1. Southern, Northern, western Blot
MB Microbial Biotechnology Presentation
Southern, Northern, western Blot
Presented by: Group 8
@ 23 March 2009

2. OBJECTIVES * To understand the techniques of molecular
searching ( Western, Northern, Southern Blots)
* To differentiate the advantages and
disadvantages of the techniques
* To determine the applications of the techniques
used

3. INTRODUCTION

4. Southern, Northern and Western Blot:
Techniques of Molecular Searching
- determine by analyzing cellular molecules (DNA, RNA
and protein)
- transfer the cellular molecules onto a carrier (membrane)
- i.e. after the gel electrophoresis - transfer the molecules
from the gel to the blotting membrane - the transferred
cellular molecules can be detected

5. It can be used as analytical tools based on;
Complementarity and Hybridization
Complementarity
Hybridization
sequence-specific or shape-specific molecular recognition that occurs when two molecules bind together
result in probe-target complex
i.e. complementary DNA sequences
antibody binds to antigen
(complementary shapes)
a process of combining complementary, single stranded nucleic acids into a single molecule
reactions are specific i.e. probes would only binds to target with complementary sequence
occur in the presence of large quantities of molecules similar but not identical to the target
hybrids that can exist (solution); DNA-DNA, DNA-RNA,protein-protein

6. Southern, Northern and Western Blot

7. Southern Blots
invented by the English Molecular Biologist Edwin Southern (1975)
determine the presence of a specific DNA sequence within a large, complex DNA sample
Probe with radioactive DNA
DNA cut with restriction enzymes is separated by molecular weight
Identify which DNA fragments obtained from a digest of a larger DNA clone that contains sequence complementary to a specific probe
Determine the number of copies of a particular DNA sequence presented in the genome
Can identify related sequences in the genome

9. Northern Blot Developed by James Alwine, David Kemp, and George
Stark (1977)
Similar to Southern Blotting
Detects the presence a specific mRNA in a total RNA extract
Can determine whether the gene is transcribed or not
Identify where and when it is transcribed
Probed with radioactive DNA or RNA
RNA denatured with formaldehyde (separated by molecular weight)

11. Western Blots Developed by W. Neal Burnette (1981)
Also known as immunoblot
Detects the presence of specific proteins in a given sample of protein extract
The procedures are rather similar to Southern and Northern except that the cellular content extracted is protein
Protein probed with radioactive or enzymatically-tagged antibodies
Gives information on the size of proteins and expression amounts of the protein
Based on protein-protein interaction; Enzyme Link Immunosorbant Assay (Elisa)
Protein denatured with SDS (separated by molecular weight)

12. Courtesy of www.molecularstation.com

13. METHODOLOGY
SOUTHERN BLOTTING
NORTHERN BLOTTING
WESTERN BLOTTING

14. Restriction Fragments Transfer separated DNA
are separated
by size by agarose gel 2
Blotting.
Transfer separated DNA
onto membrane for
further analysis 3
Digest DNA with
restriction enzymes 1
Wash the filter
and expose
the film to x-ray 6
Synthesis of
labelled probe 4
Hybridize the
target DNA
with specific
labeled probe 5

15. Southern Blotting Steps: DNA Fragmentation Agarose Gel Electrophoresis
Depurination (optional)
Neutralization
Blotting
Prehybridization and Hybridization
Removal of Unbound Probe
Autoradiograph

16. 1. DNA Fragmentation - bacterial enzymes
DNA is digested by one or several restriction enzymes or restriction endonucleases
- bacterial enzymes
- cut at specific sequence (restriction site)
If REs with different buffer requirements are used, a prior addition of RE buffer before the second enzyme is used.

17. 2. Agarose Gel Electrophoresis
Restriction fragments are separated electrophoretically by size on agarose gel.
- negatively charged
DNA fragments migrate into gel toward the anode (+ve electrode) under the influence of electric field.

18. Rate of movement is determined by size of fragment
the largest
molecule
has the lowest
mobilities

19. 3. Depurination Optional Occurs before neutralization
When DNA fragments is > 15 kilobases, it is too hard to be transferred to filter
The gel is treated with dilute acid (0.2 M HCl for 15 minutes)
depurinate DNA fragment into smaller pieces and promote higher efficiency transfer to filter.

