Optimized Multiplex Polymerase Chain Reaction

Abstract

Implementation of the whole-genome shotgun sequencing approach to prokaryotes and eukaryotes necessitates methods for rapid gap closure. During a whole-genome shotgun sequencing project, DNA sequences are aligned against each other to create continuous assemblies or contigs (1). However, some areas of the genome are not represented owing to repeats, secondary structure, or toxicity in Escherichia coli. When no cloned template is available for sequencing, these gaps must be resolved by polymerase chain reaction (PCR), testing primers from each contig end against primers from all the other contig ends in all the possible combinations. When dealing with large genomes, generating templates for numerous gaps by only combinatorial PCR is cumbersome and inefficient. Optimized Multiplex PCR (2) provides an effective new method for the rapid closure of a large number of gaps. Its framework for minimizing the number of PCR pipetting steps is achieved by combining Multiplex PCR (3) with a mathematical method (4). Unlike combinatorial PCR, Optimized Multiplex PCR minimizes the number of reactions by combining primers into pools and testing these pools against each other. This method has been successfully implemented in the closure of numerous genomes at The Institute for Genomic Research. In this chapter, we provide a simple guide to the Optimized Multiplex PCR method by describing in detail the steps involved in primer design, primer pooling, Multiplex PCR, and multiplex sequencing.