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DNA SEQUENCING
by Nancy Ridenour

LAB #______
NAME ___________________________

Introduction:

In 1975, the art of DNA sequencing advanced explosively when new techniques were developed that made the task astonishingly simple. The crucial technique involves finding the gene you want to sequence, cutting it out of the chromosome with restriction enzymes, making many copies of that gene through PCR or gene amplification in a yeast or bacteria, and exposing the many copies of the gene to a radioactive label at the end of the DNA strands that are called 3 prime ends. This technique was designed by Allan Maxam and Walter Gilbert. The strands are separated, and one specific strand of the DNA is kept for analysis. The multiple copies of the strand are separated into equal quantities and placed into four test tubes. Each tube is treated with a different chemical that selectively destroys one of the nitrogen bases in the DNA strand. The concentration of the chemical is such that it does not destroy all the bases on all of the copies of the DNA single strand. Therefore, the fragments in any given chemical treatment will have strands of DNA that come in many lengths. The fragments are placed in a gel electrophoresis and separated according to their size and charge, the smallest fragments moving the furthest through the gel. The fragments that contain the labeled pieces will show up on an x-ray film and can be read to determine the sequence of the nucleotides in the DNA that makes of the gene. From this sequence, the scientist can determine the sequence of amino acids that make up the protein that is coded by that DNA. If someone has a defective gene, the scientist can determine this and note what amino acids are substituted in the protein for the correct amino acid that would be in the non mutated protein.

Objectives:

  • learn to read a gel electrophoresis sequence
  • learn a technique of DNA sequencing through gel electrophoresis
  • covert the DNA sequence into amino acid sequence


Materials:

  • 24 xerox copies of DNA sequence per team
  • colored pencils
  • 3 scissors per team
  • large poster board or newsprint per team
  • tape
  • mRNA/amino acid chart
  • 1 meter stick per team

Procedure:


Part A:

  1. The following diagram represents an x-ray of the gel from the electrophoresis of segments of a DNA strand. Each letter at the top represents one of the four bases in a nucleotide of a DNA molecule. The marks under each of the bases represent segments of DNA that migrated through the gel. (The radioactive probes attached to the segments "burn" these marks into the x-ray film when it is exposed to the gel.) The numbers represent the relative distances traveled by the segments, with "1" being the furthest distance the segment traveled and "6" being the shortest distance. The smallest segments of DNA move the furthest while the longer segments of DNA move the shortest distance. When you read a gel, read it from the bottom to the top as shown in the following example. This sequence is read as TTCGGA, T being the shortest segment and A being the longest segment.


    GAT C
    6
    --

    5--

    4--

    3

    --
    2

    --
    1

    --


  2. "Read" the DNA sequence for Watson from the bottom to the top of the diagram of a gel from the electrophoresis of segments of a DNA strand. Each line represents a segment of labeled DNA that migrated through the gel. The smallest pieces migrated the furthest and the larger ones migrated the shortest distance. Record your results in the Data section of this lab.


    GAT C
    12 --


    11
    --

    10
    --

    9
    --

    8

    --
    7
    --

    6

    --
    5


    --
    4


    --
    3 --


    2
    --

    1

    --


Part B:

  1. Working as a team, obtain 4 sheets of the single strand of DNA as shown below. Your teacher will copy the 4 sheets, each containing 6 copies of the strand show. This will give you 24 copies of the single strand.



  2. Cut out the strands of DNA with the radioactive probe at each end. You will have six strands from each sheet.

  3. Place six (6) strands of the DNA in each of the four "test tubes" that contain the chemicals that will selectively destroy the nucleotides in the DNA sequence. Use a tray for your test tube. Label the trays left to right "G", "A", "T", and "C"as shown below:



  4. On your poster board, construct an electrophoresis gel template with the wells for G-A-T-C at the top of the "gel." Number the side from 1-21 because we are sequencing 21 nitrogen bases. "1" will represent the distance traveled of one nucleotide in the DNA segment, while "21" will represent the distance traveled of a 21 nucleotide DNA segment. Each number should be of equal distance from the previous one. Use a meter stick to measure the scale. Make a grid as shown in the diagram. This will make it easier to move your DNA segments through the gel to their final position. Set the "gel" up as follows:

    G A T C
    21



    20



    19



    18



    17



    16



    15



    14



    13



    12



    11



    10



    9



    8



    7



    6



    5



    4



    3



    2



    1



  5. In "test tube" G, cut out the "G"s, but for the clarity of this lab, make a cut AFTER the G, one from each strand. Make sure you cut after a different G on each of the strands. You will then result in pieces of several lengths from the probe end. You should have six different sized pieces of DNA, each starting with the probe and ending with a G. Throw away the portions of the DNA segment that do not contain the probe, since they would not show up on the x-ray film.

  6. Repeat the procedure #5 for "test tubes" A, T and C. There may be extra strands for some of the treatments. these can be thrown away.

  7. Take the segments from "test tube" G that have a radioactive label attached to one end. The segments without the label would migrate through the gel but would not show up on the x ray film. Do not use the unlabeled segments for this demonstration. From well G, move the segments through the gel according to their size. The smallest piece will move the furthest, the larger parts will move the shortest distance. In other words, a segment with only one nucleotide would move to position 1, one with two nucleotides to position 2, etc.

  8. Repeat this for the other "test tubes"

  9. Show your finished product to your teacher.

Data and Observations:

Part A:

  1. Record Watson's DNA sequence read from bottom to top.



Part B:

  1. Record the DNA sequence read from bottom to top that you produced with the DNA fragments.



Analysis:

Part A:

  1. What is the mRNA sequence that can be transcribed from Watson's DNA sequence?


  2. Refer to the amino acid chart in your book. Give the amino acid sequence that is coded by the m RNA sequence.



Part B:

  1. What is the mRNA sequence that can be transcribed from the above DNA sequence?


  2. Refer to the amino acid chart in your textbook. Give the amino acid sequence that is coded by the mRNA sequence.


  3. What m RNA sequence codes for a stop codon?


  4. What would happen if there were a mutation and the stop codon was found in the middle of the mRNA that coded for a protein?








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