The study was conducted to detect the porcine DNA in pharmaceutical products in local market using polymerase chain reaction (PCR) and southern-hybridization on the biochip. A total of 113 (n=113) of hard (82 samples) and soft gel (31 samples) capsules from pharmaceutical products were purchased and tested for the presence of porcine DNA for Halal authentication. All capsules were gelatin-based purchased from local over the counter (OTC) markets. Of all samples tested, 37.2% (42/113) contained porcine DNA. While, none porcine DNA band was detected for 62.8% (71/113) of capsules tested. All samples which were positive toward porcine DNA were imported pharmaceutical products with none Halal logo. Results in the presence study demonstrated that the PCR techniques and southern-hybridization on the biochip is suitable tool for monitoring the Haram component in highly processed product of soft and hard capsule.
Mitochondrial DNA (mtDNA) deletions are a major cause of chronic progressive external ophthalmoplegia (CPEO) and Kearns-Sayre syndrome (KSS). We analyzed single mtDNA deletions in 11 CPEO and one KSS patients by means of Southern blot and long polymerase chain reaction (PCR) assays. The deletion sizes ranged from 3.4 kb to 6.9 kb whereas the heteroplasmy level varied from 18.8% to 85.5%. Two unique deletions sized 4320 bp and 4717 bp were found. This study represents the first genetic screen of mtDNA disorders in Malaysia, and it follows the data seen in other published reports on CPEO and KSS genetic aetiology.
The belief that beekeepers live longer than anyone else is present since ages. However, no research has been done to explore the longevity of life in beekeepers. Here, we investigated the telomere length in 30 male beekeepers and 30 male non-beekeepers and associated them with the longevity of life using Southern analysis of terminal restriction fragments (TRFs) generated by Hinf I/Rsa I digestion of human genomic DNA using TeloTAGGG Telomere Length Assay. Interestingly, we found that the telomere length of male beekeepers was significantly longer than those of male non-beekeepers with a p value of less than 0.05, suggesting that beekeepers may have longer life compared to non-beekeepers. We further found that the consumption of bee products for a long period and frequent consumption of bee products per day are associated with telomere length. An increase of year in consuming bee products is associated with a mean increase in telomere length of 0.258 kbp. In addition, an increase in frequency of eating bee products per day was also associated with a mean increase of 2.66 kbp in telomere length. These results suggested that bee products might play some roles in telomere length maintenance.
Beta-thalassemia major is one of the commonest genetic disorders in South-East Asia. The spectrum of beta-thalassemia mutations in the various ethnic sub-populations on the island of Borneo is unknown. We studied 20 Dusun children from the East Malaysian state of Sabah (North Borneo) with a severe beta-thalassemia major phenotype, using a combination of Southern analysis, polymerase chain reaction analysis and direct sequencing. We found the children to be homozygous for a large deletion, which has a 5' breakpoint at position -4279 from the cap site of the beta-globin gene (HBB) with the 3' breakpoint located in a L1 family of repetitive sequences at an unknown distance from the beta-globin gene. This was similar to a recent finding of a large deletion causing beta-thalassemia first described in unrelated beta-thalassemia heterozygotes of Filipino descent. This report describes the first 20 families with homozygosity of the deletion causing a severe phenotype. It provides the first information on the molecular epidemiology of beta-thalassemia in Sabah. This finding has implications for the population genetics and preventative strategies for beta-thalassemia major for nearly 300 million individuals in South-East Asia.
The DNA genomes of isolates of rice tungro bacilliform virus from Bangladesh, India, Indonesia, Malaysia and Thailand were cloned and compared with that of the type isolate from the Philippines. Restriction endonuclease maps revealed differences between the isolates and cross-hybridization showed that they fell into two groups, those from the Indian subcontinent and those from south-east Asian countries. The genomes of isolates from the Indian subcontinent contained a deletion of 64 bp when compared with those from south-east Asia. The implications of this variation are discussed.
