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How to amplify 4.1Kb gene from genomic DNA? - (Nov/13/2007 )

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Hello Mol. Bio. Experts

I need to amplify a gene that is 4.1kb long (no introns) from genomic DNA.

Preliminary PCR amplifications have failed.

I am trying to amplify it as 3 fragments with overlapping ends and then pool them together and do another PCR with end primers.

Does anyone have a tested, proven and simple alternative to amplifying such large genes?? special polymerases?? solvent conditions??

thanks very much in advance.

Rami

-brami-

Use Phusion polymerase. It is a protein fusion of Taq and Pfu so it is robust and accurate and is easily the best polymerase on the market. I would also suggest BLASTing your primers in order to make sure that the 3` end of each primer is only going to anneal to your target of interest. There is so much sequence in genomic DNA that it is very easy for the 3` end of you primers to anneal to secondary sites. If the sequence is GC-rich, add some betaine (final concentration 0.5 - 2.0M) to the reaction. It will help dissociate the strong GC hydrogen bonds.

Good luck,
Rob

-killerkoz17-

If your primers are not working, then try a gradient PCR for annealing temperature. I nothing works, then redesign the primers. Life is too short to try to optimize reactions with poor primers, and their cost is low. I agree that betaine is useful for high GC. For very low GC, lower the extension temperature.

-phage434-

you can use the EXPAND LONG RANGE Taq Polymerase from ROCHE. In addition you could use DMSO in your reaction tube. good luck!

-vit72-

Overlapping PCR is making things more difficult, try using "long template" enzyme mixes, most companies have one. 4,1 kb is not too large to amplify.
Are your shorter PCR's positive and do you have any control for the quality of your DNA?

-vairus-

I agree with killerkoz17 use Phusion polymerase and try the amplification.
All the best.

-newarray-

QUOTE (vairus @ Nov 15 2007, 01:34 AM)
Overlapping PCR is making things more difficult, try using "long template" enzyme mixes, most companies have one. 4,1 kb is not too large to amplify.
Are your shorter PCR's positive and do you have any control for the quality of your DNA?

Yeah, 4.1 kb is borderline for me. Definitely attempt it but if it doesn't work well go straight to amplifying it in two or three parts. Splice overlap isn't that difficult. Design the overlap regions so that they contain unique RE sites (unique for the insert) in them and you can do 3-way or 4-way ligations as a back up if the splice overlaps are proving difficult.

-killerkoz17-

QUOTE (killerkoz17 @ Nov 14 2007, 05:31 PM)
Design the overlap regions so that they contain unique RE sites (unique for the insert) in them and you can do 3-way or 4-way ligations as a back up if the splice overlaps are proving difficult.

if you insert the restriction sites you will be altering the sequence and, if in the translated part of the gene, altering the product.

the intent of overlapping segments is to bind complementary ends to each other and extend along the resulting construct (in both directions) finally reproducing the gene with the desired sequence.

-mdfenko-

I agree with mdfenko that putting in unique Rest. enzyme sites to generate fragments and then stitching them together will be very tricky, unless one can find unique sites within the gene.

best is ofcourse, if a polymerase can amplify the whole 4.1kb.

otherwise, it seems like one will have to do it in pieces and put them together in an overlapping pcr approach.



QUOTE (mdfenko @ Nov 15 2007, 08:49 AM)
QUOTE (killerkoz17 @ Nov 14 2007, 05:31 PM)
Design the overlap regions so that they contain unique RE sites (unique for the insert) in them and you can do 3-way or 4-way ligations as a back up if the splice overlaps are proving difficult.

if you insert the restriction sites you will be altering the sequence and, if in the translated part of the gene, altering the product.

the intent of overlapping segments is to bind complementary ends to each other and extend along the resulting construct (in both directions) finally reproducing the gene with the desired sequence.

-brami-

QUOTE (vit72 @ Nov 14 2007, 06:26 AM)
you can use the EXPAND LONG RANGE Taq Polymerase from ROCHE. In addition you could use DMSO in your reaction tube. good luck!



Used this many times with genomic DNA to make products as long as 30 kb. Haven't done it in a while, but I recall that I usually set up two tubes - one with extra Mg++ added and one without. One or the other always worked.

-smu2-

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