Polymerase Resistance to Polymerase Chain Reaction Inhibitors in Bone*

Abstract: Amplification of DNA from aged or degraded skeletal remains can be a challenging task, in part due to naturally occurring inhibitors of the polymerase chain reaction. PCR inhibitors may act by inactivating a polymerase itself, or compete with or bind other reaction components, although various polymerases may be differentially susceptible to such insult. In this study, ten thermostable polymerases from six bacterial species were examined for their ability to amplify DNA in the presence of bone‐derived or individual PCR inhibitors. Two polymerases, one from Thermus aquaticus and one from Thermus thermophilus, showed lower susceptibility to inhibition from bone, while polymerases from Thermus flavus were highly susceptible. Addition of bovine serum albumin improved the activity of most of the enzymes. Taken together, the results indicate that thermostable DNA polymerases have different susceptibility to bone‐derived PCR inhibitors, and that those most often used in forensic laboratories may not be optimal when working with DNA from skeletal remains.     

Viewing time measures and sexual interest: another piece of the puzzle

Abstract

The present study compared a viewing time (VT) measure with the Sexual Deviance Card Sort (Laws et al., 2000 Laws, D. R., Hanson, R. K., Osborn, C. A. and Greenbaum, P. E. 2000. Classification of child molesters by plethysmographic assessment of sexual arousal and a self-report measure of sexual preference. Journal of Interpersonal Violence, 15: 1297–1312. [Crossref], [Web of Science ®] , [Google Scholar]) and past sexual behaviour. Twenty-six adult males who committed a contact sexual offence (19 of whom had child victims and seven with adult victims) each completed the self-report card sort and viewed 640 slides of nude and clothed males and females, ages 5, 9, 13 years and adult. The offenders were unaware that their viewing time was being recorded. VT allowed for greater consistent classification of sexual interest: for gender preference, 79% for individuals with child victims, 86% for individuals with adult victims; for age preference, 84% for individuals with child victims, and 57% for individuals with adult victims. Results demonstrated that a combination of nude and clothed computer-modified imagery can provide accurate sexual interest classification.     

Human genomic DNA quantitation system, H-Quant: development and validation for use in forensic casework

The human DNA quantification (H-Quant) system, developed for use in human identification, enables quantitation of human genomic DNA in biological samples. The assay is based on real-time amplification of AluYb8 insertions in hominoid primates. The relatively high copy number of subfamily-specific Alu repeats in the human genome enables quantification of very small amounts of human DNA. The oligonucleotide primers present in H-Quant are specific for human DNA and closely related great apes. During the real-time PCR, the SYBR Green I dye binds to the DNA that is synthesized by the human-specific AluYb8 oligonucleotide primers. The fluorescence of the bound SYBR Green I dye is measured at the end of each PCR cycle. The cycle at which the fluorescence crosses the chosen threshold correlates to the quantity of amplifiable DNA in that sample. The minimal sensitivity of the H-Quant system is 7.6 pg/microL of human DNA. The amplicon generated in the H-Quant assay is 216 bp, which is within the same range of the common amplifiable short tandem repeat (STR) amplicons. This size amplicon enables quantitation of amplifiable DNA as opposed to a quantitation of degraded or nonamplifiable DNA of smaller sizes. Development and validation studies were performed on the 7500 real-time PCR system following the Quality Assurance Standards for Forensic DNA Testing Laboratories.     

Characterization of the variant allele 9.2 of Penta D locus

DNA profiles of forensic cases of Córdoba Province, Argentina, typed by PowerPlex 16 kit (Promega), have shown in the Penta D locus few samples with a variant allele migrating as an off ladder between alleles 9 and 10. In order to determine the molecular basis of the new variant allele, three samples were subject to polymerase chain reaction amplification of the Penta D locus by monoplex, and were further purified and sequenced. The sequence analysis revealed that the off ladder allele has ten repeats motifs AAAGA as allele 10, with three nucleotides (TAA) deletion in the 3' flanking region, 128 nucleotides after the last repeat. Therefore, the variant allele could be explained by a deletion of allele 10, and was designated 9.2. Mse I digestion assay allows to corroborate allele 9.2 without sequencing. A population study in Córdoba Province indicates that allele 9.2 of Penta D locus has a frequency of 0.0063.     

