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  1. Fulltext An alternate method for DNA and RNA extraction from clotted blood
    Zakaria Z, Umi SH, Mokhtar SS, Mokhtar U, Zaiharina MZ, Aziz AT, et al.
    Genet. Mol. Res., 2013;12(1):302-11.
    PMID: 23408417 DOI: 10.4238/2013.February.4.4
    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.
    Matched MeSH terms: DNA/isolation & purification*
  2. Fulltext Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms
    Wong YP, Othman S, Lau YL, Radu S, Chee HY
    J Appl Microbiol, 2018 Mar;124(3):626-643.
    PMID: 29165905 DOI: 10.1111/jam.13647
    Loop-mediated isothermal amplification (LAMP) amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions by using a DNA polymerase with high displacement strand activity and a set of specifically designed primers to amplify targeted DNA strands. Following its first discovery by Notomi et al. ( Nucleic Acids Res 28: E63), LAMP was further developed over the years which involved the combination of this technique with other molecular approaches, such as reverse transcription and multiplex amplification for the detection of infectious diseases caused by micro-organisms in humans, livestock and plants. In this review, available types of LAMP techniques will be discussed together with their applications in detection of various micro-organisms. Up to date, there are varieties of LAMP detection methods available including colorimetric and fluorescent detection, real-time monitoring using turbidity metre and detection using lateral flow device which will also be highlighted in this review. Apart from that, commercialization of LAMP technique had also been reported such as lyophilized form of LAMP reagents kit and LAMP primer sets for detection of pathogenic micro-organisms. On top of that, advantages and limitations of this molecular detection method are also described together with its future potential as a diagnostic method for infectious disease.
    Matched MeSH terms: DNA/isolation & purification
  3. Fulltext DNA, RNA, and protein extraction: the past and the present
    Tan SC, Yiap BC
    J Biomed Biotechnol, 2009;2009:574398.
    PMID: 20011662 DOI: 10.1155/2009/574398
    Extraction of DNA, RNA, and protein is the basic method used in molecular biology. These biomolecules can be isolated from any biological material for subsequent downstream processes, analytical, or preparative purposes. In the past, the process of extraction and purification of nucleic acids used to be complicated, time-consuming, labor-intensive, and limited in terms of overall throughput. Currently, there are many specialized methods that can be used to extract pure biomolecules, such as solution-based and column-based protocols. Manual method has certainly come a long way over time with various commercial offerings which included complete kits containing most of the components needed to isolate nucleic acid, but most of them require repeated centrifugation steps, followed by removal of supernatants depending on the type of specimen and additional mechanical treatment. Automated systems designed for medium-to-large laboratories have grown in demand over recent years. It is an alternative to labor-intensive manual methods. The technology should allow a high throughput of samples; the yield, purity, reproducibility, and scalability of the biomolecules as well as the speed, accuracy, and reliability of the assay should be maximal, while minimizing the risk of cross-contamination.
    Matched MeSH terms: DNA/isolation & purification*
  4. Rapid detection of porcine DNA in processed food samples using a streamlined DNA extraction method combined with the SYBR Green real-time PCR assay
    Tan LL, Ahmed SA, Ng SK, Citartan M, Raabe CA, Rozhdestvensky TS, et al.
    Food Chem, 2020 Mar 30;309:125654.
    PMID: 31678669 DOI: 10.1016/j.foodchem.2019.125654
    A specialized DNA extraction method and a SYBR Green quantitative polymerase chain reaction (SyG-qPCR) assay were combined to generate a ready-to-use kit for rapid detection of porcine admixtures in processed meat products. Our qPCR assay utilized repetitive LINE-1 elements specific to the genome of Sus scrofa domesticus (pig) as a target and incorporated internal controls. We improved the genomic DNA extraction method, and reduced extraction times to the minimum. The method was validated for specificity, sensitivity (0.001% w/w) and robustness, and values were compared with those of a commercially available kit. We also tested our method using 121 processed food products and consistently detected amplification only in samples containing pork. Due to its efficiency and cost-effectiveness, our method represents a valuable new method for detecting food adulteration with pork that is superior to existing quality control approaches.
