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VNTR-PCR 995

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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

    The polymerase chain reaction (PCR) is used to amplify particular regions of DNA molecules, using primers (shown in green in the Figure above) that are complementary to the sequences of regions flanking the DNA sequence to be amplified. The two primers, which have different DNA sequences as shown above, serve as starting sites for the addition of nucleotides during synthesis of new DNA strands (shown in pink in the Figure) by DNA polymerases such as Taq.

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

     II. EXPERIMENTAL PROCEDURES:

    Be sure to use aerosol-resistant A. Isolation of Your Buccal Cells using a Catch-All DNA pipette tips. Swab: Each person should put on gloves and isolate his/her own

    buccal cells following the procedure described below:

    Bottled water is available. 1. Each person should rinse her/his mouth thoroughly with water before collecting her/his buccal cells.

     2. Obtain an Isotherm and some ice before beginning.

     3. Obtain a Catchall DNA swab and a clear tube containing Quick- Extract DNA solution from the front bench.

     4. Collect your buccal cells by rolling the swab firmly against the inside of your cheek.

    The more cells you collect, the 5. Roll the swab about 20 times against the inside of each cheek, higher your yield of DNA will making sure you move the swab over your entire cheek. be.

     6. Place the collection swab into the tube containing Quick-Extract DNA extraction solution.

    Rotating the swab between 5 7. Rotate the swab in this solution a minimum of 5 times. and 10 times dislodges the cells from the brush.

    This ensures that most of the 8. Press the swab against the side of the tube and rotate the swab liquid and cells remain in the while removing it from the tube. tube.

    This ensures that the cells and 9. Screw the tube cap tightly closed and vortex the tube for 10 the solution are well mixed. seconds.

     10. Use a black marker to label this tube with “Buc DNA” and your photo ID number, not your lab pair number.

     B. Isolation of Your Genomic DNA:

    This incubation breaks open or 1. Incubate your tube at 65? C for 30 min in a water bath set to this lyses the cells. temperature. Photograph your gel which destained overnight.

     2. After this incubation ends, vortex your tube for 15 seconds.

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

    This incubation inactivates 3. Transfer your tube to a heating block set at 98?C and incubate enzymes (including nucleases, for 8 min. which degrade DNA) as well as compounds that could inhibit

    the PCR reactions.

     4. After this incubation ends, vortex your tube for 15 seconds.

     5. Transfer your tube to the heating block set at 98? C again and incubate for another 8 min.

     6. After this incubation ends, vortex your tube for 15 seconds again.

    This DNA should be stored at 7. Place your tube in the Isotherm to keep cold until you are ready 20? C to set up your PCR reactions.

     Congratulations! You have now isolated a small quantity of

    your own genomic DNA!

     C. Setting up Three PCR Reactions Specific for your D1S80 Alleles: Each person will now set up three PCR reactions specific

    for D1S80 using your buccal cell DNA. You will use Ready-To-Go-Bead Tubes for these reactions.

    Continue to use ARTs for 1. Change your gloves before beginning to set up this reaction. pipetting.

    The Ready-To-Go PCR Bead 2. Obtain three clear 0.5-ml Ready-To-Go PCR Bead Tubes from contains Taq polymerase, the the front bench and put it in your Isotherm. Make sure bead is at four dNTP’s, MgCl, KCl, and 22the bottom of the tube. Tris-HCl buffer.

     3. Each person should write her/his photo ID number on the top and side of each 0.5-ml Bead Tube.

    Take your Bead Tubes and 4. Using an ART and the "PCR ONLY" P200 Pipetman on the front Isotherm to the bench at the bench, add 22.5 l of the prepared D1S80 primer mixture to each front of the lab to add the PCR of your bead tubes. Do not touch the bead with the tip. primer mix.

     BE SURE TO LEAVE THE “PCR ONLY” P200 AT THE

    FRONT BENCH.

    Keep these tubes cold in ice as 5. Flick your Bead Tubes gently with your fingers until the PCR often as possible. bead is dissolved.

     6. Flick your tube of buccal cell DNA with your fingers to mix.

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

     7. Using your P20 and a 20 l ART, transfer 2.5 l of your buccal cell DNA preparation to each of your Bead Tubes.

    8. Flick your Bead Tubes gently with your fingers to mix.

    9. Spin your Bead Tubes 10 sec in the microfuge.

     10. Place one Bead Tube in the Techne thermal cycler.

    Label the side and hinge (if 11. Obtain two 0.2 ml pink PCR tubes from the front bench. possible) of these tubes with your photo ID number.

     12. Using your P200 set to “0-3-5”, transfer the contents of one

    bead tube to a pink PCR tube you just labeled.

