1. Southern, Northern and Western blotting
生理所 黃阿敏

2. Comparison of Southern, Northern, and Western analyses of Gene X

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

4. Southern hybridization
Transfer buffer

5. Detection of an RFLP by Southern blotting

6. Detection of the sickle-cell globin gene by Southern blotting
More than 3000 human genetic diseases are attributable to single-gene defects. In most of these the mutation is recessive: that is, it shows its effect only when an individual inherits two defective copies of the gene, one from each parent. One goal of modern medicine is to identify those fetuses that carry two copies of the defective gene long before birth so that the mother, if she wishes, can have the pregnancy terminated. In sickle-cell anemia, for example, the exact nucleotide change in the mutant gene is known (the sequence GAG is changed to GTG at a specific point in the DNA strand that codes for the β chain of hemoglobin). For prenatal diagnosis, two DNA oligonucleotides are synthesized - one corresponding to the normal gene sequence in the region of the mutation and the other corresponding to the mutant sequence. If the oligonucleotides are kept short (about 20 nucleotides), they can be hybridized with DNA at a temperature selected so that only the perfectly matched helix will be stable. Such oligonucleotides can thus be used as labeled probes to distinguish between the two forms of the gene by Southern blotting on DNA isolated from fetal cells collected by amniocentesis. A fetus carrying two copies of the mutant β-chain gene can be readily recognized because its DNA will hybridize only with the oligonucleotide that is complementary to the mutant DNA sequence.

7. Checking of the gene knockout mice
Figure 1. Targeting strategy and confirmation of gene targeting event. (A) Map of the K5 gene locus, the targeting vector, and the
recombinant K5 locus. The core promoter and the first two exons of the K5 gene up to the 59 EcoRI (R*) site were replaced by the HPRT
minigene. The arrow in the HPRT minigene indicates the direction of transcription. In addition, an HSV/TK minigene was inserted as a
negative selectable marker. The NotI restriction site was used to linearize the vector for transfection. Probes A and B mark the position of the
59 and 39 probe used in Southern blotting. Arrows above the K5 gene locus and the recombinant allele indicate primer positions for
PCR-based genotyping. Letters indicate restriction sites: A, ApaI; C, AccIII; H, HindIII; N, NotI; R, EcoRI; X, XbaI. (B) Example of Southern blot
analysis of ES cells. To confirm the correct targeting event, genomic DNA was digested with ApaI for detection with the 59 probe, which led
to a 5.6-kb band for the targeted allele and a 4.4-kb band for the wild-type allele. For detection with the 39 probe, an AccIII digest was
performed resulting in a 6.6-kb fragment for the targeted allele and a 7.8-kb fragment for the wild-type allele. (C) Identification of genotypes
by PCR. Primers designed to identify wild-type and mutant alleles (A) were used for genotyping of the litters. The wild-type allele resulted
in a product of ;1.8-kb size, the targeted allele in a 1.4-kb product. (D) Neonatal homozygous K5 2/2 mouse. The fragile epidermis almost
completely lost contact with the dermis after the mechanical stress of birth. Paws were sometimes denuded (arrow). K5 2/2 animals died
within 1 h after birth.
Molecular Biology of the Cell
Vol. 12, 1775–1789, June 2001
Complete Cytolysis and Neonatal Lethality in Keratin
5 Knockout Mice Reveal Its Fundamental Role in Skin
Integrity and in Epidermolysis Bullosa Simplex
Bettina Peters,* † Jutta Kirfel,* † Heinrich Bu¨ ssow, ‡ Miguel Vidal, § and
Thomas M. Magin* †i

8. Flow chart of Southern hybridization
Preparing the samples and running the gel
Southern transfer
Probe preparation
Prehybridization
Hybridization
Post-hybridization washing
Signal detection
Isotope
Non-isotope

9. Preparing the samples and running the gel
Digest 10 pg to 10 g of desired DNA samples to completion.
Prepare an agarose gel, load samples (remember marker), and electrophorese.
Stain gel ethidium bromide solution (0.5 g/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 mg x 3 Kb/3 x 106 Kb = 10 mg x 1/106 = 10 pg
Plasmid DNA or PCR products
0.1 mg of a 3 Kb plasmid DNA 100 ng

11. Gel treatment Acid treatment Denaturation Neutralization
0.2 N HCl solution
Denaturation
NaOH solution
Neutralization
Tris-Cl buffer (pH8.0)

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

16. Nick translation

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.

18. In vitro transcription

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.

20. T4 polynucleotide kinase activity

21. Non-isotope labeling Digoxigenin-11-dUTP (DIG-dUTP) labeling
DNA labeling
Oligonucleotide labeling
RNA labeling

22. PCR Labeling, Random Primed Labeling, and RNA Labeling

23. Prehybridization
Add prehybridization solution and prehybridize at hybridization temperature for 2-4 hr

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 = (log Ci) [% (G+C)] (% formamide)- 600/n (% 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 membranesNC
Nylon
Hydrophobic binding
Covalent binding
Fragile
Durable
Probe length > 200 ~ 300 bp
< 200 ~ 300 bp is O.K.
Lower background
Higher background
Cannot be exposed to basic solution
Can be exposed to basic solution
Not easily reprobed
Can be reprobed several times

28. Signals detection Autoradioragraphy Non-isotope detection system
Chemiluminescent detection
Colorimetric detection
Multicolor detection

29. Autoradiography
Exposure to x-ray film

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
Prepare RNA samples and run RNA gel
Northern transfer
Probe preparation
Prehybridization
Hybridization
Post-hybridization washing
Signal detection
Isotope
Non-isotope

32. Preparation of agarose/formaldehyde gel
E.g. Prepare a 350 ml 1.2% agarose/formaldehyde gel
4.2 g agarose in 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 l of 10x MOPS running buffer
8.75 l of 37% formaldehyde
25 l of formamide.
Prepare RNA samples:
38.75 l of premix
RNA (0.5 to 10 g)*
water to 50 l
*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 l 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
RNA gel
28 S
18 S

36. Western blotting, or immunoblotting
Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies.

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

38. SDS polyacrylamide-gel electrophoresis (SDS-PAGE)

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