Malays. Appl. Bwl. (1997) 26(2): 13-19
PCR-AMPLIFICATION OF THREE mtDNA GENES FOR DETERMINING THE MOLECULAR PHYLOGENY OF THE GENUS BETTA
A. M. MAJID1, ORIS SANJUR2, CAROL DIMEO2, M. B. BLACK2 and R. C. VRUENHOEK2
1 Department of Genetics, Faculty of Life Sciences, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia
2 Center for Theoretical and Applied Genetics, Cook College, P. O . Box 231 Rutgers, The State University of New Jersey,
New Brunswick , New Jersey 08903 - 023 J, U. S. A.
ABSTRACT
Total genomic DNA from muscle tissues of Betta splendens and Betta pugnax preserved in 90% ethanol was extracted b) the phenol extraction method. The amount of DNA obtained from the samples was too little for direct analysis. Polymerase chain reaction (PCR) amplification of three genes found in the mitochondria! DNA (mtDNA) genome was attempted b) using various sets of primers. The cytochrome b (cyt b) gene was amplified using three different sets of primers: LA/HA LC/HA and LA/HB. The D-loop gene amplification was attempted by using the HTDK/LPro primers and the ATPase gene by means of the H6989/L8331 primers. Of these, the LC/HA for cyt b, the HTDK/LPro for the D-loop and the H6989/L8331 for ATPase yielded amplification products sufficient for sequencing purposes. A decision was made to stud} the potential of the cyt b gene for constructing a phylogenetic relationship of the Betta species. The ATPase gene was deemed as being less suitable for this purpose as it is highly-conserved. A 900-bp fragment of the cyt b gene was purifiec from agarose gel. Sequencing of a section of this fragment was accomplished by the cycle sequencing technique. The sequences for the B. splendens and B. pugnax were found to be identical.
ABSTRAK
Jumlah DNA di dalam genom daripada tisu otot ikan Betta splendens dan Betta pugnax yang di simpan di dalam 90 % stanol telah diekstrak dengan keadah ekstraksi fenol. Jumlah DNA yang diperolehi daripada sampel-sampel ini adalal" terlalu rendah untuk dianalisis. Tindakbalas rantaian polimerase (PCR) lelah digunakan untuk menggandakan tiga ger yang terdapat pada DNA mitokondria (mtDNA). Gen sitokrom b (cyt b) telah digandakan dengan menggunakan tiga sei primer: LA/HA, LC/HA dan LA/HB. Gen D-loop telah digandakan dengan menggunakan set primer HTDK/LPro sementarE gen ATPase pula dengan set primer H6989/L83331. Set-set primer LC/HA untuk cyt b, HTDK/LPro untuk D-loop dar H6989/L8331 untuk ATPase telah menghasilkan produk-produk penggandaan yang mencukupi untuk penjujukan gen-gen ini. Satu keputusan telah di ambil untuk mengkaji potensi gen cyt b untuk pembentukan kaitan filogenetik antars spesies-spesies Betta. Gen ATPase dianggap terlalu terpelihara (conserved) untuk tujuan ini. Satu serpihan gen cyt b yans panjangnya lebih kurang 900 bp telah ditulenkan. Penjujukan sebahagian daripada serpihan ini telah dicapai melalui teknik penjujukan berkitar. Didapati bahawa jujukan untuk B. splendens dan B. pugnax tidak berbeza.
Key words: mtDNA genes, molecular phylogeny, genus Betta
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