A coding measure scheme numerically translates the DNA sequence to a time domain signal for protein coding regions identification. A number of coding measure schemes based on numerology, geometry, fixed mapping, statistical characteristics and chemical attributes of nucleotides have been proposed in recent decades. Such coding measure schemes lack the biologically meaningful aspects of nucleotide data and hence do not significantly discriminate coding regions from non-coding regions. This paper presents a novel fuzzy semantic similarity measure (FSSM) coding scheme centering on FSSM codons׳ clustering and genetic code context of nucleotides. Certain natural characteristics of nucleotides i.e. appearance as a unique combination of triplets, preserving special structure and occurrence, and ability to own and share density distributions in codons have been exploited in FSSM. The nucleotides׳ fuzzy behaviors, semantic similarities and defuzzification based on the center of gravity of nucleotides revealed a strong correlation between nucleotides in codons. The proposed FSSM coding scheme attains a significant enhancement in coding regions identification i.e. 36-133% as compared to other existing coding measure schemes tested over more than 250 benchmarked and randomly taken DNA datasets of different organisms.
Three species of otter can be found throughout Malay Peninsula: Aonyx cinereus, Lutra sumatrana, and Lutrogale perspicillata. In this study, we focused on the A. cinereus population that ranges from the southern and the east coast to the northern regions of Malay Peninsula up to southern Thailand to review the relationships between the populations based on the mitochondrial D-loop region. Forty-eight samples from six populations were recognized as Johor, Perak, Terengganu, Kelantan, Ranong, and Thale Noi. Among the 48 samples, 33 were identified as A. cinereus, seven as L. sumatrana, and eight as L. perspicillata. Phylogenetically, two subclades formed for A. cinereus. The first subclade grouped all Malay Peninsula samples except for samples from Kelantan, and the second subclade grouped Kelantan samples with Thai sample. Genetic distance analysis supported the close relationships between Thai and Kelantan samples compared to the samples from Terengganu and the other Malaysian states. A minimum-spanning network showed that Kelantan and Thailand formed a haplogroup distinct from the other populations. Our results show that Thai subspecies A. cinereus may have migrated to Kelantan from Thai mainland. We also suggest the classification of a new subspecies from Malay Peninsula, the small-clawed otter named A. cinereus kecilensis.
Blood cockles are among the most economically important brackish water invertebrates found in Malaysia. However, our knowledge of blood cockle phylogeny and systematics is rudimentary, especially for the species Tegillarca granosa. It is unclear, for instance, whether the cockles occurring on the west coast of peninsular Malaysia constitute a single species, or multiple, phylogenetically distinct species. We performed the first DNA molecular phylogenetic analysis of T. granosa to distinguish it from other related species found in other parts of the world and to create a DNA database for the species. An approximately 585-nucleotide fragment of the mitochondrial DNA (cytochrome oxidase I, COI) was sequenced for 150 individual cockles, representing 10 populations: three from the north, four from the central part and three from the southern part of peninsular Malaysia. Phylogenetic analyses of the resulting dataset yielded tree topologies that not only showed the relationship between T. granosa and its closest relatives but its position in the evolutionary tree. Three mitochondrial clades were evident, each containing an individual genus. Using the mutation rate of the COI gene, the divergence time between T. granosa and its closest related species was estimated to be 460 thousand years ago. This study provides a phylogenetic framework for this ecologically prominent and commercially important cockle species.
Anopheles (Cellia) maculatus Theobald is a major malaria vector in southern Thailand and peninsular Malaysia, and previous population genetic studies suggested that mountain ranges act as barriers to gene flow. In this study, we examine the genetic variance among 12 collections of natural populations in southern Thailand by analyzing 7 microsatellite loci. Based on analysis of molecular variance (AMOVA), three geographic populations of An. maculatus are suggested. The southern population exists in western Thailand north of 12 degrees north latitude. Mosquitoes to the south fall into two genetic populations: 1) the middle southern collections located on the west side of the Phuket mountain range between 8 degrees and 10 degrees north latitude, and 2) the southern collections located on the east of the Phuket mountain range located between approximately 6.5 degrees and 11.5 degrees north latitude. AMOVA revealed significant genetic differentiation between northern and middle southern and southern populations. The middle southern population was moderately differentiated from the southern population. Furthermore, gene flow was restricted between proximal collections located on different sides of the Phuket mountain range. Collections separated by 50 km exhibited restriction of gene flow when separated by geographic barriers, whereas greater gene flow was evident among collections 650 km apart but without geographic barriers.
