An alternate method for extracting DNA from environmentally challenged teeth for improved DNA analysis.

A grinding-free method to extract DNA from teeth via a direct minimal-invasive retrograde approach to the pulp cavity and dentine was compared to a standard grinding/pulverisation method. This alternate method uses endodontic dental files to access the root canals and pulp cavity for tissue and dentine harvest via the apical end of the roots and avoids mechanical damage to the crown and root morphology. In contrast, other methods require pulverisation of the whole root or tooth, transection or destruction of the occlusal surface to gain access to the DNA in the root canals and pulp chamber.

Linear amplification of target prior to PCR for improved low template DNA results.

Forensic analysis of genetic material is often limited by the quantity and quality of DNA available for examination. Stochastic effects associated with low amounts of starting template can lead to a reduction in the quality of the result, making interpretation difficult. This paper presents an amplification method to copy target DNA in a linear fashion prior to short tandem repeat (STR) analysis to increase the available starting template without introducing the amplification bias seen in other methods used to increase the sensitivity of PCR.

Comment on Kokshoorn, B, and Blankers, BJ 'Response to Grisedale, KS and van Daal, A: comparison of STR profiling from low template DNA extracts with and without the consensus profiling method'.

Kokshoorn and Blankers responded to our recent article by saying that replicate analysis and consensus profiling of low template samples was best in terms of reliability and objectivity. We agree that the consensus approach has benefits, particularly in eliminating non-repeating spurious alleles from the final profile. However, with the development of statistical models that can accommodate stochastic effects and allele drop in, it may be beneficial to perform a single amplification with three times the amount of template, since much information is lost from the profile using the consensus approach.

Comparison of STR profiling from low template DNA extracts with and without the consensus profiling method.

Background: The consensus profiling method was introduced to overcome the exaggerated stochastic effects associated with low copy number DNA typing. However, little empirical evidence has been provided which shows that a consensus profile, derived from dividing a sample into separate aliquots and including only alleles seen at least twice, gives the most informative profile, compared to a profile obtained by amplifying the entire low template DNA extract in one reaction. Therefore, this study aimed to investigate the quality of consensus profiles compared to profiles obtained using the whole low template extract for amplification.

DRD2 C957T and TaqIA genotyping reveals gender effects and unique low-risk and high-risk genotypes in alcohol dependence.

Aims: As recent conflicting reports describe a genetic association between both the C- and the T-alleles of the dopamine D2 receptor (DRD2) C957T polymorphism (rs6277) in alcohol-dependent subjects, our aim was to examine this polymorphism and TaqIA (rs1800497) in Australian alcohol-dependent subjects.

Methods: The C957T polymorphism was genotyped in 228 patients with alcohol dependence (72 females and 156 males) and 228 healthy controls.

Results: The C-allele and C/C genotype of C957T was associated with alcohol dependence, whereas the TaqIA polymorphism was not.

A DRD2 and ANKK1 haplotype is associated with nicotine dependence.

To test the importance of the dopamine D2 receptor (DRD2) region in nicotine dependence, 150 smokers and 228 controls were genotyped for the DRD2 C957T, -141delC and ANKK1 TaqIA polymorphisms (rs6277, rs1799732 and rs1800497, respectively). The -141delC SNP did not show any association but both the C957T and TaqIA SNPs showed association at the allele, genotype, haplotype and combined genotype levels. The 957C/TaqI A1 haplotype was more than 3.

Assessment of DNA degradation and the genotyping success of highly degraded samples.

DNA becomes progressively more fragmented as biological tissue degrades resulting in decreasing ability to gain a complete DNA profile. Successful identification of samples exhibiting very high levels of DNA degradation may be complicated by presenting in minute quantities. The industry standard method for human DNA identification utilising short tandem repeats (STR) may produce partial or no DNA profile with such samples.

Genetics and cardiovascular disease: Design and development of a DNA biobank.

Coronary artery disease (CAD) is a complex disease with environmental and genetic determinants. Many other cardiovascular (CV) conditions also have a genetic basis. A positive family history of CV disease in first-degree relatives is a strong independent risk factor for CAD as well as several other cardiac disorders.

Extracting evidence from forensic DNA analyses: future molecular biology directions.

Molecular biology tools have enhanced the capability of the forensic scientist to characterize biological evidence to the point where it is feasible to analyze minute samples and achieve high levels of individualization. Even with the forensic DNA field's maturity, there still are a number of areas where improvements can be made. These include: enabling the typing of samples of limited quantity and quality; using genetic information and novel markers to provide investigative leads; enhancing automation with robotics, different chemistries, and better software tools; employing alternate platforms for typing DNA samples; developing integrated microfluidic/microfabrication devices to process DNA samples with higher throughput, faster turnaround times, lower risk of contamination, reduced labor, and less consumption of evidentiary samples; and exploiting high-throughput sequencing, particularly for attribution in microbial forensics cases.

Validity of low copy number typing and applications to forensic science.

Low copy number (LCN) typing, particularly for current short tandem repeat (STR) typing, refers to the analysis of any sample that contains less than 200 pg of template DNA. Generally, LCN typing simply can be defined as the analysis of any DNA sample where the results are below the stochastic threshold for reliable interpretation. There are a number of methodologies to increase sensitivity of detection to enable LCN typing.

The DRD2 gene 957C>T polymorphism is associated with posttraumatic stress disorder in war veterans.

