Wei Qing, Liu Sanzhen, Huang Jianfeng, Mao Xueying, Chu Xiaohui, Wang Yu, Qiu MinYan, Mao Yumin, Xie Yi, Li Yao
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai 200433, P.R. China.
J Biochem Mol Biol. 2004 Jul 31;37(4):439-44. doi: 10.5483/bmbrep.2004.37.4.439.
Double stranded targets on the cDNA microarray contain representatives of both the coding and noncoding strands, which will introduce hybridization competition with probes. Here, the effect of double and single strands of targets on the signal intensity and the ratios of Cy5/Cy3 within the same slide were compared. The results show that single stranded targets can increase the hybridization efficiency without changing the Cy5/Cy3 ratio. Based on these results, a new strategy was established by generating cDNA targets with asymmetric PCR, instead of conventional PCR, to increase the sensitivity of the cDNA microarray. Furthermore, the feasibility of this approach was validated. The results indicate that the cDNA microarray system based on asymmetric PCR is more sensitive, with no decrease in the reliability and reproducibility as compared with that based on conventional symmetric PCR.
cDNA微阵列上的双链靶标包含编码链和非编码链的代表，这会与探针产生杂交竞争。在此，比较了同一载玻片上靶标的双链和单链对信号强度以及Cy5/Cy3比率的影响。结果表明，单链靶标可提高杂交效率，而不改变Cy5/Cy3比率。基于这些结果，建立了一种新策略，即通过不对称PCR而非传统PCR生成cDNA靶标，以提高cDNA微阵列的灵敏度。此外，该方法的可行性得到了验证。结果表明，基于不对称PCR的cDNA微阵列系统更灵敏，与基于传统对称PCR的系统相比，可靠性和可重复性没有降低。
