Amplify a large sequence of DNA.

[sharik]

Im working on Alpha thalassaemia. it is 4 Kb gene and my target fragment is on 2 kb.
but i'm getting bands below 1 kb, and i have run my thermal runs and getting same results
please help me out.

can someone tell me PCR buffer recipe for larger Fragment of DNA ?

Regards
Sharik Ahmed

[mchlbrmn]

What does "thermal runs" mean?
If you are using taq polymerase, it can usually handle this, but some specific sequences are more difficult than others, and perhaps a different polymerase will work better. You could try a polymerase that specifies that it can PCR up to 5 or 8 kb. Proofreading polymerases, Phusion, Pfu, etc, or a taq polymerase "long pcr" mix might be worth trying.

For using the polymerase you already have, you can try the usual optimizations:
First, a couple of new primer sets can be tried, or used nested if they fail individually.
The anneal temp can be adjusted up (probably) and/or down, depending on the primer Tm.
The extension time should be ample.
Added cosolvent helps for some templates. Betaine, or 3-10% DMSO can be tried, also glycerol, and formamide work in some cases.
Various of these variations can all be run at once (particularly if you have two machines, or a gradient machine) and run on a gel.
In terms of buffer itself, the only thing I do is use the alternate "GC Buffer" that my Phusion polymerase provides (contains a cosolvent, I assume), or I used to try changing the Mg concentration, but I stopped messing with that.

[tluebke]

For better results you can lower the Elongation temperature to 68 °C. I would recommend you TaKaRa LA Taq. It always worked fine for long amplifications up to 5 kb.

Have you checked your primers for cross reactions? This could be another reason. What is your template? Pure Plasmid-DNA or a whole genome? You can blast against your target sequence.

[CrowSan]

Just wanted to say that for long fragments give a 1 min extension time per kb. (i.e. 2 kb = 2 min extension).

[sharik]

Thanks for your support.
I meant, I have run multiple PCR reactions.

@tluebke.. im working on whole genome.

and im using Accuprim Taq polymerase of Invitrogen company, it can amplify upto 4 kb.
i am using this recipe( 200um dNTP, 1.9mM mgcl2, 10% DMSO, 67 mM tris HCL, 16.6mM NH2So4, 66uM EDTA, 10mM betamarcaptoethanol, and BSA). if i need some amendment please correct me, but this recipe it isnt working.
im not getting where is problem :$

and i will follow your instructions. im beginner how can i check cross reaction ?
and one thing more my amplified product would be act as DNA for nested pcr ?

[mchlbrmn]

EDTA!! Was that a typo? EDTA would remove all the Mg and cause a negative reaction. But, you got smaller bands, so maybe it's a typo?

Invitrogen gurarantees the enzyme, so why don't you call them and turn the problem over to their eager hands. Invitrogen web site:

PCR so reliable, we guarantee your results. Get a successful PCR reaction the first time with AccuPrime™ enzymes and primers from Invitrogen. Put an end to waste: no more rework or optimization and repeat reactions. Start getting used to PCR you can count on. And if something should go wrong, we’ll troubleshoot and make it right. Even redesign and synthesize primers at no charge.

[sharik]

@ Thanx mchlbrmn

yeah My sample type is EDTA..you are right but its very difficult to call invitrogen company because we are belong to Asia , we have their distributor but dont have support.
and it would be easy to shift on Pfu taq.
one thing more i wana show my gel picture how can i send my gel pictures ?

[mchlbrmn]

I think the "Img" button above is to post images, or you can paste a link to photograph web site.
So, you have pfu? I've used variants of pfu from Stratagene/(Invitrogen now) that can do up to 15 or 20kb, they say, but check the instructions with your enzyme to see if it's good for large sizes (although 2kb is not large, maybe you should treat as if is is larger since the sequence may be troublesome).
Oh, I thought it said 66mM EDTA, but now I see it's uM, so that shouldn't chelate more than 0.13mM Mg, so that probably won't block the reaction, but I don't know why EDTA is there, unless it's leftover from an upstream procedure.
You could take a couple ul your PCR product into a new nested reaction and PCR more cycles if the new primers are within the PCR product (although this will increase the chance of mutation by increasing the cycle number).
Why are you not using the Accuprime buffer and thermostable protein?

[mchlbrmn]

It just occurred to me, is it possible you are PCRing a 2kb cDNA size DNA from the genome? If so, introns could make it far larger, and impossible to PCR.
If you would like to share the primer sequences, and the PCR conditions, we might have suggestions on the conditions.
The problem may not be related to the size or the enzyme mix, but could be the PCR cycling conditions for the primers

[Labrat_5]

Hi Sharik,

What do you know..I also work on alpha thalassemia. As far as trying to amplify a large region and only getting the smaller fragments you have mentioned, I suspect the problem is the specificity of your primers. The alpha globin gene cluster is extremely G-C rich with repetitive elements scattered throughout the region from zeta globin to theta. Alpha globin gene region has homologous duplicated regions in and around both alpha 2 and alpha 1, specifically labelled as X, Y and Z. That is why most mutations that cause alpha thalassemia are mainly due to deletions. We use a modified protocol of Dode's paper (Brit J Haematol 1990 76(2):275-281) to amplify the genes and with great success. I suspect trying to amplify large segments by long distance PCR regardless of DNA polymerase used will be futile since you will have to deal with many pseudo-genes, repetitive regions, alu repeat units etc. We don't used the buffer system you mentioned instead we use betaine/5% DMSO combination mchlbrmn suggested.

[sharik]

@ labrat...
i got some results but i want more good results can you email me Dode's paper (Brit J Haematol 1990 76(2):275-281) ???
i didnt find on search engine.

Thanks im looking forward your reply.