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VNTR-PCR 995

By Robin Stevens,2014-12-23 13:45
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VNTR-PCR 995

Laboratory 2A: Isolation of Your Own Buccal Cell DNA;

    Amplification of Your D1S80 Alleles

    (Tuesday Morning)

Background Reading and Links: nd1. PCR: DNA Science, 2 Ed.: Chapter 6: pp. 192 195 nd2. DNA Polymorphisms and Human Genetics: DNA Science, 2 Ed.: Chapter 8: pp. 277 293

    3. Polymerase Chain Reaction: http://www.dnalc.org/ddnalc/resources/pcr.html

    4. Polymerase Chain Reaction: http://www.youtube.com/watch?v=_YgXcJ4n-kQ

Objectives of Laboratory 2A:

    1. Photograph your gel from yesterday

    2. Isolate your own genomic DNA from your buccal cells using a DNA swab

    3. Set up three PCR reactions with this DNA to amplify your D1S80 alleles

    4. Streak JM109 for transformation and observe epidermal microbes

Flow Chart of Laboratory 2A:

    Collect Buccal Cells Isolate Your Set Up D1S80 Streak Observe Epidermal Using a DNA Swab Genomic DNA PCR Reactions JM109 Microbes

I. INTRODUCTION: As you will learn, the polymerase chain reaction (PCR) is a method

    by which a small, defined region of DNA can be synthesized from a minute amount of DNA, as little as a single molecule, to yield quantities of DNA sufficient for detailed analyses such as gel electrophoresis or sequencing. Today, you will collect your buccal cells using a DNA swab and isolate your own genomic DNA from these cells. You will use your DNA preparation to set up three PCR reactions specific for your D1S80 alleles. Your samples will be amplified this morning, and after lunch you will analyze your D1S80 PCR products by agarose gel electrophoresis. The technique we will use for amplification of your D1S80 alleles is a modification of a procedure previously used by the FBI for human identification. (We thank Ms. Judy Brown, Edison Career Center, Wheaton, MD 20906, for making an earlier version of this procedure available.)

     Maternal D1S80 DNA

     Repeat

     5’ 3’

     3' 5'

Primer 1

     Primer 2

     5’ 3’

     3’ 5’

    Paternal D1S80 DNA

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    2009 HHMI Summer Workshop, Dept. of Molecular Biology, Princeton University

    As shown above, each of us has two copies of the D1S80 locus, one of which we inherit from our mother and one from our father. Alleles of the D1S80 locus consist of varying numbers of a 16 bp repeat.

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    2009 HHMI Summer Workshop, Dept. of Molecular Biology, Princeton University

    The polymerase chain reaction (PCR) has revolutionized not only molecular biology but also

    several other scientific fields. PCR is a method by which a defined region of DNA is synthesized from minute amounts, even as little as a single DNA molecule, to yield quantities of DNA sufficient for detailed studies and analyses. This technique has become widely used in genetic diagnosis and forensics, as well as in innumerable basic research applications. The requirements for PCR include: a DNA polymerase to synthesize DNA, a DNA template for the polymerase to copy, the four

    deoxynucleoside triphosphates (dATP, dGTP, dCTP and dTTP) that are the building blocks of

    DNA, short DNA molecules (oligonucleotides) to serve as starting points or primers for DNA

    synthesis, and suitable reaction conditions for the DNA polymerase to synthesize DNA. PCR is

    usually performed using a thermally stable DNA polymerase known as Taq polymerase, which was

    isolated from Thermus aquaticus, a thermophilic bacterium that inhabits hot springs in Yellowstone National Park. In the reactions you will set up today, the template will be the DNA you will isolate this morning from your buccal cells. The primers are short (15-25 bp) DNA molecules that function as starting sites for Taq polymerase to begin synthesizing DNA and are specific for the

    chromosomal region being amplified, in this case the D1S80 locus. The sequences of the primers are very important: they must be the exact complements (A pairing with T and G pairing with C)

    of sequences that flank the chromosomal region to be amplified.

    The basic PCR cycle is composed of three steps or reactions, each of which is performed at a different temperature that can vary according to the nucleotide sequences of the primers employed. In the first step of reactions specific for the D1S80 genes, the template DNA is denatured at high o C for 15 sec in our reactions). In the second step, the temperature temperature for a short time (94

    ois lowered to allow the primers to anneal to the template DNA, again for a short time (15 sec at 68

    C). In the third step, the temperature is raised to the optimal temperature for Taq polymerase to o synthesize DNA (72C for 15 sec). These steps are diagrammed in the Figure on the next page. Although the procedure is very rapid compared to many other techniques (a single three-reaction cycle usually requires less than four minutes), it is necessary to repeat this cycle thirty times to synthesize enough DNA for cloning and analysis by agarose gel electrophoresis.

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    2009 HHMI Summer Workshop, Dept. of Molecular Biology, Princeton University