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Nested PCR

Nested PCR - PCR - Polymerase Chain Reaction Forum

Nested PCR - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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  #1  
Old 09-19-2008, 03:03 PM
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Default Nested PCR



I am quite new to PCR....
Want to know why in Nested PCR a second set of different primers are used.... why don't the primers that are initially added work?
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Old 09-19-2008, 04:25 PM
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Default Re: Nested PCR

In nested PCR, you use two sets of primers. This is done so that the reactions work in conjunction to produce a detectable amount of PCR product (visible on stained gel) from either a very small amount of starting DNA or of a DNA product that is rare (present in few copies).

I personally used a lot of nested PCR to detect infectious pathogens (like viruses); so in the initial PCR reactions a lot of the DNA belonged to the host and very little DNA belonged to the virus; this first PCR run usually didn't produce visible bands on a gel. But the second nested PCR worked extremely well and made the sensitivity (and specificity) of detecting the viral DNA very high.

Another reason for using nested PCR is that it improves the specificity of the assay, since you have four primers amplifying the same DNA segment. Very useful when you have sequences with a lot of homology that result in non-specific bands.
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Old 09-21-2008, 07:28 AM
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Default Re: Nested PCR

Thanks a lot. I was also wondering why in nested PCR the amplification doesn’t occur (why the reaction terminates) when the same set of primers are added in the second stage.
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  #4  
Old 09-23-2008, 08:32 PM
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Default Re: Nested PCR

Quote:
Originally Posted by Sharika View Post
Thanks a lot. I was also wondering why in nested PCR the amplification doesn’t occur (why the reaction terminates) when the same set of primers are added in the second stage.
It does occur if you have good quality target DNA in the first place. But this is in an ideal situation. You can verify this if you have a positive control, known positive etc.
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Old 11-27-2008, 05:03 PM
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Default Re: Nested PCR

I wanna ask you somebody. Can we go to the real time pcr with stage I Nested PCR product? I think we get the problem with stage 1 Nested PCR primers...
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Old 11-27-2008, 09:50 PM
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Default Re: Nested PCR

Yes, you can do real-time PCR on the first-stage nested PCR product, so you can perform the second stage of nested PCR using real-time PCR.

This is often done so when co-amplifying IAC (internal amplification control) with tested sample - first stage of nested PCR is performed (on IAC) and after the first stage longer product is used as an IAC template for the real-time PCR.

The most important thing - you have to have some product after the first stage of nested PCR to work with.
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Old 11-28-2008, 08:23 AM
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Default Re: Nested PCR

You are absolutely right,I have some real time PCR products with different length...
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Old 12-28-2008, 09:27 PM
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Default Re: Nested PCR

I wanna ask you a question about low copy cdna analysis by the taqman probes. I found the my gene amplification with nested PCR. Do you think I can succes with taqman probe, too? not nested Taqman PCR
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Old 12-30-2008, 10:28 AM
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Default Re: Nested PCR

Quote:
Originally Posted by omerkacar View Post
I wanna ask you a question about low copy cdna analysis by the taqman probes. I found the my gene amplification with nested PCR. Do you think I can succes with taqman probe, too? not nested Taqman PCR
Give it a try
I suppose that if you need to have nested PCR your taqman probes will result in very low amplification and efficiency of the reaction. But if you don't try you'll never know.
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  #10  
Old 12-31-2008, 01:34 PM
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Default Re: Nested PCR

I think I will not give any expressio data with Taqman probe...
But I have to try it....
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