Download DETERMINING THE METHOD OF DNA REPLICATION LAB

DETERMINING THE METHOD OF DNA REPLICATION LAB
BACKGROUND
In 1953 Watson and Crick published a paper in the journal Nature that described the
structure of the DNA molecule. The authors acknowledged the shape of the molecule
was conducive to replication; "It has not escaped our notice that the specific pairing we
have postulated immediately suggests a possible copying mechanism for the genetic
material". It was not until 1958 that Meselson and Stahl conducted a now classic
experiment to determine the method of DNA replication.
DESCRIPTION OF REPLICATION
After the publication of the structure of DNA, several possible hypotheses were
advanced to describe how the DNA replicated. Three hypotheses were considered the
most likely candidates to correctly explain replication: conservative, semiconservative,
and dispersive. During conservative replication, the hypothesis held, the DNA molecule
splits along the H bonds so that new nitrogenous bases could be brought in for
complementary pairing. Next the parent (old) strand was thought to separate from the
daughter (new), and then both parent strands would reconnect. Daughter strands
connected as well, resulting in a complete old parent and new daughter DNA molecule.
In semiconservative replication, the DNA molecule was believed to separate along its H
bonds while the parent strands served as a pattern for making new daughter strands. In
effect, both DNA molecules were assumed to be composed of old and new strands of
DNA. Dispersive replication involved fragmenting the parent DNA prior to copying and
then realignment of the daughter molecule, each strand, thus, being a hybrid of parent
and daughter DNA.
GOALS AND METHODS
Meselson and Stahl wanted to determine which of the competing hypotheses
best described the process of DNA replication. In order to perform an experiment they
needed to overcome two technical obstacles: marking the DNA with "heavy" nitrogen
(15N), and devising a method of differentiating between "heavy" nitrogen and "light" or
normal nitrogen (14N). The first difficulty was overcome by growing bacteria on "heavy"
nitrogen media until they reproduced for several generations; at this point their DNA
contained the "heavy" nitrogen. Density gradient centrifugation proved to be the key to
overcoming the second difficulty; in this procedure DNA was placed in a cesiumchloride (cs-cl) solution and centrifuged for 20 hours. This procedure would serve to
differentiate the "heavy" and "light" nitrogen into different bands suspended in the cs-cl
solution. The "heavy" DNA would form a band near the bottom of the tube while the
"light" DNA band would be found near the top of the tube.
EXPERIMENTAL PROCEDURE
1. Grow bacteria in 15N to allow A,T,C and G to incorporate the isotope.
2. After 12 hours, take sample for later analysis (Tube 1 – bacteria grown on 15N).
3. Transfer remaining culture to 14N media. Bacterial DNA incorporates 14N for
replication of DNA.
4. After 4 hours (population doubles), take sample of 14N solution for later analysis
(Tube 2 – bacteria grown on 14N).
5. Obtain sample from 14N culture 4 hours later than Tube 2(Tube 3 – bacteria of 2nd
generation on 14N).
6. Obtain sample of bacteria grown in 14N (Tube 4 – bacteria grown only in 14N).
7. Examine DNA density with cesium-chloride centrifugation.
MODELING EXPECTED PARENTAL AND DAUGHTER DNA FOR TWO TYPES OF
REPLICATION
1. Obtain a laminated sheet (Possible Modes of Replication) on which you can
arrange DNA segments.
2. Use dark colored pipe cleaner strips to represent “heavy” DNA and lighter colored
strips to represent “light” DNA.
3. Place two strands of “heavy” DNA in the upper left corner of the sheet to
represent the parental generation (P1).
4.Using your understanding of how conservative replication was expected to occur,
position two pairs of appropriately colored pipe cleaners on the board for the F1
(1st ) generation. Only “light” nitrogen was available to this generation.
5. Continue modeling conservative replication for an F2 (2nd) generation Again, only
“light” nitrogen was available to this generation.
6. Similarly, model the semi-conservative replication process for the F1 and F2
generations. As before, only the “light” nitrogen is available for use in replication
of the DNA.
7. Fill in the diagrams below based on arrangement of “light” and “heavy” DNA as
shown on the laminated sheet (ex. HH or LL or HL).
DNA BANDING PATTERNS FOLLOWING DENSITY GRADIENT CENTRIFUGATION
FOR TWO POSSIBLE MODES OF DNA REPLICATION
1.Obtain a centrifuge tube diagram sheet (Prediction of DNA Banding Following
Density Gradient Centrifugation) to be used to model the location of the density
dependent DNA.
2. Transfer the DNA molecules from the replication sheet to the centrifuge tube
sheet, and place over the tube in a position determined by DNA density. Be
careful to keep conservative DNA strands separated from semi-conservative
strands.
3. Sketch and label the banding pattern noted on the sheet in the space provided
below.
ANALYSIS AND CONCLUSION
1. Examine the DNA banding pattern predictions above and the actual results below
to determine the method by which DNA replicates. Give the method of replication
_____________________________.
2. What is happening in the bacteria to cause the line for first generation to be at the
midpoint of the tube?
What about the second generation?
3. What information do the control tubes provide?
Below is a dispersive replication schematic which will be helpful in answering
question:
4. Other replication schematics have been completed earlier in this exercise. Why
did Meselson and Stahl allow the bacteria to divide into an F2 generation? Why not
stop at F1 to analyze the banding pattern (hint assume the F1 would appear as a
hybrid line in the tube)?