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This might be a stupid question!! But I will ask anyways :D

Genetics as it applies to evolution, molecular biology, and medical aspects.

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This might be a stupid question!! But I will ask anyways :D

Postby zox9xoz » Thu Jun 09, 2011 10:29 pm

I just want to make sure whether I have understood right or not.

In a single drop of blood there might be like 1 cell which in turn might have lets say 1 DNA (assuming 1 cell = 1 dna). Now this DNA is extracted from the blood sample and run through PCR.

During the PCR, this 1 DNA is broken down into lets say 10 different fragments (sequences of base pairs with different sizes) using many copies of primers, nucleotides and enzyme. And these 10 fragments are amplified to create millions of copies. So now we have millions of copies of these 10 DNA fragments of different sizes.

After this we run Gel Electrophoresis process where these millions of copies of these 10 fragments are aligned according to their sizes. We should see 10 bands in the gel with different thickness where thickness indicates the number of copies of that specific fragment. So if a particular band is thick, it means that we have more number of copies of that fragment and thin band means less number of copies.

Please tell me whether this is correct. I hope what I have written makes sense!! I am not a biology student so i just need the surface level information, like a layman.

Thanks
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Postby canalon » Thu Jun 09, 2011 10:47 pm

There would be more DNA molecules than that in a drop of blood, but since they are all identical, that does not matter. But there might not be enough to yield enough to be visualized on a gel, so yes after extraction of DNA you can run a PCR to amplify certain portions of the DNA.

This is where you turn wrong. A PCR do not break the DNA with primer, it just uses the marker to amplify some sections of the DNA. The Primers are somr sort of Bookmark that tells where the section amplified start adn finished. A more accurate and nice explication of the principle of PCR is in this movie.

Generally PCR amplify one or only a few fragment (multiplex PCR) in one reaction, although some can go way beyond either because of the awesome technical skill of the designer of the experiment (I have seen up to 25 specific fragment at one time, generally above 3 definig the right conditions to have only what you want and always have it start to get tricky), or because you use poorly (by design) specific primers.
Anyway, independantly of the number of fragment, you do not expect to see the starting DNA in the gel (too little), but the massively amplified product will show as bright bands. The intensity of fluorescence, will depend indeed of the amount of DNA (the more DNA, the more molecule of fluorescent dye it can bind, the stronger the fluorescence). But in a gel this just a rough idea of the actual DNA concentration and it is hard to get precise estimation of the DNA obtained. But you are right. Now what can influence the number of copies made in the PCR is a long post in itself. Even more so if the PCR amplified multiple bands at the same time (multiplex PCR).
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Re: This might be a stupid question!! But I will ask anyways :D

Postby zox9xoz » Thu Jun 09, 2011 11:57 pm

Hello thanks for replying and the video, but my doubt is that after PCR why do we have different sizes of DNA fragments?

Since we are amplifying only a part of a dna (target sequence) we should have all the copies with the similar sizes. Or is it that after the PCR we have millions of copies of dna target sequence (all of same size) plus fewer copies of the dna sequence which was not targeted and they are of different sizes?

For example in the video which you sent me, after cycle 3 (at 2:47) he says 6 longer length molecules and 2 target molecules. i.e their sizes are not equal. So probably after 30 cycles we will have many copies of the pure target sequence(all of same sizes) and other dna fragments which might or might not contain the target sequence and will be of different sizes.

Also since there are many copies of the target sequence, the band width in the gel should be thick.
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Postby canalon » Fri Jun 10, 2011 2:14 am

Yes there is a mix of different sizes. The template is (very) long, the product from the first step, and made from the template in the subsequent steps are also longer than the expected product (they are not as long as the template since there is only so much DNA that can be made by the polymerase during the extension step), but the vast majority are going to be the size comprised in between the 2 primers. And that is basically all you can see on the gel.
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Postby JackBean » Fri Jun 10, 2011 7:39 am

well, in each run, the polymerase starts with one primer and runs until it has proper temperature (it can usually do about 1-2 kbp) or until it reaches end of the template. Thus, if the polymerase copies the original strand, you get long strands of various lengths, but these are formed in a linear manner. On the other hand, when you use either these long or already exactly long pieces, you get always these short pieces, which you want and these are produced exponentially. Thus after several cycles there is much more of these "exact short pieces" than the long ones (and even more, the long have different lengths, so they are not focused in one band as the shot ones;)
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