PCR for long DNA fragment

[roniadam]

I just have a query about using the traditional PCR. If we have a long gene, lets say about 6 kb, and we want to amplify it. How can we amplify this gene using conventional PCR. I was thinking that we can cut it into small fragments using RE and then amplify these fragments using designed primers for each exon.
1. Is this idea right for long gene and normally done in the lab?

2. If my idea is right, we amplify these fragments, do we have to ligate these fragments to each other and then express the gene. OR we can just express each fragment alone?

3. What is the best and most advanced technique for DNA sequencing? Is it pyrosequencing?

[canalon]

Depnds what you want. If you want just a presence/absence kind of result, regular PCR might work without many tweakings. Just a long amplification time.
If you need to sequence your fragment, you can just use mix of polymerase optimised for "long PCR" (look for this keyword, there are plenty) which are usually a mix of regular Taq (for speed) and a slower but more accurate polymerase (like Pfu) for accuracy. That should work. Then you can clone your fragment directly without bothering to religate, And that would be much easier.

Pyrosequencing allow high throughput during sequencing, but gives much shorter read length (100-200 bp) than the Sanger method. And it is not widely available yet, so depending on your needs it is often easier to have regular sequencing done. In fact best would probably depend on your application.

[zakia]

I HAVE A QUESTION OVER THIS QUESTION. How much controlled is this reaction?

[canalon]

what do you mean controlled?

[aviprem]

For amplification of long templates, use specific long PCR amplification polymerases with proof reading activity, like Expand Hi Fi Polymerase (Roche). If you have trouble in amplifying the gene as a 6 kb single fragment , try designing primers to amplify gene as fragments ( eg two 3 Kb fragments or three 2 kb fragments).

The ligation of individual fragments can be avoided if you can use an overlap PCR strategy to splice together the individual fragments. Design primers in such a way that there will be an overlap of at least 20-25 bp between the adjacent fragments. The gel purified fragments can then be spliced together by overlap PCR to generate the 6 kb gene.

all the best

Regards
Avinash

http://aviprem.gq.nu