Dried blood spots (DBS) are currently the recommended sample collection method for newborn screening programmes in America. Early diagnosis of beta-thalassaemia screening is essential as it provides an added advantage especially in sickle cell disease. Beta-thalassaemia frequency is high in many poor countries, and the cost of using commercial DNA extraction kits can be prohibitive. Our study assessed three methods that use minimal reagents and materials to extract DNA from DBS for beta-thalassaemia identification.
PCR has been extensively used for amplification of DNA sequences. We conducted a study to obtain the best amplification conditions for cytochrome b (Cyt b), cytochrome c oxidase I (COI) and 12S rRNA (12S) gene fragments of Malayan gaur mtDNA. DNA from seven Malayan gaur samples were extracted for PCR amplification. Various trials and combinations were tested to determine the best conditions of PCR mixture and profile to obtain the best PCR products for sequencing purposes. Four selected target factors for enhancing PCR, annealing temperature, concentration of primer pairs, amount of Taq polymerase, and PCR cycle duration, were optimized by keeping the amount of DNA template (50 ng/μL) and concentration of PCR buffer (1X), MgCl(2) (2.5 mM) and dNTP mixture (200 μM each) constant. All genes were successfully amplified, giving the correct fragment lengths, as assigned for both forward and reverse primers. The optimal conditions were determined to be: 0.1 μM primers for Cyt b and COI, 0.3 μM primers for 12S, 1 U Taq polymerase for all genes, 30 s of both denaturation and annealing cycles for Cyt b, 1 min of both stages for 12S and COI and annealing temperature of 58.4 ° C for Cyt b, 56.1 ° C for 12S and 51.3 ° C for COI. PCR products obtained under these conditions produced excellent DNA sequences.
Infection with Chloromyxum careni Mutschmann, 1999 was found in the Asian horned frog Megophrys nasuta from Malaysia and Indonesia. Kidney was the only organ infected. Coelozoic plasmodia up to 300 μm were localized in Bowman's space, embracing the glomerulus from all sides, or rarely in lumina of renal tubules. Plasmodia are polysporic, containing disporic pansporoblasts. Myxospores observed by light microscopy are colorless, variable in shape and size, measuring 6.0-8.5 × 5.0-6.5 μm, composed of two symmetrical valves joined by a meridian suture, containing four pyriform polar capsules 3.0-4.0 × 2.5-3.0 μm and a single sporoplasm. Each valve possesses 14-24 (median 21) fine longitudinal ridges clearly visible only in scanning electron microscopy. Rarely, atypical spores with a markedly pointed posterior pole and only 6-10 surface ridges are present in plasmodia together with typical spores. Both small subunit (SSU) and large subunit (LSU) rRNA gene sequences possess extremely long GU-rich inserts. In all SSU and LSU rDNA-based phylogenetic analyses, C. careni clustered as a distinct basal branch to the Myxobolus+Myxidium lieberkuehni clade, out of the marine Chloromyxum clade containing Chloromyxum leydigi, the type species of the genus. These morphological and phylogenetic data suggest erection of a new genus for the C. careni lineage, but we conservatively treat it as a Chloromyxum sensu lato until more information is available.
Nasopharyngeal carcinoma (NPC) is one of the most common cancers in Malaysia, mainly occurring among the Chinese population. To detect common genetic alterations in NPC, we screened seven cases of NPC using the comparative genomic hybridization (CGH) technique. Before proceeding to the CGH technique, the tumors were first confirmed to consist of 75% tumor cells or more. In brief, the technique consists of binding tumor DNA with normal DNA and human Cot-1 DNA, which is then hybridized to normal metaphase spreads. The slides were then counterstained with 4,6 diamino-2-phenylindole (DAPI II) for detection. Analyses were performed using CGH software (Cytovision). We found genetic alterations in all seven NPC samples. The common chromosomal gains (57%, four cases) were found on chromosome arms 1q, 4p, 5, 7q, 11, 14p, 15q, 18p, and 21p, and common chromosomal losses (43%, three cases) were found on chromosome arm 16p. Our results showed chromosomal alterations in all seven NPC cases in the Malaysian population. This result provides the platform for further investigations to locate tumor suppressor genes and oncogenes at specific chromosomal regions in Malaysian NPC patients.
We have analysed DNA fingerprinting patterns by pulsed-field gel electrophoresis (PFGE) of 52 unrelated Burkholderia pseudomallei strains isolated from septicemic and localized infections from Malaysian subjects. A total of 38 PFGE types were observed among 36 septicemic and 16 localized strains with no predominant pattern. Type 25 was seen in 2 epidemiologically related strains, suggesting human to human transmission. Twelve PFGE types were shared among 26 strains (21 septicemic and 5 localized) showing close genetic relatedness with coefficient of similarity of 0.81 to 1.0. The other 26 strains (15 septicemic and 11 localized) were unrelated as shown by the similarity coefficient of < 0.8. This study showed that our B. pseudomallei strains in Malaysia were mainly heterogenous with no predominant type both in septicemic or localized strains.
