Browsing by Subject "DNA"

Now showing 1 - 12 of 12
  • Thumbnail Image
    Item
    Open Access
    Circular dichroism as a means to follow DNA gymnastics:on the shoulders of giants
    (2009) Mills, M; Lin, C; Chauhan, M; Klump, H H
    This is the first report of DNA stem-loops self-assembled by 'foot-loop' interactions into either two-dimensional strings or three-dimensional spirals, distinguished by circular dichroism spectroscopy. All subunits are linked by cooperative Watson-Crick hydrogen bonds
  • Thumbnail Image
    Item
    Open Access
    DNA repair in Bacteroides fragilis Bf-2
    (1987) Abratt, Valerie Rose; Woods, David R; Jones, David T
    Repair deficient mutants of Bacteroides fragilis have been isolated in order to study the responses of this organism to various DNA damaging agents at the physiological and molecular levels. Two types of mutants were isolated by ethyl methane sulphonate mutagenesis of B.fragilis followed by selection for sensitivity to mitomycin C. One mutant (UVS9) showed sensitivity to both mitomycin C and far-UV irradiation. The other (MTC25) was more sensitive to mitomycin C than UVS9, but showed wild-type resistance to UV radiation. Both mutant strains had wild-type resistance to methyl methane sulphonate.
  • Thumbnail Image
    Item
    Open Access
    DNA-mediated biomineralization of a new planar Pt-complex
    (2006) Klump, H H; Koch, K; Lin, C T
    The crystal growth morphology of a coordination complex of Pt(II) that crystallizes from solution can be controlled by using a second molecular species such as peptides or other organic compounds. Examples of crystal growth controlled by nucleic acids are few. In this article we describe the use of branched three-way junction (3WJ) DNA to influence the crystal growth of a planar platinum compound, cis-[(2, 2′-bipyridyl)N,N-di(2-hydroxyethyl)-N′-benzoylthioureatoplatinum(II)]chloride. Platinum complexes with extended planar aromatic residues are capable of stacking in the absence as well as in the presence of linear DNA double helices. This feature is based on the interaction of the compound with DNA through intercalation, resulting in the prevention of binding of DNA polymerase. Microscopic one-dimensional crystals were observed under these conditions. In the presence of the branched 3WJ DNA, however, additional nucleation sites are present, resulting in extended crystal growth of unique Pt compounds. At least two different crystal modifications were observed using transmission electron microscopy.
  • No Thumbnail Available
    Item
    Open Access
    Evaluation of forensic DNA profiling success on teeth that have been submerged in the ocean
    (2025) Mphaka, Francina Dimpho; Heathfield, Laura; Martin, Donna-Lee
    Deceased humans that are recovered from marine environments contribute to the burden of unidentified bodies, both globally and in South Africa. Compromised conditions of these bodies make identification difficult. In these instances, DNA analysis becomes an invaluable tool for identification. Nonetheless, there is a notable gap in existing literature regarding the use of DNA from teeth for identifying human remains recovered from marine environments. This study therefore aimed to evaluate forensic DNA profiling success on human teeth samples (n = 90) that were submerged in two different marine locations along Cape Town's coastline. Thirty adult volunteers each donated three wisdom teeth, where each tooth per individual was subjected to a different condition for 20 days: one was submerged in False Bay, one was submerged in Table Bay and the remaining tooth was kept as an unsubmerged, matched control in the laboratory. DNA was extracted and quantified using quantitative polymerase chain reaction before undergoing DNA profiling. No significant difference was observed in the likelihood ratio between DNA recovered from samples submerged in Table Bay (median: 1.031 × 1013; range: 4.221 to 5.633 × 1031) compared to False Bay (median: 3.501 × 1015 range: 1.557 × 103 to 2.578 × 1035). As expected, matched control samples yielded significantly higher DNA concentrations than submerged samples and showed significantly higher Likelihood ratios (median: 3.094 × 1028; range: 11.11 to 1.614 × 1033) than submerged samples (p < 0.001). Three DNA profiles from False Bay samples showed no allele detection and therefore were uninformative. Further, allele drop-ins were observed in three DNA profiles from False Bay samples but none in Table Bay samples. These were hypothesised to be due to the presence of marine microbial DNA and could potentially confound DNA interpretations. Overall, the results suggested that the protocol used was suitable for DNA profiling of teeth samples recovered from Table Bay and further research is required to obtain insight into aquatic conditions affecting samples submerged in False Bay.
