Preliminary trials of low volume AmpFlSTR Profiler Plus amplification using AmpliGrid (AG480F) slides

Low volume PCR using the AmpliGrid (480F) slide system can potentially enhance the generation of more complete profiles from trace samples, in addition to providing a more cost-effective alternative for typing standard samples. Based on our preliminary results, implementation will require a reasonable investment in optimisation and validation for the intended purpose.     

Population genetics for Y-chromosomal STRs haplotypes of Chinese Tujia ethnic group

Allele and haplotype frequencies of 11 Y-chromosomal short tandem repeats (STRs) included in the PowerPlex^® Y Systems (Promega) were determined in a sample of 215 unrelated healthy male individuals of Chinese Tujia ethnic group living in Chongqing (Southwest of China). The gene diversity values of the Y-STRs loci ranged from 0.3757(DYS391) to 0.9170 (DYS385a/b). A total of 195 haplotypes were identified in the Y-STR loci, among which 180 were unique, 11 were found in two individuals, 3 were shared in three individuals and 1 was shared in four individuals. The observed haplotype diversity value and discrimination capacity were 0.9942 and 0.9070, respectively.     

Analysis of AmpFlSTR MiniFiler™ loci and its forensic application

The allele frequencies of eight MiniFiler™ loci have been analyzed in 101 Japanese individuals living in Kanagawa with informed consent by means of ABI 310 Genetic Analyzer. A total of 7 alleles for D13S317, 8 alleles for D7S820, 11 alleles for D2S1338, 11 alleles for D21S11, 5 alleles for D16S539, 14 alleles for D18S51, 8 alleles for CSF1PO, and 13 alleles for FGA were observed. The polymorphic profiles of these MiniFiler™ loci in the present study were essentially the same as those obtained by using the AmpFlSTR^® Identifiler^® PCR Amplification kit. The combined matching probability of eight MiniFiler™ loci and cumulative probability of paternity exclusion were estimated as 1.97 × 10⁻¹⁰ and 0.9996, respectively. The MiniFiler™ kit was useful for individual identification in forensic analysis.     

Post-injection hybridization of complementary DNA strands on capillary electrophoresis platforms: A novel solution for dsDNA artifacts

Several laboratories have reported the occurrence of a split or n − 1 peak at the vWA locus in PowerPlex^® 16 and PowerPlex^® ES amplification products separated on 4- and 16-capillary electrophoresis instruments. The root cause of this artifact is post-PCR reannealing of the unlabeled, unincorporated vWA primer to the 3′-end of the tetramethylrhodamine (TMR)-labeled strand of the vWA amplicon. This reannealing occurs in the capillary post-electrokinetic injection. The split peak is eliminated by incorporation into the loading cocktail of a sacrificial hybridization sequence (SHS) oligonucleotide that is complementary to the vWA primer. The SHS preferentially anneals to the primer instead of the TMR-labeled strand of the vWA amplicon. In addition, the n − 10/n − 18 artifact that may be seen at the vWA locus was determined to be due to double-stranded amplicon formed post-electrokinetic injection into the capillary. This was also eliminated by adding in two Complementary Oligo Targets (COT1 and COT2) in addition to the SHS oligonucleotide into the loading cocktail. These three oligonucleotides are complementary to the 33 bases at the 5′-end of the unlabeled vWA amplicon strand and the 60 bases at its 3′-end and therefore compete for hybridization to the TMR-labeled amplicon strand. Incorporation of these three oligonucleotides in the Internal Lane Standard 600 (ILS600) eliminate both the split peak and n − 10/n − 18 artifact in PowerPlex^® 16 and PowerPlex^® ES amplification products without affecting sizing of alleles at the vWA locus or any locus in the PowerPlex^® 16, PowerPlex^® Y, PowerPlex^® ES, AmpFlSTR^® Profiler Plus^® ID, AmpFlSTR^® Cofiler^®, and AmpFlSTR^® SGM Plus^® kits.     

An analysis of single and multi-copy methods for DNA quantitation by real-time polymerase chain reaction

The goal of this paper was to examine and compare two different commercially available approaches to the determination of the relative quantities of autosomal and Y chromosomal DNA using real-time PCR. One, Quantifiler^® Duo, utilizes a TaqMan^® assay with single copy probes for both autosomal human and Y quantification. The other method, Plexor HY^® utilizes a primer quenching assay with multi-copy probes for its quantification of autosomal human and Y chromosomal DNA. To test these approaches we have utilized the NIST Human DNA Quantitation Standard Reference Material 2372, a set of three different NIST human DNA quantification standards, to examine the precision, accuracy and sensitivity of the real-time PCR assays. We also examined data from both systems utilizing casework samples. The results show that both systems produced linear estimates for DNA quantity over a broad range of input DNA. However we did observe some apparent copy number effects when comparing the three different NIST standards which we attributed to issues with sequence variations in the different standards. Overall, the single copy approach provided better accuracy while the multi-copy approach produced better sensitivity. Thus the choice of which system to use should depend upon the goals of the user.     