Genes involved in the 4-aminobenzenesulfonate (4-ABS) degradation pathway of Hydrogenophaga sp. PBC were identified using transposon mutagenesis. The screening of 10,000 mutants for incomplete 4-ABS biotransformation identified four mutants with single transposon insertion. Genes with insertions that impaired the ability to utilize 4-ABS for growth included (1) 4-sulfocatechol 1,2-dioxygenase β-subunit (pcaH2) and 3-sulfomuconate cycloisomerase involved in the modified β-ketoadipate pathway; (2) 4-aminobenzenesulfonate 3,4-dioxygenase component (sadA) involved in aromatic ring hydroxylation; and (3) transposase gene homolog with a putative cis-diol dehydrogenase gene located downstream. The pcaH2 mutant strain accumulated brown metabolite during growth on 4-ABS which was identified as 4-sulfocatechol through thin layer chromatography and HPLC analyses. Supplementation of wild-type sadA gene in trans restored the 4-ABS degradation ability of the sadA mutant, thus supporting the annotation of its disrupted gene.
Humans reached present-day Island Southeast Asia (ISEA) in one of the first major human migrations out of Africa. Population movements in the millennia following this initial settlement are thought to have greatly influenced the genetic makeup of current inhabitants, yet the extent attributed to different events is not clear. Recent studies suggest that south-to-north gene flow largely influenced present-day patterns of genetic variation in Southeast Asian populations and that late Pleistocene and early Holocene migrations from Southeast Asia are responsible for a substantial proportion of ISEA ancestry. Archaeological and linguistic evidence suggests that the ancestors of present-day inhabitants came mainly from north-to-south migrations from Taiwan and throughout ISEA approximately 4,000 years ago. We report a large-scale genetic analysis of human variation in the Iban population from the Malaysian state of Sarawak in northwestern Borneo, located in the center of ISEA. Genome-wide single-nucleotide polymorphism (SNP) markers analyzed here suggest that the Iban exhibit greatest genetic similarity to Indonesian and mainland Southeast Asian populations. The most common non-recombining Y (NRY) and mitochondrial (mt) DNA haplogroups present in the Iban are associated with populations of Southeast Asia. We conclude that migrations from Southeast Asia made a large contribution to Iban ancestry, although evidence of potential gene flow from Taiwan is also seen in uniparentally inherited marker data.
Human enterovirus 71 (EV-71) is genotyped for molecular epidemiological investigation mainly using the two structural genes, VP1 and VP4. Based on these, EV-71 is divided into three genotypes, A, B and C, and within the genotypes B and C, there are further subgenotypes, B1-B5 and C1-C5. Classification using these genes is useful but gives incomplete phylogenetic information. In the present study, the phylogenetic relationships amongst all the known EV-71 and human enterovirus A (HEV-A) isolates with complete genome sequences were examined. A different tree topology involving EV-71 isolates of subgenotypes, C4 and B5 was obtained in comparison to that drawn using VP1. The nucleotide sequence divergence of the C4 isolates was 18.11% (17-20%) when compared to other isolates of subgenotype C. However, this positions the C4 isolates within the cut-off divergence value of 17-22% used to designate the virus genotypes. Hence, it is proposed here that C4 should be designated as a new genotype D. In addition, the subgenotype B5 isolates had an average nucleotide divergence of only 6.14% (4-8%) when compared to other subgenotype B4 isolates. This places the B5 isolates within the subgenotype B4. It is proposed here that the B5 isolates to be redesignated as B4. With the newly proposed genotype D and inclusion of subgenotype B5 within B4, the average nucleotide divergence between genotypes was 18.99% (17-22%). Inter- and intra-subgenotype average divergences were 12.02% (10-14%) and 3.92% (1-10%), respectively. A phylogenetic tree built using the full genome sequences is robust as it takes into consideration changes in the sequences of both the structural and non-structural genes. Similar nucleotide similarities, however, were obtained if only VP1 and 3D RNA polymerase genes were used. Furthermore, addition of 3D RNA polymerase sequences will also show recombination events. Hence, in the absence of full genome sequences, it is proposed here that a combination of VP1 and 3D RNA polymerase gene sequences be used for initial genotyping of EV-71 isolates.
Array-based comparative genomic hybridization (array CGH) is a new molecular technique that has the potential to revolutionize cytogenetics. However, use of high resolution array CGH in the clinical setting is plagued by the problem of widespread copy number variations (CNV) in the human genome. Constitutional microarray, containing only clones that interrogate regions of known constitutional syndromes, may circumvent the dilemma of detecting CNV of unknown clinical significance.
