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  1. Fulltext Overlooked: Extrachromosomal DNA and Their Possible Impact on Whole Genome Sequencing
    Dennin RH
    Malays J Med Sci, 2018 Mar;25(2):20-26.
    PMID: 30918452 DOI: 10.21315/mjms2018.25.2.3
    Extrachromosomal (ec) DNA in eukaryotic cells has been known for decades. The structures described range from linear double stranded (ds) DNA to circular dsDNA, distinct from mitochondrial (mt) DNA. The sizes of circular forms are described from some hundred base pairs (bp) up to more than 150 kbp. The number of molecules per cell ranges from several hundred to a thousand. Semi-quantitative determinations of circular dsDNA show proportions as high as several percentages of the total DNA per cell. These ecDNA fractions harbor sequences that are known to be present in chromosomal DNA (chrDNA) too. Sequencing projects on, for example the human genome, have to take into account the ecDNA sequences which are simultaneously ascertained; corrections cannot be performed retrospectively. Concerning the results of sequencings derived from extracted whole DNA: if the ecDNA fractions contained therein are not taken into account, erroneous conclusions at the chromosomal level may result.
    Matched MeSH terms: DNA, B-Form
  2. Fulltext Rapid identification of melioidosis agent by an insulated isothermal PCR on a field-deployable device
    Chua KH, Tan EW, Chai HC, Puthucheary SD, Lee PC, Puah SM
    PeerJ, 2020;8:e9238.
    PMID: 32518734 DOI: 10.7717/peerj.9238
    Background: Burkholderia pseudomallei causes melioidosis, a serious illness that can be fatal if untreated or misdiagnosed. Culture from clinical specimens remains the gold standard but has low diagnostic sensitivity.

    Method: In this study, we developed a rapid, sensitive and specific insulated isothermal Polymerase Chain Reaction (iiPCR) targeting bimA gene (Burkholderia Intracellular Motility A; BPSS1492) for the identification of B. pseudomallei. A pair of novel primers: BimA(F) and BimA(R) together with a probe were designed and 121 clinical B. pseudomallei strains obtained from numerous clinical sources and 10 ATCC non-targeted strains were tested with iiPCR and qPCR in parallel.

    Results: All 121 B. pseudomallei isolates were positive for qPCR while 118 isolates were positive for iiPCR, demonstrating satisfactory agreement (97.71%; 95% CI [93.45-99.53%]; k = 0.87). Sensitivity of the bimA iiPCR/POCKIT assay was 97.52% with the lower detection limit of 14 ng/µL of B. pseudomallei DNA. The developed iiPCR assay did not cross-react with 10 types of non-targeted strains, indicating good specificity.

    Conclusion: This bimA iiPCR/POCKIT assay will undoubtedly complement other methodologies used in the clinical laboratory for the rapid identification of this pathogen.

    Matched MeSH terms: DNA, B-Form
  3. Fulltext A/T Run Geometry of B-form DNA Is Independent of Bound Methyl-CpG Binding Domain, Cytosine Methylation and Flanking Sequence
    Chia JY, Tan WS, Ng CL, Hu NJ, Foo HL, Ho KL
    Sci Rep, 2016 08 09;6:31210.
    PMID: 27502833 DOI: 10.1038/srep31210
    DNA methylation in a CpG context can be recognised by methyl-CpG binding protein 2 (MeCP2) via its methyl-CpG binding domain (MBD). An A/T run next to a methyl-CpG maximises the binding of MeCP2 to the methylated DNA. The A/T run characteristics are reported here with an X-ray structure of MBD A140V in complex with methylated DNA. The A/T run geometry was found to be strongly stabilised by a string of conserved water molecules regardless of its flanking nucleotide sequences, DNA methylation and bound MBD. New water molecules were found to stabilise the Rett syndrome-related E137, whose carboxylate group is salt bridged to R133. A structural comparison showed no difference between the wild type and MBD A140V. However, differential scanning calorimetry showed that the melting temperature of A140V constructs in complex with methylated DNA was reduced by ~7 °C, although circular dichroism showed no changes in the secondary structure content for A140V. A band shift analysis demonstrated that the larger fragment of MeCP2 (A140V) containing the transcriptional repression domain (TRD) destabilises the DNA binding. These results suggest that the solution structure of MBD A140V may differ from the wild-type MBD although no changes in the biochemical properties of X-ray A140V were observed.
    Matched MeSH terms: DNA, B-Form/chemistry*
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