aid in the analysis of extremely degraded samples in which allelic drop out of larger fragments is observed.     

Genetics and genomics of core short tandem repeat loci used in human identity testing

Over the past decade, the human identity testing community has settled on a set of core short tandem repeat (STR) loci that are widely used for DNA typing applications. A variety of commercial kits enable robust amplification of these core STR loci. A brief history is presented regarding the selection of core autosomal and Y-chromosomal STR markers. The physical location of each STR locus in the human genome is delineated and allele ranges and variants observed in human populations are summarized as are mutation rates observed from parentage testing. Internet resources for additional information on core STR loci are reviewed. Additional topics are also discussed, including potential linkage of STR loci to genetic disease-causing genes, probabilistic predictions of sample ethnicity, and desirable characteristics for additional STR loci that may be added in the future to the current core loci. These core STR loci, which form the basis for DNA databases worldwide, will continue to play an important role in forensic science for many years to come.     

Validation and casework testing of the BioPlex-11 for STR typing of telogen hair roots

A new STR typing strategy has been developed allowing the simultaneous amplification and subsequent analysis of 11 polymorphic systems with amplicon sizes smaller than 270bp. The multiplex amplification reaction includes six STR loci from the European standard set of loci (ESS) for DNA databases (D3S1358, D8S1179, D21S11, THO1, FGA and VWA) as well as four additional STR systems selected for their robustness (D2S1338, D12S391, TPOX and D5S818) together with the sex-specific locus amelogenin. After PCR amplification, the multiplex reaction is splitted into two sets of STR multiplexes by using biotin labelled primers only for one set. Using streptavidin-coated Sepharose beads five STR systems are separated from the other six systems prior to being analysed in two different runs on a capillary gel electrophoresis instrument. The multiplex system was developed and tested especially for the use in forensic casework if only limited amounts or highly degraded DNA is available, for instance, when isolated from telogen hair roots.     

Validation of the Short Amplicon Multiplex Q8 Including the German DNA Database Systems*

Abstract: We have developed a concept to enable the analyzing of degraded stains with limited DNA template quantity. Therefore we have constructed a short tandem repeat (STR) multiplex including the German DNA database systems (Q8). The amplicon lengths are smaller than 280 bp. For the validation of Q8 over 50 degraded samples were investigated. Amplifications were performed with “low copy number” PCR, the number of PCR cycles was increased to 33 and the reaction volume was decreased to 12.5 μL. Compared with the MPX2 and Nonaplex kit, the average success rate was increased using the Q8 kit by approximately 20% and 30%, respectively. The efficiency of a sensitive STR multiplex with reduced amplicon lengths was confirmed in comparing the success rates of Q8 for typing degraded samples and samples with limited amount of DNA template while partial profiles were observed with the majority of the samples using commercially available kits.     

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.     

Validation of a 21-locus autosomal SNP multiplex for forensic identification purposes

A single nucleotide polymorphism (SNP) multiplex has been developed to analyse highly degraded and low copy number (LCN) DNA template, i.e. <100 pg, for scenarios including mass disaster identification. The multiplex consists of 20 autosomal non-coding loci plus Amelogenin for sex determination, amplified in a single tube PCR reaction and visualised on the Applied Biosystems 3100 capillary electrophoresis (CE) system. Allele-specific primers tailed with shared universal tag sequences were designed to speed multiplex design and balance the amplification efficiencies of all loci through the use of a single reverse and two differentially labelled allele denoting forward universal primers. As the multiplex is intended for use with samples too degraded for conventional profiling, a computer program was specifically developed to aid interpretation. Critical factors taken into account by the software include empirically determined extremes of heterozygous imbalance (Hb) and the drop-out threshold (Ht) defined as the maximum peak height of a surviving heterozygous allele, where its partner may have dropped out. The discrimination power of the system is estimated at 1 in 4.5 million, using a White Caucasian population database. Comparisons using artificially degraded samples profiled with both the SNP multiplex and AMPFISTR SGM plus (Applied Biosystems) demonstrated a greater likelihood of obtaining a profile using SNPs for certain sample types. Saliva stains degraded for 147 days generated an 81% complete SNP profile whilst short tandem repeats (STRs) were only 18% complete; similarly blood degraded for 243 days produced full SNP profiles but only 9% with STRs. Reproducibility studies showed concordance between SNP profiles for different sample types, such as blood, saliva, semen and hairs, for the same individual, both within and between different DNA extracts.     

Developmental validation of reduced-size STR Miniplex primer sets

This paper describes a developmental validation study of three Miniplex sets covering 12 of the 13 CODIS loci. As these new sets will be used for the analysis of degraded and low level DNA, the validation studies were performed using 100-125 pg of DNA, the lowest input level at which peak balance, peak intensity, and allele consistency were stable. To demonstrate the applicability of the Miniplex sets to forensic casework, these validation studies were completed in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM). A range of tests were performed including studies of concordance with standard multiplex kits, sensitivity and reproducibility, and PCR amplification conditions. Additionally, studies of mixtures, nonhuman and environmentally degraded DNA, and simulated forensic samples were performed. Our results demonstrate that Miniplex STR amplification procedures are a robust and sensitive tool for the analysis of degraded DNA.