用Bubble　PCR法对一个新的锌指基因进行外显子划界

Identification of Exon Boundaries in a Novel ZincFinger Gene Using Bubble-PCR Method

介绍了一种确定基因外显子一内含子交界点的快速、准确的方法，应用该方法对本室克隆的一个新的锌指基因ZNF191进行了外显子划界。将含有ZNF191全长CDNA的基因组DNA经限制酶酶切后与退火的Bubblelinker连接，以该连接产物为模板，用依据cDNA序列设计的引物和Bubblelinker上的特异引物进行PCR扩增，继以相同的扩增条件对扩增产物进行测序，从而确定了外显子－内含子的交界点。结果确认出ZNF191基因由4个外显子和3个内含子组成，这一结论为后来完成的ZNF191基因的全长测序结果所证实。

A rapid and accurate method to identify the exon-nitron boundaries in a gene is described.The exon boundaries of a novel zinc finger gene ZNF191 cloned in the laboratory was identifiedwith this method. Genomic DNAs containing the sequence of ZNF191 cDNA were digested withproper restriction enzymes and then ligated to an annealed bubble linker. The ligation productwas used as the template to carry out PCR with the primers of ZNF191 cDNA and the bubblelinker. Then exon-nitron boundaries were identified by sequencing of PCR products, with thesame amplification condition. It has been found that the zinc finger gene (ZNF191 ) has 4 exonsand 3 nitrons. This result was confirmed by the whole sequencing of genomic DNA of theZNF191 performed afterword.