    Matched MeSH terms: DNA/isolation & purification
  5. Reproductive success of two male morphs in a free-ranging population of Bornean orangutans
    Tajima T, Malim TP, Inoue E
    Primates, 2018 Mar;59(2):127-133.
    PMID: 29387973 DOI: 10.1007/s10329-017-0648-1
    The reproductive success of male primates is not always associated with dominance status. For example, even though male orangutans exhibit intra-sexual dimorphism and clear dominance relationships exist among males, previous studies have reported that both morphs are able to sire offspring. The present study aimed to compare the reproductive success of two male morphs, and to determine whether unflanged males sired offspring in a free-ranging population of Bornean orangutans, using 12 microsatellite loci to determine the paternity of eight infants. A single flanged male sired most of the offspring from parous females, and an unflanged male sired a firstborn. This is consistent with our observation that the dominant flanged male showed little interest in nulliparous females, whereas the unflanged males frequently mated with them. This suggests that the dominant flanged male monopolizes the fertilization of parous females and that unflanged males take advantage of any mating opportunities that arise in the absence of the flanged male, even though the conception probability of nulliparous females is relatively low.
    Matched MeSH terms: DNA/isolation & purification
  6. Universal mini COI barcode for the identification of fish species in processed products
    Sultana S, Ali ME, Hossain MAM, Asing, Naquiah N, Zaidul ISM
    Food Res Int, 2018 03;105:19-28.
    PMID: 29433207 DOI: 10.1016/j.foodres.2017.10.065
    Species substitution, the use of a low value fish in place of a high value fish, is the biggest problem in international trade and the leading cause of fraud in the fisheries arena sector. Current DNA barcoding systems have partly solved this problem but also failed in many instances to amplify PCR targets from highly processed products because of the degradation of a longer barcode marker (~650bp). In the present study, a novel mini barcode marker (295bp) was developed to discriminate fish species in raw and processed states forms. The barcode primers were cross-tested against 33 fish species and 15 other animal species and found to be universal for all the tested fish varieties. When 20 commercial fish products of five different categories were screened, all commercial fish sample yielded positive bands for the novel fish barcode. PCR product was sequenced to retrieve the species IDs that reflected 55% (11/20) of Malaysian fish products were mislabeled.
    Matched MeSH terms: DNA/isolation & purification
  7. The Challenges of DNA Extraction in Different Assorted Food Matrices: A Review
    Sajali N, Wong SC, Hanapi UK, Abu Bakar Jamaluddin S, Tasrip NA, Mohd Desa MN
    J Food Sci, 2018 Oct;83(10):2409-2414.
    PMID: 30184265 DOI: 10.1111/1750-3841.14338
    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.
    Matched MeSH terms: DNA/isolation & purification*
  8. Fulltext Measuring the Electronic Properties of DNA-Specific Schottky Diodes Towards Detecting and Identifying Basidiomycetes DNA
    Periasamy V, Rizan N, Al-Ta'ii HM, Tan YS, Tajuddin HA, Iwamoto M
    Sci Rep, 2016 07 20;6:29879.
    PMID: 27435636 DOI: 10.1038/srep29879
    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.
    Matched MeSH terms: DNA/isolation & purification
  9. Challenges and strategies in the preparation of large-volume polymer-based monolithic chromatography adsorbents
    Ongkudon CM, Kansil T, Wong C
    J Sep Sci, 2014 Mar;37(5):455-64.
    PMID: 24376196 DOI: 10.1002/jssc.201300995
    To date, the number of published reports on the large-volume preparation of polymer-based monolithic chromatography adsorbents is still lacking and is of great importance. Many critical factors need to be considered when manufacturing a large-volume polymer-based monolith for chromatographic applications. Structural integrity, validity, and repeatability are thought to be the key factors determining the usability of a large-volume monolith in a separation process. In this review, we focus on problems and solutions pertaining to heat dissipation, pore size distribution, "wall channel" effect, and mechanical strength in monolith preparation. A template-based method comprising sacrificial and nonsacrificial techniques is possibly the method of choice due to its precise control over the porous structure. However, additional expensive steps are usually required for the template removal. Other strategies in monolith preparation are also discussed.
