Primer design for Whole Genome Amplification using genetic algorithms.
Whole Genome Amplification (WGA) is an important process to increase limiting amounts of genomic DNA prior to genomic analyses. Current amplification methods based on primer extension or strand displacement principles employ primers of partially or totally random sequence. In this paper, we present a method using Genetic Algorithms to optimize a single primer design to be used in a primer extension reaction to achieve unbiased WGA. Computational simulation and prediction of a suitable primer proposed two candidates NYP6-1 (ATCTCA) and NYP6-2 (TGAGAT). NYP6-1 amplified to a maximum length of 2537 base pairs (bp), had genome coverage of approximately 45.62%, with an average of 493 and variance of 163 amplicons per 1 megabasepairs (Mb). NYP6-2 amplified to a maximum length of 2926 bp and covered 54.35% of the genome with an average of 579 and a variance of 191 amplicons per Mb. In contrast, the original primer used in Degenerate Oligonucleotide-Primed PCR (DOP-PCR) had coverage of 20.93%, an average of 74 and variance of 188 amplicons per Mb when extended up to a length of 2000 bp. Successful WGA of miniscule amounts of genomic DNA requires the amplification method used to resolve issues on efficiency, accurate representation of the whole genome and ability to degraded DNA. The sequence NYP6-2 discovered using our method can be confidently used in a primer extension based protocol to perform quantitatively unbiased WGA.
Png
Adrian E H
AE
Bioinformatics Group, Nanyang Polytechnic, 180 Ang Mo Kio Avenue 8, Singapore 569830, Republic of Singapore.
Choo
Keng Wah
KW
Lee
Cheryl I P
CI
Leong
Siew Hong
SH
Kon
Oi Lian
OL
D000465:Algorithms; D002877:Chromosomes, Human / Q000235:genetics; D003198:Computer Simulation; D017931:DNA Primers / Q000235:genetics*; D005784:Gene Amplification; D023281:Genomics / Q000379:methods* / Q000706:statistics & numerical data; D006801:Humans; D008957:Models, Genetic; D016133:Polymerase Chain Reaction / Q000379:methods / Q000706:statistics & numerical data; D012985:Software Design
D017931:DNA Primers