Making deletions with PCR

Malte Buchholz mbuchho at med.uni-goettingen.de
Mon Feb 22 08:12:48 EST 1999


Another, slightly different, method that I have used successfully and
which I find somewhat less complicated is Overlap Extension PCR. You
generate your desired construct in two steps (any ol` Taq polymerase
will do):


                                        /
                             primer 1 
/                                             primer 3
                               
<-----/                                              <-----
 --------------------------------------I---to be
deleted---I------------------------------- plasmid
 ------>                                                   /------> 
 primer 2                                                 /  primer 4
                                                         /
                                      
       
                      product a                              
   
-------------------------------------------------                                
primer 3
    ------------------------------------------------- ....>      product
b             <-----
    ------>                          
<.....-------------------------------------------------
    primer 2                               
-------------------------------------------------
                                                                        


You first amplify 2 fragments with primers pairs 1/2 and 3/4. Primers 1
and 4 have additional sequences which are complementary to each other.
In the second step you combine the two fragments and add primers 2 and
3. In the first few rounds of amplification, the complementary
(overlapping) sequences will hybridize and serve as primers, and the
full length product will be amplified by the two oligonucleotide
primers. I have found this method to be very effective, not only for
deletions, but for introducing any mutation that one might desire.

Hope this helps,

Malte



More information about the Methods mailing list