D1.1 Dna replication

Question 1

Describe the process of DNA replication

Answer 1

1. DNA helicase unzips the DNA double helix at replication forks by breaking complementary hydrogen bonds between complementary nucleotides. The 2 strands (leading and lagging) act as a template to synthesise new DNA strands
2. Single strand binding proteins keep bases from reforming hydrogen bonds btwn them.
3. DNA primase adds a RNA primer to each strand of DNA
4. DNA polymerase III attaches to the RNA primer, moves in a 5’ to a 3’ direction along each strand of DNA, forming a growing chain of DNA nucleotides, using the pre-existing strands as a template. Nucleotides are added by complementary base pairing.
5. DNA polymerase III moves towards the replication fork on the leading strand (DNA replication is CONTINUOUS), and away from the replication fork on the lagging strand (DNA replication is DISCONTINUOUS).
6. Okazaki fragments are formed on the lagging strands = RNA primers have to be inserted regularly
7. DNA polymerase I replaces RNA primer with DNA nucleotides
8. Ligase joins the Okazaki fragments together with DNA strands

Question 2

Properties of DNA replication

Answer 2

• ## semi-conservative, as daughter DNA molecules have one parent strand and one newly synthesised strand

Question 3

Describe the process of polymerase chain reaction

Answer 3

PCR uses cycles of heating and cooling to amplify a sample of DNA - used to AMPLIFY DNA sequences from a small DNA sample.

1. Denaturation: DNA sample heated to 95 degrees to break the hydrogen bonds and separate the 2 DNA strands
2. Annealing: Temperature reduced to 54 degrees = allows DNA primers to bind to both strands of DNA, next to the sequence to be copied
3. DNA synthesis: temperature raised to 72 degrees = allows taq DNA polymerase to replicate both strands, starting at the primer = 2 identical double-stranded DNA molecules produced, exact copies of the original
4. steps 1-3 repeated many times to produce many copies of DNA

Question 4

Explain the purpose of using primers, the temperature change, and the use of Taq polymerase in PCR

Answer 4

primers - to show taq polymerase where to begin DNA synthesis

temp change - 95 (break hydrogen bonds), 54 (low enough for primers to bind to template), 72 (optimum temp for taq DNA polymerase activity)

Taq polymerase - obtained from a bacteria living in hot springs = enzyme is not denatured at the temperatures during PCR. (normal human proteins e.g. DNA polymerase are denatured at high temp)

Question 5

Purpose and process of gel electrophoresis

Answer 5

Purpose: SEPARATE charged molecules like DNA - produce DNA profiles (DNA fingerprints) to find origin

Process:
- DNA is an acid and dissociates in water to become negatively charged in water
1. restriction endonuclease enzymes used to chop up DNA into varying sizes of negatively charged fragments.
- desired size is 250-30000 base pairs in length
- restriction endonuclease enzymes cut the backbone of the DNA double helix at highly specific sequences = shorted DNA sequences produced, highly distinctive fragment patterns.
- these patterns used to produce DNA profiles (combinations of DNA unique to the individual)
2. DNA fragments inserted into wells in placed in agar gel which is in a salt solution
3. Placed in electrophoresis chamber
4. Electricity run through the salt solution
5. (-ve) DNA fragments move from the negative electrode to positive electrode in the electrophoresis chamber.
6. Smaller (lighter) DNA fragments move faster and hence further than larger (heavier) DNA fragments
7. Dye added, pattern becomes visible

Question 6

Process of DNA proofreading

Answer 6

• DNA polymerase III proofreads growing DNA chain
• Removes any mismatched bases at the 3’ end, replacing them with the correct nucleotides
• after the removal of the nucleotide mismatch, it reverses direction (once again) and continues its synthesis of the DNA strand in the 5’ to 3’ direction

Question 7

Why is one strand slower to form than the other in DNA replication?

Answer 7

• DNA replication can only take place in the 5’ to 3’ direction
• The 2 strands of DNA are antiparallel
• One strand forms continuously as the replication fork progresses (leading strand)
• the other strand forms discontinuously in fragments (forming Okazaki fragments) as replication progresses, this discontinuous function takes longer (lagging strand)

Question 8

tRNA structure

Answer 8

three leafed clover