D1.1 DNA replication

What is DNA replication required for?

1. Reproduction

2. Growth

3. Tissue replacement

What are the parts of a nucleotide?

Phosphate attached to 5th carbon of sugar

Pentose sugar (deoxyribose for dna and ribose for rna)

Base attached to 1st carbon (purine: A, G or pyramidine: C, T, U)

Where does dna replication happen?

In the nucleus

How are nucleotides connected from top to bottom and side to side respectively?

Covalent bonds

Hydrogen bonds

Why is DNA replication semi-conservative?

One strand(template strand) of the ‘parent’ DNA is kept in the ‘daughter’ molecule

The other half is determined by code on template strand is built from free nucleotides

What is the importance of semi conservative replication(keep one og dna strand)

Ensures genetic continuity with high accuracy

What is the role of helicase in DNA replication

Unwinds the DNA(like untwisting a twisted rope)

Breaks hydrogen bonds between base pairs(like unzipping a zipper)

What is the role of DNA polymerase III in DNA replication

Links new nucleotides together in 5’ to 3’ direction

Proofreads(recognizes wrong nucleotides and replaces with correct one)

What is the direction of the leading strand towards the replication fork?

3’ to 5’

What is the direction of the leading strand towards the replication fork?

3’ to 5’

In which direction is dna replicated?

5’ to 3’ always

Describe the replication of the leading strand (continuous)

Note: direction of leading strand=3’→5’, therefore, new strand synthesised=5’→3’(towards replication fork)

1. Helicase unwinds and separates dna strands forming replication fork

2. Gyrase releases tension from unwinding(ahead of rep fork)

3. SSBP(single strand binding proteins) hold the 2 strands apart

4. RNA primase adds a primer once

5. DNA polymerase III binds to primer n adds new nucleotides to 3’ end of leading strand in 5’→3’(extends primer)

6. DNA polymerase I removed primer and replaced it with DNA nucleotide

Describe the replication of lagging strand(discontinuous)

New strand synthesized 5’→3’(away from replication fork therefore discontinous)

1. Helicase unwinds and unzips DNA double helix

2. Gyrase releases tension from unwinding(ahead of rep fork)

3. SSBP keep both strands apart

4. RNA primase adds several primers as replication occurs away from replication fork

5. DNA polymerase III binds to primers n adds nucleotides to 3’ end of primer. Makes Okazaki fragments

6. DNA polymerase I removed primers and replaces with DNA nucleotides

7. Ligase joined okazaki fragments together by forming phosphodiester bonds

Which enzyme adds RNA primers to the strands for dna replication?

RNA primase (distinct from RNA polymerase which is involved in transcription)

What does Ligase do?

Links Okazaki fragments together in replication of lagging strand

What are Okazaki fragments?

Short sections of dna formed on lagging strand during dna replication

What does DNA polymerase I do?

Removes rna primer and replaces it with dna nucleotide

Which structure prevents nucleotides to bind together again after unwinding of the strand?

SSBP (single strand binding proteins)

Which enzyme releases tension after unwinding of the dna strand?

Gyrase

True or false: both strands of the parent dna molecule are template strands

True

What is DNA profiling/fingerprinting

Process of matching an unknown sample of DNA to known sample of DNA to see if they correspond

Describe the process of PCR(Polymerase Chain reaction)

Artificial way to replicate DNA

Stuff needed:

Target DNA, Taq polymerase, free nucleotides, DNA primer

1. Denaturation: Thermal cycle set at 95°C, high heat breaks H-bonds to separate strands

2. Annealing: Thermal cycle cooled to 55°C→allows primers to bind to start of target sequences on each strand

3. Elongation: Thermal cycled set at 72°C, Taq polymerase adds and joins new nucleotides(similar to DNA polymerase III)

How many copies of DNA does polymerase chain reaction (PCR) create in terms of n (number of cycles)?

2^n

What makes taq polymerase better than dna polymerase in polymerase chain reaction (PCR)?

It doesn’t denature at high temperatures

What is gel electrophoresis

Process to separate fragments of dna on an agarose gel according to size&overall charge

True or false: the largest and heaviest dna fragment travels the furthest in gel electrophoresis

False, it travels the least

Describe the process of gel electrophoresis

1. DNA cut at specific base sequences with enzyme restriction endonuclease

2. Samples of dna injected into well at top of gel

3. One well filled with DNA ladder (contains fragments with known lengths→to determine length of samples)

4. Electric current runs through gel

5. DNA(-ve) moves towards +ve pole at diff speeds(shorter=faster&further)

6. After smallest molecule reaches bottom, voltage turns off, gel removed and strained with dye

7. DNA bonds fluoresce n r compared with DNA ladder to determine length

What r the applications of PCR & gel electrophoresis

1. DNA profiling/fingerprinting(identifying individuals with DNA)

   Forensic science & paternity test

True or false: dna polymerase forms hydrogen bonds between bases of new nucleotides and the template strand

False: hydrogen bonds form spontaneously without an enzyme

Instead, dna polymerase covalently bonds new DNA nucleotides together

True or false: in gel electrophoresis, dna fragments move towards the -vely charged cathode

False: dna is -vely charged so it moves towards the +vely charged anode (link to electrolysis in chemistry)

What did crick and Watson propose for dna replication?

The semi-conservative nature of replication

Who provided experimental evidence for the semi-conservative nature of dna replication?

Meselson and Stahl

True or false: dna and rna primase are the same

True

How do u know if a gene or homozygous or heterozygous in gel electrophoresis?

Homozygous: only one band appears as they are identical in length so they land in the same spot

Heterozygous: 2 separate bands appear