Name_______________________

Date________________
Section__

Testing for Fragile X

In this lab you will run a DNA fingerprint analysis on a sample of Cindys DNA to determine if
she is a carrier of Fragile X. Restriction enzymes will be used to cut the DNA into fragments and
those fragments will be run through a gel during gel electrophoresis. Cindys DNA will be
compared with a DNA sample from a person with the Normal DNA. Normal DNA has
approximately 29 CGG repeats in front of the FMR1 gene. Individuals with 50 -200 repeats
have a permutation. Individuals who have between 200-700 CGG repeats have the Fragile X
Syndrome. The more repeats there are, the more severe the symptoms may be.

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Methylation & Restriction Enzymes

Methylation occurs when a methyl group (CH3) is attached to nucleotides on the DNA
and may inhibit the expression of a gene in a mammalian cell probably by affecting the
binding properties or the active site on the DNA when transcription occurs. In particular,
methylation is known to affect the activity of some restriction enzymes found in bacterial
cells. Restriction enzymes cut DNA at specific sites based on recognition of a particular
sequence of four to six base pairs (bp). The recognition sequences for Hind III and EcoRl
are shown below. The symbol (^) shows where each DNA backbone is cut.
For enzyme EcoRl: G^AATTC.
CTTAA^G

For enzyme Hind III: A^AGCTT

TTCGA^A

EcoRl enzyme activity is inhibited if its recognition site is methylated. Hind III, on the
other hand, is not affected by methylation. We can exploit these properties of restriction
enzymes to determine whether DNA has been methylated or not.

PART I RESTRICT DNA SAMPLES

1. As a table, label 6 tubes according to the table below.
2. Split into partners and make sure each set has 3 tubes of the labeled tubes.
3. Travel to each table with a partner. Put the correct reagents in each tube as listed in the table
below.
TUBE 1
8 L NORMAL DNA
2 L STERILE H2O

TUBE 2
8 L NORMAL DNA
2 L EcoRl enzyme

TUBE 3
8 L NORMAL DNA
2 L HindIII enzyme

TUBE 4
8 L CINDY DNA
2 L STERILE H2O

TUBE 5
8 L CINDY DNA
2 L EcoR1 enzyme

TUBE 6
8 L CINDY DNA
2 L HindIII enzyme

4. Once the entire table is back together, spin all 6 tubes in the microcentrifuge.
5. Double Check the amount of liquid in each tube (they should all be the same) and make note
of any differences that you observe.
6. Give your tubes to your teacher to be put in the warm water bath.

PART II CAST AGAROSE GEL

1. Prepare the agarose gel. (Be sure the gates are up, tightened, and the comb is in the correct
place.)
2. Send one member to get gel from your teacher and pour. Let stand while you prepare the
samples.
3. Add 2 L of loading dye to each of your 6 tubes.
4. Spin all the tubes together in the centrifuge.
5. When the gel has solidified, lower and secure the gates at both ends of the casting tray and
place it in the gel box. The comb should be located at the cathode end (black - ).

PART III LOAD AN AGAROSE GEL

6. Fill your gel box with 300 mL of buffer.
7. Carefully remove the comb by pulling directly up on it when it is in the gel box with buffer.
8. Load 12 L of your sample into each well. How much of your sample should be left in the
tube? Tube A should be loaded into well number one. Note if any samples are in excess or
deficient to explain possible experimental error.

PART IV GEL ELECTROPHORESIS

9. If there is already a gel box that is plugged into the power source, turn the power supply off
before securely closing the lid of the gel box, plugging the electrodes (red and black wires)
into the power source and then turning the power supply on at 100 V.
10. Double check to be sure that your box is working. What can you look for to be sure
electricity is running through your gel box? Check for mAmps.
11. Label the plastic tray with your names, time the gel box was turned on, and your class period.

PART V STAIN AND VIEW THE GEL

YOUR TEACHER WILL STAIN THE GEL, BUT YOU ARE RESPONSIBLE FOR
KNOWING WHAT SHE DID AND WHY!
Access your photo in the Live Binder to include in your lab report.