Question

In an experiment, E.coli cells have been genetically modified through site directed mutagenesis. This is a process through which specific locations in an organism’s genome can be altered in order to study the function of such locations, or proteins coded for by genes found in a location. There are many techniques available for site directed mutagenesis. (You have access to PCR ingredients, any primer you wish, laboratory equipment, Lambda phage, and the ability to alter Lambda genome, a commercially available plasmid, such as Puc19, E.coli K-12, plates, media, etc..)

4. Using the tools given above, come up with a way to achieve insertion of your DNA into the genome (step by step process, no short cuts. Remember I don’t know what your experiment is, you do.)(4 points)

Answer 1

Site Directed Mutagenesis (SDM) is an in-vitro procedure wherein custom designed oligonucleotide primers are used to confer a desired mutation in a double-stranded DNA plasmid. Now, I’m writing step by step procedure of doing DNA insertion using SDM technique.

Step 1- Primer design for incorporation of an insertion into plasmid DNA

Insertions are incorporated into plasmid DNA through the use of specifically designed forward and reverse primers. Insertions less than or equal to 6 nucleotides are incorporated into the 5´ end of the forward primer while the reverse primer anneals back-to-back with the 5´ end of the complementary region of the forward primer. Larger insertions can be created by incorporating half of the desired insertion into the 5´ ends of both primers.

Step 2- Exponential amplification of DNA using PCR

In this step, the amplification of the plasmid DNA is done using the above designed primers and a mix of commercially available (compatible) DNA polymerases.

Step 3 – Treatment and enrichment

In this step step the PCR products are incubated with a unique mix of enzyme containing a kinase, a ligase and DpnI. Together, these enzymes allow for rapid circularization of the PCR product and removal of the template DNA.

Step 4 – Transformation

In this step the transformation of template DNA into chemically competent cells is done by using transformation vehicle such as Lambda phage.

Thanks!