Recombinant PCR - (Aug/25/2006 )

I have a problem with recombinant PCR. I know there are many variants on this theme, so let me
explain what I am attempting to do. I am using recombinant PCR to generate
a chimeric gene construct...specifically in the first round of PCR I amplify
the two distinct halves of the chimera, e.g. DNA A and DNA B. I phenol
extract, ethanol precipitate or gel isolate. I should note that DNA A and
DNA B have a common overlap sequence at one end of each molecule (10-15
nucleotides). In the second round of PCR, the two pieces are allowed to
anneal and then extended by the polymerase. After 1-5 cycles, primers
complementary to the extremities of the new chimeric construct are added and
amplification allowed to proceed for 25-29 cycles.

My problem is with the second round of PCR (I have no problem with the first
round of PCR and I get good yields of a single product). In the second round
of PCR my problems include the following: 1) no amplification OR a smear.

Any input on procedure, conditions, would be greatly appreciated.

Thanks in advance.
DIVYA

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I'm well versed in this technique...

Allow your two 'halves' of the chimera to amplify for 5-30 full cycles. Dilute this product and use it as a template for your final amplification rxn with primers. Use an extension time that's long enough to favor the final chimera.

The 1-5 cycles may not be enough to create sufficient chimeric template for the final amplification.

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HI,
thanks for the suggestion. i'll definately try it but do you mean that i should allow them to amplify without any primers for say about 20 cycles. Also note that i have to keep the extention time to minimum 2.5 minutes coz my final recombinant amplicon is 2.7kb.

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>2.5 is not a very good idea. For a 2.7kb amplicon I'd go up to 3.5min. I've used this technique for 5-7kb constructs before.

You are correct . Try the second amplification step without primers. I've done it with and without primers and its worked. The real key is to not carry over any first amplification primers (those used to amplify each half)
Good luck.

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I do things a bit differently. I do normal PCR of the two fragments, then 1000x dilute the reaction and do two PCR reactions with only a single primer -- the outside primer. I cycle 8 cycles. This produces linear amounts of single stranded DNA of the size of each half-product. I mix these two reactions together, giving a normal PCR reaction with outside primers and template formed from the extension and overlap of the single stranded pieces.

But I think your real problem is that 10 bp overlaps are not long enough. The overlap should be the same size as a good primer -- 18-24 bp. Any of these schemes will work if the primers are right.

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Well I agree with vasussci.

I also allow the fragments to join for 15 cycles and further 30 cycles with pull-through primers. The first time I did this experiment I prepared about five different dilutions of the 15 cycle product and choose the one that yielded high amount of PCR product.

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