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%.     

Validation of the MiniFiler™ Kit in archaeological samples

We evaluate the usefulness of MiniFiler™ Kit in the field of ancient DNA. A set of samples belonging to different locations from Iberian Peninsula, with ages ranging from Neolithic to XVII century, was tested. Results could be replicated in only one burial site, probably due to the taphonomic conditions. Other cases could only produce partial or none genetic profiles.     

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.     

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.     

Sexual assault cases related to unknown perpetrator: Almost 50% of the analyzed cases corresponded to serial offenders

As a collaborative effort in solving sexual assault cases where there is no suspect, the DNA profiles associated with 273 cases were analyzed. Such cases were submitted from different Criminal Investigation Units belonging to eight judicial departments of Buenos Aires Province. A single NN male profile was recovered from the evidences by differential lysis with DNA IQ System (Promega) and typing with IdentiFiler kit. Comparative analysis of the compiled DNA profiles showed that in 45% of the cases the evidence DNA profile matched in at least two unrelated cases. Associations between groups of unsolved cases provided a valuable tool in aiding law enforcement investigations.     

The problem of DNA contamination in forensic case work—How to get rid of unwanted DNA?

The PCR technique has become a powerful and very sensitive tool in a broad field of research, that is, molecular biology, medical diagnostics, population genetics, ancient DNA analysis and forensic casework.However, the high sensitivity down to single molecules can easily cause false-positive PCR results due to different types of contamination. In this study, artificial DNA contaminations (saliva and pure DNA) were treated with UV irradiation and other decontamination procedures. A satisfactory DNA removal could not be achieved, emphasizing the necessity of contamination avoidance.     

命案现场重建解析—“7·06”灭门惨案现场重建

2004年云南“7·06”灭门惨案得以及时告破,得益于办案人员凭借对案发现场的精勘细验和认真分析研究;是一起典型的重建案发现场,进而一举破案的成功案例。