To date, three species of the genus Glischropus are recognized from the Indomalayan zoogeographic region-G. bucephalus from the Indochinese subregion, G. tylopus from the Sundaic subregion (Peninsular Thailand and Malaysia, Borneo, Sumatra, Moluccas) and G. javanus, restricted to Java. The investigation of the holotype and three topotype specimens of G. batjanus supported the view that the name was previously correctly regarded as the junior subjective synonym of G. tylopus. During review of material recently collected in southwestern Sumatra, Indonesia, one specimen of a yet undescribed species of Thick-thumbed bat was identified. G. aquilus n. sp. markedly differs from its congeners by its dark brown pelage, nearly black ear and tragus, and in skull proportions. The phylogenetic analysis based on cytb sequences also supports the specific distinctness of G. aquilus n. sp. Its discovery brings the count to 88 species of bats known from Sumatra.
Mackerel (Scombridae; Rastrelliger) are small commercially important pelagic fish found in tropical regions. They serve as a cheap source of animal protein and are commonly used as live bait. By using a truss morphometrics protocol and RAPD analysis, we examined morphological and genetic variation among 77 individual mackerel that were caught using long lines and gillnets at 11 locations along the west coast of Peninsular Malaysia. Nineteen morphometric traits were evaluated and genetic information was estimated using five 10-base RAPD random primers. Total DNA was extracted from muscle tissue. Morphometric discriminant function analysis revealed that two morphologically distinct groups of Rastrelliger kanagurta and a single group of R. brachysoma can be found along the west coast of Peninsular Malaysia. We also found that the head-related characters and those from the anterior part of the body of Rastrelliger spp significantly contribute to stock assessment of this population. RAPD analysis showed a trend similar to that of the morphometric analysis, suggesting a genetic component to the observed phenotypic differentiation. These data will be useful for developing conservation strategies for these species.
Iranihindia martellata (Senior-White, 1924) is recorded from peninsular Malaysia for the first time. Male and female specimens in the recent collections of forensically important sarcophagid flies were examined and identified based on morphology and DNA sequencing analysis. Male genitalia offer unambiguous species identification characteristics in the traditional taxonomy of flesh flies but the female flies are very similar to one another in general morphology. Female of I. martellata was determined by DNA sequencing (COI and COII) and PCR-RFLP (COI) analysis. Identified females were carefully examined and compared with the morphologically similar species, Liopygia ruficornis (Fabricius, 1794). Female genitalia are re-described and illustrated in this paper.
The Old World screwworm fly (OWS), Chrysomya bezziana Villeneuve (Diptera: Calliphoridae), is a myiasis-causing blowfly of major concern for both animals and humans. Surveillance traps are used in several countries for early detection of incursions and to monitor control strategies. Examination of surveillance trap catches is time-consuming and is complicated by the presence of morphologically similar flies that are difficult to differentiate from Ch. bezziana, especially when the condition of specimens is poor. A molecular-based method to confirm or refute the presence of Ch. bezziana in trap catches would greatly simplify monitoring programmes. A species-specific real-time polymerase chain reaction (PCR) assay was designed to target the ribosomal DNA internal transcribed spacer 1 (rDNA ITS1) of Ch. bezziana. The assay uses both species-specific primers and an OWS-specific Taqman((R)) MGB probe. Specificity was confirmed against morphologically similar and related Chrysomya and Cochliomyia species. An optimal extraction protocol was developed to process trap catches of up to 1000 flies and the assay is sensitive enough to detect one Ch. bezziana in a sample of 1000 non-target species. Blind testing of 29 trap catches from Australia and Malaysia detected Ch. bezziana with 100% accuracy. The probability of detecting OWS in a trap catch of 50 000 flies when the OWS population prevalence is low (one in 1000 flies) is 63.6% for one extraction. For three extractions (3000 flies), the probability of detection increases to 95.5%. The real-time PCR assay, used in conjunction with morphology, will greatly increase screening capabilities in surveillance areas where OWS prevalence is low.
The discovery of semiconducting behavior of deoxyribonucleic acid (DNA) has resulted in a large number of literatures in the study of DNA electronics. Sequence-specific electronic response provides a platform towards understanding charge transfer mechanism and therefore the electronic properties of DNA. It is possible to utilize these characteristic properties to identify/detect DNA. In this current work, we demonstrate a novel method of DNA-based identification of basidiomycetes using current-voltage (I-V) profiles obtained from DNA-specific Schottky barrier diodes. Electronic properties such as ideality factor, barrier height, shunt resistance, series resistance, turn-on voltage, knee-voltage, breakdown voltage and breakdown current were calculated and used to quantify the identification process as compared to morphological and molecular characterization techniques. The use of these techniques is necessary in order to study biodiversity, but sometimes it can be misleading and unreliable and is not sufficiently useful for the identification of fungi genera. Many of these methods have failed when it comes to identification of closely related species of certain genus like Pleurotus. Our electronics profiles, both in the negative and positive bias regions were however found to be highly characteristic according to the base-pair sequences. We believe that this simple, low-cost and practical method could be useful towards identifying and detecting DNA in biotechnology and pathology.
