A Study on the Effects of Degradation and Template Concentration on the Amplification Efficiency of the STR Miniplex Primer Sets.

References

Laird, R., Dawkins, R., & Gaudieri, S. (2005), Use of the genomic matching technique to complement multiplex STR profiling reduces DNA profiling costs in high volume crimes and intelligence led screens. Forensic Science International, 151 (2), 249-257.

Chung, D., Drabek, J., Opel, K., & Butler, J. (2004), A study on the effects of degradation and template concentration on the amplification efficiency of the STR miniplex primer sets. Journal of Forensic Science, 49 (4), 733-740.

Nicklas, J., Noreault, T., & Buel, E. (2012), Development of a fast, simple profiling method for sample screening using high resolution melting (HRM) of STRs. Journal of Forensic Science, 57 (2), 478-488.

Phillips, C., Prieto, Lourdes., Fondevila, M., & Salas, A. (2009), Ancestry analysis in the 11-m Madrid bomb attack investigation. PLoS ONE, 4 (8), 1-10.

Roeper, A., Reichert, W., & Mattern, R. (2007), The Achilles tendon as a DNA source for STR typing of highly decayed corpses. Forensic Science International, 173 (2), 103-106.

Balogh, M., Burger, J. & Bender, K. (2003), STR genotyping and mtDNA sequencing of latent fingerprint on paper. Journal of Forensic Science, 137 (2), 188-190.

Use of the genomic matching technique to complement multiplex STR profiling reduces DNA profiling costs in high volume crimes and intelligence led screens.
The genomic matching technique (GMT) targets duplicated polymorphic sequences within genomic blocks in the human major histocompatibility complex (MHC), differentiating between individuals at the DNA level using a single primer pair per block. The GMT is currently used to supplement human leukocyte antigen (HLA) typing to match donor and recipient pairs for bone marrow transplantation and has the potential to be employed as a powerful exclusion tool in forensic biology. The GMT is highly reproducible, produces DNA profiles from less than 1ng of DNA and was successfully employed to profile a range of forensic samples including buccal swabs, handled objects and fingerprints. Furthermore, GMT profiles from a single genomic block in the MHC are likely to be more discriminatory than known highly polymorphic short tandem repeat (STR) loci such as ACTBP2. As such, the GMT can reduce the cost of investigations that require profiling of multiple suspects or samples from one or more crime scenes and could be extended to profile genomic blocks in other polymorphic genetic systems in the human genome.

A Study on the Effects of Degradation and Template Concentration on the Amplification Efficiency of the STR Miniplex Primer Sets.
In forensic DNA analysis, the samples recovered from the crime scene are often highly degraded leading to poor PCR amplification of the larger sized STR loci. To avoid this problem, we have developed STR markers with redesigned primer sequences called “Miniplexes” to produce smaller amplicons. To assess the effectiveness of these kits, we have tested these primer sets with enzymatically degraded DNA and compared the amplifications to a commercial kit. We also conducted sensitivity and peak balance studies of three Miniplex sets. Lastly, we report a case study on two human skeletal remain samples collected from different environmental conditions. In both types of degraded DNA, the Miniplex primer sets were capable of producing more complete profiles when compared to the larger sized amplicons from the commercial kit. Correct genotypes were obtained at template concentrations as low as 31 pg/25 μL. Overall, our data confirm that our redesigned primers can increase the probability of obtaining a usable profile in situations where standard kits fail.

Development of a Fast, Simple Profiling Method for Sample Screening Using High Resolution Melting (HRM) of STRs.
: A screening assay has been developed to provide preliminary individualization of crime scene samples thus eliminating expensive, time-consuming short tandem repeat (STR) profiling of non-probative samples. High resolution melting performed in a real-time PCR instrument is used to detect the slight melting differences between the length and sequence variations of 22 forensic STRs. Three STRs (vWA, D18S51, THO1) were chosen to develop an assay which was optimized for Mg++ concentration, annealing ⁄ extension time ⁄ temperature, assay volume, and bovine serum albumin addition. The assay was tested for reproducibility, uniformity for genotype, melting profile consistency, effects of inhibitors, and mixture effects. The assay could be used to determine DNA concentration when a standard curve is run simultaneously. Calculations of costs show that the assay can save significant time and money for a crime with many samples or suspects.

