DNA Replication, PCR & Cloning Flashcards
What is the S phase of the cell cycle?
DNA replication.
Give the 4 stages of the cell cycle.
G1, S, G2, M.
What are telomeres?
Highly repetitive sections of DNA that protect the ends of chromosomes from degradation, recombination & fusion with other chromosomes.
What is the centromere?
Repetitive DNA which forms the spindle attachment site in mitosis.
What is the origin of replication?
Site where duplication of DNA begins, each chromosome will have many origins.
What is semi-conservative replication?
The new DNA helix has one new strand and one old strand.
What is needed for DNA synthesis?
DNA polymerase & Mg^2+
dNTPs
Single stranded DNA template.
Primer 3’ -OH
Describe the process of DNA replication.
DNA polymerase unwinds the double helix.
Single stranded binding protein prevents base-pairing until DNA polymerase arrives, they leave one face open so it is still accessible to DNA pol.
Primase synthesises short RNA primer copied from DNA.
Sliding clamp loaded onto DNA by clamp loader enzyme, opens up the sliding clamp allowing it to encircle the DNA, and it is then locked around the DNA by ATP hydrolysis, this also releases clamp loader.
DNA polymerase elongate RNA primers with new DNA.
Nucleases remove RNA at 5’ end of neighboring fragment & DNA polymerase fills the gap.
DNA ligase connects adjacent Okazaki fragments on lagging strand.
What is the directionality of DNA synthesis?
New strand synthesised 5’ to 3’
Why are replication bubbles necessary in eukaryotes?
Multiple points of origin are needed for each chromosome as replicating the whole chromosome from one origin would take too long.
Bubbles meet eventually & replication is complete.
Describe the process of bacterial replication.
One origin.
An intermediate theta structure forms, the replication forks move away from the origin & eventually meet.
Both strands are copied at fork.
Synthesis of new strand 5’ to 3’
What is the directionality of the leading strand?
5’ to 3’
What is the role of histone chaperones in eukaryotic DNA replication?
Load histones onto newly-synthesised DNA.
What is the problem caused when helicase unwinds the DNA helix and how is it fixed?
Causes supercoiling ahead of the replication fork.
Topoisomerases unwind this supercoiling by breaking & reforming phosphodiester bonds using a swivel motion.
Give the rate of DNA replication in eukaryotes vs E.coli.
~50bp s^-1 : ~1000bp s^-1
What is the combined accuracy of eukaryotic DNA replication?
1 in 10^10
Why is DNA pol. more accurate than RNA pol.?
DNA pol. must undergo a conformational change to close around the DNA strand, so if base pairing is wrong the conformational change is less likely to occur.
DNA pol. can also proofread when it ‘chews back’ to remove an incorrect base pair an replace with the correct one.
What is the function of mismatch repair proteins?
MutS cans newly synthesised strands & detects kinks caused by mismatched base pairs.
MutL binds to a nick in newly synthesised strand, The DNA between the nick and the kink is removed & then replaced by correct sequence.
What are errors in mismatch repair proteins are associated with?
Predisposition to some cancers.
What is PCR?
Polymerase chain reaction, amplification of DNA.
What does PCR require?
- template DNA
- primers
- dNTPs
- Mg 2+ buffer
- Taq polymerase (from organisms that live in very hot environments)
Give the 3 steps in PCR.
Denaturation, double DNA strand melts open at 95 degrees C.
Annealing, primers bind to DNA and polymerase attaches and starts copying DNA at ~50 degrees C.
Extension, at 72 degrees C Taq polymerase can extend the fragment from the primers.
How many cycles does PCR go through?
~30-40
How can you calculate the number of DNA copies for a certain PCR cycle number?
2n (n = cycle no.)