DOG(R)1, which encodes 2-deoxyglucose-6-phosphate phosphatase, has been used as a selectable marker gene to produce transgenic plants. In this study, a transformation vector, pBIDOG, which contains the DOG(R)1 gene, was transformed into oil palm embryogenic calli (EC) mediated by Agrobacterium tumefaciens strain LBA4404. Transformed EC were exposed to 400 mg l(-1) 2-deoxyglucose (2-DOG) as the selection agent. 2-DOG resistant tissues were regenerated into whole plantlets on various regeneration media containing the same concentration of 2-DOG. The plantlets were later transferred into soil and grown in a biosafety screenhouse. PCR and subsequently Southern blot analyses were carried out to confirm the integration of the transgene in the plantlets. A transformation efficiency of about 1.0% was obtained using DOG(R)1 gene into the genome of oil palm. This result demonstrates the potential of using combination of DOG(R)1 gene and 2-DOG for regenerating transgenic oil palm.
This study was conducted to investigate the sensitivity and detection of porcine DNA in raw materials, ingredients and finished bakery products by polymerase chain reaction (PCR) - southern hybridization on chip analysis. A total of 20 samples (n=20* 3) with three replicates for each samples were obtained from a bakery factory located in Bangi, Selangor from January to December 2012. The sensitivity level of PCR-southern hybridization on chip was 0.001 ng. The species-specific oligonucleotide primers used in PCR-southern hybridization were targeted on the mitochondria DNA (mtDNA) of cytochrome b (cyt b) gene sequence, namely cty b biotin-labeled oligonucleotide primers. The amplicon from PCR amplification was 276 bp in size. None of the raw materials, ingredients and finished bakery product samples was positive towards porcine DNA, except for the positive control. The results in the present study demonstrated that the PCR- southern-hybridization technique on the gene chip (OliproTM Porcine gene chip) is a sensitive tool for monitoring the porcine component in highly processed ingredients and finished bakery products.
Kajian ini dijalankan untuk mengesahkan kemampuan teknologi DNA mikroaturan cip gen OliproTM FoodPATH bagi mengesan bakteria patogen makanan. Sebanyak 9 jenis DNA bakteria patogen makanan telah digunakan iaitu Bacillus cereus, Escherichia coli O157:H7, Staphylococcus aureus, Vibrio cholerae, Vibrio parahaemolyticus, Listeria monocytogenes, Salmonella spp., Shigella spp. dan Campylobacter spp. Sebanyak 36 kombinasi templat DNA bakteria patogen makanan telah digunakan. Pengesahan bagi mengesan bakteria patogen makanan dilakukan dengan menggunakan kaedah reaksi berantai polimerase (PCR) dan penghibridan Southern-blotting di atas cip gen untuk mengesahkan kemampuannya. Keputusan daripada analisis hibridasi di atas cip gen telah dibandingkan dengan hasil gel elektroforesis 2.0% (w/v). Lima saringan diperlukan untuk menghabiskan 36 kombinasi templat DNA bakteria patogen makanan. Setiap saringan, satu cip gen telah digunakan sebagai kawalan negatif tidak diinokulasikan dengan sebarang kombinasi DNA bakteria patogen makanan. Daripada hasil kajian, didapati bahawa semua kombinasi templat DNA bakteria patogen makanan telah dapat dikesan. Cip yang digunakan sebagai kawalan negatif tidak menunjukkan kehadiran DNA. Oleh itu, daripada kajian ini cip gen OliproTM FoodPATH didapati memberikan keputusan yang lebih baik berbanding dengan 2.0% (w/v) gel elektroforesis.
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.
A total of 11 Vibrio cholerae isolates from 1996-1998 outbreaks in Malaysia and 4 V. alginolyticus were analyzed. Isolates were characterized by polymerase chain reaction (PCR) and Southern hybridization for the presence of the gene encoding zonula occludens toxin (zot). Screening of zot gene by PCR revealed the presence of this gene in V. cholerae and V. alginolyticus. The zot gene from one V. cholerae Ogawa isolate that was cloned in a pCR 2.1 TOPO vector was sequenced. The sequences obtained were 99% homologous to the zot gene sequence from the Gene Bank.
