title: A simple one-step PCR walking method and its application of bacterial rRNA for sequencing identification.
abstract: There are many PCR walking methods applied currently, and they all have examples of successful application in organisms which are more complex than bacteria. However, to a certain extent, it will be more convenient for researchers if the complicated operation and poor specificity for bacteria can be improved. Here, we introduced an improved one-step PCR walking method of bacteria. Using a specific primer of the known sequence together with a universal semi-random primer, the unknown sequence adjacent to a known sequence can be obtained easily by just one ordinary round PCR. The products can be gel-purified and directly sequenced. Specific primers were designed according to the gene sequence of bacterial rRNA, and the variable and adjacent gene sequences were obtained by this method. The sequence analysis of the product showed that it can improve the resolution of bacterial identification to the species level.
authors.affiliation: State Key Laboratory of Dairy Biotechnology, Technology Center of Bright Dairy & Food Co., Ltd, Shanghai, China, zhanghongfa1977@sina.com.
authors.forename: Hongfa
authors.initials: H
authors.lastname: Zhang
authors.forename: Chunping
authors.initials: C
authors.lastname: You
authors.forename: Jing
authors.initials: J
authors.lastname: Ren
authors.forename: Dan
authors.initials: D
authors.lastname: Xu
authors.forename: Mei
authors.initials: M
authors.lastname: Han
authors.forename: Wenyan
authors.initials: W
authors.lastname: Liao
mesh_terms: D001419:Bacteria / Q000145:classification* / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D017422:Sequence Analysis, DNA
chemical_list: D012329:RNA, Bacterial; D012335:RNA, Ribosomal