Presented by: Presented by: Group 8Group 8@ 23 March @ 23 March 20092009

MB 206 Microbial Biotechnology Presentation

Southern, Northern, western Blot

* 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

OBJECTIVES

INTRODUCTION

• 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

Southern, Northern and Western Blot:

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 moleculereactions are specific i.e. probes would only binds to target with complementary sequenceoccur 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

It can be used as analytical tools based on; Complementarity and Hybridization

Southern, Northern and Western Blot

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

Southern Blots

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

Digest DNA with restriction enzymes

1 Blotting. Transfer separated DNA

onto membrane for further analysis

3

Hybridize the target DNA

with specific labeled probe

5

Restriction Fragments are separated

by size by agarose gel

2

Synthesis of labelled probe

4Wash the filter

and expose the film to x-ray

6

Southern Blotting

Steps:1. DNA Fragmentation 2. Agarose Gel Electrophoresis3. Depurination (optional)4. Neutralization5. Blotting6. Prehybridization and Hybridization7. Removal of Unbound Probe8. Autoradiograph

1. DNA Fragmentation

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 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

Only ssDNA can be transferred to filter

5. Blotting

Exert pressure evenly to a gel to ensure even contact between gel and membrane

Transfer is done by capillary action which draws buffer (binds ssDNA) up onto the membrane

The binding of DNA to membrane is due to ion exchange interactions

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

5. Blotting (continued)

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

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 (b) Preparation of Labeled-probe

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.

Comparison of Nitrocellulose, Nylon membranes

Nitrocellulose membrane

Nylon membrane

DNA- binding capacity

100 µg/cm 500 µg/cm

Stability fragile less fragilethan 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:

1. 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 BlottingGel electrophoresis

1

Electroblotting2

Labeling with primary antibody

3

Labeling with 2nd antibody

4

Blocking step

Visualization5

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.

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

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

Blocking Step (Continued)

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.

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.

The advantages and disadvantages of Southern, Northern and Western Blots (Techniques of molecular

searching)

Gene Cloning • 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: 1. Southern blotting2. Northern blotting3. Western blotting

Screening of Gene Library

1. 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

• 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

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

Application (continue)

Zoo blot 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

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:

1. Southern BlottingUsed to identify specific restricted DNA fragments of interest.

2. Northern BlottingUsed to detect cellular RNA.

3. Western BlottingUsed to detect proteins of particular specificity.

Use a very similar methodology with some exceptions:

1. Sample preparation DNA cut with restriction enzyme – Southern RNA denatured with formaldehyde – Northern Protein denature with SDS – Western

2. Separation Agarose gel – Southern & Northern SDS polyacrylamide - Western

3. Blotting Capillary action – Southern & Northern Electrophoresis – Western

4. Hybridization Radioactivelly labelled DNA probes – Southern Radioactivelly labelled RNA probes – Northern Complementary antibody probes – Western

Advantages 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:

1. Screening of gene library Southern Blotting

Construction of restriction map of specific genes Northern Blotting

Determination of mRNA size and quality control of cloned cDNA

2. Study of gene evolution Southern Blotting

Identification of structurally related genes among same or different species and showing of evolutionary gene conservation degree.

3. Study of gene expressions

Southern BlottingUnderstand various biological processes

Northern BlottingComparison of gene transcriptional activites, analysis of gene expression patterns and regulated genes

Western BlottingAnalysis of expression of recombinant protein

4. Clinical dianogsis

Southern BlottingDetection of point mutation (Hemophilia A), gene arrangement (leukemia) and gene amplification (Charcot-Marie-Tooth syndrome).

Western BlottingImmunogenic response caused by infectious agents, alcohol abuse, abnormal cellular proteins and auto-antibodies.

QUESTIONS

1. 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.C. 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.

D. All of the above.

2. What is the type of probe used in Western Blotting?A. RNA B. Antibody C. DNA D. Protein

3. Which of the following is not the functions of southern blotting?

A. Detect the restiction site.B. Detect the specific DNA sequence.C. Determine the length of the restriction fragment which carries the

sequence.D. Study a specific gene expression.

4. What is the gel used in the gel electrophoresis of Northern Blotting?

A. SDS polyacrylamide gelB. Agarose gelC. Polyvinylpyrrolidone gelD. None of the above

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

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