%0 Journal Article
%9 ACL : Articles dans des revues avec comité de lecture répertoriées par l'AERES
%A Emonet, Sébastien
%A Grard, G.
%A Brisbarre, N.
%A Moureau, G.
%A Temmam, S.
%A Charrel, R.
%A de Lamballerie, Xavier
%T LoPPS : A long PCR product sequencing method for rapid characterisation of long amplicons
%D 2006
%L fdi:010037790
%G ENG
%J Biochemical and Biophysical Research Communications
%@ 0006-291X
%K sequencing ; PCR ; RNA viruses ; LoPPS
%M CC:0002376792-0004
%N 4
%P 1080-1085
%R 10.1016/j.bbrc.2006.04.015
%U https://www.documentation.ird.fr/hor/fdi:010037790
%> https://www.documentation.ird.fr/intranet/publi/2007/suppl/010037790.pdf
%V 344
%W Horizon (IRD)
%X 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.
%$ 052 ; 020