Comparison of hybridization behavior between double and single strands of targets and the application of asymmetric PCR targets in cDNA microarray.
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.
Wei
Qing
Q
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai 200433, P.R. China.
Liu
Sanzhen
S
Huang
Jianfeng
J
Mao
Xueying
X
Chu
Xiaohui
X
Wang
Yu
Y
Qiu
MinYan
M
Mao
Yumin
Y
Xie
Yi
Y
Li
Yao
Y
D002232:Carbocyanines / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006824:Hybridization, Genetic; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results
D002232:Carbocyanines; D018076:DNA, Complementary; D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; C089187:cyanine dye 3; C085321:cyanine dye 5; D012313:RNA; D004247:DNA