Evidence for DNA replication Flashcards
What did Watson and Crick discover? What was the hypothesis that they made?
- Discovered structure of DNA.
- Hypothesis: DNA replicated by semi- conservative replication rather than conservative replication.
What experiment proved Watson and Crick’s hypothesis that DNA replicated by semi- conservative replication?
- Meselson and Stahls experiment.
What is semi conservative replication vs conservative replication?
- Semi- conservative: Each daughter DNA copy has one original DNA strand and one newly synthesised DNA strand.
- Conservative: During replication, original DNA remains intact/ two newly- synthesised strands join together.
What element do DNA bases contain?
- Nitrogen
What are the two isotopes of nitrogen used in the mediums in Meselson and Stahl’s experiment? Which isotope is heavier/ which is lighter?
- ¹⁴N: lighter
- ¹⁵N: heavier
What do the bacteria in Meselson and Stahl’s experiment do?
- The bacteria takes in the nitrogen isotopes (that would be in the mediums they are grown in) to make new DNA nucleotides.
What would the weight be like of the DNA of bacteria grown in medium containing ¹⁴N isotope vs medium containing ¹⁵N isotope?
- Bacteria grown in medium containing ¹⁴N will have DNA that only contains this isotope/ the DNA will be lighter.
- Bacteria grown in medium containing ¹⁵N will have DNA that only contains this isotope/ the DNA will be heavier.
What can we do to DNA samples to confirm their density (ie. to confirm whether their DNA contains ¹⁴N or contains ¹⁵N isotope?) What would results be for DNA containing ¹⁴N isotope? (how about ¹⁵N ?)
- DNA samples can be spun in centrifuge to confirm density
- ¹⁵N DNA will setlle lower down as it is more dense.
- ¹⁴N DNA will settle higher up as it is less dense.
Outline the main steps of Meselson and Stahl’s experiment.
1.) Grew E coli in growth medium that only contained ¹⁵N.
2.) Transferred E coli to growth medium that only contained ¹⁴N (allowed bacteria- and therefore the DNA- to divide once only.)
3.) E coli left in ¹⁴N growth medium/ allowed bacteria/ DNA to replicate for 2nd time.
4.) E coli left in ¹⁴N growth medium/ allowed bacteria/ DNA to replicate for 3rd time.
In the first stage of Meselson and Stahl’s experiment, analyse position of DNA after centrifugation and therefore the DNA sample?
- DNA settled at bottom (after centrifugation.)
- 100% of DNA made up of 2 strands of ¹⁵N DNA.
In the second stage (1st round of division in ¹⁴N medium) of Meselson and Stahl’s experiment, analyse position of DNA after centrifugation and therfore the DNA sample? Explain what happens to the DNA during this 1st round of DNA replication
- DNA settles at a higher position (after centrifugation.)
- 100% of DNA contains 1 ¹⁵N strand and 1 ¹⁴N strand.
- Doube helix unwinds into 2 separate strands (each strand is ¹⁵N DNA.)
- These strands act as a template for newly synthesised DNA (both strands will be ¹⁴N as this is only isotope available.)
- So, each of the two copies will have one ¹⁴N strand and one ¹⁵N strand.
In the third stage (2nd round of division in ¹⁴N medium) of Meselson and Stahl’s experiment, analyse position of DNA after centrifugation and therfore the DNA sample? Explain what happens to the DNA during this 2nd round of DNA replication
- 2 bands in centrifuge. 1 band (lower in centrifuge –> heavier ) = 50% of DNA has one strand ¹⁴N/ one strand ¹⁵N. Other 50% of DNA (2nd band = higher up in centrifuge –> lighter) has two strands of ¹⁴N.
- 4 strands on 2 DNA molecules separate/ act as templates.
- All of the newly synthesised strands will be ¹⁴N/ lighter strands.
In the fourth stage of Meselson and Stahl’s experiment (3rd round of division in ¹⁴N medium) , analyse position of DNA after centrifugation and therfore the DNA sample? Explain what happens to the DNA during this 2nd round of DNA replication
- 2 bands in centrifuge. 1 band = (lower in centrifuge –> heavier) = 25% of DNA has one strand ¹⁴N/ one strand ¹⁵N.
- 2nd band = (higher in centrifuge –> lighter) =75% of DNA has one strand ¹⁴N/ one strand ¹⁴N (lighter.)
- 8 strands on 4 DNA molecules separate/ act as templates.
- All of the newly synthesised strands will be ¹⁴N/ lighter strands.
What would’ve happened in the first round of replication in ¹⁴N medium of the experiment if it was conservative replication/ not semi- conservative replication?
- 1 DNA molecule will have both of its strands as ¹⁵N/ other DNA molecule will have both of its strands as ¹⁴N.
- After centrifugation, there hypothetically would be 2 bands.
- 1st band would be higher: contains 50% of DNA with both strands ¹⁴N and 2nd band would be lower: contains 50% of DNA with both strands ¹⁵N.
- THIS IS NOT WHAT WAS FOUND - RATHER 100% of DNA was formed in middle band in test-tube.
When we say a DNA strand is ¹⁴N, what does this exactly mean?
- This means the DNA strand’s nucleotides’ bases contain the isotope of nitrogen, ¹⁴N.