  • Thumbnail Image
    Item
    Open Access
    Fat mass and obesity associated (FTO) gene influences skeletal muscle phenotypes in non-resistance trained males and elite rugby playing position
    (2017) Heffernan, S M; Stebbings, G K; Kilduff, L P; Erskine, R M; Day, S H; Morse, C I; McPhee, J S; Cook, C J; Vance, B; Ribbans, W J; Raleigh, S M; Roberts, C; Bennett, M A; Wang, G; Collins, M; Pitsiladis, Y P; Williams, A G
    Abstract Background FTO gene variants have been associated with obesity phenotypes in sedentary and obese populations, but rarely with skeletal muscle and elite athlete phenotypes. Methods In 1089 participants, comprising 530 elite rugby athletes and 559 non-athletes, DNA was collected and genotyped for the FTO rs9939609 variant using real-time PCR. In a subgroup of non-resistance trained individuals (NT; n = 120), we also assessed structural and functional skeletal muscle phenotypes using dual energy x-ray absorptiometry, ultrasound and isokinetic dynamometry. In a subgroup of rugby athletes (n = 77), we assessed muscle power during a countermovement jump. Results In NT, TT genotype and T allele carriers had greater total body (4.8% and 4.1%) and total appendicular lean mass (LM; 3.0% and 2.1%) compared to AA genotype, with greater arm LM (0.8%) in T allele carriers and leg LM (2.1%) for TT, compared to AA genotype. Furthermore, the T allele was more common (94%) in selected elite rugby union athletes (back three and centre players) who are most reliant on LM rather than total body mass for success, compared to other rugby athletes (82%; P = 0.01, OR = 3.34) and controls (84%; P = 0.03, OR = 2.88). Accordingly, these athletes had greater peak power relative to body mass than other rugby athletes (14%; P = 2 x 10 -6 ). Conclusion Collectively, these results suggest that the T allele is associated with increased LM and elite athletic success. This has implications for athletic populations, as well as conditions characterised by low LM such as sarcopenia and cachexia.
  • No Thumbnail Available
    Item
    Open Access
    Investigation into implementing a massively parallel sequencing workflow for forensic human identification in South Africa
    (2025) Martin, Donna-Lee; Heathfield, Laura
    South Africa faces grave challenges with high crime rates and associated unidentified bodies each year. DNA profiling using capillary electrophoresis (CE) is typically utilised for human identification purposes but is limiting when applied to degraded post-mortem samples. The ForenSeqTM DNA Signature Prep kit was the first massively parallel sequencing (MPS) workflow validated on the MiSeq FGxTM system, addressing several challenges identified in CE-based methods. With forensic laboratories in developing regions showing proclivity towards a seemingly impossible adoption of MPS, sequence-based studies in Africa are sorely needed to leverage emerging advancements for forensic human identification. This study proposed a four-phased approach for laboratories to facilitate the implementation of MPS for forensic human identification, and included: optimisation, population data generation, internal validation and demonstration of applicability. An optimisation study was carried out to ensure high first-time success rates of analysing reference samples (crude buccal swab lysates) with the ForenSeqTM DNA Signature Prep kit. This entailed systematic adjustments to a direct PCR approach and the development of a lysate purification method. This optimised approach was subsequently used to conduct a population study comprising 463 consenting South African volunteers, wherein the first sequence-based allele frequency data pertaining to autosomal short tandem repeat (A-STR) markers were generated for South African populations. Rich variation was observed, where 80 novel allele sequences were recorded. An increase of 86% was observed in length- to sequence-based allele counts across several A-STR markers, with additional variation recorded in flanking regions. Furthermore, a concordance rate exceeding 99% was achieved. The novel findings and abundance of variation observed in the South African population surpasses that which has been previously characterised on a global scale, warranting further research into characterising sequence data for other forensically relevant markers. The final facet of this study involved the internal validation of the optimised MPS workflow, from sample preparation to sequencing. The workflow was deemed fit for purpose and reported the first performance parameters for post-mortem crude buccal swab lysates. The validated workflow was then applied to a forensic cold case to generate investigative leads from a severely decomposed body, demonstrating the comprehensive capability of the workflow. The synthesis of results obtained in this study have led to key recommendations for under-resourced laboratories to maximise resources for large-scale studies.