     13. Repeat for the second pink PCR tube.

     14. Place both pink PCR tubes in the labeled rack on the front

    bench.

     The PCR conditions for D1S80 are outlined below.

    This step brings the samples to o Soak: 94C 2 min the correct temperature.

    This temperature denatures the o 30 Cycles: 94C 15 sec DNA.

    Primers anneal to the template o 68C 15 sec during this incubation.

    New DNA is synthesized during o 72C 15 sec this incubation.

     o Termination: 72C 10 min

    This low temperature ensures o 4C Until sample removal the stability of the PCR

    products.

    Your DNA will be stored until Please place the microtube that contains the remainder of your we are sure that the buccal cell DNA into the numbered rack on the front bench in amplifications of your D1S80 numerical order for storage. alleles have been successful.

     The sequences of the two primers used to amplify D1S80 are:

    Primer 1 5'-GAAACTGGCCTCCAAACACTGCCCGCCG-3'

    Primer 2 5'-GTCTTGTTGGAGATGCACGTGCCCCTTGC-3'

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

Lab 2: Background Information: D1S80 & Trinucleotide Repeats

    As you will soon prove to yourselves, the Polymerase Chain Reaction or PCR is one of the

    most important techniques in molecular biology today. PCR is so powerful that it is possible to derive enough DNA for amplification and analysis from a gentle swabbing of buccal (cheek) cells from your mouth. The oligonucleotide primers that you will use today span a region or locus within

    the genome known as D1S80, which is highly variable or polymorphic within the human

    population. The variability at the D1S80 locus is caused by the presence of Variable Numbers of

    Tandem Repeats or VNTRs. VNTRs are DNA sequences composed of different numbers of a

    repeated “core” sequence arranged sequentially. The size of the core sequence can vary from 8 to 100 bp in different VNTRs, and the number of repeats present at a VNTR locus also varies widely. Although many different VNTRs have been identified in the human genome, their function is not currently known.

    The D1S80 locus is located in chromosome 1, the largest human chromosome, and the

    repeating sequence at D1S80 is 16 bp in length. An example of one D1S80 sequence, including

    the primer sites flanking the repeats, is shown on the next page. To date, twenty-nine different

    alleles of D1S80, which range in size from 200 bp to 700 bp because of their different numbers of repeats, have been identified. Thus, 435 different allelic combinations are theoretically possible. Because of the polymorphic nature of genetic loci like the VNTR D1S80, it is theoretically highly unlikely for more than a few people in the world to have the same alleles of several polymorphic loci. Thus, several of these loci can be used as the “DNA fingerprint” or “DNA profile” of an

    individual. DNA fingerprints are used to identify individuals or to prove relationships among individuals. You will understand and appreciate this concept better after seeing the class gel containing the D1S80 samples of everyone in the Workshop.

    As shown in the Figure below, each of us has two copies of the D1S80 locus, one inherited from each of our parents. Approximately 86% of the population is heterozygous at this particular locus because a different VNTR allele has been inherited from each parent, as illustrated in the Figure below. Following agarose gel electrophoresis, the amplified D1S80 PCR products will appear as one band in a homozygous individual or two bands in a heterozygous individual. How this great sequence diversity is generated at VNTR loci among different individuals is still unknown, but could be caused by genetic recombination or errors in DNA replication or repair.

     Maternal D1S80 DNA

     Repeat

     5’ 3’

     3' 5'

Primer 1

     Primer 2

     5’ 3’

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

     Paternal D1S80 DNA

     3’ 5’

    5’ACCGGCCCCT CACGGTGCCA AGGAAACAGC CCCACCATGA GGCGCTGAGA

    GAAACTGGCC TCCAAACACT GCCCGCCGTC CACGGCCGGC CGGTCCTGCG

    TGTGAATGAC TCAGGAGCGT ATTCCCCACG CGCCAGCACT GCATTCAGAT

    AAGCGCTGGC TCAGT

    GTCAGCCCAA GGAAGA

    CAGACCACAG GCAAGG

    AGGACCACCG GAAAGG

    AAGACCACCG GAAAGG

    AAGACCACCG GAAAGG

    AAGACCACAG GCAAGG

    AGGACCACCG GAAAGG

    AAGACCACCG GCAAGG

    AGGACCACCG GCAAGG

    AGGACCACCA GGAAGG

    AGGACCACCA GCAAGG

    AGGACCACCA GCAAGG

    AGGACCACCA GGAAGG

    AGGACCACCA GGAAGG

    AGGACCACCG GCAAGG

    AGGACCACCA GGAAGG

    AGGACCACCA GGAAGG

    AGGACCACCG GCAAGG

    AGGACCACCA GGAAGG

    AGAACCACCA GGAAGG

    AGGACCACCA GGAAGG

    AGGACCACCA GGAAGG

    AGGACCACTG GCAAGG

    AAGACCACCG GCAAGC

    CTGCAAGGGG CACGTGCATC TCCAACAAGA CAAAATAAAC AAGCCAGAGA GGGCTTGTGA

    CCAGTGTGGC ATTTGTCAC 3’

