Emonet Sébastien, Grard G., Brisbarre N., Moureau G., Temmam S., Charrel R., de Lamballerie Xavier. (2006). LoPPS : A long PCR product sequencing method for rapid characterisation of long amplicons. Biochemical and Biophysical Research Communications, 344 (4), p. 1080-1085. ISSN 0006-291X.
Titre du document
LoPPS : A long PCR product sequencing method for rapid characterisation of long amplicons
Emonet Sébastien, Grard G., Brisbarre N., Moureau G., Temmam S., Charrel R., de Lamballerie Xavier
Source
Biochemical and Biophysical Research Communications, 2006,
344 (4), p. 1080-1085 ISSN 0006-291X
Here, we propose an optimised protocol (LoPPS, long PCR product sequencing) which allows the fast, cost-attractive, and high-throughput sequencing of long PCR products. LoPPS constitutes an alternative to the primer-walking technology which is expensive and time consuming but remains the current standard procedure. It is based on the ultrasonic shearing, polishing, and cloning of PCR or RT-PCR products and is compatible with 96- or 384-well microplate systems in which bacterial growth, preparation of plasmid DNA, and sequencing can be automated. We present results obtained from 24 different RT-PCR products (2.5-4.8 kbp long) obtained from various RNA viruses and fully sequenced using LoPPS. The method proved to be robust and fast. It was successfully used on a low amount of DNA and allowed each target nucleotide position to be controlled twice or more, with a final cost which is one-third of that of primer-walking.
Plan de classement
Sciences fondamentales / Techniques d'analyse et de recherche [020]
;
Entomologie médicale / Parasitologie / Virologie [052]
