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How two fragments joint together when doing fusion PCR?

Fusion PCR

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4 replies to this topic

#1 Johnson Wu

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Posted 12 August 2014 - 06:03 AM

As everyone knows that fusion PCR is an efficient way to joint two fragment when doing cloning.
But I always have a question. How new strands joint with the old one? There are suppose a nick between them. (as attch files)

Thank you all smile.png

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#2 Trof

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Posted 13 August 2014 - 06:13 AM

You mean in a plasmid? They don't. They stay nicked. Bacteria will join them after successful transformation. The bigger the fragment inbetween, the better chance they will stay nicked and transform. Otherwise they may of course detach


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#3 Johnson Wu

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Posted 13 August 2014 - 09:25 AM

Hi, thank you for the reply. And sorry about that I don't type my question clearly.
My question is mainly in the "PCR REACTION".
And my concern is that, if the nick still exist after finished the first extension (PCR program),
we will only get gray fragments(as picture) since second extension (and in the next 28 runs).
So we will never have the fusion fragment.(? 



#4 Trof

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Posted 13 August 2014 - 10:06 AM

I don't get the picture much, to be honest, grey fragments mainly.

 

What do you have on the start? The red and blue fragment, right?

 

So, when you align primers, they will complement the red fragment completely, as well as the blue one.They don't stop at the "Here" marker you made there, because at the time of elongation, the red and blue fragments won't be aligned, they will still be denatured (because of course I suppose they are double stranded in the first place).

 

I think you don't have the right picture.

 

300px-Splicing_by_Overlap_Extension_PCR.

 

(taken from Wikipedia OE-PCR page, which is essentially the thing you do)

 

You can see, the compatible single strands of both fragments here anneal and since they have intact 3' end, the 3' end serves as a primer to complete the whole "fusion". The fused sequence is then further amplified by the "outer" parimer pair.


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#5 Johnson Wu

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Posted 13 August 2014 - 11:26 PM

yeah, thanks Trof smile.png  , I got it.
I just confuse with 5' and 3' end, and now I known that.







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