Analysis of solid-phase allele-specific primer extension characteristics on biochip in combination with modified primers and PicoGreen staining method.
DNA microarray technology offers potential for future high-throughput variation genotyping. Allele-specific primer extension procedure on microarray has been considered as an efficient method for single nucleotide polymorphism(SNP) genotyping. However, the high cost of the fluorescent-labeled dNTP used for signal detection in this method limits its application. In the present study, we evaluated the characteristics of solid-phase allele-specific primer extension, in terms of specificity and efficiency and demonstrated that compared to liquid-phase reaction, it requires lower annealing temperature, and higher template and Mg2+ concentrations. The extension efficiency and specificity were though linked, behave diametric during the gradient change of template and Mg2+ concentrations or annealing temperature. To obtain both optimal signal intensity and specificity, we introduced an artificial mismatched base at the third position from the primer 3'end, which enhanced the specificity significantly. The PicoGreen staining method, which could decrease the cost greatly, was then introduced to replace the fluorescent-labeled dNTP for signal detection.
School of Life Science & Technology, Shanghai Jiaotong University, 1954 Huashan Road, Shanghai 200030, China.
Shengying
S
Qin
Jianhua
J
Liu
D000483:Alleles; D004247:DNA / Q000032:analysis* / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry* / Q000235:genetics; D005456:Fluorescent Dyes; D005838:Genotype; D020411:Oligonucleotide Array Sequence Analysis; D009841:Oligonucleotides / Q000737:chemistry* / Q000235:genetics; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013194:Staining and Labeling / Q000379:methods*
D017931:DNA Primers; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D009930:Organic Chemicals; C099476:PicoGreen; D004247:DNA