    Matched MeSH terms: DNA/isolation & purification
  10. Unusually high genetic diversity in the Bornean Limnonectes kuhlii-like fanged frogs (Anura: Dicroglossidae)
    Matsui M, Kuraishi N, Eto K, Hamidy A, Nishikawa K, Shimada T, et al.
    Mol Phylogenet Evol, 2016 09;102:305-19.
    PMID: 27374495 DOI: 10.1016/j.ympev.2016.06.009
    A fanged frog Limnonectes kuhlii was once thought to be wide-ranging in Southeast Asia, but is now confined to its type locality Java through recent phylogenetic studies, which clarified heterospecific status of non-Javanese populations, and monophyly of Bornean populations. However, large genetic differences among Bornean populations suggest occurrence of cryptic species, which we test using dense geographic sampling. We estimated the phylogenetic relationships among samples of Bornean populations together with their putative relatives from the continental Southeast Asia, using 2517bp sequences of the 12S rRNA, tRNA(val), and 16S rRNA of mitochondrial DNA, and 2367bp sequences of the NCX1, POMC, and RAG1 of nuclear genes. In the mtDNA trees, Bornean L. kuhlii-like frogs formed a monophyletic group split into 18 species lineages including L. hikidai, with the deepest phylogenetic split separating L. cintalubang from the remaining species. Almost all of these lineages co-occur geographically, and two to three lineages were found syntopically in each locality. Co-occurrence of more than one lineage may be maintained by differential morphology and microhabitat selection. These syntopic lineages should be regarded as distinct species. Our results clearly indicate that taxonomic revision is urgent to clarify many evolutionary problems of Bornean L. kuhlii-like frogs.
    Matched MeSH terms: DNA/isolation & purification
  11. Molecular characterization of Phytophthora palmivora responsible for bud rot disease of oil palm in Colombia
    Maizatul-Suriza M, Dickinson M, Idris AS
    World J Microbiol Biotechnol, 2019 Feb 27;35(3):44.
    PMID: 30810828 DOI: 10.1007/s11274-019-2618-9
    Bud rot disease is a damaging disease of oil palm in Colombia. The pathogen responsible for this disease is a species of oomyctes, Phytophthora palmivora which is also the causal pathogen of several tropical crop diseases such as fruit rot and stem canker of cocoa, rubber, durian and jackfruit. No outbreaks of bud rot have been reported in oil palm in Malaysia or other Southeast Asian countries, despite this particular species being present in the region. Analysis of the genomic sequences of several genetic markers; the internal transcribe spacer regions (ITS) of the ribosomal RNA gene cluster, beta-tubulin gene, translation elongation factor 1 alpha gene (EF-1α), cytochrome c oxidase subunit I & II (COXI and COXII) gene cluster along with amplified fragment length polymorphism (AFLP) analyses have been carried out to investigate the genetic diversity and variation of P. palmivora isolates from around the world and from different hosts in comparison to Colombian oil palm isolates, as one of the steps in understanding why this species of oomycetes causes devastating damage to oil palm in Latin America but not in other regions. Phylogenetic analyses of these regions showed that the Colombian oil palm isolates were not separated from Malaysian isolates. AFLP analysis and a new marker PPHPAV, targeting an unclassified hypothetical protein, was found to be able to differentiate Malaysian and Colombian isolates and showed a clear clade separations. Despite this, pathogenicity studies did not show any significant differences in the level of aggressiveness of different isolates against oil palm in glasshouse tests.
    Matched MeSH terms: DNA/isolation & purification
  12. Phylogeny of the most species-rich freshwater bivalve family (Bivalvia: Unionida: Unionidae): Defining modern subfamilies and tribes
    Lopes-Lima M, Froufe E, Do VT, Ghamizi M, Mock KE, Kebapçı Ü, et al.
