Ong, Eng Keat
(2009)
cDNA Clones Construction Of Dengue Virus Type-2
(New Guinea C-Strain).
Masters thesis, Universiti Sains Malaysia.
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, pGEMR-
T vector and transformed into Escherichia coli (E. coli) JM109 strain. The identity
of each clone was verified using DNA sequencing. The seven overlapping fragments
were successfully joined together in pGEMR-T vector and transformed into E. coli
JM109 strain at 25°C with the presence of ampicillin (25 pg/ml) to produce a cDNA
clone carrying the full-length genome of DEN-2.
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