title: Randomly broken fragment PCR with 5' end-directed adaptor for genome walking.
abstract: Many genome walking methods based on polymerase chain reaction (PCR) are available, including those with and without restriction enzyme modification. Nevertheless, these methods suffer from low reproducibility, inefficiency, and non-specificity. Here, we present a traceable and efficient PCR strategy: randomly broken fragment PCR with 5' end-directed adaptor for genome walking. The genome is first fragmented randomly. After blunting ends, the fragments are ligated to the 5' end-directed adaptors. Semi-nested PCR is then performed. Thus, we can obtain an unknown sequence by cloning the fragments of interest, followed by sequencing. This method effectively bypasses the above-mentioned obstacles and offers the advances: 1) genome fragmentation without using restriction enzymes; 2) enhancement of primer specificity and the prevention of self-ligation between the adaptors by employing a 5' end-directed adaptor. All of the steps in this new method are straightforward, and the unknown sequence can be definitively obtained by merely applying the method once.
authors: 1] Laboratory of Food Safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China [2] The Supervision, Inspection & Testing Center of Genetically Modified Food Safety, Ministry of Agriculture, Beijing 100083, China [3].
authors: Wentao
authors: W
authors: Xu
authors: Ying
authors: Y
authors: Shang
authors: Pengyu
authors: P
authors: Zhu
authors: Zhifang
authors: Z
authors: Zhai
authors: Jing
authors: J
authors: He
authors: Kunlun
authors: K
authors: Huang
authors: Yunbo
authors: Y
authors: Luo
mesh_terms: D016386:Chromosome Walking / Q000379:methods*; D017931:DNA Primers; D023281:Genomics / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D003313:Zea mays / Q000235:genetics
chemical_list: D017931:DNA Primers