An alternative method for direct sequencing of PCR products, for epidemiological studies performed by nucleic sequence comparison. Application to rabbit haemorrhagic disease virus.
A sequencing strategy based on the use of commercially available fluorescent-labeled universal primers for directly sequencing polymerase chain reaction (PCR) amplified material has been developed. The PCR reactions were performed with hybrid primers, specific to the viral sequence and possessing the sequence of the standard sequencing primers at their 5' end (M13 and reverse primers). These amplified fragments were sequenced by a classical dye-primer kit on an automated sequencer. As opposed to the use of fluorescent dideoxynucleotides, this sequencing method yielded accurate, high-grade sequences and had several advantages. First, the intensity of the sequencing peaks was much more homogeneous with the dye primer method. In addition, the problem of altered electrophoretic mobility, which may occur during the sequencing of custom-synthesised fluorescent primers, was avoided. This method was successfully reproduced using several different sets of chimeric primers. We believe that it is suitable for epidemiological studies conducted by nucleic sequence comparison, as in the case of rabbit haemorrhagic disease virus, as well as in other systems.
Vende
P
P
INRA, unité de virologie et d'immunologie moléculaires, domaine de Vilvert, Jouy-en-Josas, France.
Le Gall
G
G
Rasschaert
D
D
D000818:Animals; D001483:Base Sequence; D004279:DNA, Viral; D005456:Fluorescent Dyes; D017929:Hemorrhagic Disease Virus, Rabbit / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000662:veterinary*; D011817:Rabbits; D017422:Sequence Analysis, DNA / Q000379:methods / Q000662:veterinary*
D004279:DNA, Viral; D005456:Fluorescent Dyes