Developmental validation of a multiplex qPCR assay for assessing the quantity and quality of nuclear DNA in forensic samples

Forensic scientists are constantly searching for better, faster, and less expensive ways to increase the first-pass success rate of forensic sample analysis. Technological advances continue to increase the sensitivity of analysis methods to enable genotyping of samples containing minimal amounts of DNA, yet few tools are available that can simultaneously alert the analyst to both the presence of inhibition and level of degradation in samples prior to genotyping to allow analysts the opportunity to make appropriate modifications to their protocols and, consequently, to use less sample. Our laboratory developed a multiplex quantitative PCR assay that amplifies two human nuclear DNA target sequences of different length to assess DNA degradation and a third amplification target, a synthetic oligonucleotide internal PCR control (IPC), to allow for the assessment of PCR inhibition. We chose the two nuclear targets to provide quantity and fragment-length information relevant to the STR amplification targets commonly used for forensic genotyping. The long target (nuTH01, 170-190 bp) spans the TH01 STR locus and uses a FAM-labeled TaqMan probe for detection. The short nuclear target (nuCSF, 67 bp) is directed at the upstream flanking region of the CSF1PO STR locus and is detected using a VIC-labeled TaqManMGB probe. The IPC target sequence is detected using a NED-labeled TaqManMGB probe. The assay was validated on the Applied Biosystems 7500 Real-Time PCR system, which is optimized for NED detection. We report the results of a developmental validation in which the assay was rigorously tested, in accordance with the current SWGDAM guidelines, for precision, sensitivity, accuracy, reproducibility, species specificity, and stability.     

Attributions in a Hypothetical Child Sexual Abuse Case: Roles of Abuse Type,Family Response and Respondent Gender

The present study examines the impact abuse type, family response, and respondent gender have on attributions of blame in a hypothetical child sexual abuse (CSA) case. Three hundred and ninety three respondents read a hypothetical CSA scenario describing the sexual assault of a 14 year old girl by a 25-year-old man and completed 14 attribution items. Overall, the assault was deemed more serious, the perpetrator more culpable, and the family less culpable when CSA involved (vaginal) penetration. Contrary to expectations, respondents were more negative towards a family who denied the abuse took place versus one which blamed or supported the victim. Finally, male respondents deemed the abuse to be less serious, were more negative towards the victim and their families, and more positive towards perpetrators than were female respondents. The role these factors play in CSA attributions, together with ideas for future research, are discussed.

Evaluation of Two New Methods for DNA Extraction of “Legal High” Plant Species

A quick, simple, and high-yield nucleic acid isolation process is crucial for high-quality DNA analysis. The ability of the MicroGEM PDQeX phytoGEM system and Omega Bio-tek E.Z.N.A.® Plant DS Mini kit to extract PCR-ready DNA was evaluated by extracting the forensically relevant “legal high” plant species: Ipomoea purpurea, Artemisia absinthium, Mitragyna speciosa, Datura stramonium, and Papaver somniferum. The plant material was pulverized, processed using the manufacturer’s plant protocol for the PDQeX Nucleic Acid Extraction or the manufacturer’s protocol for the Omega extraction, quantified using the Invitrogen Qubit 2.0 Fluorometer, and analyzed for amplifiability by PCR using a Qiagen Rotor-Gene Q instrument and published assays. The DNA amplicons for the legal high species produced high-resolution melt curves concordant with the melts observed when DNA was isolated using the Qiagen DNeasy Plant Mini Kit in previous studies.