Cloning by PCR and Mutagenesis

Appreciate that there are different approaches to the cloning of specific genes.

A moment of silence here while we appreciate all the different approaches...

Okay, back to being serious. There's really just two main approaches (well, at least only two that we learned about during the lecture). They both start off with figuring out what protein you want to clone, determining some of the N-terminal amino acid sequence and from there taking a stab at what the potential DNA sequence would be (presumably taking into account that some amino acids can be coded for by more than one codon).

From there you can take one of two approaches, depending on what technology is available to you. Since several different genomes have already been sequenced, the easiest approach nowadays would generally be to search the database for the gene of interest. You might also be able to find similar genes coding for other proteins, and from there you might be able to get some kind of idea of what your protein does. To be a bit more certain, though, you can amplify the gene (and therefore protein production), allowing you to study the protein more and find out more about it. To amplify the gene, use the database to help you design some oligonucleotide primers so that you can use PCR (more on PCR in an earlier post). This amplified DNA can then be cloned into an expression vector (see the end of my most recent post) to create lots of protein.

An alternative approach can be used if you don't have access to a database for searching for genes. After working out a potential DNA sequence, you can make a DNA probe using this sequence data. This probe can then be used to find the gene of interest in a gene library (more on gene libraries in a bit). Once you've found your gene, you can clone it and so forth.

Be familiar with cDNA library construction and traditional screening by hybridisation.

cDNA libraries can be bought commercially, but just in case you wanted to make your own, I'm gonna tell you how (or rather, the theory behind it anyway). *Do not try this at home*

cDNA is essentially the complement of mRNA. To create cDNA, simply use the enzyme reverse transcriptase to transcribe the mRNA into DNA. Remove the mRNA with alkali and add a poly-G tail (this is so you can add a poly-C primer for synthesis of the other strand). Synthesise the other strand using DNA polymerase.

Next you have to do a couple more fancy things with the cDNA. Firstly, you have to methylate it so that restriction enzymes won't cut it where you don't want it to be cut. Secondly, you have to add EcoRI linkers to either side (essentially the "cutting sites" for EcoRI). Cleave them with EcoRI to form sticky ends.

Now for the recombination part! Take some bacteriophage lambda (a bacteriophage is essentially a virus that infects bacteria) and cleave its DNA with EcoRI. Ligate this to the cDNA. Now you can package this cDNA into bacteriophage lambda so that it can go ahead and infect EcoRI with some shiny new cDNA!

cDNA libraries can be used to screen by hybridisation of a probe, as alluded to in the previous section. Firstly, the library has to be plated out and a nitrocellulose membrane placed on top. The colonies or plaques will be transferred to this membrane due to the binding of DNA. Place the nitrocellulose membrane in a plastic bag along with a solution containing a radioactive probe (I don't know how long for, sorry :P). Later wash and radiograph the nitrocellulose paper to find the location of the radioactive probes, which should be located in the same place as colonies containing the gene of interest. These colonies can then be removed from the agar plate and cultured in nutrient broths.

Understand the principle of PCR.
Know how PCR can be used for the cloning of specific genes.

See my previous post on PCR.

Be familiar with how PCR can be used for site-directed mutagenesis. 

One way of testing out how a mutation affects the function of a protein is to create a primer with a mutation in it. The mutation can be located almost anywhere in a primer, except for the 3' end, because that has to fit to the DNA well for synthesis to proceed.