3. Southern hybridization
First described by E. M. Southern in 1975.
Applications of Southern hybridization
RFLP’s, VNTR’s and DNA fingerprinting
Checking of the gene knockout mice
The flow chart of Southern hybridization
8. Flow chart of Southern
hybridization
Preparing the samples and running the gel
Southern transfer
Probe preparation Isotope
Non-isotope
Prehybridization
Hybridization
Post-hybridization washing
Signal detection
9. Preparing the samples and
running the gel
Digest 10 pg to 10 µg of desired DNA samples
to completion.
Prepare an agarose gel, load samples
(remember marker), and electrophorese.
Stain gel ethidium bromide solution (0.5
µg/ml).
Photograph gel (with ruler).
10. Critical parameters (I)
Note the complexity of DNA
Genomic DNA
A single-copy of mammalian gene, 3 Kb
average in length
10 µg x 3 Kb/3 x 106 Kb = 10 µg x 1/106 = 10
pg
Plasmid DNA or PCR products
0.1 µg of a 3 Kb plasmid DNA ≅100 ng
12. Southern transfer
Measure gel and set up transfer
assembly:
Wick in tray with 20x SSC
Gel
Nitrocellulose or Nylon filters (soaked
in H2O and 20x SSC)
3MM Whatman filter paper
Paper towels
Weight
13. After Southern transfer
Dissemble transfer
pyramid and rinse
nitrocellulose in 2x SSC
Bake nitrocellulose at 80°C
for 2 hr or UV-crosslink
Nylon membrane for
seconds
14. Preparation of probes
Synthesis of uniformly labeled
double-stranded DNA probes
Preparation of single-stranded probes
Labeling the 5′ and 3′ termini of DNA
15. Synthesis of double-stranded DNA
probes
- Nick translation of DNA
- Labeled DNA probes using random
oligonucleotide primers
17. Preparation of single-stranded
probes
Synthesis of single-stranded DNA probes
using bacteriophage M13 vectors.
Synthesis of RNA probes by in vitro
transcription by bacteriophage DNA-
dependent RNA polymerase.
19. Labeling the 5′ and 3′ termini of DNA
Labeling the 3′ termini of double-stranded DNA
using the Klenow fragment of E. coli DNA
polymerase I. (lack of 5’ 3’ exonuclease
activity)
Labeling the 3′ termini of double-stranded DNA
using bacteriophage T4 DNA polymerase.
Labeling the 5′ termini of DNA with
bacteriophage T4 polynucleotide kinase.
24. Hybridization
Remove prehybridization
solution and add
hybridization solution
Add 500,000 cpm of the
probe/ml hybridization
solution.
Hybridize overnight at
appropriate temperature.
25. Post-hybridization washing
Wash twice, 15 min each, in 1x SSC, 0.1% SDS at
room temperature.
Wash twice, 15 min each, in 0.25x SSC, 0.1%SDS
at hybridization temp
26. Critical parameters (II)
Homology between the probe and the sequences
being detected
Tm = 81 +16.6 (log Ci) + 0.4 [% (G+C)] - 0.6 (%
formamide)- 600/n - 1.5 (% mismatch)
Factors can be changed:
Hybridization temp.
Washing temp.
Salt concentration during washing
High temp., low salt: high stringency
Low temp., high salt: low stringency
If 50 % formamide is used
42 oC for 95 ~ 100 % homology
37 oC for 90 ~ 95 % homology
32 oC for 85 ~ 90 % homology
27. Comparison of nitrocellulose
and nylon membranes
NC Nylon
Hydrophobic binding Covalent binding
Fragile Durable
Probe length > 200 ~ < 200 ~ 300 bp is
300 bp O.K.
Lower background Higher background
Cannot be exposed Can be exposed to
to basic solution basic solution
Not easily Can be reprobed
reprobed several times
30. Northern blotting or Northern
hybridization
Technique for detecting specific RNAs
separated by electrophoresis by hybridization
to a labeled DNA probe.
31. The flow chart of Northern
hybridization samples and run RNA gel
Prepare RNA
Northern transfer
Probe preparation
Isotope
Prehybridization Non-isotope
Hybridization
Post-hybridization washing
Signal detection
32. Preparation of
agarose/formaldehyde gel
E.g. Prepare a 350 ml 1.2%
agarose/formaldehyde gel
4.2 g agarose in 304.5 g water. Microwave, then
cool to 60°C. Add 35 ml 10x MOPS running buffer
and 10.5 ml 37% formaldehyde
33. Preparation of RNA samples
Prepare a premix:
5 µl of 10x MOPS running buffer
8.75 µl of 37% formaldehyde
25 µl of formamide.
Prepare RNA samples:
38.75 µl of premix
RNA (0.5 to 10 µg)*
water to 50 µl
*If the mRNA species of interest makes up a relatively high percentage of
the mRNA in the cell (>0.05% of the message), total cellular RNA can be
used. If the mRNA species of interest is relatively rare, however, it is
advisable to use poly(A)+ RNA.
Incubate 15 min at 55°C
34. Running the RNA gel
Add 10 µl formaldehyde loading buffer to
each sample and load gel. Run gel at 100 to
120 V for ~3hr.
Remove gel from the running tank and rinse
several times in water. Place gel in 10x SSC
for 45 min.
Do not need post-transferring gel treatment
35. An example of Northern
blotting
Northern blot
RNA gel 28 S
18 S
37. Flow chart of Western blotting
Electrophoresing the protein sample
Assembling the Western blot sandwich
Transferring proteins from gel to nitrocellulose paper
Staining of transferred proteins
Blocking nonspecific antibody sites on the nitrocellulose paper
Probing electroblotted proteins with primary antibody
Washing away nonspecifically bound primary antibody
Detecting bound antibody by horseradish peroxidase-anti-Ig conjugate and
formation of a diaminobenzidine (DAB) precipitate
Photographing the immunoblot
39. Analysis of protein samples by SDS
polyacrylamide-gel electrophoresis and
Western blotting
Protein bands
detected by
specific antibody
SDS-PAGE Western blot
40. Comparison of Southern, Northern,
and Western blotting techniques
Southern blotting Northern blotting Western blotting
Molecule DNA (ds) mRNA (ss) Protein
detected
Gel Agarose gel Formaldehyde Polyacrylamide gel
electrophoresis agarose gel
Gel Depurination, - -
pretreatment denaturation, and
neutralization
Blotting method Capillary transfer Capillary transfer Electric transfer
Probes DNA cDNA, cRNA primary antibody
Radioactive or Radioactive or
nonradioactive nonradioactive
Detection Autoradiography Autoradiography Chemiluminescent
system Chemiluminescent Chemiluminescent Colorimetric
Colorimetric Colorimetric