Alsmadi Osama, Alkayal Fadi, Monies Dorota, Meyer Brian F
Genetics Department, Research Center, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia.
BMC Res Notes. 2009 Mar 24;2:48. doi: 10.1186/1756-0500-2-48.
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.
Effects of primer length and composition on amplification quality and quantity were explored in this study at two different temperatures (30 degrees C & 40 degrees 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 degrees C & 40 degrees C, of the human whole genome via the use of GGGCAGGANG 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 degrees C and 40 degrees C.
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.
全基因组扩增（WGA）是消除与基因组DNA（gDNA）数量相关的分子分析限制的一种实用解决方案。已经开发出不同的方法来扩增全基因组，包括引物延伸预扩增（PEP）、简并寡核苷酸引物PCR（DOP-PCR）和多重置换扩增（MDA）。这些方法中的每一种都有其自身的优点和局限性。
本研究在两个不同温度（30摄氏度和40摄氏度）下探讨了引物长度和组成对扩增质量和数量的影响。通过扩增产量、基因组覆盖率和等位基因脱扣（ADO）分析测量，新的引物设计与提高的扩增温度相结合显著改善了MDA。一个显著的发现是，通过使用GGGCAGGANG热点重组共有引物，在30摄氏度和40摄氏度下对人类全基因组进行了全面扩增。通过Affymetrix 10K SNP芯片分析对扩增进行了表征。最后，新引物设计的使用在30摄氏度和40摄氏度下均抑制了模板非依赖性DNA扩增（TIDA）。
本研究中使用新引物并结合MDA中提高的孵育温度显著提高了特异性、扩增产量并抑制了TIDA。