Ancestry Analysis in the 11-M Madrid Bomb Attack Investigation
The 11-M Madrid commuter train bombings of 2004 constituted the second biggest terrorist attack to occur in Europe after Lockerbie, while the subsequent investigation became the most complex and wide-ranging forensic case in Spain. Standard short tandem repeat (STR) profiling of 600 exhibits left certain key incriminatory samples unmatched to any of the apprehended suspects. A judicial order to perform analyses of unmatched samples to differentiate European and North African ancestry became a critical part of the investigation and was instigated to help refine the search for further suspects. Although mitochondrial DNA (mtDNA) and Y-chromosome markers routinely demonstrate informative geographic differentiation, the populations compared in this analysis were known to show a proportion of shared mtDNA and Yhaplotypes as a result of recent gene-flow across the western Mediterranean, while any two loci can be unrepresentative of the ancestry of an individual as a whole. We based our principal analysis on a validated 34plex autosomal ancestry informative- marker single nucleotide polymorphism (AIM-SNP) assay to make an assignment of ancestry for DNA from seven unmatched case samples including a handprint from a bag containing undetonated explosives together with personal items recovered from various locations in Madrid associated with the suspects. To assess marker in formativeness before genotyping, we predicted the probable classification success for the 34plex assay with standard error estimators for a naïve Bayesian classifier using Moroccan and Spanish training sets (each n = 48). Once misclassification error was found to be sufficiently low, genotyping yielded seven near-complete profiles (33 of 34 AIM-SNPs) that in four cases gave probabilities providing a clear assignment of ancestry. One of the suspects predicted to be North African by AIM-SNP analysis of DNA from a toothbrush was identified late in the investigation as Algerian in origin. The results achieved illustrate the benefit of adding specialized marker sets to provide enhanced scope and power to an already highly effective system of DNA analysis for forensic identification.

The Achilles tendon as a DNA source for STR typing of highly decayed corpses.
Abstract: Forensic criminal casework often involves DNA profiling of human postmortem tissues, whereas degradation processes can affect PCR-based Short Tandem Repeat (STR) analysis. Degradation of DNA is observed to vary among different tissues and with time. Therefore, the stability of DNA in Achilles tendon samples is compared to that in muscle and kidney specimens with a variety of postmortem histories. Tissue samples from 28 autopsy cases, including 15 decomposed corpses and a control group of 13 non-decayed corpses were analyzed. DNA was isolated using the All-tissue DNA Kit (GEN-IAL, Troisdorf, Germany), quantified by spectrophotometric measurement, amplified by the multiplex PCR genRES MPX-2 (Serac, Bad Homburg, Germany), and analyzed on the ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Darmstadt, Germany). Quantitative analysis of non-decomposed tissues revealed that the recovery of DNA was highest in kidney followed by muscle, whereas Achilles tendon tissue was the poorest source of isolated DNA. Only small amounts of DNA were present in both kidney and muscle samples from decomposed corpses. However, from decayed Achilles tendon samples twice as much DNA as from non-decayed samples could be isolated on average. These results suggest DNA to be better protected in Achilles tendons. Moreover, postmortem changes in Achilles tendons may even improve DNA isolation.

STR genotyping and mtDNA sequencing of latent fingerprint on paper.
A systematic study was conducted to investigate whether DNA can be successfully extracted from latent fingerprints deposited on ordinary paper and analyzed using short tandem repeat profiling and mitochondrial DNA sequencing. In order to evaluate the performance of latent fingerprint analysis in a criminal case, experiments with varying conditions were carried out to improve our understanding of low copy number (LCN) DNA typing. After optimizing the extraction methods to achieve increased sensitivity, the examination of touched paper can routinely yield the STR profile of the individual who has touched it. A fingerprint can therefore be considered as a potential source of DNA for genetic identification. Nevertheless, the findings of our “after enhancement experiment” (using chemically or physically pre-treated fingerprints), and our “mixture experiment” (using fingerprints from three to four people on the same sheet of paper) help to define the limitations of the low copy number PCR technique in forensic casework.