  • No Thumbnail Available
    Item
    Open Access
    Investigation into implementing a massively parallel sequencing workflow for forensic human identification in South Africa
    (2025) Martin, Donna-Lee; Heathfield, Laura
    South Africa faces grave challenges with high crime rates and associated unidentified bodies each year. DNA profiling using capillary electrophoresis (CE) is typically utilised for human identification purposes but is limiting when applied to degraded post-mortem samples. The ForenSeqTM DNA Signature Prep kit was the first massively parallel sequencing (MPS) workflow validated on the MiSeq FGxTM system, addressing several challenges identified in CE-based methods. With forensic laboratories in developing regions showing proclivity towards a seemingly impossible adoption of MPS, sequence-based studies in Africa are sorely needed to leverage emerging advancements for forensic human identification. This study proposed a four-phased approach for laboratories to facilitate the implementation of MPS for forensic human identification, and included: optimisation, population data generation, internal validation and demonstration of applicability. An optimisation study was carried out to ensure high first-time success rates of analysing reference samples (crude buccal swab lysates) with the ForenSeqTM DNA Signature Prep kit. This entailed systematic adjustments to a direct PCR approach and the development of a lysate purification method. This optimised approach was subsequently used to conduct a population study comprising 463 consenting South African volunteers, wherein the first sequence-based allele frequency data pertaining to autosomal short tandem repeat (A-STR) markers were generated for South African populations. Rich variation was observed, where 80 novel allele sequences were recorded. An increase of 86% was observed in length- to sequence-based allele counts across several A-STR markers, with additional variation recorded in flanking regions. Furthermore, a concordance rate exceeding 99% was achieved. The novel findings and abundance of variation observed in the South African population surpasses that which has been previously characterised on a global scale, warranting further research into characterising sequence data for other forensically relevant markers. The final facet of this study involved the internal validation of the optimised MPS workflow, from sample preparation to sequencing. The workflow was deemed fit for purpose and reported the first performance parameters for post-mortem crude buccal swab lysates. The validated workflow was then applied to a forensic cold case to generate investigative leads from a severely decomposed body, demonstrating the comprehensive capability of the workflow. The synthesis of results obtained in this study have led to key recommendations for under-resourced laboratories to maximise resources for large-scale studies.
  • Thumbnail Image
    Item
    Open Access
    Maternal blood contamination of collected cord blood can be identified using DNA methylation at three CpGs
    (2017) Jones, Meaghan J
    Abstract Background Cord blood is a commonly used tissue in environmental, genetic, and epigenetic population studies due to its ready availability and potential to inform on a sensitive period of human development. However, the introduction of maternal blood during labor or cross-contamination during sample collection may complicate downstream analyses. After discovering maternal contamination of cord blood in a cohort study of 150 neonates using Illumina 450K DNA methylation (DNAm) data, we used a combination of linear regression and random forest machine learning to create a DNAm-based screening method. We identified a panel of DNAm sites that could discriminate between contaminated and non-contaminated samples, then designed pyrosequencing assays to pre-screen DNA prior to being assayed on an array. Results Maternal contamination of cord blood was initially identified by unusual X chromosome DNA methylation patterns in 17 males. We utilized our DNAm panel to detect contaminated male samples and a proportional amount of female samples in the same cohort. We validated our DNAm screening method on an additional 189 sample cohort using both pyrosequencing and DNAm arrays, as well as 9 publically available cord blood 450K data sets. The rate of contamination varied from 0 to 10% within these studies, likely related to collection specific methods. Conclusions Maternal blood can contaminate cord blood during sample collection at appreciable levels across multiple studies. We have identified a panel of markers that can be used to identify this contamination, either post hoc after DNAm arrays have been completed, or in advance using a targeted technique like pyrosequencing.
