A question about DNA / PCR

Discussion in 'Chemistry' started by Michelle Redford, Sep 28, 2008.

  1. Michelle Redford Registered Member

    A relative who is in college asked me to help him with an essay he's supposed to do... since he knows I like science and I'm supposed to help him.
    He's writing about DNA (basically, from A to Z). The basics aren't really a problem, but...
    There's a chapter where he's supposed to write about DNA modifications (like are they possible, how are they done, etc). I came accross this article which is about a research on our subject. I understand the whole article, except for this part: "It's all done with PCR..." What is PCR? I googled it and found this on Wikipedia. I read it and I think I understand... but I have a question: Can PCR be done on living humans?

    Sorry for the long post. I hope I explained it nicely... I appreciate any help.

    Thanks in advance.

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  3. Idle Mind What the hell, man? Valued Senior Member

    PCR takes a short section of DNA and amplifies it -- it is used for detection of a particular sequence of DNA because even if there is a single copy of that sequence it can be amplified.

    I'm not sure what you mean by "Can PCR be done on living humans?" Could you explain a little further?
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  5. Michelle Redford Registered Member

    Thanks for the quick answer.

    I thought with PCR you can modify DNA. I think I got it now... They use PCR to "scan" the DNA and then use the process of methylation to "play" with genes.

    Anyway, I needed someone to clarify what PCR is... Thank You, Idle Mind.

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  7. Roman Banned Banned

    PCR is an in vitro process- that is, it is done outside of a living organism.

    Polymerase Chain Reaction is a fast, cheap, effective way to copy small amounts of DNA into large enough quantities to run assays on, such as DNA fingerprinting or doing phylogenetics.

    In order for PCR to work, one needs several things:
    Template DNA-
    The DNA you want to copy. This could be DNA extacted from blood at a crime scene or collected from a strange new animal. Note that this extraction of DNA will be genomic- all the DNA in the organism will likely be present.

    Small pieces of DNA, called oligonucleotides, that are synthesized in a lab (you can order these off the net). They complementarily bind to single stranded DNA, and tell the DNA polymerase where to begin. If you’re looking for a cite of interest, like a microsatellite region in the case of blood from the crime scene, this will base pair with a nearby region, that doesn’t differ from individual to individual. Primers are often labeled, either radioactively or flourouscently, so that they can be visualized on a gel or other medium after PCR.

    DNA polymerase (DNA pol)-
    This is an enzyme that “reads” a single strand of DNA and “writes” down a complementary base pair. It’s found in all living organisms, since we all need to coy our DNA. In fact, we have our own little PCRs going on in our own cells right now!

    These are the raw materials that the DNA pol uses to copy the template DNA when synthesizing a new strand of DNA.

    The process works like this:
    The double stranded DNA is heated up to 94°C which melts the DNA into single strands. This allows our primers access to complementary base pair. Lowering the temperature back down allows the DNA pol to bind and begin copying. After synthesizing the sequence of interest, heating it back up to 94° melts the DNA and allows more primer to bind to the new sequences. In this manner, the amount of DNA grows exponentially. It’s theoretically possible to take a single strand of DNA and copy it until you have billions and billions of copies.

    I mentioned early that this is an extra cellular process- and this presents a problem. Every organism has DNA pol, which means it’s relatively easy to get. Unfortunately, though, we need single stranded DNA for a PCR to work, and the DNA we have is double stranded. In our bodies, enzymes unzip the DNA. In the lab, though, we have to melt the DNA, by heating it up to 94°C. The DNA pol in us is, unsurprisingly, stable at 37°C. Which means that when we melt our DNA, our DNA pol in our reaction gets ruined.

    Luckily, though, not all organisms live at 37°C. A bacterium, Thermophilus aquaticus, that lives in hotsprings at Yellowstone National Park, live in near boiling water. From this, we get Taq, a thermostable DNA polymerase. This is the main DNA polymerase used in labs today.

    Early methods of copying DNA required insertion into plasmids, then putting the plasmids into bacteria and growing those into colonies, followed by screening the bacterial colonies for ones that took up the plasmid. Then those had to be grown, followed by extraction and more screening to make sure your DNA of interest. Then that had to be purified. This method is still used for medium to large pieces of DNA, such as manipulation of genes.

    PCR is typically used to screen for whether or not something is present, usually a fragment of a gene that has been identified as a marker. Plasmids & phages inserted into bacteria are used for larger segments of DNA, such as genes. PCR loses copy fidelity with large pieces of DNA. Bacteria also can turn the DNA inserted into them into mRNA, which will then code for a funtional product. So in that way, not only do you know whether or not a gene is present, but what it codes for
  8. Idle Mind What the hell, man? Valued Senior Member

    Thank you for the thorough explanation, Roman.
  9. Michelle Redford Registered Member

    Thank you Roman very much! That really helped.

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