Question about site directed mutagenesis - (Sep/09/2009 )

Hi,

I am very naive to site directed mutagenesis. I have a vector containing my gene of interest which is a receptor. I want to mutate certain amino acids in its different domains. I also have two different restriction sites on either end of my receptor. How should I go about mutating specific residues in my gene? I am aware that I need to design primers with a single base mismatch and perform PCR. But

1) How would I know my gene has been mutated?
2) The PCR product is going to be a small segment of my gene, how can I make sure I get my entire gene with the mutated bit inside the vector?
3) Is it mandatory to use kits availabe?

Thanks!
Help much appreciated

cupidstunt on Sep 9 2009, 01:10 PM said:

Strategene's site directed mutagenesis kit is great. I highly recommend it. It is easy to use and I, and others in my lab and in my previous lab, have had 100% success rate with it.
For your primer design, use the following considerations (from the manual)
♦ Both mutagenic primers must contain the desired mutation and anneal to the same sequence on opposite strands of the plasmid

♦ Primers should be between 25 and 45 bases in length, with a melting temperature (tm) of ≥78°C. Primers longer than 45 bases may be used, but using longer primers increases the likelihood of secondary structure formation, which may affect the efficiency of the mutagenesis reaction. The following formula is commonly used for estimating the Tm of primers:
Tm = 81.5 +0.41(%GC) - 675/N - % mismatch
For calculating Tm:
• N is the primer length in bases.
• values for %GC and % mismatch are whole numbers

♦ The desired mutation (deletion or insertion) should be in the middle of the primer with 10–15 bases of correct sequence on both sides

♦ The primers optimally should have a minimum GC content of 40% and should terminate in one or more C or G bases

As for your PCR products, the kit works by PCR's your entire plasmid the mutant primers, then digesting the nicked template plasmid with DpnI.
You can check for the mutation by sequence analysis, or, if you are clever, you can engineer your mutation in such a manner that you introduce or remove a restriction site on/from the wild-type plasmid, thereby allowing to check for mutation success with a simple restriction digestion of your clones.