Exploring whole genome amplification as a DNA recovery tool for molecular genetic studies.
The whole genome amplification (WGA) protocol evaluated during this study, GenomiPhi DNA amplification kit, is a novel method that is not based on polymerase chain reaction but rather relies on the highly processive and high fidelity Phi29 DNA polymerase to replicate linear genomic DNA by multiple strand displacement amplification. As little as 1 ng of genomic DNA template is sufficient to produce microgram quantities of high molecular weight DNA. The question explored during this study is whether such a WGA method is appropriate to reliably replenish and even recover depleted DNA samples that can be used for downstream genetic analysis. A series of human DNA samples was tested in our laboratory and validated using such analytical methods as gene-specific polymerase chain reaction, direct sequencing, microsatellite marker analysis, and single nucleotide polymorphism allelic discrimination using TaqMan and Pyrosequencing chemistries. Although degraded genomic DNA is not a good template for Phi29 WGA, this method is a powerful tool to replenish depleted DNA stocks and to increase the amount of sample for which biological tissue availability is scarce. The testing performed during the validation phase of the study indicates no discernable difference between WGA samples and the original DNA templates. Thus, GenomiPhi WGA can be used to increase precious or depleted DNA stocks, thereby extending the life of a family-based linkage analysis project and increasing statistical power.
Holbrook
Jennifer Frenck
JF
Director, Biomolecular Core Lab, 1600 Rockland Road, Suite H-3B Room 336, Wilmington, DE. 19899, USA.
Stabley
Deborah
D
Sol-Church
Katia
K
D000328:Adult; D004259:DNA-Directed DNA Polymerase; D004587:Electrophoresis, Agar Gel; D005819:Genetic Markers; D015894:Genome, Human; D006801:Humans; D018895:Microsatellite Repeats; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques
D005819:Genetic Markers; D004259:DNA-Directed DNA Polymerase