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Salters-Nuffield Advanced Biology Resources

Activity 2.16 Student Sheet

MESELSON AND STAHL’S EXPERIMENT ON DNA REPLICATION Purpose 

To explain the method of DNA replication by considering the different theories originally proposed to explain the process and the evidence which supports the theory that is now accepted.

Procedure Complete the interactive tutorial associated with the activity and then complete this worksheet. Matthew Meselson and Frank Stahl worked at the California Institute of Technology. In 1958 they grew bacteria in growth medium containing ammonium ions (NH4+) as the source of nitrogen. The type of DNA made by the cells depended on the type of nitrogen present in the bacteria’s growth medium. They used two isotopes of nitrogen – 14N and 15N. 14N is the common, light form (isotope) of nitrogen. 15N is the heavier form. They then extracted DNA from the bacterial cells and centrifuged the resulting solution to isolate the DNA. The DNA made with 14N and the DNA made with 15N accumulated at different levels in the centrifuged solutions, according to the DNA’s density.

Questions Q1

In each of the label boxes in Figure 1 below, fill in the number to show the type of nitrogen present in each band. a) The band of DNA towards the bottom, is Nitrogen – 15, and the one towards the top is Nitrogen – 14.

Q2

In each magnified circle in Figure 1, colour in the sections of the DNA molecules found in each band to show the type of DNA present. Use your own colour code, selecting one colour for DNA made with 14N and another colour for DNA made with 15N. You will need an intermediate colour for medium DNA later, so choose colours carefully.

A) All the DNA molecules at the bottom DNA band, will be red, since it’s conservative DNA, also, all of the DNA molecules at the top DNA band will be blue, since it’s also conservative DNA. Figure 1 Position of labelled nitrogen bands in the centrifuge tubes.

Q3

How did Meselson and Stahl produce bacterial cells containing only DNA made with 15N? A) They placed the DNA in a medium containing only Nitrogen-15, and allowed the bacteria to replicate for 1 generation (20 minutes), so the nitrogenous bases, of the new DNA, contained only 15N.

All users will need to review the risk assessment information and may need to adapt it to local circumstances. © 2015 University of York, developed by University of York Science Education Group. This sheet may have been altered from the original.

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Salters-Nuffield Advanced Biology Resources

Activity 2.16 Student Sheet

Bacteria containing DNA made with 15N were allowed to replicate once in a solution containing only N. Any new DNA made would contain 14N. After a single replication the DNA was extracted and centrifuged. 14

Q4

Colour in the band(s) in the centrifuge tube in Figure 2 to show the density of the DNA that Meselson and Stahl found after this first replication. Use your colour codes for heavy, medium and light DNA. Heavy – red Medium – red / blue Light - blue Figure 2 DNA after the first replication.

Q5

Figure 3 shows DNA replication according to the theory of conservative replication. Explain why the bands found by Meselson and Stahl after one replication (shown in Figure 2) refute (do not support) the theory of conservative replication.

Figure 3 Conservative replication.

A) The bands found, refute the theory, because when the DNA was replicated, the N-15 and N14, DNA, mixed, forming DNA molecules, which contained a N-15 strand and a N-14 strand which proves semi-conservative replication, however, in the theory we see that one DNA molecule, is all N-15, and One is all N-14, therefore their discovery refutes this theory. Bacteria containing DNA made with 15N were allowed to replicate twice in a solution containing only 14N. Any new DNA made would contain 14N. After two replications the DNA was extracted and centrifuged. Q6

a

Figure 4 shows DNA replication according to the ‘dispersive’ theory, where new ( 14N) and original (15N) DNA are dispersed throughout any new DNA molecules synthesised. This is theory 1 in the interactive tutorial. Colour in the DNA and DNA nucleotides to show the distribution of DNA with 15N and DNA with 14N. Use your colour code from question 2.

All users will need to review the risk assessment information and may need to adapt it to local circumstances. © 2015 University of York, developed by University of York Science Education Group. This sheet may have been altered from the original.

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Salters-Nuffield Advanced Biology Resources

Activity 2.16 Student Sheet

Figure 4 The dispersive theory of replication.

b

Draw bands on the centrifuge tubes in Figure 5 to show the DNA present after the first and second ‘dispersive’ replications.

Figure 5 Bands that would occur after dispersive replication.

All users will need to review the risk assessment information and may need to adapt it to local circumstances. © 2015 University of York, developed by University of York Science Education Group. This sheet may have been altered from the original.

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Salters-Nuffield Advanced Biology Resources Q7

a

Activity 2.16 Student Sheet

Figure 6 shows the semi-conservative theory of replication where new DNA molecules have one strand of the original (15N) DNA and one strand of the (14N) DNA. This is theory 2 in the interactive tutorial. Colour in the DNA and DNA nucleotides using your colour code from question 2.

Figure 6 The semi-conservative theory of replication.

First Replication:

Second Replication:

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Salters-Nuffield Advanced Biology Resources b

Activity 2.16 Student Sheet

Draw bands on the centrifuge tubes in Figure 7 to show the DNA present after each replication.

Figure 7 Bands that would occur after each replication.

Q8

Meselson and Stahl found equal amounts of light and intermediate density (medium) DNA present after two DNA replications. Explain which of these three theories for DNA replication is supported by this evidence and which is refuted. Use a separate sheet of paper if you need more space for your answer. A)

All users will need to review the risk assessment information and may need to adapt it to local circumstances. © 2015 University of York, developed by University of York Science Education Group. This sheet may have been altered from the original.

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