Reduction of Powerplex(®) Y23 reaction volume for genotyping buccal cell samples on FTA(TM) cards.

PowerPlex(®) Y23 is a novel kit for Y-STR typing that includes new highly discriminating loci. The Israel DNA Database laboratory has recently adopted it for routine Y-STR analysis. This study examined PCR amplification from 1.2-mm FTA punch in reduced volumes of 5 and 10 µL. Direct amplification and washing of the FTA punches were examined in different PCR cycle numbers. One short robotically performed wash was found to improve the quality and the percent of profiles obtained. The optimal PCR cycle number was determined for 5 and 10 µL reaction volumes. The percent of obtained profiles, color balance, and reproducibility were examined. High-quality profiles were achieved in 90% and 88% of the samples amplified in 5 and 10 µL, respectively, in the first attempt. Volume reduction to 5 µL has a vast economic impact especially for DNA database laboratories.

Long allele designations at D2S1338 and D19S433 loci as influenced by various multiplex STR kits.

European forensic laboratories are replacing the STR multiplex kits with the new generation 16/17 STR kits. This study examines the influence of the new generation kits and the new Applied Biosystems 3500xL Genetic Analyzer on the designation of long D2S1338 and D19S433 off-ladder alleles. Different allele calls were obtained using the new NGM™ (Applied Biosystems) and PowerPlex(®) ESI™ (Promega) kits compared with AmpFℓSTR(®) SGM Plus™ kit (Applied Biosystems). Sequence analysis was used to determine accurate allele designation. The new multiplex kits and the 3500xL Genetic Analyzer improved accuracy of long allele designations. DNA databases worldwide include countless profiles obtained by previous kits. Discrepancies between the new and former technologies may cause failure to detect hits. Discordance is expected due to primer sequence differences between various kits. An additional discordance, occurring in long alleles, independent of primer sequence is reported in this study.

Can brothers share the same STR profile?

This report demonstrates the limits of DNA identification when siblings are involved. The Israeli DNA database routinely amplifies suspects samples using the PowerPlex(®) ESI16 system (Promega). While uploading a series of suspects into the database software, we found an unusual high number of shared alleles between two suspects 31 out of 32 alleles. Verification of their demographic data identified them as brothers. After confirmation of their paternity affiliation using the AmpFlSTR(®)YFiler™ (Applied Biosystems), we used two other multiplexes kits to improve the differentiation rate. The PowerPlex(®) ESX17 System (Promega) added one locus, SE33, who exhibits four different alleles. The second kit, the AmpFlSTR(®)MiniFiler™ (Applied Biosystems) added three more loci. Only one allele difference was found. In order to increase the discrimination power between related and unrelated individuals, we recommend that the DNA laboratories consider using a larger multiplex typing kit in cases like the one informed here.

A search for obligatory paternal alleles in a DNA database to find an alleged rapist in a fatherless paternity case.

A sexual assault case resulted in a pregnancy, which was subsequently aborted. The alleged father of the fetus was unknown. Maternal and fetal types were obtained using the 11-locus AmpFℓSTR(®) SGM Plus(®) kit. The national DNA database was searched for the paternal obligatory alleles and detected two suspects who could not be excluded as father of the male fetus. Additional typing using the AmpFℓSTR(®) Minifiler(™) kit, containing three additional autosomal loci, was not sufficient to exclude either suspect. Subsequent typing using the PowerPlex(®) 16, containing four additional loci, and Y-Filer(™) kits resulted in excluding one suspect. Searching a database for paternal obligatory alleles can be fruitful, but is fraught with possible false positive results so that finding a match must be taken as only preliminary evidence.

The Israel DNA database--the establishment of a rapid, semi-automated analysis system.

The Israel Police DNA database, also known as IPDIS (Israel Police DNA Index System), has been operating since February 2007. During that time more than 135,000 reference samples have been uploaded and more than 2000 hits reported. We have developed an effective semi-automated system that includes two automated punchers, three liquid handler robots and four genetic analyzers. An inhouse LIMS program enables full tracking of every sample through the entire process of registration, pre-PCR handling, analysis of profiles, uploading to the database, hit reports and ultimately storage. The LIMS is also responsible for the future tracking of samples and their profiles to be expunged from the database according to the Israeli DNA legislation. The database is administered by an in-house developed software program, where reference and evidentiary profiles are uploaded, stored, searched and matched. The DNA database has proven to be an effective investigative tool which has gained the confidence of the Israeli public and on which the Israel National Police force has grown to rely.

Discordance at D3S1358 locus involving SGM Plus™ and the European new generation multiplex kits.

During the course of routine database sample analysis in the Israel Police DNA database, an off-ladder D3S1358 allele, calculated to be >22.1, extending into the adjacent vWA locus was observed using Applied Biosystems SGM Plus™ kit. To verify the size of this D3S1358 long allele and to ensure it was not part of a trialle pattern in the neighboring locus, the sample was amplified using three of the European new generation STR multiplex kits: NGM(TM) (Applied Biosystem), Powerplex™ ESX and ESI (Promega). The results of these amplifications determined the variant to be a 22 allele. Subsequent sequencing confirmed this designation and revealed a nucleotide polymorphism. Ten additional SGM Plus™ profiled samples with D3S1358 alleles larger than 19, were re-analyzed using NGM(TM) and Powerplex™ ESX which also showed discordance in the calculated results between original SGM Plus™ designations and those obtained with the European new generation multiplexes.

A Y-chromosome STR marker should be added to commercial multiplex STR kits.

