2.7 + 7.1 Replication

Question 1

Why is DNA replication semi-conservative?

Answer 1

Semi-conservative:

• in new helix one strand is from template
• another strand is newly synthesised

Question 2

Steps of DNA replication + enzymes

Answer 2

1. Uncoils double strand, breaks H bonds - helicase (uses ATP) and DNA gyrase (topoisomerase)- replication fork created
2. Single stranded binding proteins attach - prevent strands from pairing
3. DNA polymerase III (always 5’->3’) attaches to leading strand and continuously synthesises new strand 5’ to 3’ end (free nucleotides)
4. DNA (RNA) primase adds RNA primer to lagging strand - DNA polymerase III synthesises in Okazaki fragments

5. DNA polymerase I removes primers - DNA ligase seals fragments

Question 3

Evidence for semi-conservative DNA replication

Answer 3

Meselson-Stahl experiment

• ¹⁵N - radioactive isotope in medium - bacteria cultured
• almost all N were radioactive
• transferred bacteria to ¹⁴N medium - after some time samples of DNA collected
• DNA density measured by centrifugation with CsCl
• after 2 generations there were no DNA strands with only ¹⁵N

= this pattern can only be seen if replication is semi-conservative

Question 4

DNA helicase structure

Answer 4

From six globular proteins - donut shape

Uses ATP to break H bonds between complementary bases

Question 5

DNA polymerase

Answer 5

• brings nucleotides into the position where they can form H bonds
• links the nucleotide to the strand by covalent bonds between free nucleotide phosphate and strang sugar
• pentose is 3’ end, phosphate 5’ end

Question 6

PCR process

Answer 6

Components:

• Taq polymerase (heat resistant from themophilic bacteria)
• primers
• free nucleotides
• DNA sample
• buffer

Process:

• Denaturing: strands separate
• Annealing: primers bind
• Elongation: syntehsis of new strand

Question 7

Enzymes involved in DNA replication

Answer 7

Question 8

How DNA sequencing is completed?

Answer 8

Sanger method

• Different fluorescent dyes attached to ndideoxynucleotides (after they attached - strand no longer elongated)
• Length of fragment (electropohoresis) determines position of that nucleotide (A, C, G, T)
  1. Four free nucleotide mixes prepared - many normal + one ddA/ddC/ddG/ddT
  4. PCR
  5. Gel electrophoresis

Question 9

Types of non-coding DNA

Answer 9

Question 10

DNA profiling technique

Answer 10

• uses: paternity tests, forensic investigations
• introns unique in each individual - satellite DNA - short tandem repeats

Procedure:

1. DNA sample
2. PCR
3. Electrophoresis
4. Comparison of STR bands

Question 11

Eukaryotic DNA packing

Answer 11

Question 12

Nucleosome structure

Answer 12