Long PCR Product Sequencing (LoPPS): a shotgun-based approach to sequence long PCR products.
Here we describe a practical procedure for sequencing long PCR products. The method relies on ultrasonic shearing of PCR products, resulting in fragments 700-1,000 nt long. Termini are subsequently repaired to obtain blunt ends and 3' A-overhangs are added before TA cloning. A predetermined number of clones are sequenced using an insert-independent primer to obtain an overlapping contig covering the full length of the PCR product. This method is cost effective and enables the complete sequencing of any large PCR product in a high-throughput format. Processing of amplified DNA requires 3 h handling time prior to the ligation step, and the clone library is available 2 d later. The complete sequence information is obtained approximately 5 d after the PCR step, depending on the sequencing procedure adopted.
Emonet
Sébastien F
SF
Unité des Virus Emergents (EA3292, IFR48, IRD UR034), Faculté de Médecine, 27 boulevard Jean Moulin, 13005 Marseille, France.
Grard
Gilda
G
Brisbarre
Nadège M
NM
Moureau
Grégory N
GN
Temmam
Sarah
S
Charrel
Rémi N
RN
de Lamballerie
Xavier
X
D001483:Base Sequence / Q000235:genetics*; D018274:Electroporation; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods*; D017423:Sequence Analysis, RNA / Q000379:methods*; D014465:Ultrasonics