Ty1-copia-like retrotransposons have been identified and investigated in several plant species. Here, the internal region of the reverse transcriptase (RT) gene of Ty1-copia-like retrotransposons was amplified by PCR from total genomic DNA of 10 varieties of banana. Two to four clones from each variety were sequenced. Extreme heterogeneity in the sequences of Ty1-copia-like retrotransposons from all the varieties was revealed following sequence analysis of the reverse transcriptase (RT) fragments. The size of the individual RT gene fragments varied between 213 and 309 bp. Southern blots of genomic DNA digested from Musa acuminata and other banana varieties probed with W8 clone from M. acuminata and A4 clone from Pisang Abu Nipah showed similar strong, multiple restriction fragments together with other faint hybridization band patterns with variable intensities indicating the presence of many copies of the Ty1-copia-like retrotransposons in the genomes. There was no correlation between retroelement sequence and the banana species (with A or B genomes) from which it arose, suggesting that the probes are not useful for tracking genomes through breeding populations.
A polymerase chain reaction assay based on the enzyme-linked immunosorbent assay (PCR-ELISA) has been developed to detect Brugia malayi infection in an area of low endemicity in Malaysia. Blood samples from 239 subjects were tested: 192 amicrofilaraemic individuals, 14 microfilaraemic persons and 3 chronic elephantiasis cases from endemic areas and 30 city-dwellers (non-endemic controls). PCR products were examined by ELISA and Southern hybridization. In the PCR-ELISA, digoxigenin-labelled PCR products were hybridized to a biotin-labelled probe. This was followed by incubation in streptavidin-coated microtitre wells and detection using anti-digoxigenin-peroxidase and ABTS [2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonic acid)]. All microfilaraemic samples were positive by PCR-ELISA and Southern hybridization and all samples from non-endemic subjects and chronic elephantiasis patients were negative. The PCR-ELISA detected 12 times as many B. malayi infections as did thick blood film examination. Nineteen of the 194 samples from the endemic area gave positive results by both PCR-ELISA and Southern hybridization, and an additional 5 samples were positive by PCR-ELISA only. The PCR-ELISA was specific and sensitive, detected more infections, and was more reproducible than Southern hybridization.
The variation in the sequence of the coat protein genes of four isolates of rice tungro spherical virus from different countries, Malaysia, Thailand, India and Bangladesh, was compared with an isolate from the Philippines. The evidence from RT-PCR, Southern blot hybridization and sequences of the coat protein genes indicated that the isolates appeared to fall into two groups. One comprised the Philippine and Malaysian isolates (about 95% sequence similarity) and the other the Bangladeshi and Indian isolates, the sequences of which differed by about 15% from that of the Philippine isolate. The Thai isolate seemed to be a mixture of these two subgroups.
Forty-six Malaysian patients with chronic granulocytic leukaemia were found to be rearranged in the breakpoint cluster region (BCR) of chromosome 22, molecular evidence of Philadelphia chromosome (t9.22) translocation. Through the use of a 1.2 kb 3' BCR probe and two restriction enzyme digests, patients' breakpoints could be localized either to 5' or 3' regions of the BCR. Breakpoint site localization at the time of DNA sampling did not show any positive statistical association to clinical status defined as chronic phase, chronic phase with less than 6 months to blast crisis, accelerated phase and blast crisis. This was in contrast to earlier reports which indicated that patients with breakpoint at 3' site were at a higher biologic risk for entering blast crisis.
