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Analysis of solid-phase allele-specific primer extension characteristics on biochip in combination with modified primers and PicoGreen staining method

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Title Analysis of solid-phase allele-specific primer extension characteristics on biochip in combination with modified primers and PicoGreen staining method
 
Creator Qin, Shengying
Liu, Jianhua
 
Subject Single nucleotide polymorphism
Allele-specific primer extension
DNA microarray
Extension efficiency and specificity
Genotyping
PicoGreen staining
 
Description 308-314
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.
 
Date 2009-03-30T08:45:25Z
2009-03-30T08:45:25Z
2005-10
 
Type Article
 
Identifier 0301-1208
http://hdl.handle.net/123456789/3534
 
Language en_US
 
Relation C07H21/04; C12N15/00; C1215/90
 
Publisher CSIR
 
Source IJBB Vol. 42(5) [October 2005]