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BCH5425 Molecular Biology and Biotechnology

BCH5425 Molecular Biology and Biotechnology
Spring 1998
Dr. Michael Blaber

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