20. 4. Neutralization Only ssDNA can be transferred to filter
DNA is placed into an alkaline solution containing 0.5mM NaOH to denature the dsDNA into ssDNA and neutralize the acid in previous step.
Function:
(1) improve binding of the –vely charged DNA to
+vely charged filter
(2) ssDNA strands for hybridization
(3) destroy any remaining RNA present in the sample

21. 5. Blotting
Transfer is done by capillary action which draws buffer (binds ssDNA) up onto the membrane
Exert pressure evenly to a gel to ensure even contact between gel and membrane
The binding of DNA to membrane is due to ion exchange interactions

22. 5. Blotting (continued)
To permanently attach the transferred DNA to the membrane, the blot can be:
- baked in a vacuum or regular oven at 80 °C for hours
- exposed to UV radiation

23. 6 (a). Prehybrization
Prevent the labeled probe from binding nonspecifically to DNA fragments on the membrane
Non-specific ssDNA is added such as salmon or herring sperm DNA; deionized formamide, and detergents such as SDS to reduce non-specific binding of the probe

24. Probe
It can be a purified RNA, a cloned cDNA, or a short synthetic oligonucleotide with a reporter substance attached to it.
- is a radioactive element like (32P) that induces light
production
It contains a short segment of ssDNA that is complementary to the DNA sequence of interest and tags the sequence of interest.
Usually prepared by making a radioactive copy of a DNA fragment.
- E.g 32P-labeled probe

25. 6 (b) Preparation of Labeled-probe
Treat with DNase (causes double stranded nick in DNA)
Add 32P, dATP, and other dNTPs to DNA polymerase I
32P becomes incorporated into, and thus labels, the DNA
Heat and on ice to prevent two strands from reannealing

27. 6 (c). Hybridization Use the same buffer as for prehybridization
The filter is removed and hybridized with a radioactively labeled probe at 65oC and incubated for several hours to allow the probe molecules to find their targets.

28. 7. Removal of Unbound Probe
Unbound probe is washed off and the membrane is exposed to x-ray film.

29. 8.Autoradiograph
The location of the probe is revealed by converting a colorless substrate to a colored product that can be seen or gives off light which will expose X-ray film.
If you used a radiolabeled
32 P probe, then you would visualize by autoradiograph.
The bands indicate the number and size of the DNA fragments complementary to the probe.
Autoradiograph is an image on x-ray film produced by a decay emission from a radioactive substances.

30. Comparison of Nitrocellulose, Nylon membranes
Nitrocellulose membrane
Nylon membrane
DNA- binding capacity
100 µg/cm
500 µg/cm
Stability
fragile
less fragile
than nitrocellulose filter
Remarks
Non-specific binding site is easily blocked
Protein staining is difficult owing to the +ve charged membrane. Blocking of unoccupied binding sites may be a problem

31. Northern Blotting

32. Steps in Northern Blotting:
Extraction of RNA
- The RNA sample can be:
i. total RNA isolated from particular samples
ii. RNA containing poly(A) tails, i.e messenger RNA(mRNA)

33. 2. Gel Electrophoresis
- agarose gel
3. The RNA molecules in the gel are transferred to nitrocellulose or nylon.
The principle and procedure for Northern Blotting is similar, except
you are working with RNA instead of DNA.

34. Western Blotting 1 3 Gel electrophoresis
Labeling with primary antibody 3
Blocking step
Electroblotting 2
Labeling with 2nd antibody 4
Visualization 5

35. 1. Gel electrophoresis

36. 2. Electroblotting
uses an electric current to drive the protein (polypeptide) bands onto the nitrocellulose membrane
It is often be used with gels made of polyacrylamide rather than that of agarose since polyacrylamide has a higher melting temperature.
Protein binding is based upon hydrophobic interactions, as well as charged interactions between the filter and protein.

37. Blocking Step
The nitrocellulose is then soaked into a concentrated nonantigenic protein solution (blocking solution containing nonfat dried milk [BLOTTO])
The protein in the solution will bind nonspecifically to all areas on nitrocellulose that do not absorb protein from the SDS-polyacrylamide gel
- The antibodies are diluted in this nonantigenic protein solution before applying to the nitrocellulose

38. Blocking Step (Continued)
Functions:
- prevent the antibodies from binding non- specifically to
the nitrocellulose and unrelated proteins on
nitrocellulose
- increase the probability that they bind to immobilized
antigenic proteins

39. 3. Labeling with Primary Antibody
forms an antibody-protein complex with the protein of interest

40. 4. Labeling with Secondary Antibody
Is conjugated to HRP (horseradish peroxidase)
Acts as antibody against primary antibody
Antigens can be visualized through coloured reaction
Advantage:
signal of both minor and major antigens can be visualized and optimized on single blot by varying the exposure time

41. 5. Visualization
The position of protein of interest is marked by visible band, forming protein-primary antibody-secondary antibody-enzyme complex
A flash light is observed which expose x-ray film. The light is due to the release of protons by catalyzing the oxidation luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) by HRP.

42. The advantages and disadvantages of Southern, Northern and Western Blots (Techniques of molecular searching)
Advantages
Disadvantages
able to identify infectious agents present in the sample and inherited disease
can be applied to mapping restriction sites in single copy gene
widely accepted method
adaptable protocol - it allows the usage of many types of probes
The processes are complex and time consuming
requires electrophoretic separation.
only one gene can be analysed at a time
gives information about presence of DNA, RNA or proteins but does not give information about regulation and gene interaction.

