title: A New Method for Rapid Screening of End-Point PCR Products: Application to Single Genome Amplified HIV and SIV Envelope Amplicons.
abstract: PCR is the most widely applied technique for large scale screening of bacterial clones, mouse genotypes, virus genomes etc. A drawback of large PCR screening is that amplicon analysis is usually performed using gel electrophoresis, a step that is very labor intensive, tedious and chemical waste generating. Single genome amplification (SGA) is used to characterize the diversity and evolutionary dynamics of virus populations within infected hosts. SGA is based on the isolation of single template molecule using limiting dilution followed by nested PCR amplification and requires the analysis of hundreds of reactions per sample, making large scale SGA studies very challenging. Here we present a novel approach entitled Long Amplicon Melt Profiling (LAMP) based on the analysis of the melting profile of the PCR reactions using SYBR Green and/or EvaGreen fluorescent dyes. The LAMP method represents an attractive alternative to gel electrophoresis and enables the quick discrimination of positive reactions. We validate LAMP for SIV and HIV env-SGA, in 96- and 384-well plate formats. Because the melt profiling allows the screening of several thousands of PCR reactions in a cost-effective, rapid and robust way, we believe it will greatly facilitate any large scale PCR screening.
authors.affiliation: Inserm U 1085-IRSET, Université de Rennes 1, Structure Fédérative Recherche Biosit, Campus de Beaulieu, Rennes, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Houzet
authors.affiliation: Inserm U 1085-IRSET, Université de Rennes 1, Structure Fédérative Recherche Biosit, Campus de Beaulieu, Rennes, France.
authors.forename: Claire
authors.initials: C
authors.lastname: Deleage
authors.affiliation: Inserm U 1085-IRSET, Université de Rennes 1, Structure Fédérative Recherche Biosit, Campus de Beaulieu, Rennes, France.
authors.forename: Anne-Pascale
authors.initials: AP
authors.lastname: Satie
authors.affiliation: Inserm U 1085-IRSET, Université de Rennes 1, Structure Fédérative Recherche Biosit, Campus de Beaulieu, Rennes, France.
authors.forename: Laetitia
authors.initials: L
authors.lastname: Merlande
authors.affiliation: Inserm U 1085-IRSET, Université de Rennes 1, Structure Fédérative Recherche Biosit, Campus de Beaulieu, Rennes, France.
authors.forename: Dominique
authors.initials: D
authors.lastname: Mahe
authors.affiliation: Inserm U 1085-IRSET, Université de Rennes 1, Structure Fédérative Recherche Biosit, Campus de Beaulieu, Rennes, France.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Dejucq-Rainsford
mesh_terms: D000818:Animals; D020029:Base Pairing / Q000235:genetics; D001826:Body Fluids / Q000821:virology; D016679:Genome, Viral / Q000235:genetics*; D006678:HIV / Q000235:genetics*; D006801:Humans; D019169:Jurkat Cells; D008252:Macaca fascicularis; D008297:Male; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D015302:Simian Immunodeficiency Virus / Q000235:genetics*