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BCH5425 Molecular Biology and Biotechnology

BCH5425 Molecular Biology and Biotechnology
Spring 1998
Dr. Michael Blaber
blaber@sb.fsu.edu


Lecture 24

Cloning PCR Products

Introduction of restriction sites

The potential problems with this method include:

Generation of half sites


The advantages of this method are:

Blunt end ligation

Ahd I restriction endonuclease:

             |  G A C N N N N N G T C   C T G N N N N N C A G           |  

Ahd I will cut this sequence to produce:

  G A C N N N                 N N G T C   C T G N N                 N N N C A G  

We could design an oligonucleotide with two Ahd I restriction sequences, with slightly different sequences in the interrupted region of the palindrome, to give:

             |                     |  G A C N N T N N G T C G A C N N A N N G T C  C T G N N A N N C A G C T G N N T N N C A G           |                     |  

If this this were inserted into a vector, and the vector then was cut with Ahd I, it would have the following sequence at the ends of the linearized vector:

  -G A C N N T                       N N G T C-  -C T G N N                       T N N C A G-  

In other words, a 3' 'T' overhang at both ends of the vector. A PCR product, with 3' A overhangs could thus be inserted into such a 'T' vector

Adding promoters, ribosome binding sites, start codons, and stop codons


PCR Mutagenesis

Gene fusion



Creation of deletions within a gene


Generation of point mutation(s) - i.e. base substitution mutations

Introduction of base substitutions via asymmetric PCR:

Insertion mutagenesis



"Random" mutagenesis with PCR


1998 Dr. Michael Blaber