    Mol Phylogenet Evol, 2017 01;106:174-191.
    PMID: 27621130 DOI: 10.1016/j.ympev.2016.08.021
    Freshwater mussels of the order Unionida are key elements of freshwater habitats and are responsible for important ecological functions and services. Unfortunately, these bivalves are among the most threatened freshwater taxa in the world. However, conservation planning and management are hindered by taxonomic problems and a lack of detailed ecological data. This highlights the urgent need for advances in the areas of systematics and evolutionary relationships within the Unionida. This study presents the most comprehensive phylogeny to date of the larger Unionida family, i.e., the Unionidae. The phylogeny is based on a combined dataset of 1032bp (COI+28S) of 70 species in 46 genera, with 7 of this genera being sequenced for the first time. The resulting phylogeny divided the Unionidae into 6 supported subfamilies and 18 tribes, three of which are here named for the first time (i.e., Chamberlainiini nomen novum, Cristariini nomen novum and Lanceolariini nomen novum). Molecular analyses were complemented by investigations of selected morphological, anatomical and behavioral characters used in traditional phylogenetic studies. No single morphological, anatomical or behavioral character was diagnostic at the subfamily level and few were useful at the tribe level. However, within subfamilies, many tribes can be recognized based on a subset of these characters. The geographical distribution of each of the subfamilies and tribes is also presented. The present study provides important advances in the systematics of these extraordinary taxa with implications for future ecological and conservation studies.
    Matched MeSH terms: DNA/isolation & purification
  13. Quantity and quality assessment of DNA extracted from saliva and blood
    Looi ML, Zakaria H, Osman J, Jamal R
    Clin. Lab., 2012;58(3-4):307-12.
    PMID: 22582505
    Saliva has been suggested as an attractive resource for evaluating physiological and pathological conditions in humans. This study aims to evaluate saliva sampling as an alternative to blood sampling for molecular testing.
    Matched MeSH terms: DNA/isolation & purification
  14. Francisella spp. detected in Dermacentor ticks in Malaysian forest reserve areas
    Koh FX, Nurhidayah MN, Tan PE, Kho KL, Tay ST
    Vet Parasitol Reg Stud Reports, 2019 08;17:100315.
    PMID: 31303231 DOI: 10.1016/j.vprsr.2019.100315
    Limited information is available on tropical ticks and tick-borne bacteria affecting the health of humans and animals in the Southeast Asia region. Francisella tularensis is a tick-borne bacterium which causes a potentially life-threatening disease known as tularemia. This study was conducted to determine the occurrence of Francisella spp. in questing ticks collected from Malaysian forest reserve areas. A total of 106 ticks (mainly Dermacentor and Haemaphysalis spp.) were examined for Francisella DNA using a Polymerase chain reaction (PCR) assay targeting the bacterial 16S rDNA. Francisella DNA was detected from 12 Dermacentor ticks. Sequence analysis of the amplified 16S rDNA sequences (1035 bp) show >99% identity with that of Francisella endosymbiont reported in a tick from Thailand. A dendrogram constructed based on the bacterial 16S rDNA shows that the Francisella spp. were distantly related to the pathogenic strains of F. tularensis. Three Francisella-positive ticks were identified as Dermacentor atrosignatus, based on sequence analysis of the tick mitochondrial 16S rRNA gene. Further screening of cattle and sheep ticks (Haemaphysalis bispinosa and Rhipicephalus microplus) and animal samples (cattle, sheep, and goats) did not yield any positive findings. Our findings provide the first molecular data on the occurrence of a Francisella strain with unknown pathogenicity in Dermacentor questing ticks in Malaysia.
    Matched MeSH terms: DNA/isolation & purification
  15. Fulltext Temperature and magnetic field driven modifications in the I-V features of gold-DNA-gold structure I-V
    Khatir NM, Abdul-Malek Z, Banihashemian SM
    Sensors (Basel), 2014;14(10):19229-41.
