Ribosomal DNA fingerprinting of Pyrodinium bahamense var. compressum in Sabah

Chan, Kher Xing (2005) Ribosomal DNA fingerprinting of Pyrodinium bahamense var. compressum in Sabah. Universiti Malaysia Sabah. (Unpublished)


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Ribosomal DNA fingerprinting was used to detect restriction fragment length polymorphism (RFLP) of the 18S small subunit (SSU) rDNA of Pyrodinium bahamense var. compressum. Four samples, Pyro, PG, G1 and PS were cultured in three types of media, f/2 medium, L1 medium and modified von Stosch's medium, respectively. Cells in f/2 medium thrived in high cell density after two weeks and were harvested for DNA extraction using two methods, protein precipitation method and chloroform/isoamyl alcohol extraction method. PCR reaction was carried out using primers TPL1F and TPL1R. Only DNA samples extracted using chloroform/isoamyl alcohol extraction method were successfully amplified with product size of about 1.6 kb. After purification, the PCR product was subjected to restriction enzyme digestion using enzyme HaeIII and also direct sequencing. The restriction fragments produced after digestion were of 1,007 bp, 228 bp, 219 bp and 173 bp, resolved as three visible bands. There was no difference in the rDNA fingerprint patterns of these four strains. Direct sequencing reactions using primers TPL1F, TPL1R and P1 generated three partial sequences for all four samples and an additional partial sequence was generated for sample Pyro using primer TPL1PyR. The size of the assembled sequences for samples Pyro, PG, G1 and PS were 1,538 bp, 1,542 bp, 1,544 bp and 1,545 bp, respectively. Multiple sequence alignment carried out using CLUSTALW discovered a few nucleotide differences between these four strains. However, when compared with the restriction fragment analysis, it is concluded that the nucleotide differences were not at the restriction sites for enzyme HaeIII on the SSU rDNA sequence of P. bahamense var. compremum. The highly conserved nature of SSU rDNA could lead to the use of rDNA as a detection probe for red tide in Sabah.

Item Type: Academic Exercise
Uncontrolled Keywords: ribosomal, DNA fingerprint, polymorphism, extraction method, PCR, CLUSTALW
Subjects: Q Science > QH Natural history > QH301 Biology
Divisions: SCHOOL > School of Science and Technology
Date Deposited: 11 Feb 2014 02:35
Last Modified: 11 Oct 2017 05:20
URI: http://eprints.ums.edu.my/id/eprint/8171

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