We describe a convenient, versatile and safe method for preparing bacterial DNA for ribotyping analysis. In this method, extraction of bacterial DNA from Salmnonella typhi and Burkholderia pseudomallei. and subsequent restriction endonuclease digestion, was performed in agarose blocks/plugs thus minimizing shearing and loss of DNA, problems commonly associated with liquid phase phenol extraction. Digested DNA in the plugs was then electrophoresed directly, transferred to nylon membranes and hybridized with labeled rDNA probes in the usual manner to provide reproducible restriction patterns. This method is particularly useful for bacterial species where standard DNA extraction in the liquid phase using phenol has been problematic (e.g. B. pseudomallei) but can be used for any bacterial species. The DNA extracted within the agarose plugs can be stored for long periods and can be used in other, widely-used typing methods such as pulsed-field gel electrophoresis (PFGE) and PCR-based techniques. Embedding live cells directly in agarose plugs also minimizes the risk of exposure to these virulent human pathogens among laboratory workers.
Lactobacillus plantarum PA18, a strain originally isolated from the leaves of Pandanus amaryllifolius, contains a pR18 plasmid. The pR18 plasmid is a 3211bp circular molecule with a G+C content of 35.8%. Nucleotide sequence analysis revealed two putative open reading frames, ORF1 and ORF2, in which ORF2 was predicted (317 amino acids) to be a replication protein and shared 99% similarity with the Rep proteins of pLR1, pLD1, pC30il, and pLP2000, which belong to the RCR pC194/pUB110 family. Sequence analysis also indicated that ORF1 was predicted to encode linA, an enzyme that enzymatically inactivates lincomycin. The result of Southern hybridization and mung bean nuclease treatment confirmed that pR18 replicated via the RCR mechanism. Phylogenetic tree analysis of pR18 plasmid proteins suggested that horizontal transfer of antibiotic resistance determinants without genes encoding mobilization has not only occurred between Bacillus and Lactobacillus but also between unrelated bacteria. Understanding this type of transfer could possibly play a key role in facilitating the study of the origin and evolution of lactobacillus plasmids. Quantitative PCR showed that the relative copy number of pR18 was approximately 39 copies per chromosome equivalent.
A small plasmid designated pAR141 was isolated from Lactococcus lactis subsp. lactis M14 and its complete 1,594 base pair nucleotide sequence was determined. Analysis of the sequence indicated that this plasmid does not carry any industrially important determinants besides the elements involved in plasmid replication and control. The transcriptional repressor CopG and replication initiation protein RepB appeared as a single operon. A small countertranscribed RNA (ctRNA) coding region was found between the copG and repB genes. The double strand origin (dso) and single strand origin (sso) of rolling circle replicating (RCR) plasmids were also identified in pAR141, suggesting that this plasmid replicates by rolling circle (RC) mode. This observation was supported by S1 nuclease and Southern hybridization analyses.
In accordance with detection of a few phospholipase A2 (PLA2) isozyme genes by Southern blot analysis, only two cDNAs, named NnkPLA-I , and NnkPLA-II, encoding group I PLA2s, NnkPLA-I and NnkPLA-II, respectively, were isolated from the venom gland cDNA library of Elapinae Naja naja kaouthia of Malaysia. NnkPLA-I and NnkPLA-II showed four amino acid substitutions, all of which were brought about by single nucleotide substitution. No existence of clones encoding CM-II and CM-III, PLA2 isozymes which had been isolated from the venom of N. naja kaouthia of Thailand, in Malaysian N. naja kaouthia venom gland cDNA library was verified by dot blot hybridization analysis with particular probes. NnkPLA-I and NnkPLA-II differed from CM-II and CM-III with four and two amino acid substitutions, respectively, suggesting that their molecular evolution is regional. The comparison of NnkPLA-I, NnkPLA-II and cDNAs encoding other group I snake venom gland PLA2s indicated that the 5'- and 3'-untranslated regions are more conserved than the mature protein-coding region and that the number of nucleotide substitutions per nonsynonymous site is almost equal to that per synonymous site in the protein-coding region, suggesting that accelerated evolution has occurred in group I venom gland PLA2s possibly to acquire new physiological functions.
