R.J. Mitchell

Secondary DNA transfer of biological substances under varying test conditions.

This research investigates factors that may influence the secondary transfer of DNA. These include the type of biological substance deposited, the nature of the primary and secondary substrate, moisture content of the deposit and type of contact between the surfaces. Results showed that secondary transfer is significantly affected by both the type of primary substrate and the moisture (wetness) of the biological sample. Porous substrates and/or dry samples diminished transfer (with on average only 0.36% of biological material being transferred from one site to another), whereas non-porous substrates and/or wet samples facilitated transfer events (approximately 50-95% of biological material was transferred from one site to another). Further, the type of secondary substrate also influenced transfer rate, with porous surfaces, absorbing transferred biological substances more readily than non-porous ones. No significant differences were observed among the biological substances tested (pure DNA, blood and saliva). Friction contact between the two substrates significantly enhanced secondary transfer compared to either passive or pressure contact. These preliminary results will assist in developing general assumptions when estimating probability of a secondary DNA transfer event under simple conditions.

Transcript levels of the intermediate size or grey zone fragile X mental retardation 1 alleles are raised, and correlate with the number of CGG repeats

Background: Grey zone or intermediate alleles are one of the three recognised classes of the X-linked fragile X mental retardation 1 (FMR1) gene showing intergenerational instability. These classes are defined according to the number of CGG repeats in the FMR1 5′-untranslated region. Although large CGG expansions (>200 repeats) cause a neurodevelopmental anomaly through silencing of the gene, resulting in a deficit of FMR1 specific protein, smaller expansions (approximately 55–200 repeats) are associated with an increased transcription and late-onset specific phenotypes. Those alleles with a CGG repeat number ranging between approximately 41 and 55 are relatively poorly defined with regard to both transcriptional and translational activity, and also potential phenotypic effects. Methods and results: Based on a sample of 33 males carrying FMR1 alleles within the grey zone range, defined here as 41–60 CGGs, we show an increased transcriptional activity relative to that seen in common alleles (5–40 CGGS). This is the first study to report a significant relationship between FMR1 mRNA levels and CGG repeat number within the grey zone range (p<0.001). From a piecewise linear regression model, the threshold for onset of the increase in mRNA levels as a function of CGG repeat size has been determined at approximately 39 repeats (standard error (SE) 3.24), and that for the reduction in the rate of this increase at approximately 54 repeats (SE 4.27). Conclusions: The ambiguities associated with the definition and transcription dynamics of the FMR1 gene within the grey zone range are dealt with. There may be specific phenotypes associated with the toxic “gain-of-function” effect of raised mRNA.

An investigation of sequence deletions of amelogenin(AMELY), a Y-chromosome locus commonly used for gender determination.

Background: Accurate gender determination is crucial in many scientific disciplines, but especially so in prenatal diagnosis of X-linked diseases and forensic investigations. Today, molecular techniques, especially typing for a length variation in the X–Y homologous amelogenin gene (AMELX and AMELY), are used for gender assignation. This amelogenin test is an integral part of most PCR multiplex kits that are used for DNA profiling, but in 1998 there was a report of two normal males being typed as female with this test. Subsequently, a small number of amelogenin negative (or AMELY null) males have been reported in various populations but little data are available characterising these deletions. Aims: The study aims to determine the size of the deletion in five AMELY null males by typing DNA samples for markers surrounding this gender-determining locus. The possible relationships among the AMELY null samples are examined through analysis of their deletion size and associated Y-chromosome microsatellite haplotypes. We also attempt to determine the frequency of AMELY negative males in Australia. Subjects and methods: DNA samples from five AMELY null males, from different geographical regions, were made available for this study. The samples were typed for eight sites, all located on the short arm of the Y chromosome, using PCR and gel electrophoresis. Eleven Y-chromosome specific microsatellites were also typed on each sample in order to generate haplotypes for phylogenetic analysis. A questionnaire was sent to all Australian forensic centres requesting information on the frequency of AMELY negative males observed in their laboratories. Results: Two different sized deletions were seen in the five AMELY null samples. One deletion (in two samples) has a size of between 304 and 731 kbp, whereas the other (in three samples) ranges between 712 and 1001 kbp. Y-microsatellite haplotypes indicate that the smaller deletion is probably identical in the two samples, but this is not the case with the larger deletion. The frequency of AMELY negative is rare in Australia, with an overall frequency of 0.02%. Conclusion: Comparisons of both deletion size and haplotypes with published data suggest that most AMELY nulls are the result of independent evolutionary events, even in those populations where the frequency is relatively high. Although AMELY null males are extremely rare in most populations, typing an additional gender-determining locus should be considered in forensic investigations where the reference sample is of unknown gender.

Beware of the Possibility of Fingerprinting Techniques Transferring DNA

Fingerprinting brushes have the potential to collect and transfer DNA during powdering. Squirrel-hair fingerprint brushes exposed to specific sets of saliva stains and brushes used in routine casework were tested for their ability to collect and transfer DNA containing material using standard DNA extraction procedures and AmpFlSTR Profiler Plus amplification and typing procedures. The tests found that the risk of transferring DNA during powdering and having a detrimental impact on the analysis increases if the examiner powders over either biological stains (such as blood or saliva) or very fresh prints and uses more sensitive PCR amplification and typing procedures. We advocate caution when powdering prints from which DNA may also be collected and provide options for consideration to limit the risk of transferred DNA contamination while fingerprinting.

