Primer extension dideoxy chain termination nucleotide sequencing of partially purified RNA virus genomes: a technique for investigating low titre viruses with extensive genome secondary structure.
A modified version of the primer extension dideoxy chain termination nucleotide sequencing technique (Sanger et al., 1977) is described. This method has advantages over existing molecular cloning and primer extension techniques in that it allows the genome of RNA viruses to be directly sequenced from partially purified RNA preparations. Thus, viruses growing at unacceptably low titres in tissue culture can now be partially purified from infected mouse brain and sequenced. The technique also incorporates steps for the denaturation of secondary structure which has previously provided difficulties for primer extension sequencing.
Gibson
C A
CA
Department of Microbiology, University of Surrey, Guildford, U.K.
Dunster
L M
LM
Bouloy
M
M
Minor
P D
PD
Sanders
P G
PG
Barrett
A D
AD
D000818:Animals; D001921:Brain / Q000382:microbiology; D005260:Female; D005416:Flavivirus / Q000235:genetics*; D051379:Mice; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D009710:Nucleotide Mapping; D012367:RNA, Viral / Q000737:chemistry; D013698:Templates, Genetic; D014036:Togaviridae Infections / Q000175:diagnosis*
D012367:RNA, Viral