AmpFl STR Research Articles

Comparison between Two Different DNA Extraction Techniques Taken from Buccal Swabs Suitable for Genetic Analyzer

This study aimed to validate two DNA extraction methods collected from buccal cells to gain a genomic DNA and to determine the effect of process time (from collection time to amplification). Total number of 116 buccal swab samples from unrelated healthy volunteers were subjected to DNA extraction by using two methods: modified (phenol chloroform) (Organic) and Prep Filer Forensic DNA Extraction kit. DNA was evaluated according to the: quantity, integrity and suitability for genetic analysis. The DNA yield and purity were measured by Nano drop Spectrophotometer. Organic method was modified from the classical method (addition Dithiothreitol to lysis buffer), The purity average values increased from 1.4 to 1.6 and it had the highest average concentration (73.3ng/µl) while the highest purity was observed in Prep Filer Forensic DNA Extraction kit with average (1.82). Delayed in sample processing effect both quantity and integrity of the extracted DNA. All samples were genetically analyzed at 16 STR with amelogenin (DNA markers) included in the Amp FlSTR® Identifiler™ PCR Amplification Kit panel from Applied Biosystems. Buccal cell swabs were very good source of genomic DNA especially for large population studies.

Y-STR haplotype diversity among the Khandayat population of Odisha, India

We analyzed seventeen Y-chromosomal short tandem repeat (Y-STR) loci in a population sample of Khandayat community residing in Odisha, India in order to find out the haplotype diversity. Blood samples were collected from 136 unrelated male individuals and genomic DNA isolation was carried out by the standard organic extraction method followed by multiplex PCR amplification using an AmpFl STR Y-filer PCR amplification kit and genotyping. A total of 130 haplotypes were observed among the studied samples, out of which 126 were unique. Allele frequency and gene diversity were calculated. Haplotype diversity and discrimination capacity were found to be 0.999128 and 0.95588 respectively. Haplotypes of Khandayat population were compared with that of other Indian populations using AMOVA (analysis of molecular variance) tool to measure the genetic relatedness between various populations of India.

Test of the rapid PCR method using AmpF lSTR Identifiler kit

Current forensic DNA typing is conducted in approximately 8–9 h. Whole steps included DNA extraction step, Quantification, PCR amplification step, electrophoresis process through capillary separation with fluorescence detection, data analysis and DNA profile interpretation. Among them, we have tested rapid PCR method of AmpF lSTR Identifler PCR to reduce running time. We have altered several PCR conditions of ordinary AmpF lSTR Identifler PCR method and used 9947A control DNA to cut the time for the PCR reaction. In the results of this study, the critical step of the PCR reaction was the annealing step and also we reduced PCR running times by 1/3 to 2/3 (approximately 60–90 min) with complete concordance of STR allele calls using standard reference material 9947A.

Evaluation of three new forensic DNA profiling kits on PCR-inhibitory crime scene samples

In 2009–2010, several forensic DNA profiling kits customised for Europe and the Prüm Treaty were commercially released. The manufacturers have made efforts to increase the PCR inhibitor tolerance compared to previous kits, as shown by their increased resistance to known molecular inhibitors such as humic acid and haematin. We have evaluated three new 15 STR-marker profiling kits (Amp FlSTR NGM, PowerPlex ESI16 and PowerPlex ESX16) on various PCR-inhibitory crime scene samples. All three kits produced usable DNA profiles from most samples. However, the kits were affected by inhibitory compounds from some of the samples, resulting in partial DNA profiles.

Applying a PCR inhibitor tolerant DNA polymerase blend in forensic DNA profiling

Crime scene samples often contain extraneous compounds that may interfere with PCR-based DNA analysis, resulting in imbalanced, partial or blank/negative DNA profiles. Customising the chemical content of the PCR reaction is a strategy that may increase PCR inhibitor tolerance without manipulating the sample. We have validated a modified version of Amp FlSTR SGM Plus, replacing Ampli Taq Gold DNA polymerase with a customised blend of two alternative polymerases, Ex Taq Hot Start and PicoMaxx High Fidelity. Allele calls are identical to standard analysis. Stutter sizes and balance values are indistinguishable. The modified chemistry provides increased resistance to PCR inhibitors, resulting in an elevated number of detected alleles for various problematic crime scene samples.

