Gene capture and random amplification for quantitative recovery of homologous genes.
The polymerase chain reaction (PCR) is instrumental in molecular analysis of microorganisms, allowing for the selective amplification of nucleic acids directly from clinical and environmental samples. However, the principles that allow for targeted amplification of DNA become a hindrance when attempting to simultaneously discriminate and quantify complex mixtures of homologous genes. Here we present a simple solution to the quantitative problem by separating the enrichment and amplification aspects of a conventional PCR reaction. In this assay, genes are enriched using a DNA oligonucleotide capture probe and subsequently amplified in a two-step random amplification protocol. In order to evaluate the quantitative aspects of the gene capture assay, we used real-time quantitative-PCR to measure initial and final concentrations of homologous genes from constructed mixtures of genomes. Upon sampling for the universal DNA-dependent RNA polymerase gene, rpoC, we were able to demonstrate quantitative recoveries from a mixed DNA sample despite differences in gene copy number ranging up to 4 orders of magnitude. This suggests that minority populations as low as 0.01% of the total community are represented as accurately as populations at higher abundance. These results offer new possibilities for accurately and quantitatively monitoring diverse mixtures of microorganisms.
Crosby
Laurel D
LD
Environmental Engineering & Science Program, Stanford University, Stanford, CA 94305, USA. laurel@stanford.edu
Criddle
Craig S
CS
D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D017931:DNA Primers; D015342:DNA Probes; D005784:Gene Amplification; D016133:Polymerase Chain Reaction / Q000379:methods; D012689:Sequence Homology, Nucleic Acid
D017931:DNA Primers; D015342:DNA Probes; D004247:DNA