Png Adrian E H, Choo Keng Wah, Lee Cheryl I P, Leong Siew Hong, Kon Oi Lian
Bioinformatics Group, Nanyang Polytechnic, 180 Ang Mo Kio Avenue 8, Singapore 569830, Republic of Singapore.
In Silico Biol. 2006;6(6):505-14.
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.
全基因组扩增（WGA）是在基因组分析之前增加有限量基因组DNA的重要过程。目前基于引物延伸或链置换原理的扩增方法使用部分或完全随机序列的引物。在本文中，我们提出了一种使用遗传算法优化单个引物设计的方法，该引物用于引物延伸反应以实现无偏差的WGA。对合适引物的计算模拟和预测提出了两个候选引物NYP6-1（ATCTCA）和NYP6-2（TGAGAT）。NYP6-1扩增的最大长度为2537个碱基对（bp），基因组覆盖率约为45.62%，每1兆碱基对（Mb）平均有493个扩增子，方差为163。NYP6-2扩增的最大长度为2926 bp，覆盖基因组的54.35%，每Mb平均有579个扩增子，方差为191。相比之下，简并寡核苷酸引物PCR（DOP-PCR）中使用的原始引物在延伸至2000 bp长度时，覆盖率为20.93%，每Mb平均有74个扩增子，方差为188。成功扩增微量基因组DNA需要所使用的扩增方法解决效率、全基因组准确代表性以及降解DNA能力等问题。使用我们的方法发现的序列NYP6-2可以可靠地用于基于引物延伸的方案中，以进行定量无偏差的WGA。
