Fig. 2.
Intron insertion using sequence overlap extension PCR (SOE-PCR). (a) Selection of an intron and intron insertion site matching the consensus. The insertion site should be situated between restriction sites SiteA and SiteB, which are suitable for reinserting the intron containing fragment in the complete virus sequence. (b) Primers are designed on the basis of an in silico construct. Primers indicated as arrows above the construct are virus sense, while primers below are antisense. Primers 1FW and 6RV contain the sequence of SiteA and SiteB, respectively. Complementary primers 3FW and 2RV at the 5′ exon-intron border and 5FW and 4RV at the 3′ intronexon border are approximately 50 nucleotides in length containing approximately 20–25 nucleotides of virus sequence and 20–25 nucleotides of intron sequence. (c) Three separate PCR reactions are performed to amplify overlapping fragments of the region 5′ to the intron (primers 1FW and 2RV), the intron (primers 3FW and 4RV), and the region 3′ to the intron (primers 5FW and 6RV). (d) SOE-PCR is performed on a mixture of the three purified PCR fragments and primers 1FW and 6RV. (e) The PCR fragment of the virus sequence with intron. This fragment is separated from primers and unspecific fragment by gel electrophoresis, cloned, and checked by sequencing before it is reinserted into the complete virus sequence