MBIO 3410 lecture Nov 6 2007

MBIO 3410 lecture Nov 6 2007 - Last Lecture Overview PCR...

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1 Last Lecture Overview PCR Key concepts Denaturation Annealing Extension PCR cycle #1 PCR cycle #2 PCR cycle #3 Important PCR Factors PCR Reaction Conditions Tris-HCl KCl or KCl Thermostable DNA Polymerases Taq DNA polymerase Pfu DNA polymerase Oligonucleotide Primers
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2 The primers that are used in a PCR experiments need not match the target sequence exactly The only place within the primer sequence that must match the target sequence exactly is the extreme 3’-end of the primer the polymerase will not efficiently extend the primer unless it matches The primers initiate the DNA replication process and are incorporated into each strand of the final amplified product Base changes can be introduced into the 5’-end of the primer to amplify these changes PCR Primer Mismatches
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3 PCR- based Mutagenesis Deliberate mutations may be introduced into the final PCR product by altering the sequence of the primer: alter the coding sequence of a gene to change the amino acid sequence of a protein or if you want to introduce a restriction enzyme recognition site without altering the amino acid sequence of the resulting protein Gene X Gene X
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4 The primers contain the recognition sequence for the EcoR I restriction enzyme at the 5’-end of the sequence used to recognize gene X The EcoR I recognition sequence does not match the template sequence exactly but the mismatches are not sufficient to prevent specific primer binding Primer 2 contains the recognition site for the BamH I restriction enzyme which will also be incorporated into the final product These fragments can them be cloned into a vector using the same restriction enzymes Note: ensure that there are no EcoR I or BamH I restriction enzyme sites in the PCR product itself To cleave efficiently three to six additional residues are usually added to the 5’-end of the primer before the restriction enzyme recognition site These are often G or C (termed a GC clamp ) to provide the maximum level of annealing between the two DNA stands and efficient cleavage by restriction enzymes
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5 PCR to search for homologous genes The second major use of mismatched primers is in the search for genes encoding a particular protein and in the search for homologous genes If you isolate a protein and sequence its amino terminal end to find the following amino acids: Met–Ile–Trp–Pro–Phe The degeneracy of the genetic code means that this amino acid sequence could be encoded for by the following DNA sequences:
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6 To PCR amplify the gene encoding this protein sequence a degenerate primer must be used The primer must be able to bind to all possible combinations that could encode the protein sequence The primer below could be synthesized to perform this function: where N is an equimolar mixture of A, T, C and G The primer above would be produced as a mixture of 24
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This note was uploaded on 07/05/2008 for the course MBIO 3410 taught by Professor Richardson during the Fall '07 term at Manitoba.

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MBIO 3410 lecture Nov 6 2007 - Last Lecture Overview PCR...

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