Comparison of DNA yield and STR success rates from different tissues in embalmed bodies.

Formalin fixation is commonly used to preserve tissue sections for pathological testing and embalming cadavers for medical dissection or burial. DNA extracted from formalin-fixed tissues may also provide an alternative source of genetic material for medical diagnosis and forensic casework, such as identifying unknown embalmed human remains. Formaldehyde causes DNA damage, chemical modifications, and degradation, thereby reducing the quantity and quality of DNA available for downstream genetic analyses. By comparing the DNA yield, level of DNA degradation, and short tandem repeat (STR) success of various tissue types, this study is the first of its kind to provide some guidance on which samples from embalmed bodies are likely to generate more complete STR profiles. Tissue samples were dissected from three male embalmed cadavers and included bone, cartilage, hair, muscle, internal organs, skin, teeth, and nail clippings. DNA was purified from all samples using the QIAamp® FFPE Tissue Kit (Qiagen), quantified using the QuantiFiler® Trio DNA Quantification kit (Life Technologies), and genotyped using the GlobalFiler® PCR Amplification Kit (Life Technologies). Results of this study showed variation in DNA quantity and STR success between different types of tissues and some variation between cadavers. Overall, bone marrow samples resulted in the highest DNA yields, the least DNA degradation, and greatest STR success. However, several muscle, hair, and nail samples generated higher STR success rates than traditionally harvested bone and tooth samples. A key advantage to preferentially using these tissue samples over bone (and marrow) and teeth is their comparative ease and speed of collection from the cadaver and processing during DNA extraction. Results also indicate that soft tissues affected by lividity (blood pooling) may experience greater exposure to formalin, resulting in more DNA damage and reduced downstream STR success than tissues under compression. Overall, we recommend harvesting from selected muscles (gastrocnemius, rectus femoris, flexor digitorum brevis, masseter, brachioradialis) or fingernails for human identification purposes.

Modified DOP-PCR for improved STR typing of degraded DNA from human skeletal remains and bloodstains.

Forensic and ancient DNA samples often are damaged and in limited quantity as a result of exposure to harsh environments and the passage of time. Several strategies have been proposed to address the challenges posed by degraded and low copy templates, including a PCR based whole genome amplification method called degenerate oligonucleotide-primed PCR (DOP-PCR). This study assessed the efficacy of four modified versions of the original DOP-PCR primer that retain at least a portion of the 5' defined sequence and alter the number of bases on the 3' end. The use of each of the four modified primers resulted in improved STR profiles from environmentally-damaged bloodstains, contemporary human skeletal remains, American Civil War era bone samples, and skeletal remains of WWII soldiers over those obtained by previously described DOP-PCR methods and routine STR typing. Additionally, the modified DOP-PCR procedure allows for a larger volume of DNA extract to be used, reducing the need to concentrate the sample and thus mitigating the effects of concurrent concentration of inhibitors.

Assessment of the role of DNA repair in damaged forensic samples.

Previous studies on DNA damage and repair have involved in vitro laboratory procedures that induce a single type of lesion in naked templates. Although repair of singular, sequestered types of DNA damage has shown some success, forensic and ancient specimens likely contain a number of different types of lesions. This study sought to (1) develop protocols to damage DNA in its native state, (2) generate a pool of candidate samples for repair that more likely emulate authentic forensic samples, and (3) assess the ability of the PreCR(TM) Repair Mix to repair the resultant lesions. Complexed, native DNA is more difficult to damage than naked DNA. Modified procedures included the use of higher concentrations and longer exposure times. Three types of samples, those that demonstrated damage based on short tandem repeat (STR) profile signals, were selected for repair experiments: environmentally damaged bloodstains, bleach-damaged whole blood, and human skeletal remains. Results showed trends of improved performance of STR profiling of bleach-damaged DNA. However, the repair assay did not improve DNA profiles from environmentally damaged bloodstains or bone, and in some cases resulted in lower RFU values for STR alleles. The extensive spectrum of DNA damage and myriad combinations of lesions that can be present in forensic samples appears to pose a challenge for the in vitro PreCR(TM) assay. The data suggest that the use of PreCR in casework should be considered with caution due to the assay's varied results.

INNULs: A novel design amplification strategy for retrotransposable elements for studying population variation.

OBJECTIVES: Retrotransposable elements (REs), consisting of long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs), are a group of markers that can be useful for human identity testing. Until now, however, due to the inherent size difference (up to 6 kb in some instances) associated with insertion and null alleles (or INNULs), the use of REs for facilitated population studies has not been sought or practical. The size of the insertion elements (from a few hundred to several thousand bp) has proven to limit their utility as a marker because of the inefficient amplicon yield with PCR. A novel primer design now facilitates INNUL marker testing. A preliminary panel of single-locus markers was developed to evaluate the potential of typing these insertion elements. Nine INNULs (5 Alu and 4 LINEs) were typed in three major North American populations and analyzed for population genetic features. In addition, the variation of each marker among the sample populations provides insight of its potential use as individual identification or ancestral marker. METHODS: INNUL markers were developed into fluorescently labeled single-loci PCR. Nine markers were developed with amplicons that were less than 180 bp in length, and, depending on the locus amplicons of the INNULs, alleles varied in size from 50 to 1 bp. This allele size is noteworthy because the insertion alleles of the 9 loci range in size from 297 to 6,195 bp. The allele distribution of the INNULs was assessed and analyzed in three major North American populations. RESULTS: Upon observation of the distribution of the alleles in three major North American populations, the markers generally met Hardy-Weinberg expectations, and there was little evidence of detectable levels of linkage disequilibrium. Due to varying distributions of the alleles in the major population groups tested, some of the markers might be better suited for use as an individual identification marker, while others are better suited for bio-ancestral studies. CONCLUSIONS: Using the primer design strategy described in our work, SINEs and (for the first time, to our knowledge) LINEs can be utilized as markers for studying population genetic variation that is more amenable to the limitations of the PCR technique. This study lays the foundation for future work of developing a multiplex panel of INNUL markers that can be used as a single-tube assay for human identity testing utilizing small amplicons (<180 bp), which could be useful for ancient or degraded forensic DNA samples.