Fragile X syndrome is a result of an unstable expansion of (CGG)n trinucleotide sequences in the FMR-1 (Fragile X Mental Retardation 1) gene site at Xq27. In a normal person, n ranges from 6 to 40 repeats with an average of 30 repeats, whereas in a mutated FMR1 gene the sequence is repeated several times over (stuttering gene). Full mutation occurs when n equals 200 repeats or more. Where n equals 50 to 200 repeats, it is a premutation. Fragile X occurs when the FMR-1 gene is unable to make normal amounts of usable Fragile X Mental Retardation Protein, or FMRP. The amount of FMRP in the body is one factor that determines the severity of the Fragile X syndrome. A person with nearly normal levels of FMRP usually has mild or no symptoms, while a person with very little or no normal FMRP has more severe symptoms. The mechanism for the role of the FMRP gene is still being researched upon. However, it has been observed that large numbers of repeats (more than 200) inactivates the gene through a process of methylation and when the gene is inactivated, the cell may make little or none of the needed FMRP. Inheritance is X-linked with reduced penetrance and the frequency of occurrence goes up through generations. The phenotypic manifestations of fragile-X syndrome vary and are largely dependent on the size of the mutation or premutation. The identification of the fragile site on G banded metaphases is a time consuming and delicate process requiring experience and skill, however, molecular diagnosis using DNA analysis and Southern blotting, even though expensive, is more specific in determining the presence or absence of the gene. This study was aimed to establish a rapid polymerase chain reaction (PCR) based - touch down PCR, as a screening method for fragile X syndrome. A total of six cases were analysed. Of these, one was a known case of Fragile X (T1) diagnosed by conventional cytogenetics, two were from the latter’s family members namely, his mother (T2) and father (T3), and the other two (T4 and T5) were randomly selected from patients presenting with dysmorphic features and delayed development respectively. One normal control (TC) was included. Cytogenetic analyses for detection of the fragile site was carried out in all cases. Two culture systems were used, namely the synchronised lymphocyte culture and the folate - thymidine deficient culture. Stained metaphases from the fragile X cultures were screened for the presence of the fragile site on the X chromosome. G-banded karyotyping was done using an image analyser to exclude presence of chromosomal abnormalities. DNA was extracted from these samples and amplified by touch-down PCR. Cytogenetic analysis revealed a folate-sensitive fragile site in the affected male, but none in the other five samples. G-banded karyotyping exhibited no additional chromosomal abnormalities. All extracted DNA samples were successfully amplified. Five of the samples showed presence of the product at the expected band at 552bp, excluding the presence of an expansion of CGG segment of the FMR-1 gene. The absence of a band in an affected individual, suggested a fully mutated allele of FRAXA (Folate Sensitive Fragile Site at Xq28). We succeeded in establishing a slightly modified touch-down PCR analysis. Our study indicates that PCR testing offers a rapid and specific method for screening of normal allele and full mutation of the fragile X gene. We suggest this technique to be applied as a complementary tool for cytogenetic analysis to detect the FRAXA gene.
We previously identified an expressed sequence tag clone, Der f 22, showing 41% amino acid identity to published Der f 2, and show that both genes are possible paralogues. The objective of this study was to characterize the genomic, proteomic and immunological functions Der f 22 and Der f 2. The full-length sequence of Der f 2 and Der f 22 coded for mature proteins of 129 and 135 amino acids respectively, both containing 6 cysteine residues. Phylogenetic analysis of known group 2 allergens and their homologues from our expressed sequence tag library showed that Der f 22 is a paralogue of Der f 2. Both Der f 2 and Der f 22 were single gene products with one intron. Both allergens showed specific IgE-binding to over 40% of the atopic patients, with limited of cross-reactivity. Both allergens were detected at the gut region of D. farinae by immunostaining. Der f 22 is an important allergen with significant IgE reactivity among the atopic population, and should be considered in the diagnostic panel and evaluated as future hypoallergen vaccine therapeutic target.