Applications of Whole Genome Amplification by Phi29 DNA Polymerase
in Conservation Genetics
Sichen Shao
Jan E. Janecka
Dr. Rodney Honeycutt
The DNA polymerase Phi29 provides a means of
amplifying whole genomes from relatively small samples of starting template DNA,
consequently providing conservation geneticists with potential means of
optimizing their experiments that might involve DNA samples of low quantity
and/or low quality. In this study, controlled PCRs designed to amplify three
microsatellite and two mitochondrial DNA loci were performed using DNA template
samples of varying quantities and qualities and versions of these samples that
have been amplified using the GenomiPhi DNA Amplification Kit by Amersham
Biosciences® that uses the Phi29 DNA polymerase. DNA was extracted from five
bobcat blood samples followed by quantification and dilution to four specific
concentrations, thus simulating varying quantities of DNA that may be found in
the field. Furthermore, each of the five DNA samples were sheared to two average
lengths (~2 kb and ~3.5 kb) on the HydroShear® DNA Shearing Device by
GeneMachines® to simulate DNA of varying quality. All of these samples were then
amplified using the GenomiPhi DNA Amplication Kit, and both the original and
amplified samples were used as templates for amplification via the polymerase
chain reacton (PCR). Analysis of the PCR products on 1% agarose gels and
comparisons of the yields produced by the original DNA and amplified DNA
templates indicated that the whole genome amplification using Phi29 DNA
polymerase enhanced the performance of DNA of low quantity, but may not improve
the results from DNA of low quality.
PowerPoint Presentation (8.5 MB)