Several published checklists of bat species have covered Peninsular Malaysia as part of a broader region and/or in combination with other mammal groups. Other researchers have produced comprehensive checklists for specific localities within the peninsula. To our knowledge, a comprehensive checklist of bats specifically for the entire geopolitical region of Peninsular Malaysia has never been published, yet knowing which species are present in Peninsular Malaysia and their distributions across the region are crucial in developing suitable conservation plans. Our literature search revealed that 110 bat species have been documented in Peninsular Malaysia; 105 species have precise locality records while five species lack recent and/or precise locality records. We retrieved 18 species from records dated before the year 2000 and seven species have only ever been recorded once. Our search of Barcode of Life Datasystems (BOLD) found that 86 (of the 110) species have public records of which 48 species have public DNA barcodes available from bats sampled in Peninsular Malaysia. Based on Neighbour-Joining tree analyses and the allocation of DNA barcodes to Barcode Index Number system (BINs) by BOLD, several DNA barcodes recorded under the same species name are likely to represent distinct taxa. We discuss these cases in detail and highlight the importance of further surveys to determine the occurences and resolve the taxonomy of particular bat species in Peninsular Malaysia, with implications for conservation priorities.
High-quality DNA extracts are imperative for downstream applications in molecular identification. Most processed food products undergo heat treatments causing DNA degradation, which hampers application of DNA-based techniques for food authentication. Moreover, the presence of inhibitors in processed food products is also problematic, as inhibitors can impede the process of obtaining high qualities and quantities of DNA. Various approaches in DNA extraction and factors in structure and texture of various food matrices affecting DNA extraction are explained in this review.
Polymerase chain reaction (PCR) is an oft-used preparatory technique in amplifying specific DNA regions for downstream analysis. The size of an amplicon was initially limited by errors in nucleotide polymerization and template deterioration during thermal cycling. A variant of PCR, designated long-range PCR, was devised to counter these drawbacks and enable the amplification of large fragments exceeding a few kb. In this chapter we describe a protocol for long-range PCR, which we have adopted to obtain products of 6.6, 7.2, 13, and 20 kb from human genomic DNA samples.
Wider availability but lack of legal market trades has given feline meat a high potential for use as an adulterant in common meat and meat products. However, mixing of feline meat or its derivatives in food is a sensitive issue, since it is a taboo in most countries and prohibited in certain religions such as Islam and Judaism. Cat meat also has potential for contamination with of severe acute respiratory syndrome, anthrax and hepatitis, and its consumption might lead to an allergic reaction. We developed a very short-amplicon-length (69 bp) PCR assay, authenticated the amplified PCR products by AluI-restriction digestion followed by its separation and detection on a lab-on-a-chip-based automated electrophoretic system, and proved its superiority over the existing long-amplicon-based assays. Although it has been assumed that longer DNA targets are susceptible to breakdown under compromised states, scientific evidence for this hypothesis has been rarely documented. Strong evidence showed that shorter targets are more stable than the longer ones. We confirmed feline-specificity by cross-challenging the primers against 10 different species of terrestrial, aquatic and plant origins in the presence of a 141-bp site of an 18S rRNA gene as a universal eukaryotic control. RFLP analysis separated 43- and 26-bp fragments of AluI-digest in both the gel-image and electropherograms, confirming the original products. The tested detection limit was 0.01% (w/w) feline meat in binary and ternary admixed as well as meatball matrices. Shorter target, better stability and higher sensitivity mean such an assay would be valid for feline identification even in degraded specimens.
This paper describes a short-amplicon-based TaqMan probe quantitative real-time PCR (qPCR) assay for the quantitative detection of canine meat in chicken nuggets, which are very popular across the world, including Malaysia. The assay targeted a 100-bp fragment of canine cytb gene using a canine-specific primer and TaqMan probe. Specificity against 10 different animals and plants species demonstrated threshold cycles (Ct) of 16.13 ± 0.12 to 16.25 ± 0.23 for canine DNA and negative results for the others in a 40-cycle reaction. The assay was tested for the quantification of up to 0.01% canine meat in deliberately spiked chicken nuggets with 99.7% PCR efficiency and 0.995 correlation coefficient. The analysis of the actual and qPCR predicted values showed a high recovery rate (from 87% ± 28% to 112% ± 19%) with a linear regression close to unity (R(2) = 0.999). Finally, samples of three halal-branded commercial chicken nuggets collected from different Malaysian outlets were screened for canine meat, but no contamination was demonstrated.
