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.
Highlights
Analysis of SIV/HIV quasispecies genetic diversity is used to assess virus compartmentalization between fluids and organs in infected individuals and to detect drug resistant viruses
A melting analysis of PCR amplification products is usually performed with a low resolution after a standard SYBR Green Quantitative PCR (QPCR) reaction or a high resolution in more specialized High Resolution Melting (HRM) studies, and results in a melting profile specific for the amplicon
In the widely used SIV env Single genome amplification (SGA) method developed by Keele et al [1], a very long amplicon (3.2-kb) containing the full length env gene is generated by non quantitative PCR (Fig 1A) and only a small amount of the SGA PCR reaction is available for the selection of positive reactions in order to keep the largest amount of material possible for direct sequencing
Summary
Analysis of SIV/HIV quasispecies genetic diversity is used to assess virus compartmentalization between fluids and organs in infected individuals and to detect drug resistant viruses. Advantages of SGA method over other approaches (e.g. bulk PCR or NGS) include enhanced specificity for the detection of rare individuals, compatibility with short and long amplicons, absence of bulk PCR induced artefacts (e.g. recombination and nucleotide misincorporations), and no requirement for extensive bioinformatics analysis. This methodology has two drawbacks: 1) the high number of PCR reaction replicates to be performed; 2) the requirement for a very labor intensive and time consuming gel electrophoresis screening step to identify the positive reactions amongst the numerous negative reactions. The development of absolute quantification by qPCR of viral cDNAs in samples facilitated the determination of the optimal working dilution, reducing this number from an average of 245 reactions to 135 reactions [11], the need for extensive gel electrophoresis analyses of PCR reaction products remains an important issue in large scale studies involving numerous samples, or in SGA core facilities, as it is labor intensive, tedious and generates lots of chemical waste
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