Mutagenesis+Summary

GN's Bioinformatics page GN 2009 - '10 Logbook

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Generating mutations lies at the heart of my project. It is the summation of all of my research thus far and the key to unlocking just how NPC2 functions. As stated elsewhere, generating mutations in areas which we think are structurally and functionally significant and observing the results is the most logical and efficient approach to determining the function of the different faces of NPC2.

Mutagenesis is undertaken by completing four basic steps: Plasmid preparation, temperature cycling, digestion, and transformation. In these four steps, a bacterial plasmid (in our case, of e. coli) is combined with an artificial oligonucleotide* primer, via temperature cycling. Digestion removes the methylated* parent strand, and the resulting vector is placed in the vicinity of (in agar with) bacteria, which every once in a while (about once in a million) uptake the plasmid and produce large quantities of the coded mutant protein.
 * These definitions are found under the DNA section

Step 1: Plasmid preparation/ Primer development

 * The initial step is to prepare the primer that contains the desired mutation. The method by which it is added to the plasmid will be explained in step two. However, one must identify the region that one wishes to mutate.
 * To begin with, we have chosen a lysine - a positively charged amino acid. This particular lysine is located in an evolutionarily conserved region of NPC2 on the surface of the protein. This leads us to believe that it is functionally significant. We will experiment with changing it to a hydrophobic (uncharged) amino acid, as well as to a negative one and a different positive one.
 * Having isolated the target amino acid, we can begin selecting the primer. Creating the primer itself will be left to a hired company, but they will generate it according to our standards. A primer is recommended to be 25-40 nucleotide bases long, with 10-15 bases on either side of the area to be changed. Concerns when making the primer include melting temperature and secondary structure complications. The melting temperature of the primer is itself composed of two main factors: GC content and percent mismatch - how large the mutation is. GC content, or Guanine-Cytosine content, is important because it affects the melting temperature of the primer. Bonds between Guanine and Cytosine are triple hydrogen bonds, and therefore require more energy to break than Adenine and Thymine, which have only two hydrogen bonds. Proper GC content is around 40%. Secondary structure complications can arise particularly in longer primers, which can twist and bond with themselves. This of course makes it far less likely that successful bonds will be made with the target plasmid.
 * The final factor is that two variants of the primer must be made - ones to anneal to opposite strands of the plasmid. One must be made for the Watson strand and one for the Crick strand (i.e. one for the 5`3` and one for the 3`5`).

Step 2: Mutant strand synthesis reaction (thermal cycling)

 * Herein lies the concern of temperature. In order to anneal the primer to the plasmid, the double helix of the plasmid must be unwound. This is accomplished by breaking the hydrogen bonds connecting the helices to each other; the Guanines to the Cytosines and such. Rather than do it via enzymes, it is far easier to simply heat the entire batch of plasmids, which provides enough ambient energy for the bonds to be broken.