43. Gene Cloning DNA selection
Basic Steps in Gene Cloning:
DNA selection
Cut DNA and vectors with restriction endonuclease
Insert DNA fragments into vectors
Seal the vector and DNA fragment with ligase
Transferred the recombinant DNA into bacterial cells
Plate the cells on agar plate with antibiotics

44. Gene Library Cloned genes with different DNA fragments from 1 organism
Gene library of the organism
Gene library  used to screen for gene of interest

45. Gene Library
Different types of gene library

46. Screening of Gene Library
Uses:
To identify the gene of interest
Techniques:
Southern blotting
Northern blotting
Western blotting

47. Screening of Gene Library
Southern blotting
~ Detect gene fragments of interest
2. Northern blotting
~ Detect transcription (mRNA) level
3. Western blotting
~ Detect the fusion protein (target protein) with a specific antibody
~ Mostly used in libraries in phage λ expression vectors

48. Southern Blotting Main functions
Detect the specific DNA sequence (gene) of interest
Determine the length of the restriction fragment carrying the sequence
Detect the restriction site

49. Southern Blotting Application Diagnosis of human disease
Detect point mutation, gene rearrangement or gene amplification
Mutated gene  change in the size (hemophilia A)
Gene rearrangement  change in size and pattern (leukemia)
Amplification  increase in gene copy number (Charcot-Marie-Tooth syndrome)

50. Southern Blotting Application (continue)
Identify structurally related genes in the same species or among other species
Understand various biological processes
Discovery of RNA splicing, genomic rearrangement to form antibodies and T cell receptors and etc.
Construct a restriction map of a specific gene
By performing RE digestion to the specific gene

51. Southern Blotting Zoo blot Application (continue)
A southern blot of genomic DNA from different species
Show the degree of evolutionary gene conservation
E.g. Identifying genes in yeasts related to oncogenes in human tumor cells

52. Northern Blotting Main functions Detect mRNA transcriptional activity
Quantifying the transcription
Determine the size of the mRNA
Determine mRNA level

53. Northern Blotting Application Analysis of regulated genes
Indicates which tissues express the gene
Investigate factors controlling the gene expression
Checking if the cloned cDNA is full length
Comparing the size of mRNA with the size of cloned cDNA

54. Northern Blotting Application (continue)
Measuring the size of a gene’s mRNA
Compare with the marker RNA of known size
Study the patterns of gene expression in embryonic and adult tissues

55. Northern Blotting Application (continue)
Comparing the transcriptional activities of genes in different cells, tissues and organisms
By measuring the density of band
Amount of transcribed RNA , density of the RNA band 

56. Western Blotting (Immunoblotting)
Main functions
Study a specific gene expression
Analyze endogenous protein level
Determine the mass of protein
Compare with protein molecular weight standards

57. Western Blotting (Immunoblotting)
Application
Analyze recombinant protein expression
Detect contaminant proteins
Determine alcohol abuse
Measure carbohydrate-deficient transferrins level in blood

58. Western Blotting (Immunoblotting)
Application (continue)
Clinical diagnosis
Detect immunogenic responses by infectious agents (bacteria, parasites)
E.g. Human immunoglobulin in serum binds to the parasitic proteins that are given externally, indicate parasitic infection

59. Western Blotting Application (continue) Clinical diagnosis
Double conform the presence of abnormal cellular proteins, e.g. prion proteins, human immunoglobulin bound to the HIV protein
Detect auto-antibodies that causes autoimmune disease
Auto-antibody: fight against normal human proteins

60. Ans: Run PCR to amplify the desired gene
Q: After we get our desired gene using blotting techniques, what can we do to it?
Ans: Run PCR to amplify the desired gene 
Generate recombinant DNA products

61. CONCLUSION

62. General functions: Southern Blotting
Used to identify specific restricted DNA fragments of interest.
Northern Blotting
Used to detect cellular RNA.
Western Blotting
Used to detect proteins of particular specificity.

63. Use a very similar methodology with some exceptions:
Sample preparation
DNA cut with restriction enzyme – Southern
RNA denatured with formaldehyde – Northern
Protein denature with SDS – Western
Separation
Agarose gel – Southern & Northern
SDS polyacrylamide - Western

64. Blotting Hybridization Capillary action – Southern & Northern
Electrophoresis – Western
Hybridization
Radioactivelly labelled DNA probes – Southern
Radioactivelly labelled RNA probes – Northern
Complementary antibody probes – Western

65. Advantages Disadvantages Involve many types of probes.
Identify inherited disease and infectious agents.
Applicable in single copy gene form.
Widely accepted.
Disadvantages
Time consuming.
Complicated process.
Cannot analyze sample of more than one gene.
Require separation by electrophoresis.
Only detect presence of targets but not interactions or regulations of targets.

