I have a gene of interest approximately 2000 bp long coding for a protein approx. 670 amino acids in length. I wish to excise one of the proteins domains from the gene. To do so I need to remove about 350 base pairs from the gene in the middle.
what is the smartest way to do this?
My original thought was to amplify the beginning region of the gene, and the end region of the gene and adding restriction enzymes to both ends and then ligating them together. However this will introduce some unwanted base pairs from the restriction sites in the middle of my gene? I believe there are better techniques to accomplish this?
Hi there,
Applying overlapping PCR technique allows you to perform deletion with 2 PCR reactions. See the following:
http://www.methods.info/Methods/Mutagenesis/PCR_splicing.html
Primers 1 and 4 can be the primers initially used for the cloning of the full length gene.
Hi there,
Applying overlapping PCR technique allows you to perform deletion with 2 PCR reactions. See the following:
http://www.methods.info/Methods/Mutagenesis/PCR_splicing.html
Primers 1 and 4 can be the primers initially used for the cloning of the full length gene.
Thank you for your responses.
Dominique I was coming to say that I had found just that technique, thank you. I found a paper: Gene splicing by overlap extension: tailor made genes using polymerase chain reaction by R.M Horton in BioTechniques that describes that very method. Such an ingenious and yet ultimately simple solution.
Please tell me whether this technique worked. How would the second round of amplification work. Though this looks the only possible way for deletion mutant.
Even if I am very late with my answer I would recommend the system applied in NEB's Q5 site directed mutagenesis system (https://www.neb.com/products/e0554-q5-site-directed-mutagenesis-kit#Product%20Information). For a deletion you just create two primers. The forward aligns at the sequence immediately following the deletion and the reverse primer immediately before the deletion. You run one PCR that creates one single fragment (gene fragment-vector-gene fragment). The ends are then treated with kinase, ligase and DpnI (KLD mix, all in one step). Super easy, super fast. Primer design is also very easy because NEB offers a tool on its website. And no, I don't work for NEB.
This is also coming late, but perhaps someone might later find it useful...
There are two ways you can go about it:
1. You can amplify the two fragments of interest and the vector backbone (into which you intend to eventually clone the resultant gene), with each fragment having a 5' overlapping region with the adjacent fragment. Then use a recombinase (we use Exnase II in my lab) to put the constructs and the vector together in a homologous recombination step before transforming the product into the desired competent host.
2. As it had been suggested in previous answers, you can use overlap PCR to join the two terminal fragments by designing primers to first amplify each fragment separately to have 5'-3' overlap, and then joining them in a second PCR (primerless). A third PCR step then amplify the joined product using the F-primer of the first fragment and R-primer of the second fragment. Then you clone the PCR product in a recipient vector followed by transformation...
The 1st approach seems easier and time-saving that the 2nd though...
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