单细胞引物延伸预扩增分析多个基因

Analysis of several genes using primer extension preamplification based on individual cell

目的 建立一种单细胞 (individual cell)水平引物延伸预扩增 (primer extensionpreamplification,PEP)方法 ,利用单个细胞同时分析多个基因。方法 采用 10 bp的随机引物先对单细胞的基因组预扩增 ,取其产物 5μl分别对 SRY、ZP3、Rh CE、Rh D、F 5个基因进行套式 PCR扩增。结果 单细胞 PEP后的产物能同时扩增 5个基因 ;对 4种已知基因型的白细胞进行诊断 ,正确率为 95 .6 %。结论PEP技术可以在单细胞水平分析 5个基因 。

Objective: To analyze several genes at the same time in one individual cell using primer extension preamplification (PEP). Methods: The whole genome of individual cell was preamplified by PEP using 10 base random oligonucleotides. The aliquots (5 μl) from PEP were used to amplify SRY, ZP3, RhCE, RhD and F VIII genes by nested PCR. Results: The five genes could be amplified following PEP-PCR. Lymphocyte of four genotypes were diagnosed by PEP-PCR; correct diagnoses were obtained in 95. 6%. Conclusion At least five genes can be analyzed by PEP-PCR based on one individual cell in this laboratory. This method could be used in preimplantation genetic diagnosis and noninvasive prenatal diagnosis.