Autosomal short tandem repeat (STR) analysis has become highly relevant in the identification of victims from mass disasters and terrorist attacks. In such events, gender misidentification can be of grave consequences, yet the list reporting amelogenin amplification failure using STR multiplex kits continues to grow. Presented here are three such examples. In the first case, we present two male suspects who demonstrated amelogenin Y-deficient results using two commercial kit procedures. The presence of their Y chromosomes was proven by obtaining a Y-haplotype. The second case demonstrated a profile from a third male suspect where only the Y homolog of the XY pair was amplified. In events such as mass disasters or terrorist attacks, timely and reliable high throughput DNA typing results are essential. As the number of reported cases of amplification failure at the amelogenin gene continues to grow, we suggest that the incorporation of a better gender identification tool in commercial kits is crucial.

DNA profiling from heroin street dose packages.

A large amount of heroin street doses are seized and examined for drug content by the Israel police. These are generally wrapped in heat-sealed plastic. Occasionally it is possible to visualize latent fingerprints on the plastic wrap itself, but the small size of the plastic item and the sealing process makes the success rate very low. In this study, the possibility of extracting and profiling DNA from the burnt edge of the plastic wrap was investigated. The idea was based on the assumption that epithelial cells might be trapped during the sealing process. The results show that there are sufficient quantities of DNA deposited at the "amorphic" burnt edges of sealed street doses for DNA profiling to be carried out. A controlled experiment using a known donor was performed. This subject carried out sealing of "street drug" packages and consequent DNA extractions were performed to show that known DNA profiles could be recovered from such packages, as a result of handling by the "packer." "Square-like" burnt edges did not yield DNA profiles, probably because of differences in the sealing process. It was also shown that DNA could be recovered from the plastic wrap itself and not only from the amorphic burnt edges. As heroin dealers and drug users are often involved in other crimes and run-ins with the law, the effective extraction and addition of their DNA profiles from such items of evidence to the newly established DNA database in Israel provides new avenues in the continued fight against crime and drug traffickers.

A possible source of reference DNA from archived treated adhesive lifters.

During the course of a double murder trial, it became apparent that the two adhesive lifters from the two cadavers had been mislabeled before being presented in court. The question was raised whether DNA testing from the biological material remaining attached to the lifters could resolve this mix-up. In fatal shooting cases where a bullet has been fired through a body surface, an adhesive lifter is applied directly to the entrance wound. The total nitrite residues, as well as biological material surrounding the wound (blood, hair, tissue) are transferred to the adhesive lifter. The nitrite residues are used for estimating firing distance. In a worst-case scenario, the biological material on the lifter may be the only remaining reference material from a victim. In this paper, we examined whether the biological material retrieved from adhesive lifters could be used for DNA typing after the lifters had been treated for GSR pattern. In as much as the biological material found on the lifters can be typed and profiled following physical and chemical treatment, we submit that archived adhesive lifters can be used as a future source of reference DNA from cadavers where no other sample is available.

'The story of Abraham, Isaac and Jacob" or "am I my brother's keeper?".

Presented is a case report of a violent sexual assault where the DNA profile obtained from an item of evidence was compared to a suspect's profile. The profiles did not match, but the sharing of such a large number of alleles raised the suspicion that perhaps the real perpetrator was a blood relative of the suspect. The investigators requested a sample from the suspect's brother, and a match was defined. In an era of technological breakthroughs in the field of forensic DNA analysis, the importance of the scientist's attention to the evidence presented in each case is stressed.

Presentation of a three-banded allele pattern--analysis and interpretation.

The Israel police forensic biology laboratory received as an item of evidence in an attempted murder case, a pair of trousers belonging to a suspect. A bloodstain was observed on the trousers and analyzed by STR typing for nine loci using the Promega GenePrint STR silver stain detection kits. The genetic profile defined was found to be identical to that of the victim's at all nine loci. Within this profile a three-banded allele pattern was observed at the D16S539 locus, both in the bloodstain and in the victim's reference blood sample. Confirmation of this phenomenon was accomplished by amplifying the extracted DNA from both the trousers and the victim's blood sample using the PowerPlex 16 kit by Promega and the AmpFlSTR SGM Plus kit by Perkin Elmer, followed by analysis of the amplification products by capillary electrophoresis on the ABI prism 310 genetic analyzer. The same three-banded allele pattern was observed at the D16S539 locus in both specimen and reference DNA, using each of the three kits. Three additional loci located on chromosome 16 (D16S3407, D16S2617 and D16S3082), not employed for forensic identification, were also analyzed and did not show three-banded allele pattern.

The effect of 1,2-indanedione, a latent fingerprint reagent on subsequent DNA profiling.

The compound 1,2-indanedione was recently introduced in our laboratory as an operational reagent for developing latent fingerprints on porous surfaces. As part of the reagent implementation, a study was carried out in order to determine whether either of the two operational 1,2-indanediones formulations interferes with further DNA profiling. Both formulations are based on HFE7100 solvent. One is acidic and the other neutral. In a controlled experiment, known donors attached stamps to envelopes by licking them. The stamped envelopes were initially treated with either one indanedione formulation or the other, and DNA was then extracted for STR typing. No differences were observed between the STR profiles obtained from treated and untreated stamps and envelopes, indicating that 1,2-indanedione does not adversely affect the extraction and subsequent amplification of the STRs examined. However, preliminary results indicate that potential DNA analysis depends on the time interval between the indanedione treatment and DNA extraction as no DNA can be recovered six days following treatment. For this reason, it is strongly recommended to extract DNA from treated items of evidence as soon as possible after indanedione treatment.