Complex casework using single nucleotide polymorphisms

Complex kinship analyses are normally a challenge for forensic laboratories, especially in cases in which the individuals involved can have criminal responsibilities. This paper presents two complex relationship caseworks studied with routine STRs and autosomal SNPs as supplementary markers. In the first case, to exclude trafficking of children, maternity investigation of a child was requested involving two alleged mothers – a 39-year-old woman, the alleged grandmother, and her absent daughter. The second one was a possible incest case with a young girl with Trisomy 21 where her father was also the alleged child's father. The individuals of these cases were typed for 17 autosomal STRs with AmpF lSTR Identifiler or IdentifilerPlus and Powerplex 16. Twenty autosomal SNPs were also typed using SNaPshot ® methodology, with two 10-plex previously revealed useful in paternity testing. Both cases gave low likelihood ratio values with STRs and a genetic incompatibility was also detected in the first case. SNP studies strongly indicated that the alleged grandmother was not the child's mother but indeed the grandmother in a real complex immigrant kinship case, while in the second casework reinforced the incest relationship. Therefore, SNPs revealed useful as additional markers in complex kinship testing.

Estimating stutter rates for Y-STR alleles

Stutter peaks are artefacts that arise during PCR amplification of short tandem repeats. Stutter peaks are especially important in forensic case work with DNA mixtures. The aim of the study was primarily to estimate the stutter rates of the AmpF lSTR Yfiler kit. We found that the stutter rates differ at the allelic level and with the parental peak height.

Direct PCR by the AmpF lSTR NGM™ kit for database purpose

To fully maximize the utility of DNA databases for tracing the origins of criminal activity, the streamlining of database sample processing must also be accompanied by high performance chemistries. Blood and buccal samples are often used for convicted-felon DNA profile databases, as these samples are easy to obtain. The AmpF lSTR NGM™ (Next Generation Multiplex) kit provides enhanced performance on degraded and inhibited samples and tolerance to PCR inhibitors, so we tested the kit ability to amplify directly blood and buccal samples deposited on special collecting cards. The ability to amplify stored DNA directly would allow the lab to get results more efficiently by eliminating the DNA purification step, minimizing the possibility of contamination reducing costs and time accelerating testing by as much as 30% so that lab faster would send consistent results to national database.

Validation of AmpF LSTR NGM SElect™ PCR amplification kit on forensic samples

The AmpF LSTR NGM SElect™ is a next generation kit developed by Applied Biosystems that contains the 5 new loci specified in the recently expanded European Standard Set (ESS) together with the remaining markers from the SGM Plus ® kit, plus the highly discriminating SE33. We performed a validation study of the NGM SElect Kit with evaluation of critical parameters as species specificity, sensitivity, degradation/inhibition study, performance on a wide variety of forensic samples, mixture sample analyses. Our feedback confirmed the multiplex shows a robust PCR chemistry and the improved performance requested by the forensic community for typing difficult samples and offers the option of a 29 or 30 cycle protocol for optimum data quality and sensitivity.

