Download Molecular biology technique (I) Southern/Northern

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
• Second strand
GGGTTTAAACCC
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 hybridization
Preparing the samples and running the gel
Southern transfer
Probe preparation
Prehybridization
Hybridization
Post-hybridization washing
Signal detection
Isotope
Non-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 80C
for 2 hr or UV-crosslink
Nylon membrane for
seconds
Preparation of probes
• Synthesis of uniformly labeled doublestranded 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 DNAdependent RNA polymerase.
In vitro
transcription
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.
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 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
Comparison of nitrocellulose and nylon
membranes
NC
Nylon
Hydrophobic binding Covalent binding
Fragile
Durable
Probe length > 200 ~ < 200 ~ 300 bp is
300 bp
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 doublestranded DNA molecule between a singlestranded 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 noncomplementary molecules.
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.
Troubleshooting
Poor 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
Troubleshooting
High 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
Isotope
Non-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 60C. 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 55C
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 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
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
Molecule
detected
Gel
electrophoresis
Gel
pretreatment
Southern blotting
DNA (ds)
Northern blotting
mRNA (ss)
Western blotting
Protein
Agarose gel
Formaldehyde
agarose gel
-
Polyacrylamide gel
Capillary transfer
cDNA, cRNA
Radioactive or
nonradioactive
Autoradiography
Chemiluminescent
Colorimetric
Electric transfer
primary antibody
Depurination,
denaturation, and
neutralization
Blotting method Capillary transfer
Probes
DNA
Radioactive or
nonradioactive
Detection
Autoradiography
system
Chemiluminescent
Colorimetric
-
Chemiluminescent
Colorimetric