Background: Variations in genes related to the dopaminergic pathway have been implicated in neuropsychiatric disorders such as schizophrenia, substance misuse, Alzheimer's disease and Post Traumatic Stress Disorder (PTSD). A single nucleotide polymorphism (SNP) (957C>T) and a deletion polymorphism (-141delC) in the DRD2 gene and a SNP (Taq1A) in a gene directly downstream of DRD2 have all been implicated in dopamine functioning in the brain.

Methods: To test the importance of these three polymorphisms in PTSD susceptibility, a genetic screen was performed in 127 war veterans diagnosed with PTSD and 228 control individuals without a history of PTSD.

Forensically relevant SNP classes.

Forensic samples that contain too little template DNA or are too degraded require alternate genetic marker analyses or approaches to what is currently used for routine casework. Single nucleotide polymorphisms (SNPs) offer promise to support forensic DNA analyses because of an abundance of potential markers, amenability to automation, and potential reduction in required fragment length to only 60-80 bp. The SNP markers will serve an important role in analyzing challenging forensic samples, such as those that are very degraded, for augmenting the power of kinship analyses and family reconstructions for missing persons and unidentified human remains, as well as for providing investigative lead value in some cases without a suspect (and no genetic profile match in CODIS).

Identification of genes differentially expressed by prematurely fused human sutures using a novel in vivo - in vitro approach.

Craniosynostosis is the premature fusion of calvarial sutures. It results from abnormal differentiation or proliferation of cells within the osteogenic fronts of growing calvarial bones. To date, research has focused on animal models and in vitro organ and tissue culture to determine the molecular mechanisms controlling calvarial suture morphogenesis.

Unravelling the molecular control of calvarial suture fusion in children with craniosynostosis.

Background: Craniosynostosis, the premature fusion of calvarial sutures, is a common craniofacial abnormality. Causative mutations in more than 10 genes have been identified, involving fibroblast growth factor, transforming growth factor beta, and Eph/ephrin signalling pathways. Mutations affect each human calvarial suture (coronal, sagittal, metopic, and lambdoid) differently, suggesting different gene expression patterns exist in each human suture.

Decreasing amplification bias associated with multiple displacement amplification and short tandem repeat genotyping.

Although multiple displacement amplification (MDA) is being used increasingly to amplify genomes, the amplification bias generated by the varphi29 polymerase can be a concern with genotyping applications. It has been noted that the bias is pronounced with small template amounts, particularly with single nucleotide polymorphism (SNP) and short tandem repeat (STR) genotyping. Bias may occur between loci, or between alleles within a locus, and may differ between sample donors at the same loci.

Promoter polymorphisms in the MATP (SLC45A2) gene are associated with normal human skin color variation.

Human pigmentation is a complex physical trait in which the membrane-associated transporter protein (MATP) plays an important role as it is involved in intracellular processing and trafficking of melanosomal proteins. Recently, pathogenic mutations in MATP have been shown to cause oculocutaneous albinism type 4, while other polymorphisms are known to have a role in normal pigmentation variation. We previously reported significant associations of two coding region polymorphisms with hair, skin, and eye color in Caucasians.

Association of functionally different RUNX2 P2 promoter alleles with BMD.

Unlabelled: RUNX2 gene SNPs were genotyped in subjects from the upper and lower deciles of age- and weight-adjusted femoral neck BMD. Of 16 SNPs in RUNX2 and its two promoters (P1 and P2), only SNPs in the P2 promoter were significantly associated with BMD. These P2 promoter SNPs were functionally different in gel-shift and promoter activity assays.

Gene polymorphisms and their effects in the melanocortin system.

In addition to its role in human pigmentation, components of the melanocortin system regulate appetite, energy homeostasis and hormone production. Recent studies have suggested possible roles of this system in immunity, transmission of pain signals, and reproductive potential. A number of polymorphisms have been identified in genes of the melanocortin system and are associated with pigmentation in humans, as well as being causative of disorders of adrenal hormone production and obesity.

Linkage disequilibrium analysis identifies an FGFR1 haplotype-tag SNP associated with normal variation in craniofacial shape.

Mutations in FGFR1 and TWIST1 have been reported to affect the timing of calvarial suture fusion resulting in craniosynostosis and facial abnormalities. We screened nonpathologic populations for genetic polymorphisms that may associate with normal craniofacial variation. We identified 17 single-nucleotide polymorphisms (SNPs) in FGFR1, 6 of which were novel (g.

Single nucleotide polymorphisms in the MATP gene are associated with normal human pigmentation variation.

Human physical pigmentation is determined by the type and amount of melanin and the process of pigmentation production probably involves more than 100 genes. A failure to synthesize melanin results in oculocutaneous albinism (OCA). A recently identified form of OCA results from mutations in the Membrane Associated Transporter Protein (MATP) gene.

The C/C genotype of the C957T polymorphism of the dopamine D2 receptor is associated with schizophrenia.

The T allele of the human dopamine D2 receptor (DRD2) gene C957T polymorphism is associated with reduced mRNA translation and stability. This results in decreased dopamine induced DRD2 upregulation and decreased in vivo D2 dopamine binding. Conversely, the C allele of the C957T polymorphism is not associated with such changes in mRNA leading to increased DRD2 expression.

Mouse models of obesity.

Insights into the etiology of human obesity have arisen from the study of animal models. Animal models of obesity are also important for the development of future treatments of obesity. An agouti mouse mutation resulting in obese, yellow mice was described over a century ago and in 1992 agouti was cloned, making it the first obesity gene characterized at the molecular level.