New validated analytical process for convicted offender samples submitted to the Canadian National DNA Data Bank

Optimization and validation of direct amplification protocols for both AmpF lSTR ® Identifiler ® Direct (IDD) and PowerPlex ® 16 HS (PP16HS) on the high-throughput 3730 DNA Analyzer (Applied Biosystems) were carried out in preparation for potential changes in Canadian legislation. Amplification conditions were optimized to perform direct amplification of single source blood, buccal and extracted hair roots collected from convicted offenders (CO) on FTA ® paper without the need for sample purification prior to amplification. Validation was performed using IDD and PP16HS on a wide collection of blood and buccal samples from volunteer donors. In addition, 111 CO blood samples were selected to assess the robustness of the new analytical process (i.e. 0.53 mm FTA disks amplified in 10 μL PCR volume) and verify the stability of DNA stored on FTA paper at room temperature for up to 10 years. Studies such as reproducibility, robustness and concordance were carried out. The quality of the STR profiles generated using the new analytical process was assessed by examining profile intensity, heterozygote peak height ratios and interlocus balance. Reliable and high quality profiles from all CO samples and volunteer samples were produced under the new process giving support to its implementation.

Analysis of severely compromised skeletal remains by the use of the latest generation kits

In recent years, European working groups (ENFSI-EDNAP) have strongly encouraged the development of new amplification kits that allow to obtain DNA profiles even on difficult samples (degraded samples, presence of inhibitory substances, LCN, mixtures, etc.). To enable the amplification of difficult biological samples the new loci were designed to achieve greater resistance to inhibitors and most robust and uniform amplification. With the aim of evaluating the performance of latest generation kits in real forensic caseworks the authors retested some bone samples previously analyzed in their laboratory over the past five years with the old commercial kits. The authors compared the performance of AmpF ℓSTR ® NGM™ PCR Amplification kit (Applied Biosystems) with PowerPlex ® ESX 17 systems (Promega). The twelve analyzed samples came from eight exhumed corpses and four bodies found outdoor in the advanced stage of putrefaction. For the genetic investigation the authors used a piece of bone taken from the femur. In conclusion, the authors can claim that the latest generation kits have proved to be decisive in all cases, including those where the previous use of traditional kits did not produce reliable and uniquely interpretable results.

Validation of the AmpF lSTR ® Identifiler ® Direct PCR Amplification kit in a laboratory accredited according to the ISO17025 standard

A total of 249 FTA ® card samples were investigated with the Identifiler ® Direct kit. PCR amplification was performed according to the manufacturer's protocol with modifications in the reaction volume (10–25 μL), the number of PCR cycles (25–27) and the number of FTA-card punches (1–2). Full concordance was observed between the results obtained with the Identifiler ® Direct kit and the washed punches typed with the Identifiler ® kit. The success rate with one 1.2 mm punch in a 15 μL PCR reaction volume and 25 PCR-cycles was 93.3%. After retyping of the failed samples, the combined success rate was 98.7%. Overall, the quality of the results was reduced when the number of punches was increased or the PCR reaction volume was decreased. The Identifiler ® Direct master mix may be used in combination with other primer sets for direct amplification of other loci, e.g. the 49 SNPs using the SNP forID multiplex assay.

Mitochondrial DNA control region variation in a Kuwaiti population sample

Mitochondrial control region (16024-576) sequences were generated from 381 Kuwaiti samples. Previously, these samples were typed with the AmpF ℓSTR ® Identifiler ® kit (Applied Biosystems, Foster City, California) [1]. Automated high throughput lab processing combined with a redundant sequencing strategy and multiple reviews of the raw electropherograms ensure the high quality of these sequences and their utility as reference population data for Kuwait.

Developmental validation of the PowerPlex ® ESX 16 and PowerPlex ® ESX 17 Systems

