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A New Method for Rapid Screening of End-Point PCR Products: Application to Single Genome Amplified HIV and SIV Envelope Amplicons.

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 gene... Full description

Journal Title: PloS one 2015, Vol.10(6), p.e0128188
Main Author: Houzet, Laurent
Other Authors: Deleage, Claire , Satie, Anne-Pascale , Merlande, Laetitia , Mahe, Dominique , Dejucq-Rainsford, Nathalie
Format: Electronic Article Electronic Article
Language: English
Subjects:
ID: E-ISSN: 1932-6203 ; DOI: 10.1371/journal.pone.0128188
Link: http://search.proquest.com/docview/1687361672/?pq-origsite=primo
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title: A New Method for Rapid Screening of End-Point PCR Products: Application to Single Genome Amplified HIV and SIV Envelope Amplicons.
format: Article
creator:
  • Houzet, Laurent
  • Deleage, Claire
  • Satie, Anne-Pascale
  • Merlande, Laetitia
  • Mahe, Dominique
  • Dejucq-Rainsford, Nathalie
subjects:
  • Animals–Genetics
  • Base Pairing–Virology
  • Body Fluids–Genetics
  • Genome, Viral–Genetics
  • HIV–Methods
  • Humans–Genetics
  • Jurkat Cells–Genetics
  • Macaca Fascicularis–Genetics
  • Male–Genetics
  • Nucleic Acid Denaturation–Genetics
  • Polymerase Chain Reaction–Genetics
  • Reproducibility of Results–Genetics
  • Simian Immunodeficiency Virus–Genetics
ispartof: PloS one, 2015, Vol.10(6), p.e0128188
description: 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.
language: eng
source:
identifier: E-ISSN: 1932-6203 ; DOI: 10.1371/journal.pone.0128188
fulltext: fulltext
issn:
  • 19326203
  • 1932-6203
url: Link


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titleA New Method for Rapid Screening of End-Point PCR Products: Application to Single Genome Amplified HIV and SIV Envelope Amplicons.
creatorHouzet, Laurent ; Deleage, Claire ; Satie, Anne-Pascale ; Merlande, Laetitia ; Mahe, Dominique ; Dejucq-Rainsford, Nathalie
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identifierE-ISSN: 1932-6203 ; DOI: 10.1371/journal.pone.0128188
subjectAnimals–Genetics ; Base Pairing–Virology ; Body Fluids–Genetics ; Genome, Viral–Genetics ; HIV–Methods ; Humans–Genetics ; Jurkat Cells–Genetics ; Macaca Fascicularis–Genetics ; Male–Genetics ; Nucleic Acid Denaturation–Genetics ; Polymerase Chain Reaction–Genetics ; Reproducibility of Results–Genetics ; Simian Immunodeficiency Virus–Genetics
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descriptionPCR 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.
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titleA New Method for Rapid Screening of End-Point PCR Products: Application to Single Genome Amplified HIV and SIV Envelope Amplicons.
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