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Protocol for in-frame deletion mutagenesis (A. Beliaev, S.B. Reed, D. Stanek, D. Saffarini, M. Romine) Step I: Generation of a deleted copy of the target gene using a two-step asymmetric/crossover PCR amplification (Fig. 1) References: M. F. Alexeyev, I. N. Shokolenko, and T. P. Croughan. 1995. Improved antibiotic-resistance gene cassettes and omega elements for Escherichia coli vector construction and in vitro deletion/insertion mutagenesis. Gene 160 (1): 63-67. M. S. Donnenberg and J. B. Kaper. 1991. Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selectionsuicide vector. Infect. Immun. 59:4310–4317. A.J. Link, D. Phillips, M. Church. 1997. Methods for Generating Precise Deletions and Insertions in the Genome of Wild-Type Escherichia coli: Application to Open Reading Frame Characterization. J Bact., 179 (20): 6228. C. W. Saltikov, D. K. Newman. 2003. Genetic identification of a respiratory arsenate reductase. PNAS, 100 (19): 10983-10988. Hu. 1993. DNA and Cell Biology. 12: 763. Mead. 1991. Biotechnology. 9: 657.  I.1. Primer Design and Amplification: Design PCR primers to amplify the regions flanking the target gene. These amplified flanking fragments should be 500-1000 bp in length. Subsequent to amplification, these flanking regions are fused together via a complementary “tag” region that is added to the 5’ end of each inner primer. This tag region should be unique, thus giving each mutant its own “barcode” for future work and identification. This fusion product will then be inserted into the cloning vector pDS3.1. The primers for the 5’-end fragment primers are 5o (outside) and 5i (inside) and the primers for the 3’-end fragment are 3i (inside) and 3o (outside). The size of the complementary tags used in our lab is generally ~21 nucleotides (tags may contain a unique restriction site to facilitate insertion of additional markers). When designing 5i and 3i primers remembe