    PMID: 25320908 DOI: 10.3390/s141019229
    The fabrication of Metal-DNA-Metal (MDM) structure-based high sensitivity sensors from DNA micro-and nanoarray strands is a key issue in their development. The tunable semiconducting response of DNA in the presence of external electromagnetic and thermal fields is a gift for molecular electronics. The impact of temperatures (25-55 °C) and magnetic fields (0-1200 mT) on the current-voltage (I-V) features of Au-DNA-Au (GDG) structures with an optimum gap of 10 μm is reported. The I-V characteristics acquired in the presence and absence of magnetic fields demonstrated the semiconducting diode nature of DNA in GDG structures with high temperature sensitivity. The saturation current in the absence of magnetic field was found to increase sharply with the increase of temperature up to 45 °C and decrease rapidly thereafter. This increase was attributed to the temperature-assisted conversion of double bonds into single bond in DNA structures. Furthermore, the potential barrier height and Richardson constant for all the structures increased steadily with the increase of external magnetic field irrespective of temperature variations. Our observation on magnetic field and temperature sensitivity of I-V response in GDG sandwiches may contribute towards the development of DNA-based magnetic sensors.
    Matched MeSH terms: DNA/isolation & purification*
  16. Potential authentication of various meat-based products using simple and efficient DNA extraction method
    Khairil Mokhtar NF, El Sheikha AF, Azmi NI, Mustafa S
    J Sci Food Agric, 2020 Mar 15;100(4):1687-1693.
    PMID: 31803942 DOI: 10.1002/jsfa.10183
    BACKGROUND: The growth of halal food consumption worldwide has resulted in an increase in the request for halal authentication. DNA-based detection using powerful real-time polymerase chain reaction (PCR) technique has been shown to be highly specific and sensitive authentication tool. The efficient DNA extraction method in terms of quality and quantity is a backbone step to obtain successful real-time PCR assays. In this study, different DNA extraction methods using three lysis buffers were evaluated and developed to recommend a much more efficient method as well as achieve a successful detection using real-time PCR.

    RESULTS: The lysis buffer 2 (LB2) has been shown to be the best lysis buffer for DNA extraction from both raw and processed meat samples comparing to other lysis buffers tested. Hence, the LB2 has been found to be ideal to detect meat and porcine DNAs by real-time PCR using pairs of porcine specific primers and universal primers which amplified at 119 bp fragment and 93 bp fragment, respectively. This assay allows detection as low as 0.0001 ng of DNA. Higher efficiency and sensitivity of real-time PCR via a simplified DNA extraction method using LB2 have been observed, as well as a reproducible and high correlation coefficient (R2  = 0.9979) based on the regression analysis of the standard curve have been obtained.

    CONCLUSION: This study has established a fast, simple, inexpensive and efficient DNA extraction method that is feasible for raw and processed meat products. This extraction technique allows an accurate DNA detection by real-time PCR and can also be implemented to assist the halal authentication of various meat-based products available in the market. © 2019 Society of Chemical Industry.

    Matched MeSH terms: DNA/isolation & purification*
  17. An assessment of three noncommercial DNA extraction methods from dried blood spots for beta-thalassaemia mutation identification
    Karthipan SN, George E, Jameela S, Lim WF, Teh LK, Lee TY, et al.
    Int J Lab Hematol, 2011 Oct;33(5):540-4.
    PMID: 21884505 DOI: 10.1111/j.1751-553X.2011.01304.x
    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.
    Matched MeSH terms: DNA/isolation & purification*
  18. Deletion in erythrocyte band 3 gene in malaria-resistant Southeast Asian ovalocytosis
    Jarolim P, Palek J, Amato D, Hassan K, Sapak P, Nurse GT, et al.
    Proc Natl Acad Sci U S A, 1991 Dec 15;88(24):11022-6.
