4 - – phenotype The procedure just outlined can be used...

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4) Use the known sequence of the end of TTTTT to PCR amplify a fragment that spans the junction between the end of TTTTT and the E. coli chromosomal site that was the target for insertion. DNA sequencing of the amplified junction fragments will give the identity of the target sequences. Since we know the DNA sequence of the entire E. coli chromosome, the gene that was the target for TTTTT insertion can be identified unambiguously. 5) The DNA sequence of the junction fragments will identify all of the genes that have been inactivated to give the His
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Unformatted text preview: – phenotype. The procedure just outlined can be used to isolate and characterize a wide variety of useful mutations. A major limitation of this method is that as stated earlier, transposon mutations usually completely disrupt the target gene and therefore lead to a complete inactivation of the gene product. Often we will want to work with point mutations (such as temperature sensitive mutations or nonsense mutations). In the next lecture we will see how transposons can also be used to facilitate analysis and manipulation of point mutations....
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