eDNA CLONES CONSTRUCTION OF DENGUE VIRUS TYPE-2 (NEW GUINEA C-STRAIN)

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Date
2009-04
Authors
KEAT, ONGENG
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Dengue virus type-2 (DEN-2) is a member of the Flaviviridae family. DEN-2 has a single-stranded positive polarity ribonucleic acid (RNA) genome of 10,723 nucleotides in length that encodes for a single polyprotein precursor. In this work, viral RNA genome was extracted from Vero and C6/36 cells infected with DEN-2 New Guinea C strain. The viral RNA genome of DEN-2 was reverse transcribed and amplified as seven overlapping fragments, covering the whole genome, by reverse transcription-polymerase chain reaction (RT-PCR). Each of these fragments was successfully cloned into a high copy number plasmid, pGEM®T vector and transformed into Escherichia coli (E. coli) JMl 09 strain. The identity of each clone was verified using DNA sequencing. The seven overlapping fragments were successfully joined together in pGEM®-T vector and transformed into E. coli JM109 strain at 25°C with the presence of ampicillin (25 !Jglml) to produce a eDNA clone carrying the full-length genome ofDEN-2.
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eDNA CLONES CONSTRUCTION OF DENGUE VIRUS TYPE-2
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