We describe the developmental validation study performed on the PowerPlex ® ESX 16 (European Standard Extended 16) and the PowerPlex ® ESX 17 Systems, part of a suite of four new DNA profiling kits developed by Promega in response to the ENFSI and EDNAP groups’ call for new STR multiplexes for Europe. The PowerPlex ® ESX 16 System combines the 11 loci compatible with the UK National DNA Database, contained within the AmpF lSTR ® SGM Plus ® PCR Amplification Kit, with five additional loci: D2S441, D10S1248, D22S1045, D1S1656 and D12S391. The multiplex was designed to incorporate these five new loci as mini- and midi-STRs while maintaining the loci found in the AmpF lSTR ® SGM Plus ® kit as standard size. The PowerPlex ® ESX 17 System amplifies the same loci as the PowerPlex ® ESX 16 System, but with the addition of a primer pair for the SE33 locus. Tests were designed to address the developmental validation guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM), and those of the DNA Advisory Board (DAB). Samples processed include DNA mixtures, PCR reactions spiked with inhibitors, a sensitivity series, and 306 United Kingdom donor samples to determine concordance with data generated with the AmpF lSTR ® SGM Plus ® kit. Allele frequencies from 242 white Caucasian samples collected in the United Kingdom are also presented. The PowerPlex ® ESX 16 and ESX 17 Systems are robust and sensitive tools, suitable for the analysis of forensic DNA samples. Full profiles were routinely observed with 62.5 pg of a fully heterozygous single source DNA template. In mixture analysis, a range of 52–95% of unique minor contributor alleles was observed at 19:1 mixture ratios where only 25 pg of the minor component was present. Improved sensitivity combined with the robustness afforded by smaller amplicons has substantially improved the quantity of information obtained from degraded samples, and the improved chemistry confers exceptional tolerance to high levels of laboratory prepared inhibitors.

Optimization and validation of a fast amplification protocol for AmpF lSTR ® 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 AmpF lSTR ® 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 AmpF lSTR ® 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 AmpF lSTR ® 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 transfer of touch DNA from hands to glass, fabric and wood

The transfer of DNA from hands to objects by holding or touching has been examined in the past. The main purpose of this study was to examine the variation in the amount of DNA transferred from hands to glass, fabric and wood. The study involved 300 volunteers (100 for glass, 100 for fabric and 100 for wood) 50% of which were male and 50% female. The volunteers held the material for 60 s. The DNA was recovered from the objects using a minitape lift, quantified using the Quantifiler kit assay, extracted using a ‘Qiagen ® QIAamp DNA mini kit’ and amplified using the AmpF lSTR ® SGM Plus™ Amplification Kit at 28 cycles. The results show that using ANOVA there was a significant difference ( F = 8.2, p < 0.05) between the three object types in the amount of DNA recovered. In terms of DNA transfer and recovery, wood gave the best yield, followed by fabric and then glass. The likelihood of success of obtaining a profile indicative of the holder was approximately 9% for glass samples, 23% for fabric and 36% for wood. There was no significant difference between the amount of DNA transferred by male or female volunteers. In this study good shedder status, as defined by obtaining useful profiles of 6 or more alleles, is estimated at approximately 22% of the population. The phenomenon of secondary transfer was observed when mixed DNA profiles were obtained but the incidence was low at approximately 10% of the total number of samples. DNA profiles corresponding to more than one person were found on objects which had been touched by only one volunteer. Although secondary transfer is possible the profiles obtained from touched objects are more likely to be as a result of primary transfer rather than a secondary source.

Genetic diversity of 17 Y-short tandem repeats in Indian population

Seventeen short tandem repeats (DYS389I, DYS390, DYS389II, DYS19, DYS385a/b, DYS393, DYS391, DYS392, DYS439, DYS438, DYS456, DYS458, DYS635, Y(GATA)H4, DYS437, and DYS448) from the non-recombining region of the human Y-chromosome were analyzed in 750 unrelated males representing four major linguistic families of India using Amp FlSTR ® Yfiler ® PCR Amplification kit. A total of 612 distinct haplotypes were observed, of which 545 were unique. Rare alleles for the loci DYS456, DYS458, DYS635, Y(GATA)H4, and duplication at the loci DYS389I and DYS389II were also observed. To understand the genetic diversity of the Indian population, and utility of Y-STRs in forensics, the locus diversity, haplotype diversity, and discrimination capacity in all populations was determined. MDS plot based on pairwise Φ st and AMOVA revealed the high genetic heterogeneity among the Indian populations due to linguistic diversity and social stratification.

