Southern, Northern and Western blotting

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

DNA

• Each individuals unique genetic blueprint is stored in material known as DNA.

• DNA is found in all cells containing a nucleus.• DNA can be extracted for analysis from hair,

bones, saliva, sperm, skin, organs, all body tissues and blood.

DNA

• The deoxyribonucleic acid, DNA, is a long chain of nucleotides which consist of:

• 1. Deoxyribose(sugar with 5 carbons)• 2. Phosphate groups• 3. Organic(nitrogenous)bases

Nitrogenous Bases

• Two classes:• Purines

– Adenine– Guanine

• Pyrimidines– Cytosine– Thymine

DNA

• DNA molecules are arranged in a double helix which resembles a tightly coiled twisted ladder.

• The sides of the ladder have alternating units of phosphate and deoxyribose sugar.

DNA

• The rungs of the ladder are formed by the nitrogenous “base pairs”.

• Hydrogen bonds hold the strands together.• The bases bind together in a complementary

fashion.

DNA

• The base adenine (A) always pairs with thymine (T).

• The base guanine (G) always pairs with cytosine (C).

DNA

• Example

• First strand GGGTTTAAACCC• Second strand CCCAAATTTGGG

DNA STORAGE AND COLLECTION

• I. Temperature Storage for DNA–Purified DNA may be refrigerated at

4°C for up to 3 years.–Samples kept over 3 years should be

frozen at -70°C.

DNA STORAGE AND COLLECTION

• II. Specimens used in DNA testing– Whole blood– Solid tissue– Serum and plasma– Urine– Bone marrow– and many others

DNA STORAGE AND COLLECTION

• III. Specimen Collection Requirements– A. Blood and Bone Marrow

• Collection tubes are EDTA or ACD• 5-15 ml • Samples should not be frozen for transport• 4-25°C

DNA STORAGE AND COLLECTION

• B. Serum– Collection tubes with no additives– 100 µl to 1 ml– Transported at 20-25°C

DNA STORAGE AND COLLECTION

• Spin the samples to separate the plasma, RBC, and buffy coat.

• Extract the buffy coat• The buffy coat is used because the WBC are

nucleated and contain DNA.

DNA STORAGE AND COLLECTION

• C. Tissue– A sterile container with no formalin or

paraffin must be used for collection.– 30 mg – Dry ice should be used for transport.

DNA STORAGE AND COLLECTION

• D. Urine– Urine container should be used for

collection.– At least 1 ml should be collected.– Transported at 4-25°C

SOUTHERN BLOTTING

• The technique was developed by E.M. Southern in 1975.

• The Southern blot is used to detect the presence of a particular piece of DNA in a sample.

• The DNA detected can be a single gene, or it can be part of a larger piece of DNA such as a viral genome.

Southern hybridization

Transfer buffer

Detection of an RFLP by Southern blotting

Detection of the sickle-cell globin gene by Southern blotting

Checking of the gene knockout mice

Flow chart of Southern hybridizationPreparing the samples and running the gel

Southern transfer

Probe preparation

Prehybridization

Hybridization

Post-hybridization washing

Signal detection

IsotopeNon-isotope

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).

Critical parameters (I)• Note the complexity of DNA

– Genomic DNA• A single-copy of mammalian gene, 3 Kb

average in length 10 g x 3 Kb/3 x 106 Kb = 10 g x 1/106 =

10 pg– Plasmid DNA or PCR products 0.1 g of a 3 Kb plasmid DNA 100 ng

Gel treatment

• Acid treatment – 0.2 N HCl solution

• Denaturation– NaOH solution

• Neutralization– Tris-Cl buffer (pH8.0)

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

After Southern transfer

• Dissemble transfer pyramid and rinse nitrocellulose in 2x SSC

• Bake nitrocellulose at 80C for 2 hr or UV-crosslink Nylon membrane for seconds

Preparation of probes

• Synthesis of uniformly labeled double-stranded DNA probes

• Preparation of single-stranded probes• Labeling the 5 and 3 termini of DNA

Synthesis of double-stranded DNA probes

- Nick translation of DNA- Labeled DNA probes using random

oligonucleotide primers

Nick translation

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.

In vitro transcription

• 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.

Labeling the 5 and 3 termini of DNA

T4 polynucleotide kinase activity

Non-isotope labeling• Digoxigenin-11-dUTP (DIG-dUTP) labeling

- DNA labeling- Oligonucleotide labeling- RNA labeling

PCR Labeling, Random Primed Labeling, and RNA Labeling

Prehybridization

• Add prehybridization solution and prehybridize at hybridization temperature for 2-4 hr

Hybridization

• Remove prehybridization solution and add hybridization solution

• Add 500,000 cpm of the probe/ml hybridization solution.

