DNA Replication

Question 1

DNA replication stages

Answer 1

Initiation: Proteins bind to DNA, unzips helix, prepares for complimentary base pairing (primer binding)

Elongation: Enzymes help complimentary base pairing to build new strands, Proof reading by other enzymes

Termination: removes RNA primers, proofreading, enzymes release strands to form double helix

Question 2

Theories of DNA replication

Answer 2

• Conservative: 2 complementary strands of DNA make identical copies for 1 pair of new DNA
• Semi-conservative: 1 strand new, 1 original per pair (correct)
• Dispersive: segments of each strand new and original

Question 3

DNA replication w enzymes

Answer 3

1) DNA Gyrase(type of topoisomerase) - undoes supercoiling to release tension
2) DNA Helicase - unzips double helix
3) single strand binding proteins (SSB) - stops rezipping
4) DNA Primase - puts 1 RNA primer on leading, many on lagging
5) DNA Polymerase III - synthesises 5’-3’ - forms Okazaki fragments for lagging & proofreads new DNA strand
6) DNA Polymerase I - replaces RNA primers w DNA
7) DNA Ligase - replaces RNA backbone gap between Okazaki fragments
8) (Eukaryotes only) Telomerase - makes non coding areas at ends

Question 4

strand names in replication

Answer 4

Leading (3’-5’ so synthesised strand 9daughter) is antiparallel in 5-3), Lagging - synthesised in 5-3’
Template, coding - respective order

Question 5

source of DNA catalytic energy

Answer 5

hydrolysis of PPPi (phosphates on 3’ of DNA chain) —> PPi - produces energy

Question 6

Polymerase Proofreading

Answer 6

polymerase comprised of polymerase domain & exonuclease domain- if mispaired base added, DNA strand flips to exonuclease domain and kicked off, strand flips back

Question 7

Extra proteins

Answer 7

SSB - single strand binding proteins
Sliding Clamp: ring to keep polymerase on strand, assembled at replication fork by clamp loader - for efficient replication

Question 8

cDNA

Answer 8

complementary DNA synthesised against mRNA, used in labs to study gene expression

Question 9

Poly(A) tail

Answer 9

chain of adenine at 3’ end of mRNA in eukaryotes after transcription to stabilise molecule

Question 10

process of reverse transcription (mRNA –> cDNA)

Answer 10

• Beads with adenine chains bind to mRNA strands so other RNA (tRNA/rRNA) are filtered out (washed away)
• Poly(T) primer binds to poly(A) tail as start codon for reverse transcriptase enzymes to transcribe cDNA from mRNA template
• RNase enzymes used to degrade RNA = single strand
• DNA polymerase used to synthesise complimentary coding strand = double stranded cDNA
• inserted into bacterial/viral vector plasmid to mass replicate/ transcription = used for research e.g, PCR

Question 11

PCR
purpose
solution contents

Answer 11

polymerase chain reaction: used to amplify a genetic sequence (portion of DNA), is used in genotyping - to check for a certain gene in an organism belonging to certain species, in forensics for DNA matching.

PCR solution includes:
- free dNTPs, polymerases - proteins for synthesising DNA, primer codons, original gene of interest, mg+, K+, water

Amplicon - gene fragment aimed to replicate

Question 12

PCR stages & temps

Answer 12

Denaturation - 95c, 20-30s denatures DNA into 2 separate strands

Annealing - 55c, 20-40s, primers anneal - bind - to each strand

Extension/Synthesis - 72c, 1 min, enzymes can bind to primers and synthesise new DNA strands (replication)

REPEATED - “thermocycling” =exponential increase of strands 20-40 cycles

extra extension stage at end to make sure all DNA is double stranded, then 4c hold stage to make DNA stable until removed

Question 13

strand names in Transcription

Answer 13

template strand = antisense strand (leading strand in replication) - the one useful as a template in transcription 3’-5’

coding strand (mRNA) = sense strand the strand coding for proteins! - makes sense 5’-3’