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. 2003 Jun;77(11):6450–6465. doi: 10.1128/JVI.77.11.6450-6465.2003

FIG. 2.

FIG. 2.

Strategies used to sequence genomic RNA of CNU/LP2. (A) Scheme for RT-PCR amplification of three overlapping cDNA amplicons representing the entire JEV genomic RNA apart from the 5′ and 3′ termini. RNA is indicated in gray, and cDNA is indicated by solid parallel lines. The top panel schematically depicts the CNU/LP2 JEV genomic RNA (10,968 bp in length). The bottom panels portray the three overlapping cDNAs, JVF (nt 1 to 3865), JVM (nt 3266 to 8170), and JVR (nt 7565 to 10893). (B) Scheme used to sequence the 3′ end of CNU/LP2 genomic RNA. The 5′-phosphorylated and 3′-blocked oligonucleotide T (Oligo T) was ligated to the 3′ end of JEV genomic RNA by T4 RNA ligase, and the resulting RNA was then used for cDNA synthesis and amplification with the primers indicated by an arrow. The resulting products were cloned and sequenced. (C) JEV-specific amplicons synthesized from the oligonucleotide T-ligated JEV genomic RNA described in B. First-strand cDNA was synthesized with oligonucleotide TR, complementary to oligonucleotide T, and the RT reaction was carried out in the presence (lane 1) or absence (lane 2) of Superscript II RT. The cDNA was amplified with oligonucleotide TR and primer J35, which is complementary to nt 10259 to 10276. The expected size of the PCR product is 727 bp. The products were separated on a 1.2% agarose gel and visualized by staining with ethidium bromide. (D) Scheme used to sequence the 5′ end of CNU/LP2 genomic RNA. The cap structure of viral genomic RNA was removed with tobacco acid pyrophosphatase, and the decapped viral RNA was then self-ligated with T4 RNA ligase and used for cDNA synthesis and amplification. The resulting amplified products were cloned and sequenced. (E) JEV-specific amplicons synthesized from the self-ligated JEV genomic RNA described in D. First-strand cDNA synthesis was carried out with primer J40, which is complementary to nt 215 to 232. The RT reaction was performed in the presence (lane 1) or absence (lane 2) of Superscript II RT. The cDNA was amplified with primer J35 and primer J39, which is complementary to nt 164 to 181. The expected size of the PCR product is 890 bp. The products were analyzed on a 1.2% agarose gel as described above. Lane M, 100-bp DNA size ladder (in base pairs).