• Hybridize overnight at appropriate temperature.

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

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 washingHigh temp., low salt: high stringencyLow 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

Comparison of nitrocellulose and nylon membranes

NC 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

Signals detection• Autoradioragraphy• Non-isotope detection system

- Chemiluminescent detection- Colorimetric detection- Multicolor detection

Autoradiography

• Exposure to x-ray film

SOUTHERN BLOTTING

• The key to this method is hybridization.• Hybridization-process of forming a double-

stranded DNA molecule between a single-stranded DNA probe and a single-stranded target patient DNA.

SOUTHERN BLOTTING

• There are 2 important features of hybridization:– The reactions are specific-the probes will only

bind to targets with a complementary sequence.– The probe can find one molecule of target in a

mixture of millions of related but non-complementary molecules.

SOUTHERN BLOTTING

SOUTHERN BLOTTING

• Steps for hybridization– 1. The mixture of molecules is separated.– 2. The molecules are immobilized on a matrix.– 3. The probe is added to the matrix to bind to the

molecules.– 4. Any unbound probes are then removed.– 5. The place where the probe is connected corresponds to

the location of the immobilized target molecule.

SOUTHERN BLOTTING

• I. DNA Purification– Isolate the DNA in question from the rest of

the cellular material in the nucleus.– Incubate specimen with detergent to

promote cell lysis.– Lysis frees cellular proteins and DNA.

SOUTHERN BLOTTING

• Proteins are enzymatically degraded by incubation with proteinase.

• Organic or non-inorganic extraction removes proteins.

• DNA is purified from solution by alcohol precipitation.

• Visible DNA fibers are removed and suspended in buffer.

SOUTHERN BLOTTING

• II. DNA Fragmentation– Cut the DNA into different sized pieces.– Use restriction endonucleases (RE)– Bacterial proteins– In vivo, they are involved in DNA

metabolism and repair or in bacterial host defense.

SOUTHERN BLOTTING

• Nucleases hydrolyze the bonds that connect bases within the strand, resulting in cleavage of the strand.

• They cleave the double stranded nucleic acid only at specific points.

SOUTHERN BLOTTING

• This allows for specific sequences to be identified more readily.

• Fragments are now easily separated by gel electrophoresis.

SOUTHERN BLOTTING

• III. Gel Electrophoresis– Sorts the DNA pieces by size– Gels are solid with microscopic pores– Agarose or polyacrimide– Gel is soaked in a buffer which controls the

size of the pores– Standards should also be run

SOUTHERN BLOTTING

• Nucleic acids have a net negative charge and will move from the left to the right. The larger molecules are held up while the smaller ones move faster. This results in a separation by size.

SOUTHERN BLOTTING

• Gels can be stained with ethidium bromide. • This causes DNA to fluoresce under UV light

which permits photography of the gel.• You can tell the exact migration of DNA

standards and the quality of the RE digestion of the test DNA.

SOUTHERN BLOTTING

• High quality intact DNA should give the appearance of a single band.

• Degraded material will smear downwards. • Only a small amount of degradation is

tolerable.

SOUTHERN BLOTTING

• IV. Blotting – Transfer the DNA from the gel to a solid

support.– The blot is usually done on a sheet of

nitrocellulose paper or nylon.

SOUTHERN BLOTTING

• DNA is partially depurinated with dilute HCL which promotes higher efficiency transfer by breaking down fragments into smaller pieces.

• DNA is then denatured with an alkaline solution such as NAOH.

• This causes the double stranded to become single-stranded.

SOUTHERN BLOTTING

• DNA is then neutralized with NaCl to prevent re-hybridization before adding the probe.

• Transferred by either electrophoresis or capillary blotting.

SOUTHERN BLOTTING

• 1) Electrophoresis- takes advantage of the molecules negative charge.

SOUTHERN BLOTTING

• 2) Capillary blotting-fragments are eluted from the gel and deposited onto the membrane by buffer that is drawn through the gel by capillary action.

SOUTHERN BLOTTING

• The blot is made permanent by:– Drying at ~80°C– Exposing to UV irradiation

SOUTHERN BLOTTING

• V. Blocking– Buffer binds to areas on the blot not

occupied by patient DNA.– Blocks the empty sites from being bound

during hybridization.

SOUTHERN BLOTTING

• VI. Preparing the probe– Small piece of DNA used to find another

piece of DNA– Must be labeled to be visualized– Usually prepared by making a radioactive

copy of a DNA fragment.

SOUTHERN BLOTTING• The DNA fragment is labeled by the Random

Hexamer Labeling Process:

– 1. The template DNA is denatured by boiling.– 2. A mixture of hexamers (6 nucleotides)

containing all possible sequences is added and allow to base pair.

– 3. DNA polymerase is added with radioactive nucleotides.