Associations between the two serum proteins haptoglobin and transferrin and leukaemia

Haptoglobin and transferrin (TF) types were determined for 134 patients with leukaemia of the four most common types: acute lymphocytic (ALL), chronic lymphocytic (CLL), acute myelocytic (AML) and chronic myelocytic leukaemia (CML). The phenotype HP1 was found to have an increased incidence in the total patient group due to an increased incidence in those with AML, ALL and CML compared with controls, but not in those with CLL. Although tests of association applied to each of the samples of the four common types of leukaemia produced no significant chi 2 values, they did indicate that the relative incidence (RI) was just under 2 for the groupings of the acute forms ALL and AML, the myelocytic forms AML and CML and for the combination of ALL, AML and CML, respectively. All these associations were statistically significant (p less than 0.05). Analysis of TF subtypes and leukaemia indicated a significantly increased frequency of TF C1C1 among leukaemia patients compared with controls (p less than 0.005). Analysis of the samples of each of the four common types suggested that while the RI was raised in all but ALL patients, the association was significant only in AML patients (p less than 0.05). However, when the two myelocytic types were combined the RI was 2.3 and the association was highly significant (p less than 0.005). No such association could be detected in the lymphocytic forms.

Swabs as DNA Collection Devices for Sampling Different Biological Materials from Different Substrates

Currently, there is a variety of swabs for collection of biological evidence from crime scenes, but their comparative efficiency is unknown. Here, we report the results of an investigation into the efficiency of different swab types to collect blood, saliva and touch DNA from a range of substrates. The efficiency of extracting blood and saliva from each swab type was also tested. Some swabs were significantly more effective than others for sampling biological materials from different substrates. Swabs with the highest sampling efficiency, however, often did not have the highest extraction efficiency. Observations were recorded regarding practicality of each swab in a variety of situations. Our study demonstrates that selection of sampling device impacts greatly upon successful collection and extraction of DNA. We present guidelines to assist in evaluation of swab choice.

DNA polymorphisms of the cholesteryl ester transfer protein (CETP) gene in Italian and Greek migrants to Australia

The distribution of two common TaqI restriction fragment length polymorphisms (RFLPs) of the cholesteryl ester transfer protein (CETP) gene were determined in 271 Italian-born and 170 Greek-born migrants to Melbourne, Australia. A much smaller number were examined for the EcoNI RFLP of the same gene. Allele frequencies of the TaqI A RFLP exhibited the least variation in both ethnic groups, and no significant regional heterogeneity in allele or genotype frequencies of either TaqI RFLP was detected for Greece or Italy. There was no difference between Italians and Greeks for the TaqI A polymorphism and the variability at the B RFLP was of borderline significance. Comparisons with other Caucasian populations revealed that allele frequencies of all three CETP RFLPs are remarkably uniform within Caucasians, with the TaqI B polymorphism being the most variable.

Aboriginal mitogenomes reveal 50,000 years of regionalism in Australia

Aboriginal Australians represent one of the longest continuous cultural complexes known. Archaeological evidence indicates that Australia and New Guinea were initially settled approximately 50 thousand years ago (ka); however, little is known about the processes underlying the enormous linguistic and phenotypic diversity within Australia. Here we report 111 mitochondrial genomes (mitogenomes) from historical Aboriginal Australian hair samples, whose origins enable us to reconstruct Australian phylogeographic history before European settlement. Marked geographic patterns and deep splits across the major mitochondrial haplogroups imply that the settlement of Australia comprised a single, rapid migration along the east and west coasts that reached southern Australia by 49–45 ka. After continent-wide colonization, strong regional patterns developed and these have survived despite substantial climatic and cultural change during the late Pleistocene and Holocene epochs. Remarkably, we find evidence for the continuous presence of populations in discrete geographic areas dating back to around 50 ka, in agreement with the notable Aboriginal Australian cultural attachment to their country.

Following the transfer of DNA: How does the presence of background DNA affect the transfer and detection of a target source of DNA?

DNA transfer is of increasing importance in crime scene situations, partly due to analytical techniques detecting profiles in ever declining amounts of DNA. Whereas the focus has previously been DNA transfer of target sources, the effects of background DNA on transfer and detection of DNA after multiple contact situations have been much less investigated. This study measured the transfer and detection rates of a specific DNA source in the presence of background DNA sources. The presence of background DNA influenced the transfer of DNA differently depending on the combination of biological material and surface type. The detection of a profile from the target DNA decreased after multiple contact situations, due to the reduced total and relative quantity of target DNA, and the increasing complexity of the mixture. The results of this study contribute to a greater understanding of the effects of background DNA sources on DNA transfer and detection.

FMR1 alleles in Tasmania: a screening study of the special educational needs population

The distribution of fragile X mental retardation‐1 (FMR1) allele categories, classified by the number of CGG repeats, in the population of Tasmania was investigated in 1253 males with special educational needs (SEN). The frequencies of these FMR1 categories were compared with those seen in controls as represented by 578 consecutive male births. The initial screening was based on polymerase chain reaction analysis of dried blood spots. Inconclusive results were verified by Southern analysis of a venous blood sample. The frequencies of common FMR1 alleles in both samples, and of grey zone alleles in the controls, were similar to those in other Caucasian populations. Consistent with earlier reports, we found some (although insignificant) increase of grey zone alleles in SEN subjects compared with controls. The frequencies of predisposing flanking haplotypes among grey zone males FMR1 alleles were similar to those seen in other Caucasian SEN samples. Contrary to expectation, given the normal frequency of grey zone alleles, no premutation (PM) or full mutation (FM) allele was detected in either sample, with only 15 fragile X families diagnosed through routine clinical admissions registered in Tasmania up to 2002. An explanation of this discrepancy could be that the C19th founders of Tasmania carried few PM or FM alleles. The eight to ten generations since white settlement of Tasmania has been insufficient time for susceptible grey zone alleles to evolve into the larger expansions.