    PMID: 1722314
    Southeast Asian ovalocytosis (SAO) is a hereditary condition that is widespread in parts of Southeast Asia. The ovalocytic erythrocytes are rigid and resistant to invasion by various malarial parasites. We have previously found that the underlying defect in SAO involves band 3 protein, the major transmembrane protein, which has abnormal structure and function. We now report two linked mutations in the erythrocyte band 3 gene in SAO: (i) a deletion of codons 400-408 and (ii) a substitution, A----G, in the first base of codon 56 leading to substitution of Lys-56 by Glu-56. The first defect leads to a deletion of nine amino acids in the boundary of cytoplasmic and membrane domains of band 3. This defect has been detected in all 30 ovalocytic subjects from Malaysia, the Philippines, and two unrelated coastal regions of Papua New Guinea, whereas it was absent in all 30 controls from Southeast Asia and 20 subjects of different ethnic origin from the United States. The Lys-56----Glu substitution has likewise been found in all SAO subjects. However, it has also been detected in 5 of the 50 control subjects, suggesting that it represents a linked polymorphism. We conclude that the deletion of codons 400-408 in the band 3 gene constitutes the underlying molecular defect in SAO.
    Matched MeSH terms: DNA/isolation & purification
  19. Graphene-Based Materials as Solid Phase Extraction Sorbent for Trace Metal Ions, Organic Compounds, and Biological Sample Preparation
    Ibrahim WA, Nodeh HR, Sanagi MM
    Crit Rev Anal Chem, 2016 Jul 03;46(4):267-83.
    PMID: 26186420 DOI: 10.1080/10408347.2015.1034354
    Graphene is a new carbon-based material that is of interest in separation science. Graphene has extraordinary properties including nano size, high surface area, thermal and chemical stability, and excellent adsorption affinity to pollutants. Its adsorption mechanisms are through non-covalent interactions (π-π stacking, electrostatic interactions, and H-bonding) for organic compounds and covalent interactions for metal ions. These properties have led to graphene-based material becoming a desirable adsorbent in a popular sample preparation technique known as solid phase extraction (SPE). Numerous studies have been published on graphene applications in recent years, but few review papers have focused on its applications in analytical chemistry. This article focuses on recent preconcentration of trace elements, organic compounds, and biological species using SPE-based graphene, graphene oxide, and their modified forms. Solid phase microextraction and micro SPE (µSPE) methods based on graphene are discussed.
    Matched MeSH terms: DNA/isolation & purification*
  20. The requirements for sterol regulatory element-binding protein (Srebp) and stimulatory protein 1 (Sp1)-binding elements in the transcriptional activation of two freshwater fish Channa striata and Danio rerio elovl5 elongase
    Goh PT, Kuah MK, Chew YS, Teh HY, Shu-Chien AC
    Fish Physiol Biochem, 2020 Aug;46(4):1349-1359.
    PMID: 32239337 DOI: 10.1007/s10695-020-00793-w
    Fish are a major source of beneficial n-3 LC-PUFA in human diet, and there is considerable interest to elucidate the mechanism and regulatory aspects of LC-PUFA biosynthesis in farmed species. Long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis involves the activities of two groups of enzymes, the fatty acyl desaturase (Fads) and elongase of very long-chain fatty acid (Elovl). The promoters of elovl5 elongase, which catalyses the rate-limiting reaction of elongating polyunsaturated fatty acid (PUFA), have been previously described and characterized from several marine and diadromous teleost species. We report here the cloning and characterization of elovl5 promoter from two freshwater fish species, the carnivorous snakehead fish (Channa striata) and zebrafish. Results show the presence of sterol-responsive elements (SRE) in the core regulatory region of both promoters, suggesting the importance of sterol regulatory element-binding protein (Srebp) in the regulation of elovl5 for both species. Mutagenesis luciferase and electrophoretic mobility shift assays further validate the role of SRE for basal transcriptional activation. In addition, several Sp1-binding sites located in close proximity with SRE were present in the snakehead promoter, with one having a potential synergy with SRE in the regulation of elovl5 expression. The core zebrafish elovl5 promoter fragment also directed in vivo expression in the yolk syncytial layer of developing zebrafish embryos.
    Matched MeSH terms: DNA/isolation & purification
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