– 4. The mixture is boiled to separate the strands and is ready for hybridization.

SOUTHERN BLOTTING

• The Random Hexamer Labeling Process produces a radioactive single-stranded DNA copy of both strands of the template for use as a probe.

SOUTHERN BLOTTING

SOUTHERN BLOTTING

• VII. Hybridization– The labeled probe is added to the blocked

membrane in buffer and incubated for several hours to allow the probe molecules to find their targets.

SOUTHERN BLOTTING

• VIII. Washing– Excess probe will have bound nonspecifically to

the membrane despite the blocking reagents.– Blot is incubated with wash buffers containing

NaCl and detergent to wash away excess probe and reduce background.

SOUTHERN BLOTTING

• IX. Detection– Radioactive probes enable autoradiographic

detection.

SOUTHERN BLOTTING

• If the probe is radioactive, the particles it emits will expose X-ray film.

• By pressing the filter and film, the film will become exposed wherever probe is bound to the filter.

• After development, there will be dark spots on the film wherever the probe bound.

TroubleshootingPoor signal Probe specific activity too low Inadequate depurination Inadequate transfer buffer Not enough target DNA Transfer time too short Inefficient transfer system Probe concentration too low Incomplete denaturation of probe and/or target DNA Final wash too stringent Hybridization time too short Inappropriate membrane

Troubleshooting

Spotty Background Unincorporated nucleotides not removed from

labeled probe Particles in hybridization buffer Agarose dried on membrane Baking or UV crosslinking when membrane contains

high salt

TroubleshootingHigh Background Insufficient Blocking Membrane allowing to dry out during hybridization or washing Membranes adhered during hybridization or washing Bubbles in hybridization bag Walls of hybridization bag collapsed on to membrane Not enough wash solution Hybridization temperature too low Labeled probe molecules are too short Probe Concentration too high Inadequate prehybridization Probe not denatured Not enough SDS in wash solution

SOUTHERN BLOTTING

• Summary of procedure– 1. Extract and purify DNA from cells– 2. DNA is restricted with enzymes– 3. Sort by electrophoresis– 4. Denature DNA– 5. Transfer to nitrocellulose paper– 6. Block with excess DNA– 7. Wash off unbound probe– 8. Autoradiograph

Watch points

• Using too little DNA-compromise the sensitivity of the test

• Using too much DNA- poor restriction enzyme digestion

• Using too high voltage setting for electrophoresis- gel to melt or appearance of artifacts

Watch points

• Improper blocking-high background and uninterpretable results.

• Insufficient washing-high background and uninterpretable results.

• Excess washing- dissociate the specific hybrids.

USES

• Identify mutations, deletions, and gene rearrangements

• Used in prognosis of cancer and in prenatal diagnosis of genetic diseases

• Leukemias• Diagnosis of HIV-1 and infectious disease

USES

• Every person has repeated sequences of base pairs which are called Variable Number Tandem Repeats (VNTRs)

• To find a particular VNTR we use a radioactive version of the one in question.

• This pattern is known as a DNA fingerprint.

USES

• Applications of DNA fingerprinting include:– Paternity and Maternity Testing– Criminal Identification and Forensics– Personal Identification

Northern blotting or Northern hybridization

• Technique for detecting specific RNAs separated by electrophoresis by hybridization to a labeled DNA probe.

The flow chart of Northern hybridization

Prepare RNA samples and run RNA gel

Northern transfer

Probe preparation

Prehybridization

Hybridization

Post-hybridization washing

Signal detection

IsotopeNon-isotope

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 60C. Add 35 ml 10x MOPS running buffer and 10.5 ml 37% formaldehyde

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 55C

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

An example of Northern blotting

Northern blot

RNA gel 28 S

18 S

Western blotting, or immunoblotting

Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies.

Flow chart of Western blottingElectrophoresing 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

SDS polyacrylamide-gel electrophoresis (SDS-PAGE)

Analysis of protein samples by SDS polyacrylamide-gel electrophoresis and Western blotting

Protein bands detected by specific antibody

SDS-PAGE Western blot

Comparison of Southern, Northern, and Western blotting techniques

Southern blotting Northern blotting Western blotting Molecule detected

DNA (ds) mRNA (ss) Protein

Gel electrophoresis

Agarose gel Formaldehyde agarose gel

Polyacrylamide gel

Gel pretreatment

Depurination, denaturation, and

neutralization

- -

Blotting method Capillary transfer Capillary transfer Electric transfer Probes DNA

Radioactive or nonradioactive

cDNA, cRNA Radioactive or nonradioactive

primary antibody

Detection system

Autoradiography Chemiluminescent

Colorimetric

Autoradiography Chemiluminescent

Colorimetric

Chemiluminescent Colorimetric