Developmental validation of the PowerPlex ® ESI 16 and PowerPlex ® ESI 17 Systems: STR multiplexes for the new European standard

In response to the ENFSI and EDNAP groups’ call for new STR multiplexes for Europe, Promega ® developed a suite of four new DNA profiling kits. This paper describes the developmental validation study performed on the PowerPlex ® ESI 16 (European Standard Investigator 16) and the PowerPlex ® ESI 17 Systems. The PowerPlex ® ESI 16 System combines the 11 loci compatible with the UK National DNA Database ®, contained within the AmpF lSTR ® SGM Plus ® PCR Amplification Kit, with five additional loci: D2S441, D10S1248, D22S1045, D1S1656 and D12S391. The multiplex was designed to reduce the amplicon size of the loci found in the AmpF lSTR ® SGM Plus ® kit. This design facilitates increased robustness and amplification success for the loci used in the national DNA databases created in many countries, when analyzing degraded DNA samples. The PowerPlex ® ESI 17 System amplifies the same loci as the PowerPlex ® ESI 16 System, but with the addition of a primer pair for the SE33 locus. Tests were designed to address the developmental validation guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM), and those of the DNA Advisory Board (DAB). Samples processed include DNA mixtures, PCR reactions spiked with inhibitors, a sensitivity series, and 306 United Kingdom donor samples to determine concordance with data generated with the AmpF lSTR ® SGM Plus ® kit. Allele frequencies from 242 white Caucasian samples collected in the United Kingdom are also presented. The PowerPlex ® ESI 16 and ESI 17 Systems are robust and sensitive tools, suitable for the analysis of forensic DNA samples. Full profiles were routinely observed with 62.5 pg of a fully heterozygous single source DNA template. This high level of sensitivity was found to impact on mixture analyses, where 54–86% of unique minor contributor alleles were routinely observed in a 1:19 mixture ratio. Improved sensitivity combined with the robustness afforded by smaller amplicons has substantially improved the quantity of data obtained from degraded samples, and the improved chemistry confers exceptional tolerance to high levels of laboratory prepared inhibitors.

Population genetic polymorphisms of 17 Y-chromosomal STR loci in South Koreans

Haplotypes and allele frequencies of 17 Y-chromosomal STR loci included in the AmpF lSTR ® Yfiler ® system were obtained from a sample of 1021 unrelated individuals living in 6 provinces of South Korea. A total of 938 haplotypes were observed in the 1021 individuals studied, of which 885 were unique. The overall haplotype diversity for the 17 Y-STR loci was 0.9998, and the discrimination capacity was 0.9187. We found 11 atypical alleles (null, duplicated, triplicated, and microvariant alleles), that have not previously been reported in South Korean populations. It seems that these 17 loci are useful genetic markers for forensic personal identification and paternity testing in the South Korean population.

Y chromosome STR allelic and haplotype diversity in five ethnic Tamil populations from Tamil Nadu, India

We have analyzed 17 Y chromosomal STR loci in a population sample of 154 unrelated male individuals of the Tamil ethnic group residing in the state of Tamil Nadu, Southern India using AmpF lSTR® Yfiler™ PCR amplification kit. The population samples consist of the following castes: Kongu Gounder (KOG), Nadar Hindu (NAH), Agamudayar (AGA), Parayar (PAR) and other Tamil individuals (MCT) of mixed castes. A total of 152 unique haplotypes were identified among the 154 individuals studied. The haplotype diversity was found to be 0.9935 or higher for all the five groups. The results of population pairwise Fst p values indicate no statistically significant differentiation between the five populations in this study, but the results were highly significant when compared with 12 other global populations ( p < 0.05). Comparison of populations in this study with other national and global populations using Principal co-ordinate analysis (PCA) using Rst distance matrix indicates a delineation of all the Indian populations from other unrelated populations.