Effect of blood stained soils and time period on DNA and allele drop out using Promega 16 Powerplex kit

Blood stained soils may be of great interest in forensic incidents. Amplification of DNA from soil is often inhibited by co-purified contaminants. Different soils types from Pakistan and Turkey were stained with blood and samples were collected systematically after specified intervals. Rapid, inexpensive, large-scale DNA extraction method involving minimal purification was developed. DNA was quantized using Spectrophotometer and Fluorometer and was confirmed by Agarose Gel Electrophoresis. DNA extracted from different soils in different periods showed a remarkable decrease in yield as well as degradation in every extraction. PCR amplification was performed using various DNA targets present in Promega 16 Powerplex^® System kit. Amplification could not carry out in all loci especially in degraded samples taken after 20 days. Allele n locus drop out was noticed which shows that DNA was degraded. For some loci more than 2 alleles were also noticed showing contamination while working with the blood stained soils.     

Haplotypes for 12 Y-chromosomal STR loci in a Korean population (the central region)

Haplotypes and allele frequencies of 12 STR loci included in the PowerPlex Y system (DYS391, DYS389I, DYS439, DYS389II, DYS438, DYS437, DYS19, DYS392, DYS393, DYS390, and DYS385a/b) were obtained from a sample of 569 unrelated individuals living in the central region of Korea. A total of 473 haplotypes were observed in the 569 individuals studied, of which 426 (90.06%) were unique. The overall haplotype diversity for the 12 Y-STR loci was 0.9985, and the discrimination capacity was 0.8313. In DYS439, we found a new intermediate-sized allele that added an A at base 3 upstream from the repeat region's first GATA motif. The allele was named 11 (U3Ains) according to its sequence structure.     

Human eye colour and HERC2, OCA2 and MATP

Prediction of human eye colour by forensic genetic methods is of great value in certain crime investigations. Strong associations between blue/brown eye colour and the SNP loci rs1129038 and rs12913832 in the HERC2 gene were recently described. Weaker associations between eye colour and other genetic markers also exist. In 395 randomly selected Danes, we investigated the predictive values of various combinations of SNP alleles in the HERC2, OCA2 and MATP (SLC45A2) genes and compared the results to the eye colours as they were described by the individuals themselves. The highest predictive value of typing either the HERC2 SNPs rs1129038 and/or rs12913832 that are in strong linkage disequilibrium was observed when eye colour was divided into two groups, (1) blue, grey and green (light) and (2) brown and hazel (dark). Sequence variations in rs11636232 and rs7170852 in HERC2, rs1800407 in OCA2 and rs16891982 in MATP showed additional association with eye colours in addition to the effect of HERC2 rs1129038. Diplotype analysis of three sequence variations in HERC2 and one sequence variation in OCA2 showed the best discrimination between light and dark eye colours with a likelihood ratio of 29.3.     

Allele frequency distribution for 21 autosomal STR loci in Bhutan

We studied the allele frequency distribution of 21 autosomal STR loci contained in the AmpFlSTR^® Identifiler™ (Applied Biosystems), the Powerplex^®16 (Promega) and the FFFL (Promega) multiplex PCR kits among 936 individuals from the Royal Kingdom of Bhutan. As such these are the first published autosomal DNA results from this country.     

Forensic applications of stable isotope analysis: Case studies of the origins of water in mislabeled beer and contaminated diesel fuel

This paper describes the use of oxygen (¹⁸O) isotope analysis of water contained in two different materials — beer and diesel fuel — involved in the resolution of two separate cases. In the first case study, it was possible to demonstrate that a sample of beer labelled as premium brand in fact belonged to a cheap brand. The second case related to the contamination of diesel fuel from a service station. The diesel fuel contained visible amounts of water, which caused vehicles that had been filled up with it to become defective. For insurance purposes, it was necessary to determine the source of water. The δ¹⁸O values for the water of nearly all samples of diesel was close to the δ¹⁸O of local tap water at the filling station.     

Validation studies of the ParaDNA® Intelligence System with artificial evidence items

Short tandem repeat (STR) profiling is one of the mostly used systems for forensic applications. In certain circumstances, STR profiling is time-consuming and costly, which potentially leads to delays in criminal investigations. LGC (Laboratory of the Government Chemist, UK) Forensics has developed a robust STR profiling platform called the ParaDNA^® Intelligence Test System which can provide early tactical intelligence and aid investigators in making informed decisions on sample prioritization for detection. Here, we validated the ParaDNA intelligence test for its application in forensic cases using a range of mock evidence items following guidelines set by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Specifically, we tested the sensitivity and accuracy of the ParaDNA intelligence test, as well as the success rates for detecting mock samples and for use in case scenarios. Our findings demonstrate that the ParaDNA intelligence test generates useful DNA profiles, especially for samples such as blood, saliva, and semen that contain ample DNA, indicating the benefits of including ParaDNA as a prior step in forensic STR profiling pipelines.     

