LoPPS: a long PCR product sequencing method for rapid characterisation of long amplicons.
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.
Emonet
Sébastien
S
Unité des Virus Emergents (EA3292, IFR48, IRD UR034), Faculté de Médecine, 27 boulevard Jean Moulin, 13005 Marseille, France.
Grard
Gilda
G
Brisbarre
Nadège
N
Moureau
Gregory
G
Temmam
Sarah
S
Charrel
Rémi
R
de Lamballerie
Xavier
X
D004247:DNA / Q000032:analysis / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D012328:RNA Viruses / Q000235:genetics; D012367:RNA, Viral / Q000032:analysis / Q000737:chemistry; D017422:Sequence Analysis, DNA / Q000379:methods*; D017423:Sequence Analysis, RNA / Q000379:methods*
D012367:RNA, Viral; D004247:DNA