Genetic polymorphism of 17 STR loci for forensic use in Chinese population from Shanghai in East China

Allele frequencies for 17 STR loci found in Identifier kit and PowerPlex^®16 Monoplex System were determined in a sample of 1000 unrelated individuals living in Shanghai in East China. The values of observed heterozygosity (Ho), power of discrimination (PD), probability of paternity exclusion (PE) and polymorphism information content (PIC) were calculated. All loci were in accordance with Hardy–Weinberg equilibrium (p < 0.05). The obtained frequency distributions were compared with other previously reported population data.     

Extraction of high quality DNA from biological materials and calcified tissues

Calcified tissues, such as bone and tooth, and some other sample types, such as those containing adhesive, present a challenge to standard extraction protocols. We have developed a lysis reagent, BTA™ lysis buffer, which is designed for use with PrepFiler™ Kit reagents. The BTA™ lysis buffer disrupts calcified tissue matrices and achieves effective extraction of DNA from pulverized bone and tooth samples. In addition, the BTA™ lysis buffer mildly but efficiently extracts DNA from challenging substrates like tape, chewing gum, and cigarette butts and, as with bone and tooth, DNA from these lysates is purified using established PrepFiler™ reagent extraction protocols.We successfully extracted DNA from powdered human bone samples, chewed gum and smoked cigarettes using BTA™ lysis buffer. Extraction yields for bone, gum and cigarette samples tested were consistent and reproducible. This extraction method efficiently removed potential PCR inhibitors from all samples tested, and C_T values for the internal PCR control of Quantifiler^® Human DNA Quantification Kit were consistent and within the normal range. The DNA extracted from these samples also provided conclusive profiles that were free of PCR artifacts when amplified using the AmpF?STR^® Identifiler^® PCR Amplification Kit. The protocol is easily adapted for automation.     

Observation of tri-allelic patterns in autosomal STRs during routine casework

We report three cases of tri-allelic patterns observed during routine forensic casework on 5964 Belgian residents. These individuals had been typed for the following 15 autosomal STRs: CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, vWA, FGA, TH01, TPOX, D2S1338 and D19S433.The first example of a tri-allelic pattern had the genotype 13;15;16 for the D8S1179 locus. In the second observation there was 16;21;22 pattern for the D18S51 locus. The third case had the alleles 10;11;13 also for D18S51.All cases belonged to the Type I tri-allelic pattern, with three uneven peaks, the sum of the heights of both smaller peaks equalling the height of the tallest peak.Three cases in 5964 typed individuals is a frequency for tri-allelic patterns in autosomal STRs of 0.05%.     

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.     

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.     

Forensic application of Y chromosome SNPs in inconclusive cases

Biallelic markers, Single Nucleotide Polymorphisms (SNPs), are nowadays a powerful tool in the analysis of degraded samples. Namely, Y chromosome SNPs allow to determine the gender of the analyzed sample and to establish its haplogroup, making possible to attribute the ethnicity of male individuals. The aim of this study is to obtain Y-SNPs in forensic samples without STRs results, checking methodologies previously used.     

Analysis of DNA profiles extracted from degraded samples from archival of formalin fixed tissue included in paraffin (FFTIP) and hairs

The possibility of studying DNA extracted from archival of formalin fixed tissue included in paraffin (FFTIP) enables valuable retrospective investigations. However, according to some authors it is difficult to obtain genomic DNA of good quality, since the process of fixation often results in fragmentation of DNA. In order to evaluate the quality and quantity of DNA extracted, necropsy samples of FFTIP (spleen/lung) and hairs, with or without bulbs, were analyzed using three methods of extraction (QIAamp DNA mini, QIAamp DNA micro-kit and phenol–chloroform followed by microcon YM-30). The amount of DNA recovered was quantified by spectrophotometer. The β-actin, amelogenin gene and the profiles of STR were analyzed. Based on experimental results, a general guideline concerning the appropriate extraction method according to the tissue and the quantity of the starting material for the analysis of DNA from FFTIP and hairs could be suggested.     

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.     

Successful STR and SNP typing of FTA Card samples with low amounts of DNA after DNA extraction using a Qiagen BioRobot EZ1 Workstation

FTA Cards (GE Healthcare) have been used for more than 4 years in Denmark for the collection of buccal cells as reference samples in crime cases. Semi-automated protocols for STR typing of DNA on punches of FTA Cards are routinely used. In average, full STR profiles were generated from approximately 95% of the FTA Cards with a standard punching protocol, while partial or no STR profile were obtained from 5% of the samples. Here, the Qiagen BioRobot^® EZ1 Workstation (Qiagen) and the EZ1 DNA Investigator Kit (Qiagen) was used to extract DNA from 29 FTA Cards from which a complete STR profile was not generated with the standard punching protocol. All 29 samples were successfully typed with the AmpF?STR^® Identifiler™ PCR Amplification Kit (Applied Biosystems) and with the SNPforID 49plex SNP assay. The lowest amount of DNA that resulted in complete STR and SNP profiles was 80 pg. The STR and SNP profiles were identical to those generated from another sample collected from each of the 29 individuals.     

Sequence polymorphisms at the DXS6789, DXS8377 and DXS101 loci in three Asian populations

Sequence analyses of X-chromosomal short tandem repeats, DXS6789, DXS8377 and DXS101 were performed for representatives of 3 Asian populations: 130 Japanese, 61 Bangladeshi and 89 Indonesian males. At DXS6789, the sequence polymorphism was found in 7 alleles in the Japanese, 3 in the Bangladeshis and 3 in the Indonesians. At DXS8377, the sequence polymorphism was found in 13 alleles in the Japanese, 9 in the Bangladeshis and in all alleles identified in the Indonesians. At DXS101, the sequence polymorphism was found in 7 alleles in the Japanese, 9 in the Bangladeshis and 8 in the Indonesians. Because sequence polymorphisms were found in most of the alleles at the DXS6789, DXS8377 and DXS101 loci, it was concluded that sequencing was essential for identifying the alleles at these loci in all 3 Asian populations.