Southern, Northern, western Blot MB 206 Microbial Biotechnology Presentation Southern, Northern, western Blot Presented by: Group 8 @ 23 March 2009

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

INTRODUCTION

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

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

Southern, Northern and Western Blot

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

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)

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)

Courtesy of www.molecularstation.com

METHODOLOGY SOUTHERN BLOTTING NORTHERN BLOTTING WESTERN BLOTTING

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

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

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.

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.

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

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.

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

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

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 2 hours - exposed to UV radiation

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

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

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

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.

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

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.

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

Northern Blotting

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)

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.

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

1. Gel electrophoresis

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.

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

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

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

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

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.

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.

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

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

Gene Library Different types of gene library

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

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

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

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)

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

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

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

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

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

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 

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

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

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

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

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

CONCLUSION

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.

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

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

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.

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.

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

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.

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

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.

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

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.

References Becker,J.M., Caldwell,G.A., and Zachgo, E.A., 1996. Biotechnology: A Laboratory Course. Academic press, pg 63-65. Gene Probe, 1997. Mondofacto [Online]. Available from: <http://www.mondofacto.com/facts/dictionary?gene%20probe> [Accessed 20 March 2009] Hockfield, S., 1993. Selected Methods for Antibody and Nucleic Acid Probes. CSHL Press, pg 293-296. Jacobson, E.R., 2007. Infectious Disease and Pathology of Reptiles. CRC Press, page 352 – 354. Kobilinsky, L.F., Liotti,T.F., Oeser-Sweat,J., and Watson, J.D., 2004. DNA: Forensic and Legal Applications. John Wiley and Sons, pg 58-60. Lutz, E., 2003. Cell and Molecular Biology: Southern or Northern Analysis [Online]. University of Strathclyde in Glasgow. Available from: <http://homepages.strath.ac.uk/~dfs99109/BB211/RecombDNAtechlect2.html> [Accessed 20 March 2009] Lyons, R.H., n.d. A Molecular Biology Glossary [Online].University of Michigan DNA Sequencing Core. Available from: <http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/mbglossary/mbgloss.html> [Accessed 20 March 2009]

References Mama Ji’s Molecular Kitchen, 2009a. Southern blotting [Online]. Arizona State University. Available from: <http://askabiologist.asu.edu/expstuff/mamajis/southern/southern.html> [Accessed 21 March 2008] Mama Ji’s Molecular Kitchen, 2009b. Northern blotting [Online]. Arizona State University. Available from: <http://askabiologist.asu.edu/expstuff/mamajis/northern/northern.html> [Accessed 21 March 2008] Mama Ji’s Molecular Kitchen, 2009c. Westhern blotting [Online]. Arizona State University. Available from: <http://askabiologist.asu.edu/expstuff/mamajis/western/western.html> [Accessed 21 March 2008] Molecular Searching Techniques, n.d. [Online]. Available from: <http://www.nsci.plu.edu/~mivey/407summer04/lectures/Molsearch.pdf> [Accessed 20 March 2009] Molecular Station, n.d. Western blot [Online]. Available from: <http://www.molecularstation.com/protein/western-blot/> [Accessed 20 March 2009] Olson,W.P., 1996. Automated Microbial Identification and Quantitation. CRC Press, page: 136 – 138.

References Patel, H., Arya, M., and Shergil, I.S., 2007. Basic Science Techniques in Clinical Practice. Springer, pg48-56. Promega Corporation, n.d. Usage Information [Online]. Available from: <http://www.promega.com/applications/cloning/REDigests.pdf> [Accessed 21 March 2008] Reece,J.B. and Campbell, N.A., 2002. Biology Sixth Edition. Pearson Education, page 384 – 385. Southern, Northern and Western Blotting, n.d. Molecular-Plant-Biotechnology info [Online]. Available from: <http://www.molecular-plant-biotechnology.info/molecular-probes-and-gene-libraries/southern-northern-western-blotting.htm> [Accessed 20 March 2009] Tietz, D., 1998. Nucleic Acid Electrophoresis. Springer, pg 5-26. Watsom,J.D., Gilman,M., Witkowski,J. and Zoller,M., 1992. Recombinant DNA, 2nd Ed. Scientific American Books, pg 127-130. White, B., 1995. Southerns, Northerns, Westerns, & Cloning: "Molecular Searching" Techniques [Online]. Available from: <http://www.sciencegateway.org/resources/biologytext/rdna/rdna.html#hybridization > [Accessed 21 March 2009]

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