DNA STRUCTURE AND REPLICATION

Question 1

explain the central dogma of molecular biology

Answer 1

DNA–> RNA–> Protein.

It describes the flow of biological information between DNA/RNA/Protein.

Question 2

what is the universal tree of life idea?

Answer 2

all life descended form one common ancestor / LUCA

Question 3

what are the 3 domains of life?

Answer 3

Bacteria, Archaea, Eukarya.

Question 4

Describe the similarities between 3 domains of life

Answer 4

bacteria and archaea have similar cellular architecture; archaea and eukarya have similar molecular architecture.

Question 5

DNA nucleotides are made of?

Answer 5

base, sugar, triphosphate

Question 6

what is a nucleoside?

Answer 6

base and sugar

Question 7

dNTP

Answer 7

deoxyribonucleoside triphosphate (DNA)

Question 8

NTP

Answer 8

ribonucleoside triphosphate (RNA)

Question 9

what are purines?

Answer 9

adenine and guanine, 2 rings

Question 10

what are pyrimidines?

Answer 10

cytosine, uracil and thymine

Question 11

what is the general structure of DNA?

Answer 11

double stranded right handed helix with minor and major grooves, each strand runs antiparallel to the other

Question 12

what are the rules / requirements for DNA replication?

Answer 12

• DNA replication is semiconservative.
• DNA polymerase synthesises DNA. needs a template strand
• polymerisation only in 5’->3’
• polymerisation needs dATP/CTP/GTP/TTP

Question 13

Describe the process of strand separation?

Answer 13

• DNA-A proteins binds and strands begin to separate
• DNA-B protein (helicase) separates fork further. one on each strand, needs ATP.
• strands are covered in single stranded binding proteins (SSBs), prevent reannealing.

Question 14

Describe the process of DNA polymerisation?

Answer 14

• lagging strand 5’–>3’
• DNA polymerase polymerises leading strand continuously as it is complementary to DNA polymerase.
• lagging strand replicated backwards through Okazaki mechanism. synthesis happens in small Okazaki fragments.
• primers added by engaging with SSBs

Question 15

E.coli DNA polymerase enzymatic functions?

Answer 15

• 5’–> 3’ DNA polymerase activity = synthesising DNA
• 5’–>3’ exonuclease activity = primer removal
• 3’–>5’ exonuclease: proofreading. will cleave incorrect nucleotide and then DNApol will add correct one.

Question 16

ddNTP

Answer 16

dideoxyribonucleotide triphosphate. chain terminating nucleotides; lack 3’ OH group needed for phosphodiester bond formation between 2 nucleotides

Question 17

What is needed for chain-termination method of DNA sequencing?

Answer 17

DNA single stranded template, DNA primer, DNA polymerase, dNTPS, ddNTPs

Question 18

describe the process of chain termination DNA sequencing?

Answer 18

• DNA samples divided into 4 separate sequencing reactions; each has all 4 dNTPs and DNA polymerase. one ddNTP added to each. each ddNTP has a radioactive label
• the formed fragments are separated by gel electrophoresis for each reaction so sequence can be ascertained
• only useful for fragmented sequences <150bp

Question 19

how was sanger sequencing improved?

Answer 19

using separation on chromatographic columns and labelling the ddNTPs with fluorescent markers and having computer detect the wavelength when sequence is terminated. useful for fragments <500 BP.

Question 20

What is next generation sequencing? (illuminati)

Answer 20

• large DNA molecules fragmented and ligated to short adaptors and attached to surface chambers
• immobilises DNA with all 4 dNTPS
• each has a specific fluorescent dye and terminator that has cleavable chemistry
• one cycle = one nucleotide added = one colour generated
• process repeated
• computer analyses light produced
• can sequence whole genomes

Question 21

what is PCR used for?

Answer 21

amplify a single copy or few copies of DNA

Question 22

stages of PCR?

Answer 22

1. denaturation (95dc) = strands separate
2. annealing (55dc) = primers base pair to complementary DNA template.
3. extension (72dc) = polymerase extends from primer to form new DNA strand