Matched MeSH terms: DNA, Complementary; DNA Copy Number Variations
A diesel-degrading bacterium from Antarctica has been isolated. The isolate was tentatively identified as Pseudomonas sp. strain DRYJ3 based on partial 16S rDNA molecular phylogeny and Biolog GN microplate panels and Microlog database. Growth on diesel was supported optimally by ammonium sulphate, nitrate and nitrite. The bacterium grew optimally in between 10 and 15 degrees C, pH 7.0 and 3.5% (v/v) diesel. The biodegradation of diesel oil by the strain increased in efficiency from the second to the sixth day of incubation from 1.4 to 18.8% before levelling off on the eighth day n-alkane oxidizing and aldehyde reductase activities were detected in the crude enzyme preparation suggesting the existence of terminal n-alkane oxidizing activity in this bacterium.
The aim of this study was to determine the genotoxicity of a locally produced dental porcelain (Universiti Sains Malaysia, Malaysia) using the Ames and Comet assays. In the Ames assay, four genotypic variants of the Salmonella strains (TA98, TA100, TA1537 and TA1535) carrying mutations in several genes were used. The dental porcelain was incubated with these four strains in five different doses both in the presence and absence of metabolic activation (S9) and the result was assessed based on the number of revertant colonies. Concurrently, appropriate positive controls were used so as to validate the test. The average number of revertant colonies per plate treated with locally produced dental porcelain was less than double as compared to that of negative control. In the Comet assay, L929 (CCL-1 ATCC, USA) mouse fibroblast cells were treated with the dental porcelain in three different concentrations along with concurrent negative and positive controls. The tail moment which was used as a measurement of DNA damage was almost equal to that of the negative control, suggesting that the locally produced dental porcelain did not induce any DNA damage. The results indicated that the locally produced dental porcelain is non-genotoxic under the present test conditions.
The VP4, VP2, and VP1 gene regions were evaluated for their usefulness in typing human enteroviruses. Three published RT-PCR primers sets targeting separately these three gene regions were used. Initially, from a total of 86 field isolates (36 HEV-A, 40 HEV-B, and 10 HEV-C) tested, 100% concordance in HEV-A was identified from all three gene regions (VP4, VP2, and VP1). However, for HEV-B and HEV-C viruses, only the VP2 and VP1 regions, and not VP4, showed 100% concordance in typing these viruses. To evaluate further the usefulness of VP4 in typing HEV-A enteroviruses, 55 Japanese and 203 published paired VP4 and VP1 nucleotide sequences were also examined. In each case, typing by VP4 was 100% in concordance with typing using VP1. Given these results, it is proposed that for HEV-A enteroviruses, all three gene regions (VP4, VP2, and VP1), would be useful for typing these viruses. These options would enhance the capability of laboratories in identifying these viruses and would greatly help in outbreaks of hand, foot, and mouth disease.
The copy numbers of 16S rRNA genes in 12 probiotic Lactobacillus strains of poultry origin were analyzed. Genomic DNA of the strains was digested with restriction endonucleases that do not cut within the 16S rRNA gene of the strains. This was followed by Southern hybridization with a biotinylated probe complementary to the 16S rRNA gene. The copy number of the 16S rRNA gene within a Lactobacillus species was found to be conserved. From the hybridization results, Lactobacillus salivarius I 24 was estimated to have seven copies of the 16S rRNA gene, Lactobacillus panis C 17 to have five copies and Lactobacillus gallinarum strains I 16 and I 26 four copies. The 16S rRNA gene copy numbers of L. gallinarum and L. panis reported in the present study are the first record. Lactobacillus brevis strains I 12, I 23, I 25, I 211, I 218 and Lactobacillus reuteri strains C 1, C 10, C 16 were estimated to have at least four copies of the 16S rRNA gene. In addition, distinct rRNA restriction patterns which could discriminate the strains of L. reuteri and L. gallinarum were also detected. Information on 16S rRNA gene copy number is important for physiological, evolutionary and population studies of the bacteria.
Matched MeSH terms: DNA Restriction Enzymes/metabolism
Hepatitis B virus (HBV) is classified into eight genotypes (A to H). In this study, three genotyping methods were compared for their sensitivity and accuracy, namely PCR-RFLP on the S region, PCR-RFLP on the pre-S region and nested PCR with type specific primers. Sixty HBV samples from infected sera were genotyped. The nested PCR with type specific primers was found to be the most sensitive and produced substantial numbers of co-infections by genotypes B and C. The sensitivities for both PCR-RFLP methods were lower and did not reveal co-infections. Generally, the three methods produced consistent genotyping results in samples infected by single genotypes but for co-infections by genotypes B and C, the two PCR-RFLP methods yielded only single genotypic results. For the cases of single genotypic infections, genotypes ascertained by sequencing were in concordance across all three methods. However, when co-infections occurred, PCR analysis on clones revealed only single genotypic infections.
