Those are really long primers, without much complexity... Other than the standard PCR tricks, which you already seem to be trying already, and trying different polymerases, etc, it's going to be a difficult experiment. There's all sorts of potential problems that can be happening -- mispriming, megapriming, hairpins, etc.

The easiest way out would be to use a synthetic gene synthesis service to make the offending region for you. This may also be the most cost effective approach, considering time, reagents, and sequencing runs that are going to be spent to make a standard PCR approach work.

You may be able to limit the size of the synthesis to 2-300 bp (if there are convenient restriction sites flanking this region); you can have the new fragment containing the modified sequence synthesized and subclone it back in to the parent vector.

Thanks for your Input Thomas. I can not go with the synthetic gene cause i have to repeat it with 3-4 other genes also. That's why i am looking for a solution which i can apply using PCR. It would be more convenient for me. Mispriming and megapriming could be happening but i am sure that hairpins are absent.

I am not understanding why there is no amplification when i am using the 1.116 kb gene fragment which is gel excised/PCR purified while i am getting a weaker band when the gene is in some expression vector. Puzzle....

There is no secret for your PCR not to succeed. I did check the sequence of your GS linker and it is OK. I guess your gene specific primers are OK. I don’t know exactly how you are performing your PCR, but I presume you are setting 2 separates PCR to amplify the 2 domains of your gene to which you add the linker from both sides of the junction. If you get the 2 fragments you will proceed to the second step PCR to generate the whole cassette.

Normally, you should also optimize the gene specific primers. Try to add additives and play with PCR conditions.

1/ Betaine (from Sigma) 1.5 to 2.5 M improves PCR especially in those high GC rich stretches. Some people add DMSO but I don’t really like it.

2/ Change your polymerase. I use to perform fusion PCR with platinum Pfx from Invitrogen (relatively cheap) which has strands displacement capacity and proofreading activity, but some polymerases from other suppliers also did work (Not all).

3/ Increase your annealing temperature above the melting temperature of your primers. I think 50 oC is low. Try 55 at least.

4/ Play a bit with MgCl2 concentration.

5/ use plasmid as template. The less PCR you perform less mutations in your cassette you will get.

Thanks Abdelhalim for your kind suggestions. Yes, i am performing two independent PCR to amplify each side of the linker and then will go to the second step PCR where i will use the gene specific primers to amplify the whole cassette.

Some of your suggestions I have already tried. But, definitely i did not try the polymerases of different sources. I am planing to set up some more PCR reactions and will let you know if any success achieved.

I welcome other suggestions also from RG members, if they have any.

Regards

-Yogendra

There are many ways suggested to improve PCR. But my first suggestion is "change enzymes". Changing Mg concentration? Maybe it works, but forget it. You might do too many conditions and get nothing. As somebody suggested, the annealing temperature is too low for such long primers. I would use shuttle PCR (anealing and extention together at 68 degrees, and denature at 94 to 98). If you can not get any bands by this, try first 3 cycles at low anneling temp, and after that apply shuttle PCR. The extension times you are using may be too long because 1min is enough for 1kbp. Especially when you get none specific bands at higher sizes than you expected, a shorter extension time could reduce such long nonspecific bands.

Finally, be careful about the amount of templates. Too much can cause troubles. 1 to 10ng would be enough, and you can decrease further.

And betain and/or DMSO could improve your PCR. It might be good idea to add them always. So you do not need to repeat PCR because you get none specific bands without them.

Thank you very much Kazuo Fushimi.

The shuttle PCR sounds interesting as i never did it. Can you please help me to write the program for this PCR. Please give me the exact sequence of program in which i may change the temp conditions as per my requirement. Please elaborate it little a bit so that i can understand it in better way. I am sure that it will not only help me but also help the others who are following this question, if they face such kind of problem in future.

step 1: 94 to 98 - 3min denaturing temp depends on your enzyme see the product sheet

step 2: 94 to 98 - 15sec (30sec would be fine but you can save your time)

step 3: 55 to 60 - 10sec (the shorter, the stricter, but the opposite can be also true)

step 4: 72 - 30sec

4 cycles of step 2 to 4

step 5: 94 to 98 - 15sec

step 6: 68 - 35sec

25-30cycles of step 5 to 6

72 - 10min

4 cycles of step 2 to 4 is optional. Initially 21 bases of your primers anneal to your templates, therefore 68 degrees may be too high. Once you get extention from primers, the whole sequences of your primers would anneal, so that 68 degrees would work.