66. Applicable in: Screening of gene library Study of gene evolution
Southern Blotting
Construction of restriction map of specific genes
Northern Blotting
Determination of mRNA size and quality control of cloned cDNA
Study of gene evolution
Identification of structurally related genes among same or different species and showing of evolutionary gene conservation degree.

67. Study of gene expressions
Southern Blotting
Understand various biological processes
Northern Blotting
Comparison of gene transcriptional activites, analysis of gene expression patterns and regulated genes
Western Blotting
Analysis of expression of recombinant protein

68. Clinical dianogsis
Southern Blotting
Detection of point mutation (Hemophilia A), gene arrangement (leukemia) and gene amplification (Charcot-Marie-Tooth syndrome).
Western Blotting
Immunogenic response caused by infectious agents, alcohol abuse, abnormal cellular proteins and auto-antibodies.

69. QUESTIONS What is a probe?
A. Nucleotide sequences present in a plasmid which are necessary for that plasmid to replicate in the bacterial host.
B. A small piece of synthetic double-stranded DNA which contains a restriction site.
A fragment of DNA or RNA that is labelled with radioactive isotopes or with a fluorescent marker that selectively binds to a specific gene so it can be isolated or identified.
All of the above.
What is the type of probe used in Western Blotting?
A. RNA B. Antibody C. DNA D. Protein

70. Which of the following is not the functions of southern blotting?
Detect the restiction site.
Detect the specific DNA sequence.
Determine the length of the restriction fragment which carries the sequence.
Study a specific gene expression.

71. What is the gel used in the gel electrophoresis of Northern Blotting?
SDS polyacrylamide gel
Agarose gel
Polyvinylpyrrolidone gel
None of the above

72. What is the disadvantage of blotting techniques?
Can identify infectious agents and inherited diseases.
It is a widely accepted method.
The process is complicated and time consuming.
It allows the using of many types of probes.

73. References
Becker,J.M., Caldwell,G.A., and Zachgo, E.A., Biotechnology: A Laboratory Course. Academic press, pg
Gene Probe, Mondofacto [Online]. Available from: < [Accessed 20 March 2009]
Hockfield, S., Selected Methods for Antibody and Nucleic Acid Probes. CSHL Press, pg
Jacobson, E.R., Infectious Disease and Pathology of Reptiles. CRC Press, page 352 – 354.
Kobilinsky, L.F., Liotti,T.F., Oeser-Sweat,J., and Watson, J.D., DNA: Forensic and Legal Applications. John Wiley and Sons, pg
Lutz, E., Cell and Molecular Biology: Southern or Northern Analysis [Online]. University of Strathclyde in Glasgow. Available from: < [Accessed 20 March 2009]
Lyons, R.H., n.d. A Molecular Biology Glossary [Online].University of Michigan DNA Sequencing Core. Available from: < [Accessed 20 March 2009]

74. References
Mama Ji’s Molecular Kitchen, 2009a. Southern blotting [Online]. Arizona State University. Available from:
< [Accessed 21 March 2008]
Mama Ji’s Molecular Kitchen, 2009b. Northern blotting [Online]. Arizona State University. Available from:
< [Accessed 21 March 2008]
Mama Ji’s Molecular Kitchen, 2009c. Westhern blotting [Online]. Arizona State University. Available from:
< [Accessed 21 March 2008]
Molecular Searching Techniques, n.d. [Online]. Available from: < [Accessed 20 March 2009]
Molecular Station, n.d. Western blot [Online]. Available from: < [Accessed 20 March 2009]
Olson,W.P., Automated Microbial Identification and Quantitation. CRC Press, page: 136 – 138.

75. References
Patel, H., Arya, M., and Shergil, I.S., Basic Science Techniques in Clinical Practice. Springer, pg48-56.
Promega Corporation, n.d. Usage Information [Online]. Available from:
< [Accessed 21 March 2008]
Reece,J.B. and Campbell, N.A., Biology Sixth Edition. Pearson Education, page 384 – 385.
Southern, Northern and Western Blotting, n.d. Molecular-Plant-Biotechnology info [Online]. Available from: < [Accessed 20 March 2009]
Tietz, D., Nucleic Acid Electrophoresis. Springer, pg 5-26.
Watsom,J.D., Gilman,M., Witkowski,J. and Zoller,M., Recombinant DNA, 2nd Ed. Scientific American Books, pg
White, B., Southerns, Northerns, Westerns, & Cloning: "Molecular Searching" Techniques [Online]. Available from: < > [Accessed 21 March 2009]

76. THE END