  • No Thumbnail Available
    Item
    Open Access
    Metabarcoding pollen to identify allergenic species of grasses (Poaceae) and ragweed (Ambrosia spp.) across six monitored sites in South Africa
    (2025) Sidla, Siyavuya; Peter, Jonathan; Berman, Dilys; Esterhuizen, Nanike; Pedretti, Sarah
    Background and aims: Aeroallergens contribute to the global burden of non-communicable diseases, causing allergic conditions such as rhinitis, conjunctivitis, and asthma. Pollen is the second most important contributor, and despite cross-reactivity between allergens, patients often experience distinct sensitivity patterns to different species. The occurrence of allergenic species varies with geography, seasonality, and their flowering periods are influenced by meteorological factors and climate. The South African Pollen Network (SAPNET) monitors aerospora in different biomes of the country, providing weekly reports on the prevalent species and their concentrations, raising awareness among allergy sufferers and healthcare providers. Grass pollen is one of the most abundant contributors to airborne allergies. However, current SAPNET methods of analysing pollen, using light microscopy, can only identify grasses up to the family level and ragweed (Ambrosia spp.) pollen to the genus level. This project aimed to introduce DNA metabarcoding techniques to classify and identify grass and ragweed species contributing to pollen allergies in various South African biomes. Methods: Samples were selected from weeks with the highest grass pollen counts between October 2022- April 2023 and included samples from sites where Ambrosia pollen was identified (Durban and Potchefstroom). DNA was isolated from environmental pollen samples using the NucleoMag bead beating method, optimised to obtain a larger quantity of double-stranded DNA (dsDNA) and further purified with an additional OneStep PCR inhibitor removal step to improve purity. DNA metabarcoding analysis was performed using two taxonomic markers: ribulose-1,5- bisphosphate carboxylase (rbcL) and internal transcribed spacer 2 (ITS2), amplified with universal primer pairs in a two-step polymerase chain reaction (PCR) library preparation for Next Generation Sequencing (NGS) with MiSeq Illumina V2 systems. The sequence reads generated were assessed for quality, pre-processed, and assigned to taxa using two bioinformatics pipelines. The first followed the method for analysing metabarcoding dual- index data described by Sickel et al. (2015), while the second was adapted from the National Botanic Garden of Wales Plant Illumina pipeline developed by Ford and Jones (2021). Results and Discussion The DNA extraction protocol was optimised by altering the bead beating method using the samples from weeks with high grass pollen concentrations. From the improved method of extraction, we were able to obtain sufficient DNA yield with an average of 21.88 ng/μL. Additional purification improved the dsDNA A280/260 purity ratio to within the ideal range for 73% of the samples, though it introduced salts that decreased the A230/260 ratio below the optimal range. Amplification of rbcL and ITS2 barcodes initially produced amplicons of 579 bp and 542 bp, respectively. However, to limit sequencing costs, an alternative set of ITS2 primers producing amplicons were explored. A total of 25 samples progressed to NGS: Cape Town (7), Kimberley (6), Bloemfontein (3), Johannesburg (3), Durban (3), and Potchefstroom (2), and six ITS2 samples were excluded during quality check due to short amplicon lengths below 100 bp. The three taxonomic classification tools; UTAX, RDP, and BLAST were used to assign taxa, with a 50-read cutoff applied as the minimum read count, with confidence scores of ≥80% for UTAX and ≥0.8 for RDP. For BLAST classifications, only sequences