[Application of oligonucleotide microarray primer extension to detection of p53 single nucleotide polymorphisms].
OBJECTIVE
Increasing evidences imply that single nucleotide polymorphisms (SNPs) are involved in etiopathology, individual therapy, and prognosis of many diseases. Rapid and accurate SNPs detection in disease genes is vitally important for genomic research. To detect p53 SNPs, the method of an arrayed primer extension based on oligonucleotide microarray was developed.


METHODS
Twelve extension primers were designed in exon 3 of p53 gene. These primers were synthesized and printed onto a microarray with 7x8 spots. The fragments of p53 DNA were extracted from K562 cells by nested polymerase chain reaction (PCR), and hybridized with the microarray. After hybridization, the primer extension reactions were carried out with DNA polymerase Klenow, labeled with Cy3-dUTP or Cy5-dCTP; the product was washed and scaned, and the fragments of p53 was sequenced by ABI3730 DNA Analyzer.


RESULTS
The scanning result showed that the primer extension reactions were successful, and single base labeled with Cy3 or Cy5 was extended correctly at the end of 3'primers. The results of microarray SNPs detection were consistent with the results of sequencing verification, while much less complicated.


CONCLUSIONS
These results indicate that the arrayed primer extension techniques are useful in parallel detecting SNPs of genes of interest, which is not only sensitive and accurate but also miniaturized the assays when analyzing multiple DNA targets with minimal reagents.
Wu
Qing-Hua
QH
Institute of Molecular Biology, Nanfang Medical University, Guangzhou, Guangdong, 510515, PR China.
Ma
Wen-Li
WL
Zhang
Bao
B
Guo
Qiu-Ye
QY
Li
Ling
L
Zheng
Wen-Ling
WL
D017931:DNA Primers / Q000235:genetics*; D016158:Genes, p53; D006801:Humans; D020014:K562 Cells; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
D017931:DNA Primers; D016159:Tumor Suppressor Protein p53