Figure 1-The new mutant PCR product contains the FCYENE sequence
Figure 2-PCR screening with the specific primer for FCYENE. The screened colonies with this method contain the mutation before the stop codon
Figure 3-In the lane containing the luciferase control, the smear is caused by too much protein loaded on the gel. The 61 kDa band observed in the lanes for Kv1.1(pcDNA3) and Kv1.4 are the result of leakage from the luciferase control lane.Background labeling of the 42 kDa band was anticipated, due to the use of a different [35S]-methionine, other than Redivue. Variations in the amount of loaded protein between the luciferase control and the mutant channels would have resulted in a better picture.
PCR is applicable to a variety of analyses. PCR amplification permits mutagenesis of cDNA templates via adjustment of the thermocycling parameters, as well as the variation of the initial concentration of the DNA template or the concentration of primers. Suggested parameters, such as a low cycle number, and high concentration of DNA template are reported to yield greater accuracy in obtaining the desired mutation. In this study, although a high cycle number was used, the results correlated with the expected size of the DNA and core proteins.
Another factor that provides some advantages, as well as disadvantages, is the use of different plasmids in mutagenesis. An advantage of the pGEM-T system is that it does not require restriction enzyme digestion for ligation. Also, the insertional inactivation of beta-galactosidase gene permits easier screening of transformants. On the other hand, to obtain the proper orientation of the insert, it is necessary to perform a secondary screen by PCR. In contrast, the pcDNA3
plasmid permits directional cloning. However, since pcDNA3 lacks insertional inactivation, colonies must be chosen randomly for screening, and then confirmed by PCR. Thus, in this study, it was relatively easy and efficient to find all the mutants in pGEM-T, while only one mutant (Kv 1.1) was recovered in pcDNA3.
Although Taq polymerase can introduce undesired mutations, the results of in vitro translation in these experiments demonstrates that mutations (e.g. insertion of a nucleotide creating a stop codon) due to the infidelity of Taq polymerase were not accidentally inserted into the sequence.
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am especially grateful to Rocco Coli, Sabrina Volpi, and Ira Daly for their patience and countless hours of assistance. I also would like to thank Dr. Thornhill for providing the wildtype cDNA constructs and for introducing me into the wonderful world of potassium channels. Thanks also to Dr. Rubin for the use of his lab and his help to make this project.
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