Being the third-largest primate population has not made macaque (Macaca fascicularis sp.) monkeys less exposed to threats and dangers. Despite wildlife protection, they have been widely hunted and consumed in several countries because of their purported nutritional values. In addition to trading as pure bush meats in several places, monkey meat has been sold in meatball and soup products in Indonesia. Thus the possibility of macaque meat trafficking under the label of common meats is quite high. This paper reports the development of a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay with the shortest amplicon length for the confirmed detection of monkey meat under compromised states which are known to degrade DNA. We amplified a 120-bp region of d-loop gene using a pair of macaque-specific primers and confirmed their specificity for the target species through cross-challenging against 17 different species using a 141-bp site of an 18 S rRNA gene as an endogenous control for eukaryotes. This eliminated the possibilities of any false-negative detection with complex matrices or degraded specimens. The detection limit was 0.00001 ng DNA in a pure state and 0.1% of meat in mixed matrices and commercial meatball products. RFLP analysis further authenticated the originality of the PCR product and distinctive restriction patterns were found upon AluI and CViKI-1 digestion. A micro-fluidic lab-on-a-chip automated electrophoretic system separated the fragments with high resolution. The assay was validated for screening commercial meatball products with sufficient internal control.
Determination of the differential DNA methylation patterns of methylenetetrahydrofolate reductase (MTHFR) that are associated with differential MTHFR activity is important to understand the pathogenesis of ischemic stroke. However, to date, no data are available on the differential DNA methylation profiles of Kelantanese Malays. Therefore, we developed a rapid and efficient serial pyrosequencing assay to determine differential DNA methylation profiles of MTHFR, which help to further our understanding of the pathogenesis of ischemic stroke. The developed assay also served as the validation platform for our previous computational epigenetic research on MTHFR.
Food falsification has direct impact on public health, religious faith, fair-trades and wildlife. For the first time, here we described a multiplex polymerase chain reaction assay for the accurate identification of five meat species forbidden in Islamic foods in a single assay platform. Five pairs of species-specific primers were designed targeting mitochondrial ND5, ATPase 6, and cytochrome b genes to amplify 172, 163, 141, 129 and 108 bp DNA fragments from cat, dog, pig, monkey and rat meats, respectively. All PCR products were identified in gel-images and electrochromatograms obtained from Experion Bioanalyzer. Species-specificity checking against 15 important meat and fish and 5 plant species detected no cross-species amplification. Screening of target species in model and commercial meatballs reflected its application to detect target species in process foods. The assay was tested to detect 0.01-0.02 ng DNA under raw states and 1% suspected meats in meatball formulation.
The ratio of genetic diversity on X chromosomes relative to autosomes in organisms with XX/XY sex chromosomes could provide fundamental insight into the process of genome evolution. Here we report this ratio for 24 cynomolgus monkeys (Macaca fascicularis) originating in Indonesia, Malaysia, and the Philippines. The average X/A diversity ratios in these samples was 0.34 and 0.20 in the Indonesian-Malaysian and Philippine populations, respectively, considerably lower than the null expectation of 0.75. A Philippine population supposed to derive from an ancestral population by founding events showed a significantly lower ratio than the parental population, suggesting a demographic effect for the reduction. Taking sex-specific mutation rate bias and demographic effect into account, expected X/A diversity ratios generated by computer simulations roughly agreed with the observed data in the intergenic regions. In contrast, silent sites in genic regions on X chromosomes showed strong reduction in genetic diversity and the observed X/A diversity ratio in the genic regions cannot be explained by mutation rate bias and demography, indicating that natural selection also reduces the level of polymorphism near genes. Whole-genome analysis of a female cynomolgus monkey also supported the notion of stronger reduction of genetic diversity near genes on the X chromosome.
We developed an alternative method to extract DNA and RNA from clotted blood for genomic and molecular investigations. A combination of the TRIzol method and the QIAamp spin column were used to extract RNA from frozen clotted blood. Clotted blood was sonicated and then the QIAamp DNA Blood Mini Kit was used for DNA extraction. Extracted DNA and RNA were adequate for gene expression analysis and copy number variation (CNV) genotyping, respectively. The purity of the extracted RNA and DNA was in the range of 1.8-2.0, determined by absorbance ratios of A(260):A(280). Good DNA and RNA integrity were confirmed using gel electrophoresis and automated electrophoresis. The extracted DNA was suitable for qPCR and microarrays for CNV genotyping, while the extracted RNA was adequate for gene analysis using RT-qPCR.
β-Thalassemia is a public health problem where 4.5% of Malaysians are β-thalassemia carriers. The genetic disorder is caused by defects in the β-globin gene complex which lead to reduced or complete absence of β-globin chain synthesis. Five TaqMan genotyping assays were designed and developed to detect the common β-thalassemia mutations in Malaysian Malays. The assays were evaluated with 219 "blinded" DNA samples and the results showed 100% sensitivity and specificity. The in-house designed TaqMan genotyping assays were found to be cost- and time-effective for characterization of β-thalassemia mutations in the Malaysian population.