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BIOL1024 Fundamentals of Biochemistry > DNA replication > Flashcards

DNA replication Flashcards

1
Q

3 models of how DNA is copied

A

conservative, semi-conservative, dispersive

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2
Q

Meselson and Stahl experiment - what is used

A

E.coli, Isotopes 14N and 15N

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3
Q

Meselson and Stahl experiment - method

A
  • cells transferred to medium (14N)
  • DNA isolated of 1,2,3….generations
  • separate DNA by density gradient centrifugation
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4
Q

at 0 generation

A

all DNA are 15N

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5
Q

1 generation

A

density between 14N + 15N (intermediate)

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6
Q

2 generation

A

14N(50%) and 50% intermediate

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7
Q

3 generation

A

14N (75%) and 25% intermediate

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8
Q

Density gradient centrifugation

A

heaviest sediment hit bottom faster

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9
Q

Caesium chloride density gradient centrifugation

A
  • DNA purified from bacteria and put in caesium chloride solution
  • spun in test tube for days at certain speed
  • DNA form equilibrium
  • higher conc of caesium at bottom
  • DNA separate depending on density (buoyant density)
  • sample reaches natural buoyancy in solution
  • bands exposed in UV light against the film
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10
Q

How DNA form equilibrium

A

density of DNA = density of surrounding

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11
Q

conclusion after M and S experiment - of mixture 14N and 15N

A

semi conservative or dispersive because they show mix of 14N and 15N

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12
Q

result of redo the experiment but can be done same in denatured DNA - single stranded

A

semi-conservative - get 15N and 14N

while dispersive - still mixed 15N and 14N

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13
Q

sucrose density centrifugation

A

sample at top of tube containing gradient of sucrose concentration
more concentrated at the bottom

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14
Q

replication of E.coli

A

circular double stranded genome

starts at a fixed point and is bidirectional

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15
Q

DNA replication in Eukaryotes

A

has multiple replication forks which are the multiple sites

strands separate and DNA polymerase forming daughter cells

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16
Q

what occurs to the duplex during replication in eukaryotes

A

opens up and new bases added at 3’ -end

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17
Q

how DNA is replicated - bonding of 2 nucleotides

A
  • deoxynucleotide triphosphate (DNT) bond to primar
  • -OH in primar attacks alpha phosphate of DNT
  • breaks bond and lose 2 phosphate group - hydrolysed
  • 2 phosphate separate which produces energy which drives reaction of addition of individual bases
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18
Q

Alpha phosphate position of DNT

A

first phosphate close to deoxyribose

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19
Q

Beta phosphate position in DNT

A

second phosphate close to deoxyribose

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20
Q

Gamma phosphate position in DNT

A

third phosphate to deoxyribose

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21
Q

Lagging strand

A

3’ - 5’ direction of other strand template

slightly slower than 5’ - 3’

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22
Q

Okazaki fragments - method

A
  • pulse labelling technique
  • using radioactive labelled DNA
  • pulse radioactive molecule and harvest DNA
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23
Q

Okazaki fragments - result

A

small fragments of DNA strand which can be joined together by DNA ligase

24
Q

helicase

A

opening up DNA stand by breaking hydrogen bonds forming the multiple replication forks

25
Q

primase

A

adding a short strip of RNA allowing polymerase to bond

26
Q

polymerase II

A

add new nucleotides at 3’ -end in 5’ - 3’ direction

27
Q

polymerase I

A

replace RNA laid down by primase

28
Q

ligase

A

reform phosphodiester linkage between lagging strands to form continuous strand

29
Q

binding proteins

A

stabilise DNA strand as it is separated as single stranded DNA is unstable

30
Q

Use of DNA polymerase for DNA sequencing

A

extend primer bound to single stranded fragment to be sequenced

31
Q

what other things needed for DNA sequencing

A

dideoxynucleoside triphosphate, normal dNTPs

32
Q

sanger method sequencing

A
  • add ddATP in chain which breaks the sequence at a specific point therefore have different size fragments
  • denature and separate products by polyacrylamid gel electrophoresis
33
Q

how to prevent formation of small fragments

A

have a lower concentration of ddNTP compared to dNTP

34
Q

Agarose

A

Physical gel used as horizontal submerge

35
Q

function of agarose

A

separate double stranded DNA fragment

36
Q

how to use agarose

A

heated and when cooled it forms a gel substance = mesh

37
Q

method of DNA sequencing using agarose gel

A

DNA is added and run through the gel to positive charge

shown by adding dye - fluoresces when bound to DNA

38
Q

polyacrylamid gel

A

chemical gel

39
Q

how to use polyacrylamid

A

gets a chemical reaction that cross polyacrylamid creating mesh network

40
Q

function of polyacrylamid

A

for small fragments

in denaturing gel at 8M urea to separate single stranded DNA fragments

41
Q

how is the DNA in polyacrylamid visualised

A

by autoradiography 32P or covalent attachment of fluorescent group

42
Q

how to read the results of DNA sequencing

A

smallest fragment at the positive side reading sequences 5’ to 3’

43
Q

increase in concentration of acrylamide

A

smaller the size of pores

44
Q

ddNTPs in sequencing to be seen

A

different colours for different bases in different or same gel

45
Q

Next generation sequencing

A
  • fragment DNA and anneal to slide using oligonucleotide adaptors
  • PCR amplified to multiple copies of that DNA
  • using fluorescent nucleotide for sequencing
  • images taken after adding new nucleotide
46
Q

what is used next generation sequencing

A

reversible terminator

47
Q

how reversible terminator is used

A

pause, take image, add nucleotide (reverse terminate)

48
Q

method of reversible terminator

A

3’ - reversible blocking group

- after image is taken, blocking group is cleaved off and adds -OH

49
Q

cluster of PCR

A

generate DNA

each cluster sequenced at the same time

50
Q

method of cluster of DNA

A

nucleotide is added to DNA template - picture taken

  • block and fluorescent dye removed
  • repeats to next nucleotide
51
Q

how DNA is amplified

A

using PCR - polymerase chain reaction

52
Q

method of PCR

A
  • DNA is denatured forming single strand by increasing temperature
  • add short primer complementary to end of sequence of interest
  • decrease temperature - anneal which binds to region to amplify
  • thermostable DNA polymerase used for DNA extension
  • repetition of denaturation, annealing and extension
53
Q

thermostable DNA polymerase

A

Taq

able to withstand high temperature and used to extend DNA

54
Q

Taq

A

originally isolated from Thermus aquaticus

55
Q

how cluster of OCR is generated

A

computer algorithms detect signals and construct sequence

BIOL1024 Fundamentals of Biochemistry (25 decks)

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