Tonooka Yuki, Mizukami Yoichi, Fujishima Masahiro
Department of Biology and Chemistry, Faculty of Science, Yamaguchi University, Yoshida 1677-1, Yamaguchi, 753-8512, Japan.
Appl Microbiol Biotechnol. 2008 Feb;78(1):173-80. doi: 10.1007/s00253-007-1289-x. Epub 2007 Dec 11.
This report describes a novel and efficient method for walking the sequence of a genomic deoxyribonucleic acid (DNA) from a known region to an unknown region based on an oligodeoxynucleotide (oligo) cassette-mediated polymerase chain reaction technique. In this method, genomic DNA is digested by a restriction enzyme that generates a sticky 5'-end, followed by ligation of a one-base excess oligo-adaptor using T4 DNA ligase. The adaptor consists of two complementary oligos that form the same sticky end as the digested genomic DNA fragments, except that the 5'-overhang base overlaps the corresponding 3'-end base of the restriction site. This overhanging terminal base prevents ligation between the adaptors, and the appropriate molar ratio of adaptor to genomic DNA enables specific amplification of the target sequence. T4 DNA ligase catalyzes both the ligation of the phosphorylated overhang base of the adaptor to genomic DNA and the excision of the corresponding 3'-terminal base of the genomic DNA. This sequence-specific exonuclease activity of T4 DNA ligase was confirmed by ligation of an alternative adaptor in which the 5'-terminal base was not consistent with the corresponding 3'-terminal base. Using this technique, the 3'- and 5'-flanking sequences of the catalase gene of the ciliate Paramecium bursaria were determined.
本报告描述了一种基于寡脱氧核苷酸（oligo）盒介导的聚合酶链反应技术，从已知区域到未知区域对基因组脱氧核糖核酸（DNA）序列进行步移的新颖且高效的方法。在该方法中，基因组DNA用产生粘性5'末端的限制性内切酶消化，然后使用T4 DNA连接酶连接一个碱基过量的oligo接头。该接头由两个互补的寡核苷酸组成，它们形成与消化后的基因组DNA片段相同的粘性末端，只是5'突出端碱基与限制性酶切位点的相应3'末端碱基重叠。这个突出的末端碱基可防止接头之间的连接，接头与基因组DNA的适当摩尔比能够实现靶序列的特异性扩增。T4 DNA连接酶催化接头的磷酸化突出端碱基与基因组DNA的连接以及基因组DNA相应3'末端碱基的切除。通过连接5'末端碱基与相应3'末端碱基不一致的替代接头，证实了T4 DNA连接酶的这种序列特异性核酸外切酶活性。利用该技术，确定了草履虫过氧化氢酶基因的3'和5'侧翼序列。
