|
NISCAIR ONLINE PERIODICALS REPOSITORY (NOPR) >
NISCAIR PUBLICATIONS >
Research Journals >
Indian Journal of Biochemistry and Biophysics (IJBB) >
IJBB Vol.42 [2005] >
IJBB Vol.42(5) [October 2005] >
| Title: | Analysis of solid-phase allele-specific primer extension characteristics on biochip in combination with modified primers and PicoGreen staining method |
| Authors: | Qin, Shengying
Liu, Jianhua |
| Keywords: | Single nucleotide polymorphism
Allele-specific primer extension
DNA microarray
Extension efficiency and specificity
Genotyping
PicoGreen staining |
| Issue Date: | Oct-2005 |
| Publisher: | CSIR |
| IPC Code: | C07H21/04; C12N15/00; C1215/90 |
| Abstract: | 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. |
| Page(s): | 308-314 |
| ISSN: | 0301-1208 |
| Source: | IJBB Vol.42(5) [October 2005]
|
|
