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BIO107 - Cell and Molecular Biology > Lesson 4 - Southern Blotting + Hybridization Method > Flashcards

Lesson 4 - Southern Blotting + Hybridization Method Flashcards

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1
Q
  • devised by Edward Southern in 1975
  • DNA fragments separated on a gel
  • blotted to a charged surface/membrane
  • hybridized with a DNA probe
A

souther blotting + hybridization

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2
Q

devised southern blotting

A

Edward Southern (1975)

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3
Q

what is done to the DNA in southern blotting

A

separated on a gel

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4
Q

how is the DNA fragments separated

A

blotting to a charged surface/ membrane

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5
Q

example of charged surface/ membrane

A

nitrocellulose membrane

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6
Q

used to find gene of interest

A

DNA probe

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7
Q

how is the location of DNA+probe on the filter detected

A

by autoradiography

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8
Q

autoradiography can detect ~5 µg single copy sequence

A

high sensitivity

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9
Q

what can the autoradiography detect

A

~5µg single copy sequence

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10
Q

applications of souther blotting/hybridization

A
  1. RFLP mapping
  2. forensic investigation
  3. genetic screening
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11
Q

RFLP

A

restriction fragment length polymorphism

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12
Q

Steps in Southern Blotting + Hybridization

A
  1. restriction digestion
  2. electrophoresis
  3. blotting and denaturation
  4. hybridization with radioactive probe
  5. autoradiography
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13
Q
  • DNA samples to be tested are prepared from the approprate sources
  • restriction enzyme is added to the samples of DNA to produce restriction fragments
A

restriction fragment preparation

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14
Q

what is produced when a restriction enzyme is added to the samples of DNA

A

restriction fragments

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15
Q
  • the mixtures of restriction fragments from each sample are separated by this
  • each sample forms a characteristic pattern of bands
A

electrophoresis

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16
Q
  • capillary action pulls an alkaline solution upward through the gel and through a sheet of nitrocellulose paper laid on top of it
  • transfers the DNA to the paper and denaturing it in the process
  • single strands of DNA stick to the paper, positioned in bands exactly as on the gel
A

blotting

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17
Q

pulls the alkaline solution upward through the gel and through a sheet of nitrocellulose paper

A

capillary action

18
Q

after blotting, what is done

A

DNA is fixed to the membrane

19
Q

how is DNA fixed to the membrane

A
  • bake at 80°C or
  • expose to UV
20
Q
  • the paper is exposed to a solution containing a radioactively labeled probe
  • probe is single-stranded DNA complementary to the DNA sequence of interest, and it attaches by base pairing to restriction fragments of complementary sequence
A

hybridization with radioactive probe

21
Q

where is the paper blot exposed to afterward

A

solution containing radioactively labeled probe

22
Q

what is a probe

A

single-stranded DNA complementary to DNA sequence of interest

23
Q

how is unhybridized probe removed

A

washing (then dry)

24
Q
  • sheet of photographic film is laid over the paper
  • the radioactivity in the bound probe exposes the film to form an image corresponding to specific DNA bands – the bands containing DNA that base pairs with the probe
A

autoradiography

25
Q

Other blotting methods

A
  1. northern blotting
  2. western blotting
  3. eastern blotting
26
Q
  • RNA blotted on membrane
  • DNA probe
A

northern blotting

27
Q

what is blotted in northern blotting?

A

RNA

28
Q

probe in northern blotting

A

DNA probe

29
Q
  • proteins blotted
  • labeled antibody specific for the protein
A

western blotting

30
Q

what is blotted in western blotting

A

proteins

31
Q

how is detection in western blotting done

A

labeled antibody specific for the protein

32
Q

used to detect glycoproteins and lipoproteins

A

eastern blotting

33
Q

what is detected in eastern blotting

A
  • glycoproteins
  • lipoproteins
34
Q

western blotting is used to verify what?

A

protein expression

35
Q

verification of ZmCA1 gene insertion

A

PCR

36
Q

verification of protein expression

A

western blot analysis

37
Q

quantification of carbonic anhydrase activity

A

enzyme assay

38
Q

what is quantified in enzyme assay

A

carbonic anhydrase activity

39
Q

detection of copy number of gene insertion

A

southern blot analysis

40
Q

what is detected in southern blot analysis

A

copy number of gene insertion

BIO107 - Cell and Molecular Biology (8 decks)

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