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Few questions about PCR

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Few questions about PCR

Postby zox9xoz » Fri May 27, 2011 12:43 pm

Hi everyone,

I am new to this subject and had few questions about PCR (Polymerase chain reaction). I hope this is the right section.

What I have understood is that the scientists extract DNA from a sample and then they amplify a part of that DNA using PCR. If it is true then:

1. How do the scientists know that which is the target sequence in a DNA? As per my knowledge the nucleotides are connected to each other via hydrogen bonds i.e A-T and G-C. Also the hydrogen bond between G-C is much stronger compared to A-T so they try to target a part of DNA which has more A-T bp(base pairs), so that they can be broken down easily while heating. But I am not sure whether it is true or not!!

2. Are primers artificially made by the scientists. If yes, then how do they know the sequence of the nucleotides to include in a primer? Is it because they already know the sequence of the DNA target?

3. What happens after the PCR? How do they analyze the results, for example detection of a disease? Do they already have a DNA sample of a virus (for example) which they match with the DNA of the subject and determine whether the subject is infected or not?

I hope what I have written makes sense :roll:
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Postby canalon » Fri May 27, 2011 2:07 pm

1-The PCR use a 95ºC step to be sure to melt the H-bonds, so there is no special need for targetting AT rich regions.

2- Yes and yes. To do a PCR you need to know the sequence beforehand.

3- You can do a lot of things with a PCR. You can just look at the presence/absence of the product, when it appears (qPCR, because that depends on the original concentration of the template), the size of the product, or clone it, sequence it, use it for other PCR (fusion, superprimer PCR, nested PCR) or many other things.
Patrick

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Postby zox9xoz » Fri May 27, 2011 2:40 pm

Hi Canalon,

Thanks for the reply. I did some more reading here and there and this is what I have understood.

For example if the scientists want to test whether a person has HIV or not:

The scientists already know the DNA sequence of a HIV virus based on which they develop the primers called HIV primers. Now during the PCR they mix the DNA sample of the person with this HIV primer (along with enzyme and nucleotides), and if this sequence matches with the target (patient's) DNA there will be amplification which means that the person is infected. And if the sequence doesn't match then there will not be any amplification which means that the person is not infected. Am I right?? Please tell me!!!

Also, if this is true then PCR is not useful for detecting unknown diseases as the scientists don't have the sequence of the DNA of the pathogen causing that disease and hence cannot artificially make the primer!!

Uff I should have taken Biology classes seriously!!
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Postby canalon » Fri May 27, 2011 2:54 pm

For HIV,
yes and no. HIV is one of the worst example that you could have selected. The virus tends to hide in a few cells deep in the patient, and PCR usually will not detect the virus in a patient. It might be useful to see if a body fluid contained the virus (ie. is contagious) but it is generally not used as a diagnostic tool. But you are right on the principle. You mix a DNA sample that you want to know if it contains your pathogen (bacteria, virus) with the PCR reagent and specific primers, and if it is present you will likely get a band in a gel. That is what I called presence/absence PCR.
And yes, if the pathogen is unknown, PCR is of little use as a quick diagnostic tool. But it can ceratinly be used as one of the tool for the identification of a novel pathogen, provided you can isolate it first.
Patrick

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Re: Few questions about PCR

Postby zox9xoz » Fri May 27, 2011 3:15 pm

Yes, it is the principle which matters right now considering my level :) But I am glad to know that you are replying which means a lot of questions in the near future :D like Real-Time PCR. But I will ask them tomorrow after studying it by myself first.

Thanks a lot for the explanation.

Regards
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