Monday, October 15, 2007

Mutating the ICAP binding site

In my last post I reasoned that I should introduce mutations into the perfect CRP binding site, called ICAP, to measure HiCRP’s preference for certain bases at certain positions within the site. This past week I did just that (I hope). However, the mutagenesis procedure presented some technical difficulties, prompting me to experiment with a new approach.

The big problem is that the ICAP site is a perfect palindrome (see the DNA sequence in the figure; the vertical dashed line shows the axis of symmetry) and the vector sequence on either side of the ICAP clone is also palindromic, resulting in a palindrome >40bp long. The problem arises because mutagenic primers are normally designed to be perfectly complementary and extend by >20 bases on either side of the mutation being introduced. I want mutations near the axis of symmetry, resulting in primers that in most cases are themselves perfect palindromes (purple primers in figure, labeled as “old method”; not to scale). Thus, it is highly favourable for primers to form hairpins and become double stranded along their entire length; these intra-molecular bonds form more readily than do inter-molecular bonds with template DNA.

Using pcr, the mutagenic primers generate full-length linear amplicons from plasmid DNA. Complementarity between the primers allows the newly synthesized sequences to anneal as double stranded circular molecules with staggered nicks, which are repaired by the host bacterium after transformation. I reasoned that the complementarity is key, but the mutagenic primers do not have to overlap for their full length. Thus I designed primers (green in the figure; not to scale) that have a region of complementarity with one another, but only one primer has the mutation and the other does not overlap this region. This allows me to design primers that contain short palindromes, thus greatly increasing their preference for annealing to template DNA and reducing the strength of intra-molecular pairing.

I was pleased to later discover that a biotech company now markets a mutagenesis system that uses the same approach of staggering primers, giving me confidence that this approach would work. I got the satisfying result today: my positive control of introducing a stop codon in pSU20’s lacZalpha gene had a 75% success rate (ie. 75% of colonies were white when plated on Xgal). This is lower than the >95% success rate I enjoyed with the old method (which used mutations in pUC18’s lacZalpha as a positive control), but all I care is that I circumvented the primer hairpin problem and got mutants. Unfortunately, my positive control mutagenesis is not exactly like my ICAP mutagenesis, so I will need to wait for sequencing results later this week to know if the experiment really worked.


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