Specific and complete human genome amplification with improved yield achieved by phi29 DNA polymerase and a novel primer at elevated temperature

Abstract

Backgrounds. Whole genome amplification (WGA) is a practical solution to eliminate molecular analysis limitations associated with genomic DNA (gDNA) quantity. Different methods have been developed to amplify the whole genome, including primer extension preamplification (PEP), degenerate oligonucleotide primed PCR (DOP-PCR), and multiple displacement amplification (MDA). Each of these methods has its own merits and limitations. Findings. Effects of primer length and composition on amplification quality and quantity were explored in this study at two different temperatures (30°C & 40°C). New primer designs combined with elevated amplification temperature has significantly improved MDA as measured by amplification yield, genome coverage, and allele drop out (ADO) analysis. A remarkable finding was the comprehensive amplification, at 30°C & 40°C, of the human whole genome via the use of GGGCAGGA*N*G hotspot recombination consensus primer. Amplification was characterized by Affymetrix 10K SNP chip analysis. Finally, the use of new primer designs has suppressed the template-independent DNA amplification (TIDA) both at 30°C and 40°C. Conclusion. The use of new primers in this study combined with elevated incubation temperatures in MDA has remarkably improved the specificity, amplification yield, and suppressed TIDA. © 2009 Alsmadi et al.