PowerPlex~(TM) 16体系在中国人群中罕见等位基因及其类型

目的 分析PowerPlexTM 16体系基因座在中国人群中的罕见等位基因及其类型。方法 应用PCR-STR和DNA序列分析技术,对4650个无关个体在15个STR基因座中的罕见等位基因进行检测。结果 在PowerPlexTM16体系中的D7S820、D16S539、Penta E基因座,检测到2种类型的罕见等位基因,而TH01、D21S11、D5S818、D13S317、Penta D、D8S1179、TPOX、FGA基因座检测出1种类型。其等位基因频率均较低(0.215‰-7.097‰)。结论 超出ladder范围的罕见等位基因序列比相邻等位基因增加(或减少)1个或数个重复单位,因碱基的插入或缺失的罕见等位基因出现在两等位基因之间。     

An updated validation of Promega's PowerPlex 16 System: high throughput databasing under reduced PCR volume conditions on Applied Biosystem's 96 capillary 3730xl DNA Analyzer

The PowerPlex 16 System from Promega Corporation allows single tube multiplex amplification of sixteen short tandem repeat (STR) loci including all 13 core combined DNA index system STRs. This report presents an updated validation of the PowerPlex 16 System on Applied Biosystem's 96 capillary 3730xl DNA Analyzer. The validation protocol developed in our laboratory allows for the analysis of 1536 loci (96 x 16) in c. 50 min. We have further optimized the assay by decreasing the reaction volume to one-quarter that recommended by the manufacturer thereby substantially reducing the total cost per sample without compromising reproducibility or specificity. This reduction in reaction volume has the ancillary benefit of dramatically increasing the sensitivity of the assay allowing for accurate analysis of lower quantities of DNA. Due to its substantially increased throughput capability, this extended validation of the PowerPlex 16 System should be useful in reducing the backlog of unanalyzed DNA samples currently facing public DNA forensic laboratories.     

Direct amplification of STRs from blood or buccal cell samples

Forensic databasing laboratories routinely analyze blood or buccal cell samples deposited on FTA^® paper. Prior to PCR amplification of the STRs, the FTA^® samples must undergo multi-step sample purification protocols to remove the PCR inhibitors present within the sample and from the FTA^® paper. The multi-step sample purification protocols are laborious, time-consuming and increase the potential for sample cross-contamination.To eliminate the need for DNA purification, we conducted studies to optimize the PCR buffer and thermal cycling parameters to allow for direct amplification of STRs from blood or buccal samples on FTA^® paper. We evaluated the effect of various factors on the DNA profile including: FTA^® disc size, blood sample load variation, and buffer formulation. The new STR assay enables the direct amplification of DNA from single source samples on FTA^® discs without sample purification. The new STR assay improves the workflow by eliminating tedious steps and minimizing sample handling. Furthermore, the new STR assay reduces cost by eliminating the need for purification reagents and expensive robots.     

Optimization and validation of a fast amplification protocol for AmpFlSTR Profiler Plus for rapid forensic human identification

The goal of this work was to optimize and validate a fast amplification protocol for the multiplex amplification of the STR loci included in AmpFlSTR^® Profiler Plus^® to expedite human DNA identification. By modifying the cycling conditions and by combining the use of a DNA polymerase optimized for high speed PCR (SpeedSTAR™ HS) and a more efficient thermal cycler instrument (Bio-RAD C1000™), we were able to reduce the amplification process from 4 h to 26 min. No modification to the commercial AmpFlSTR^® Profiler Plus^® primer mix was required. When compared to the current Royal Canadian Mounted Police (RCMP) amplification protocol, no differences with regards to specificity, sensitivity, heterozygote peak height ratios and overall profile balance were noted. Moreover, complete concordance was obtained with profiles previously generated with the standard amplification protocol and minor alleles in mixture samples were reliably typed. An increase in n − 4 stutter ratios (2.2% on average for all loci) was observed for profiles amplified with the fast protocol compared to the current procedure. Our results document the robustness of this rapid amplification protocol for STR profiling using the AmpFlSTR^® Profiler Plus^® primer set and demonstrate that comparable data can be obtained in substantially less time. This new approach could provide an alternative option to current multiplex STR typing amplification protocols in order to increase throughput or expedite time-sensitive cases.     

The BodeChecks solution: A high throughput analysis software combining GeneMapper ID, FSS-i, LIMS, and artificial intelligence

Combining key attributes of GeneMapper^® ID and FSS-i³ software packages with our internally created LIMS and some additional analytical programming has permitted us to increase quality checks on DNA profile data review while eliminating analysis time.