Genetic polymorphisms at four STR loci from the HLA region in a Venezuelan population

POPULATIONS: Whole blood samples from 74 unrelated healthy individuals were collected. The donors' sample included Venezuelan mestizos from various regions of the country, but mostly from the resident population of Caracas City. A Venezuelan mestizo is the offspring of a mating between a native Venezuelan and a person born in Europe, mainly in Spain.     

Laser microdissection separation of pure spermatozoa from epithelial cells for short tandem repeat analysis

Short tandem repeat (STR) analysis is a valuable tool in identifying the source of biological stains, particularly from the investigation of sexual assault crimes. Difficulties in analysis arise primarily in the interpretation of mixed genotypes when cell separation of the sexual assailant's sperm from the victim's cells is incomplete. The forensic community continues to seek improvements in cell separation methods from mixtures for DNA typing. The feasibility of applying laser microdissection (LMD) technology to precisely separate sexual assault cell mixtures by visual inspection coupled with laser dissection was assessed through three experiments. First, various histological stains were evaluated for use with LMD and DNA analysis. Second, different DNA isolation methods were evaluated on LMD-collected cells. Finally, STR analysis was performed on LMD-separated sperm cells from mixtures of semen and female buccal epithelial cells. The results indicated (a) hematoxylin/eosin staining performed best in its ability to differentiate sperm and epithelial cells while exhibiting the least negative effect on further downstream analysis; (b) both QIAamp and Lyse-N-Go methods were useful for recovery of DNA from LMD-collected sperm cells; and (c) LMD separation provided clear STR profiles of the male donor with the absence of any additional alleles from the female donor. This report describes an efficient, low-manipulation LMD method for the efficient separation of spermatozoa from two-donor sperm/epithelial cell mixtures.     

The application of miniplex primer sets in the analysis of degraded DNA from human skeletal remains

A new set of multiplexed PCR primers has been applied to the analysis of human skeletal remains to determine their efficacy in analyzing degraded DNA. These primer sets, known as Miniplexes, produce shorter amplicons (50-280 base pairs (bp)) than standard short tandem repeat (STR) kits, but still utilize the 13 CODIS STR loci, providing results that are searchable on national DNA databases. In this study, a set of 31 different human remains were exposed to a variety of environmental conditions, extracted, and amplified with commercial and Miniplex DNA typing kits. The amplification efficiency of the Miniplex sets was then compared with the Promega PowerPlex 16 system. Sixty-four percent of the samples generated full profiles when amplified with the Miniplexes, while only 16% of the samples generated full profiles with the Powerplex 16 kit. Complete profiles were obtained for 11 of the 12 Miniplex loci with amplicon sizes less than 200 bp. These data suggest smaller PCR amplicons may provide a useful alternative to mitochondrial DNA for anthropological and forensic analysis of degraded DNA from human skeletal remains.     

The utility of whole genome amplification for typing compromised forensic samples

Biological evidence has become invaluable in the crime laboratory; however, it may exist in limited quantity and/or quality. Given this, the ability to amplify total DNA obtained from evidence, in an unbiased manner, would be highly advantageous. Methods for whole genome amplification (WGA) have the potential to fulfill this role, resulting in a virtually unlimited supply of DNA. In the research presented, two WGA methods, improved primer extension preamplification and multiple displacement amplification (MDA), were tested using commercial kits. Control DNA, artificially degraded DNA, and DNA from fresh blood, aged blood, hair shafts, and aged bones underwent WGA, followed by short tandem repeat and mitochondrial DNA analysis. The methods did amplify DNA, but performed poorly on forensically relevant samples; the maximum amplicon size was reduced, and MDA often resulted in extraneous bands following polymerase chain reaction. Taken together, WGA appears to be of limited forensic utility unless the samples are of a very high quality.     

Simultaneous determination of total human and male DNA using a duplex real-time PCR assay

A single duplex assay to determine both the amount of total human DNA and the amount of male DNA in a forensic sample has been developed. This assay is based on TaqMan technology and uses the multicopy Alu sequence to quantitate total human DNA and the multicopy DYZ5 sequence to quantitate Y chromosomal (male) DNA. The assay accepts a wide concentration range of input DNA (2 muL of 64 ng/microL to 0.5 pg/microL), and also allows detection of PCR failure. The PCR product sizes Alu (127 bp) and DYZ5 (137bp) approximate that of the smaller short tandem repeats (STRs) which should make the assay predictive of STR success with degraded DNA. The assay was optimized for probe/primer concentrations and BSA addition and validated on its reproducibility, on its human specificity, on its nonethnic variability, for artificial mixtures and adjudicated casework, for the effect of inhibitors and for state of DNA degradation. This assay should prove very usual in forensic analyses because knowing the relative amounts of male versus female DNA can allow the examiner to decide which samples may yield the most probative value in a case or direct the samples to methods that would yield the greatest information.