寡核苷酸阵列引物延伸技术在p53基因突变检测中的应用

Application of Oligonucleotide Microarray Primer Extension to Detection of p53 Single Nucleotide Polymorphisms

背景与目的越来越多的研究表明,许多疾病的发生、个体化治疗及预后与基因的SNP密切相关,对其快速、准确的检测在基因组研究和应用中具有重要的意义。本文应用寡核苷酸阵列引物延伸法对人类肿瘤相关性最高的基因——p53基因进行SNPs的平行检测。方法根据寡核苷酸基因芯片上延伸引物的设计原则设计出12条p53基因第三外显子上的SNP检测的延伸引物,点样制备7×8点阵的寡核苷酸基因芯片,将巢式PCR扩增出的p53基因片段与芯片杂交,在Klenow酶的介导下进行荧光标记的碱基延伸反应、洗脱、扫描。同时p53基因片段进行测序分析。结果经荧光检测分析,芯片经延伸反应后出现了明显的Cy3-dUTP或Cy5-dCTP荧光标记信号,检测灵敏度好,结果与测序验证结果一致。结论采用寡核苷酸芯片的阵列引物延伸技术可以在基因水平实现对p53基因的多个位点的高灵敏度平行检测,是一种高效、价廉、准确的SNP检测方法。

BACKGROUND & 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 7×8 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. CONCLUSION: 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.