The p53 gene is a tumour suppressor gene that encodes a 393-amino-acid nuclear DNA-binding phosphoprotein. The significance of p53 detection is that p53 mutation is linked with chemo-resistance and transformation to more aggressive disease in a large number of tumour types and it was confirmed that mutant p53 is involved in neoplastic transformations. In addition, the expression of p53 has been closely correlated with clinicopathological findings. Since breast cancer has been reported as one of the most frequent malignancies in women in Malaysia, the expression of p53 was studied in 382 cases of invasive ductal carcinoma of the breast, obtained from three major hospitals in the North-East States of Malaysia. The study utilized an enzyme immunohistochemistry assay for the detection of p53. It was found that p53 was expressed in 29.6% of all the study cases. Furthermore, its expression was significantly correlated with the age and the clinical grading of the disease. No significant statistical correlations were depicted with lymph node status, tumour size, side of tumour, and expression of estrogen and progesterone receptors. Nevertheless, knowledge of the p53 status may be valuable in making clinical decisions regarding diagnosis, prognosis and therapy.
The full-length genomes of two DENV-1 viruses isolated during the 2005-2006 dengue incidents in Brunei were sequenced. Twenty five primer sets were designed to amplify contiguous overlapping fragments of approximately 500-600 base pairs spanning the entire sequence of the genome. The amplified PCR products were sent to a commercial laboratory for sequencing and the nucleotides and the deduced amino acids were determined. Sequence analysis of the envelope gene at the nucleotide and amino acid levels between the two isolates showed 92 and 96 % identity, respectively. Comparison of the envelope gene sequences with 68 other DENV-1 viruses of known genotypes placed the two isolates into two different genotypic groups. Isolate DS06/210505 belongs to genotype V together with some of the recent isolates from India (2003) and older isolates from Singapore (1990) and Burma (1976), while isolate DS212/110306 was clustered in genotype IV with the prototype Nauru strain (1974) and with some of the recent isolates from Indonesia (2004) and the Philippines (2002, 2001). In the full-length genome analysis at the nucleotide level, isolate DS06/210505 showed 94 % identity to the French Guyana strain (1989) in genotype V while isolate DS212/110306 had 96 % identity to the Nauru Island strain (1974) in genotype IV. This work constitutes the first complete genetic characterization of not only Brunei DENV-1 virus isolates, but also the first strain from Borneo Island. This study was the first to report the isolation of dengue virus in the country.
Male-specific coliphages are often used as indicators of contamination by enteric viruses. These phages can be detected in water samples by plaque assays and by polymerase chain reaction. In this study, the M13 coliphage was used to develop a real-time PCR assay for the detection of male-specific DNA coliphages. The real-time PCR was found to have a reaction efficiency of 1.45 and detection limit of 10(-3) plaque forming units per reaction mix. Repeated amplification and melting curve analyses demonstrated high specificity and reproducibility of the real-time assay. Quantitative detection with the real-time PCR should allow rapid assessment of the level of viral contamination in water.
Genetic variation due to heavy metal contamination has always been an interesting topic of study. Because of the numerous contaminants being found in coastal and intertidal waters, there is always much discussion and argument as to which contaminant(s) caused the variations in the genetic structures of biomonitors. This study used a Single Primer Amplification Reaction (SPAR) technique namely Random Amplified Polymorphic DNA (RAPD) to determine the genetic diversity of the populations of the green-lipped mussel Perna viridis collected from a metal-contaminated site at Kg. Pasir Puteh and those from four relatively' uncontaminated sites (reference sites). Heavy metal levels (Cd, Cu, Pb and Zn) were also measured in the soft tissues and byssus of the mussels from all the sites. Cluster analyses employing UPGMA done based on the RAPD makers grouped the populations into two major clusters; the Bagan Tiang, Pantai Lido, Pontian and Kg. Pasir Puteh populations were in one cluster, while the Sg. Belungkor population clustered by itself. This indicated that the genetic diversity based on bands resulting from the use of all four RAPD primers on P. viridis did not indicate its potential use as a biomarker of heavy metal pollution in coastal waters. However, based on a correlation analysis between a particular metal and a band resulting from a specific RAPD primer revealed some significant (P < 0.01) correlations between the primers and the heavy metal concentrations in the byssus and soft tissues. Thus, the correlation between a particular metal and the bands resulting from the use of a specific RAPD primer on P. viridis could be used as biomonitoring tool of heavy metal pollution.
Matched MeSH terms: Sequence Analysis, DNA; Random Amplified Polymorphic DNA Technique