Simultaneous cloning of multiple nuclear genes by pooling PCR products of variable size: a cost-effective method of improving efficiency in large-scale genetic analyses

摘要

I present a simple approach to overcome the high cost and low efficiency of cloning polymerase chain reaction (PCR) products for individuals in wide-scale population genetic analyses. The methodology reduces the number of cloning reactions per individual by engineering a suite of genetic markers that differ in size and pooling these PCR products prior to cloning. Alleles from each gene are then recovered by screening transformed bacterial colonies and identifying the inserts corresponding to each gene based on size. I demonstrate the utility of this technique by presenting the results I obtained from cloning four nuclear genes in 118 individuals from three species of sea urchins (Strongylocentrotus purpuratus, S. droebachiensis and S. pallidus). Of the 472 different PCR products I cloned, I recovered at least one allele for 432 of them (91.5%) by screening between 16 and 32 bacterial colonies for each individual. There existed a bias with respect to recovery efficiency: the two largest fragments (1130-800 bp) were recovered 100% of the time, while the two smaller fragments (580-650 bp) were recovered in 85.6% and 81.4% of the experiments, respectively. I discuss the promise of this application for wide-scale genetic analyses.