Site-Directed Mutagenesis Technique

            The original goal for my research project was to create a ligation independent cloning (LIC) vector from the plasmid pcDNA3.1+.  The basic outline of this project consisted of: digesting the plasmid using the restriction endonucleases KpnI and HindIII, annealing pre-designed oligonucleotides that were complimentary to the nucleotide overhangs on the digested vector, and combining the digested plasmid and annealed insert through the use of the enzyme T4 DNA ligase to create recombinant DNA.  This novel plasmid could then be used to perform ligation independent cloning, which I will not describe in depth in this blog post.  While the outline appears fairly straightforward in principle, I was unable successfully create my vector after nearly a year of performing research.
            It was decided to try and create a ligation independent cloning vector using a different starting plasmid, and pEGFP-C1 was chosen to fill this role.  The benefit of using the vector pEGFP-C1 is that it contains the gene for enhanced green fluorescent protein.  Cells that express this vector with the desired gene are then tagged with a fluorescent protein, and this helps with the ease of observing the transfected cells.

            There is one problem that exists with trying to use pEGFP-C1 for my LIC vector instead of pcDNA3.1+.  To activate the LIC vector, it must be cut with a restriction endonuclease at the location where the gene will be inserted.  We designed this region to be a cut site for the enzyme SacII.  This is a logical choice for the vector pcDNA3.1+ because it does not contain any other sequences where SacII can digest.  pEGFP-C1, however, contains one other SacII cut site besides the one found in the insert.  This would create difficulties when trying to use the vector because SacII would be able to make two cuts every time the LIC vector derived from pEGFP-C1 is digested.  The LIC vector with both sites digested would be made effectively inactive.

             A technique to circumvent this problem with the created LIC vector is site-directed mutagenesis.  QuickChange Lightning Site-directed Mutagenesis is described in Figure 1 (Stratagene, 2007).  The objective of this technique is to change the nucleotide sequence of the non-insert SacII restriction site.  By changing the nucleotides in this region, the sequence-dependent SacII will no longer be able to cleave the DNA molecule at this location.

            The key to site-directed mutagenesis is in designing oligonucleotides that contain the desired mutation to serve as complimentary primers to the plasmid of interest.  In order for the primers to pair with their complimentary regions by abrupt cooling, the double stranded plasmid must first be denatured with heating.  A heat resistant DNA polymerase then extends the 3’ end of each primer.  DNA ligase links the 5’ end with the 3’ of the DNA strand containing the designed primer to create a circular DNA molecule.  Another round of similar replication must be performed to produce the double-stranded plasmid with the desired mutation. This process can be repeated with the assistance of a thermocycler to create a large quantity of mutated plasmid.

            The original plasmid, or parental DNA, that remains after the replication cycles can be cleaved and inactivated through the use of the restriction enzyme DpnI.  This enzyme has the capability of cleaving methylated and hemimethylated DNA.  The parental DNA is isolated from E. coli which methylates its DNA (Stratagene, 2007).  The mutated DNA will not be susceptible to cleavage by DpnI because it has been produced in vitro without any methylation.  The plasmid of interest can be harvested by transforming it into competent cells. 

Literature Cited:

Stratagene.  (2007).  QuickChange Lightning Site-Directed Mutagenesis Kit.  Retrieved from http://www.qcbio.com/stratagene/210518.pdf

Figure 1.  QuickChange Lightning Site-Directed Mutagenesis makes use of primers that contain the desired mutation.  The primers were designed to change a SacII restriction site into a non-cut site.  The figure is provided in Stratagene’s protocol for the QuickChange Lightning Site-Directed Mutagenesis.