Lesson 4 - Southern Blotting + Hybridization Method

Question 1

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

Answer 1

souther blotting + hybridization

Question 2

devised southern blotting

Answer 2

Edward Southern (1975)

Question 3

what is done to the DNA in southern blotting

Answer 3

separated on a gel

Question 4

how is the DNA fragments separated

Answer 4

blotting to a charged surface/ membrane

Question 5

example of charged surface/ membrane

Answer 5

nitrocellulose membrane

Question 6

used to find gene of interest

Answer 6

DNA probe

Question 7

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

Answer 7

by autoradiography

Question 8

autoradiography can detect ~5 µg single copy sequence

Answer 8

high sensitivity

Question 9

what can the autoradiography detect

Answer 9

~5µg single copy sequence

Question 10

applications of souther blotting/hybridization

Answer 10

1. RFLP mapping
2. forensic investigation
3. genetic screening

Question 11

RFLP

Answer 11

restriction fragment length polymorphism

Question 12

Steps in Southern Blotting + Hybridization

Answer 12

1. restriction digestion
2. electrophoresis
3. blotting and denaturation
4. hybridization with radioactive probe
5. autoradiography

Question 13

• DNA samples to be tested are prepared from the approprate sources
• restriction enzyme is added to the samples of DNA to produce restriction fragments

Answer 13

restriction fragment preparation

Question 14

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

Answer 14

restriction fragments

Question 15

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

Answer 15

electrophoresis

Question 16

• 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

Answer 16

blotting

Question 17

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

Answer 17

capillary action

Question 18

after blotting, what is done

Answer 18

DNA is fixed to the membrane

Question 19

how is DNA fixed to the membrane

Answer 19

• bake at 80°C or
• expose to UV

Question 20

• 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

Answer 20

hybridization with radioactive probe

Question 21

where is the paper blot exposed to afterward

Answer 21

solution containing radioactively labeled probe

Question 22

what is a probe

Answer 22

single-stranded DNA complementary to DNA sequence of interest

Question 23

how is unhybridized probe removed

Answer 23

washing (then dry)

Question 24

• 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

Answer 24

autoradiography

Question 25

Other blotting methods

Answer 25

1. northern blotting 2. western blotting 3. eastern blotting

Question 26

- RNA blotted on membrane - DNA probe

Answer 26

northern blotting

Question 27

what is blotted in northern blotting?

Answer 27

RNA

Question 28

probe in northern blotting

Answer 28

DNA probe

Question 29

- proteins blotted - labeled antibody specific for the protein

Answer 29

western blotting

Question 30

what is blotted in western blotting

Answer 30

proteins

Question 31

how is detection in western blotting done

Answer 31

labeled antibody specific for the protein

Question 32

used to detect glycoproteins and lipoproteins

Answer 32

eastern blotting

Question 33

what is detected in eastern blotting

Answer 33

- glycoproteins - lipoproteins

Question 34

western blotting is used to verify what?

Answer 34

protein expression

Question 35

verification of ZmCA1 gene insertion

Answer 35

PCR

Question 36

verification of protein expression

Answer 36

western blot analysis

Question 37

quantification of carbonic anhydrase activity

Answer 37

enzyme assay

Question 38

what is quantified in enzyme assay

Answer 38

carbonic anhydrase activity

Question 39

detection of copy number of gene insertion

Answer 39

southern blot analysis

Question 40

what is detected in southern blot analysis

Answer 40

copy number of gene insertion