PCR-Based Technologies for Identifying Unknown Gene Sequences
Genome walking is a basic molecular biology technique for obtaining unknown sequences. In biology, unknown sequences are usually related to gene regulation, diseases, undiscovered functions of genes, and food safety. The first genome walking method described, inverse PCR, has been established for 28 years. Despite the shortcomings of I-PCR, it has permitted the identification of several previously unknown sequences. To overcome the drawbacks of I-PCR and enhance its efficiency, and in conjunction with recent advances in molecular biology, many genome walking methods have been developed. Essentially, these methods may be classified into the two following categories: those with or those without genome digestion involving restriction enzymes in the first step. According to the features of specific genome walking techniques, these methods are suitable for different types of templates, such as long or short genome walking, single or multiple insertion identification, or simple or complex DNA structures. As the world begins to place greater importance on food security, the future of genome walking is promising, especially in identifying the unintended consequences of GM foods.
KeywordsGenome walking Unknown sequences Flanking sequences PCR DNA sequencing
This work is supported by the National Science and Technology Major Project (2016ZX08012-001). Many thanks to Ying Shang, for her kindly help in manuscript conception and preparation.
- 17.Chen J-Y, Shen X-l, Xin Y-R, Chen X-J. Application of the modified TAIL-PCR technique in cloning of PM H~+-ATPase gene promoter from wheat genomic DNA [J]. J Henan Agric Univ. 2008;1:000.Google Scholar
- 18.Isegawa Y, Sheng J, Sokawa Y, Yamanishi K, Nakagomi O, Ueda S. Selective amplification of cDNA sequence from total RNA by cassette-ligation mediated polymerase chain reaction (PCR): application to sequencing 6 5 kb genome segment of hantavirus strain B-1. Mol Cell Probes. 1992;6(6):467–75.CrossRefPubMedGoogle Scholar
- 19.Nthangeni MB, Ramagoma F, Tlou MG, Litthauer D. Development of a versatile cassette for directional genome walking using cassette ligation-mediated PCR and its application in the cloning of complete lipolytic genes from Bacillus species. J Microbiol Methods. 2005;61(2):225–34.CrossRefPubMedGoogle Scholar
- 29.Xu W, Shang Y, Zhu P, Zhai Z, He J, Huang K, Luo Y. Randomly broken fragment PCR with 5′ end-directed adaptor for genome walking. Scientific reports 3; 2013.Google Scholar
- 31.Kovalic D, Garnaat C, Guo L, Yan Y, Groat J, Silvanovich A, Ralston L, Huang M, Tian Q, Christian A. The use of next generation sequencing and junction sequence analysis bioinformatics to achieve molecular characterization of crops improved through modern biotechnology. Plant Genome. 2012;5(3):149–63.Google Scholar
- 32.Yang L, Wang C, Holst-Jensen A, Morisset D, Lin Y, Zhang D. Characterization of GM events by insert knowledge adapted re-sequencing approaches. Scientific reports 3; 2013.Google Scholar