with 99 - 100% identity was considered, using thresholds of ≥80% query coverage and an E-value of ≤1.00×10-5. UTAX classified rbcL and ITS2 sequences identified 233 unique Poaceae species, including Lolium perenne (ryegrass), which was one of the 17 species identified by both barcodes. RDP classifications resulted in 48 Poaceae species, with Poa annua (annual bluegrass) and Cynodon dactylon (Bermuda grass) being two of the three species classified by both barcodes. BLAST classifications produced the largest biodiversity, identifying 307 unique Poaceae species with 19 species common to both barcodes including Cynodon dactylon and Lolium perenne. Additionally, Paspalum notatum (Bahia grass), Phleum pratense (Timothy grass), Phragmites australis (common reed) and Stenotaphrum secundatum (buffalo grass). Two ragweed species, Ambrosia artemisiifolia and A. trifida were positively identified from rbcL sequences with classifications using the three tools. Conclusions: This pilot study successfully used pollen metabarcoding to identify known allergenic grasses in several South African regions, including Bermuda, rye, Timothy, common reed and annual bluegrass in Cape Town; Bermuda, rye, and annual bluegrass in Kimberley; Bahia and Bermuda grass in Johannesburg; weeping love and Bermuda grass in Bloemfontein; and Ambrosia artemisiifolia and A. trifida in Durban. The short length of DNA libraries limited sequence read length, which significantly influenced the classification of operational taxonomic units (OTUs). The low confidence in taxa assignments from UTAX classifications and the identification of species via BLAST that are not recorded in South Africa raised concerns regarding the reliability of UTAX as a taxonomic classification tool and the NCBI based reference databases, which are primarily developed based on Northern Hemisphere plant data and may misclassify endemic species. Future research should address these limitations and include a larger number of SAPNET samples to better map the seasonality and distribution of Poaceae species across South African biomes.
  • Thumbnail Image
    Item
    Open Access
    No evidence for association of insulin receptor substrate-1 Gly972Arg variant with type 2 diabetes mellitus in a mixed-ancestry population of South Africa
    (2014) Vergotine, Zelda; Kengne, André Pascal; Erasmus, Rajiv Timothy; Matsha, Tandi Edith
    BACKGROUND: The most common single-nucleotide polymorphism in the insulin receptor substrate-1 (IRS1) gene is Gly972Arg, which is associated with a 25% increased risk of developing diabetes. The mixed-ancestry population of South Africa (SA) has one of the highest prevalences of type 2 diabetes mellitus (T2DM) in Africa. OBJECTIVE: To report the frequency of IRS1 Gly972Arg and investigate its associations with cardiometabolic traits. METHODS: DNA from 856 mixed-ancestry adults drawn from an urban community of Bellville South, Cape Town, SA, was genotyped by two independent laboratories. Oral glucose tolerance tests were performed and cardiometabolic risk factors measured. RESULTS: A total of 237 (24.7%) participants had T2DM. The IRS1 Gly972Arg variant was present in 7.9% of the individuals studied and only one participant (non-diabetic) carried the homozygous A/A variant. In linear and logistic regression analyses, Gly972Arg was not associated with obesity, insulin resistance/sensitivity or T2DM. CONCLUSIONS: The prevalence of the Gly972Arg variant in the mixed-ancestry population of SA is comparable to that reported in African Americans, but its presence is not associated with cardiometabolic traits. This suggests that the Gly972Arg variant may not aid diabetes risk evaluation in this setting, nor can such information help explain the high prevalence of diabetes previously reported in this population.