Extraction platform evaluations: a comparison of AutoMate Express™, EZ1® Advanced XL, and Maxwell® 16 Bench-top DNA extraction systems.

The DNA extraction performance of three low-throughput extraction systems was evaluated. The instruments and respective chemistries all use a similar extraction methodology that involves binding DNA to a coated magnetic resin in the presence of chaotropic salt, washing of the resin to remove undesirable compounds, and elution of DNA from the particles in a low-salt solution. The AutoMate Express™ (Life Technologies Corporation, Carlsbad, CA), EZ1® Advanced XL (Qiagen Inc., Valencia, CA), and Maxwell® 16 (Promega Corporation, Madison, WI) were compared using a variety of samples including: blood on swabs, blood on denim, blood on cotton, blood mixed with inhibitors (a mixture of indigo, hematin, humic acid, and urban dust) on cotton, blood on FTA® paper, saliva residue on cigarette butt paper, epithelial cells on cotton swabs, neat semen on cotton, hair roots, bones, and teeth. Each instrument had a recommended pre-processing protocol for each sample type, and these protocols were followed strictly to reduce user bias. All extractions were performed in triplicate for each sample type. The three instruments were compared on the basis of quantity of DNA recovered (as determined by real-time PCR), relative level of inhibitors present in the extract (shown as shifts in the C(T) value for the internal PCR control in the real-time PCR assay), STR peak heights, use of consumables not included in the extraction kits, ease of use, and application flexibility. All three systems performed well; however extraction efficiency varied by sample type and with the preprocessing protocol applied to the various samples.

Genetic analysis of 7 medieval skeletons from the Aragonese Pyrenees.

AIM: To perform a genetic characterization of 7 skeletons from medieval age found in a burial site in the Aragonese Pyrenees. METHODS: Allele frequencies of autosomal short tandem repeats (STR) loci were determined by 3 different STR systems. Mitochondrial DNA (mtDNA) and Y-chromosome haplogroups were determined by sequencing of the hypervariable segment 1 of mtDNA and typing of phylogenetic Y chromosome single nucleotide polymorphisms (Y-SNP) markers, respectively. Possible familial relationships were also investigated. RESULTS: Complete or partial STR profiles were obtained in 3 of the 7 samples. Mitochondrial DNA haplogroup was determined in 6 samples, with 5 of them corresponding to the haplogroup H and 1 to the haplogroup U5a. Y-chromosome haplogroup was determined in 2 samples, corresponding to the haplogroup R. In one of them, the sub-branch R1b1b2 was determined. mtDNA sequences indicated that some of the individuals could be maternally related, while STR profiles indicated no direct family relationships. CONCLUSIONS: Despite the antiquity of the samples and great difficulty that genetic analyses entail, the combined use of autosomal STR markers, Y-chromosome informative SNPs, and mtDNA sequences allowed us to genotype a group of skeletons from the medieval age.

Y-STR loci diversity in native Alaskan populations.

Y chromosome short tandem repeat (Y-STR) loci are important genetic markers for forensic biological evidence analyses. However, paternal inheritance, reduced effective population size, and lack of independence between loci can reduce Y-STR diversity and may yield greater population substructure effects on a locus-by-locus basis compared with the autosomal STR loci. Population studies are necessary to assess the genetic variation of forensically relevant markers so that proper inferences can be made about the rarity of DNA profiles. This study examined 16 Y-STRs in three sampled populations of Native Americans from Alaska: Inupiat, Yupik, and Athabaskan. Population genetic and statistical issues addressed were: (1) the degree of diversity at locus and haplotype levels, (2) determination of the loci that contribute more so to haplotype diversity, and (3) the effects of population substructure on forensic statistical calculations of the rarity of a Y-STR profile. All three population samples were highly polymorphic at the haplotype level for the 16 Y-STR markers; however, the Native Americans demonstrated reduced genetic diversity compared with major US populations. The degree of substructure indicated that the three populations were related and admixed in terms of paternal lineage. The examination of more polymorphic loci may be needed to increase the power of discrimination of Y-STR systems in these populations.

Borrelia, Ehrlichia, and Rickettsia spp. in ticks removed from persons, Texas, USA.

Data regarding the type, frequency, and distribution of tick-borne pathogens and bacterial agents are not widely available for many tick species that parasitize persons in the southern United States. We therefore analyzed the frequency and identity of pathogens and bacterial agents in ticks removed from humans and subsequently submitted to the Texas Department of State Health Services, Zoonosis Control Program, from October 1, 2004, through September 30, 2008. The data showed associations of bacterial agents and potential vectors. Tick-related illnesses may pose unidentified health risks in areas such as Texas, where incidence of human disease related to tick bites is low but well above zero and where ticks are not routinely suspected as the cause of disease. Cause, treatment, and prevention strategies can be better addressed through collecting sufficient data to establish baseline assessments of risk.