    The DNA sequence of a typical D1S80 locus is above. The 16 base pair tandem repeats are aligned in the vertical column on the left to emphasize sequence similarities. The location of the PCR primers are underlined. [Kasai, Nakamura and White. 1990. J. Forensic Sci. 35 (5): 1196 1200. Sekiguchi et al. 1994. Thesis, Nat. Res. Institute

    of Police Science.]

    As mentioned previously, because PCR requires only a small amount of DNA, this technology has also made it possible to detect numerous genetically-based human diseases, including cystic fibrosis, hemophilia, Huntington's Disease, muscular dystrophy, and abnormalities in the tumor suppressor genes p53 and Rb, which Ms. Schepis may mention in her talk “D1S80 and Beyond”. At our current state of knowledge, the many different VNTR loci such as D1S80 in human populations have no known function, but it has recently been discovered that abnormal numbers of other short

    repeating sequences actually can cause genetic diseases in humans. At least 14 diseases are caused by the presence of abnormally large numbers of repeated trinucleotide sequences within or adjacent to genes. This progressive accumulation of trinucleotide repeats could be caused by replicative instability of GC-rich nucleotide repeats, specifically CAG, CGG, and CTG. These repeated sequences might cause DNA to adopt a different conformation or form hairpin structures, which could cause DNA polymerase to slip. (Richard Epstein. 2003. Human Molecular Biology. Press

    Syndicate of the University of Cambridge. Cambridge, United Kingdom. 623 pp.)

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

    The locations of the repeats in four diseases are summarized in the Figure on the next page,

    (Morell. Science 260: 1422 1423. 1993.). Fragile-X which was adapted from the journal Science

    Syndrome is a form of mental retardation that is associated with an accumulation of CGG-rich trinucleotide repeat sequences. This abnormally high number of repeats causes methylation of a nearby CpG sequence and transcriptional repression or silencing of the FMR-1 gene. Spinobulbar

    Muscular Atrophy is characterized by degeneration of the nervous system and progressive muscular atrophy. Myotonic Dystrophy involves testicular dysfunction as well as retarded growth and sexual development. Huntington's is a degenerative disease of the central nervous system.

    In two of these diseases, Spinobulbar Muscular Atrophy and Huntington's Disease, the expanded repeats occur within the protein coding sequences, thus leading to the production of mutant proteins with altered amino acid sequences. As you know, the amino acid sequence of a protein determines its conformation, and the normal conformation of a protein is necessary for that protein to function normally. Therefore, these mutant proteins that contain abnormally large numbers of these repeats will have abnormal sequences, conformations and functions. The expanded repeats in the other two diseases, Fragile-X Syndrome and Myotonic Dystrophy, do not occur within the protein coding regions and thus do not alter the amino acid sequence or the conformation of the protein involved. However, either the level or the mode of expression of these proteins could be altered by these mutations, and this has actually been shown to be the case for both diseases (Feng et. al. 1995. Science 268: 731.). Causation of human diseases by expansion of

    trinucleotide repeat sequences in DNA within or flanking genes is a type of mutagenesis not known to occur in other species.

    Fragile-X Syndrome (FMR-1)

    5’ AAAAA

     (CGG) 7-50

Spinobulbar Muscular Atrophy (androgen receptor)

    5’ AAAAA

     (CAG) 11-31

Myotonic Dystrophy (myotonin kinase)

    5’ AAAAA

     (CTG) 5-35

Huntington’s Disease (huntingtin)

    5’ AAAAA

     (CAG) 11-34

    Although low numbers of trinucleotide repeats are present at different sites in the genome and are not deleterious, abnormally high numbers of these repeats can cause diseases. The numbers in the Figure above indicate the normal number of repeats, and the solid bars indicate coding regions (Adapted from Morell. Science 260: 1422-1423. 1993.).

    At least 50 human genes are known to have stretches of at least five trinucleotide repeats. The mechanism by which these repeats are expanded has not yet been determined, although, as explained earlier, abnormalities in basic cellular processes such as DNA replication or DNA repair are likely “culprits.” A molecular technique, known as Repeat Expansion Detection, has been

    developed to search the human genome for the presence of long stretches of trinucleotide repeats

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

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