  • No Thumbnail Available
    Item
    Open Access
    Sex estimation of unidentified human remains: Concordance between morphological anthropological assessment and DNA analysis
    (2025) Leggett, Celeste Esther; Heathfield, Laura; Gibbon, Victoria
    Identifying skeletonised human remains is a challenge worldwide, and sex determination is an important part of the process. Recently in Cape Town, there have been two medico-legal death investigations involving unidentified skeletonised remains who were estimated to be female anthropologically but were biologically sexed male based on DNA analyses. This study aimed to assess the agreement of sexing methods in a Western Cape South African forensic sample of skeletonised individuals (n=126), who were morphologically estimated to have biological sex as female (n=41). Of the 41 anthropologically estimated to be female cases, 19 were excluded for being probable or likely 'archaeological'. DNA was extracted from hard tissue samples from the remaining 22 individuals and biological sex was assessed by quantitative real-time PCR. DNA from thirteen cases (13/22; 59%) were amplified, with six showing evidence of Y-chromosomal DNA and inferred male sex. However, since the DNA concentrations were below the validated dynamic range, these results were suggestive only. DNA profiling confirmed that one case was male but did not provide further clarity on the biological sex of the remainder of cases due to low copy number (LCN) DNA. This study presents another confirmed case report of sex discordance between anthropological and DNA analysis for a sample of decedents from the Western Cape, South Africa. While qPCR suggested 54% (7/13) agreement and 46% (6/13) disagreement, confirmation in most cases was hindered by LCN DNA. The Western Cape population, influenced by San and Khoe ancestry, tends to have smaller body size and gracility, which may impact the accuracy of morphology-based assessments of male robusticity as they may appear more female or ambiguous. These results highlight the need to update anthropological data for the diverse South African population and implement improved molecular techniques for reliable DNA profiling. This study underscores the limitations of both anthropological and molecular sex methods and stresses the importance of interdisciplinary collaboration for accurate forensic identification.
  • No Thumbnail Available
    Item
    Open Access
    Structure/function analyses indicate novel roles for mycobacterial DnaQ homologs in genome maintenance
    (2025) Griffault, Dimitri; Warner, Digby
    Mycobacterial DNA metabolism is of increasing interest as both an underexplored source of new targets for anti-tuberculosis (TB) drug development and for its potential role in the emergence of drug-resistant Mycobacterium tuberculosis strains. However, the redundancy implied by the sizable complement of DNA replication and repair pathways complicates investigations of gene function. There are, moreover, multiple examples in mycobacteria of apparent fusion – or hybrid – proteins in which N-and C-terminal domains appear to provide discrete functions. Both challenges apply to the mycobacterial DnaQ homologs – comprising separate DnaQ and DnaQ-UvrC hybrid proteins – which, by analogy to model organisms such as E. coli, have traditionally been assumed to fulfil proofreading roles in DNA replication owing to the presence of conserved exonuclease domains. Phylogenetic analysis of DnaQ-like proteins revealed a unique domain composition specific to the Mycobacterium genus comprising a conserved BRCA1 C Terminus (BRCT) domain in DnaQ. Owing to the presence of the BRCT domain and based on the phenotypes observed in domain-targeted mutants, it appeared that the activity of DnaQ protein (M. tuberculosis Rv3711c; M. smegmatis MSMEG_6275) might be linked to the mycobacterial gyrases that are responsible for DNA negative supercoiling following replication. The phylogenetic analysis also revealed highly conserved nucleotide excision repair (NER) proteins among bacteria; however, some species like Actinobacteria, possess both a canonical UvrC along with a DnaQ-UvrC protein. The mycobacterial DnaQ-UvrC (M. tuberculosis Rv2191; M. smegmatis MSMEG_4259) N- terminal was shown to be structurally very similar to that of DnaQ and its C-terminal to that of UvrC, giving the ability to bind either the β-clamp or the components of the UvrABC system. Opposing phenotypes between DnaQ-UvrC (M. tuberculosis Rv2191; M. smegmatis MSMEG_4259) and uvrC deletion imply a DNA-damage specific NER in mycobacteria. This was further confirmed using a combination of gene knockout, site-directed mutants and CRISPRi of NER genes, namely uvrB and uvrC, in DNA damaging conditions. However, further work is required to elucidate the precise functions of DnaQ and DnaQ-UvrC, and their contribution to genome dynamics in a family of organisms that includes major human and animal pathogens.