Perhaps this would be helpful. I assume that for a GS-linker you're going to be using an amino acid sequence like (Gly-Gly-Gly-Gly-Ser)n or something like that. One possible codon for glycine that you can use is GGA and one possible codon you can use for serine is TCC. Put those two together and you get GGATCC. Look familiar? That's a BamHI site!

There are a lot of ways to work with this but off the top of my head the most convenient thing I can think off is simply doing two separate PCR's with two sets of primer

For gene A:

-->Forward primer = (optional overhang sequence if you want to clone this PCR fragment into a vector, an overhang sequence could be an enzyme site that matches the MCS in the vector) and homology to the beginning of gene A

-->Reverse primer = overhang sequence containing the COMPLETE G-S linker + homology to the end of gene A

For gene B:

-->Forward primer overhang sequence containing the COMPLETE G-S linker + homology to beginning of gene B

-->Reverse primer overhang sequence (optional overhang sequence if you want to clone this PCR fragment into a vector, an overhang sequence could be an enzyme site that matches the MCS in the vector) and homology to the end of gene B

After doing the two separate PCR's and purifying the PCR fragments, simply digest each PCR fragment with BamHI + (optional, if you have enzyme overhangs to clone these fragments into a vector, also digest it with this enzyme too), run it on a gel and PCR the PCR fragments. Then simply perform a ligation.

I'm sure you can adapt this or change this into whatever suits your purposes but I feel like this should be an easy way for your to join two pieces of DNA separated by a GS-linker

Hi there,

The primers have to be designed carefully as stop codon has to be removed for gene A and the reading frame has to be conserved between A, the linker and B.

Hi Ying Lam Tang I assume that you have gene A without stop codon in your plasmid, if not, you should amplify it again without it. Next for your SOE PCR toy should design one primer 5' gene A, second primer 3' gene B, and check your reading frame to avoid any unwanted stop codon. For the PCR, increase the extension time for your final product, since you will have 1.7 kb, I would try with 2 minutes to be safe, depending on your polymerase (check the manufacturer's directions).

Cheers

Hi @Ying Lam Tang

The paper you have referenced is best for overlap extension PCR cloning (a form of megaprimer PCR) for insertion and replacement of DNA sequences... However, for an ideal (splicing by) overlap PCR for joining fragments of DNA sequence, I have attached herewith two very useful papers. Actually, they are almost similar, but going through the two will give you a clearer picture of the steps.

Hint: You can design primers to amplify the two fragments A & B with

- the 5'-end of the reverse primer of A having the (complementary) linker sequence as an overlap (15-18 bp or more depending on how long your intended linker is). You can add complementary bases from the beginning part of fragment B to have the overlapping region as long as possible.

- the 5'-end of the forward primer of B having the linker sequence as an overlap appropriately complementary to that of A. The same story as above apply here.

Amplify the fragments separately in the first PCR procedure, then join the fragments in a primerless second PCR procedure. You then amplify the linked fragments in a third PCR using the forward primer of A and the reverse primer of B...

Note the precautions of stop codon exclusion for fragment A, and inclusion of your desired unique restriction sites on the 5'-end of A and 3'-end of B, as well as proper maintenance of the reading frame...