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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Abstract
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