K.

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

 First described by E. M. Southern in 1975.

 Applications of Southern hybridization
 RFLP’s, VNTR’s and DNA fingerprinting
 Checking of the gene knockout mice
 The flow chart of Southern hybridization
Southern hybridization

Transfer buffer
Detection of an RFLP by
Southern blotting
Flow chart of Southern
hybridization
Preparing the samples and running the gel

Southern transfer

Probe preparation Isotope

Non-isotope
Prehybridization

Hybridization

Post-hybridization washing

Signal detection
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

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
transcriptio
n
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 Can be exposed to
to basic solution basic solution
Not easily Can be reprobed
reprobed several times
Signals detection
 Autoradioragraphy
 Non-isotope detection system
- Chemiluminescent detection
- Colorimetric detection
- Multicolor detection
Autoradiography

 Exposure to x-ray film

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
Isotope
Prehybridization Non-isotope

Hybridization

Post-hybridization washing

Signal detection
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
Southern blotting Northern blotting Western blotting
Molecule DNA (ds) mRNA (ss) Protein
detected
Gel Agarose gel Formaldehyde Polyacrylamide gel
electrophoresis agarose gel
Gel Depurination, - -
pretreatment denaturation, and
neutralization
Blotting method Capillary transfer Capillary transfer Electric transfer
Probes DNA cDNA, cRNA primary antibody
Radioactive or Radioactive or
nonradioactive nonradioactive
Detection Autoradiography Autoradiography Chemiluminescent
system Chemiluminescent Chemiluminescent Colorimetric
Colorimetric Colorimetric
DNA FINGER PRINTING
 The basic methodology of DNA profiling in
plants involve first the extraction of DNA from
plant cells, quantification and quality
assessment of extract. The further steps are of
two types,
 1) PCR based.  -   RAPD, ISSR, SSR        
 2) Non PCR based. – RFLP
 PCR based techniques diluted DNA is mixed with a
master mix comprising the PCR buffer, DNTPS,
primer, water and the Taq polymerase enzyme in a
PCR eppendorf tube .
 The mixture is loaded into the PCR. The PCR is pre-
programmed for appropriate number of cycles and
temperature variations depending on the technique. 
 After required cycles, the samples are subjected to
electrophoresis, either AGE or PAGE, depending on
the technique. The staining is done for revealing the
banding pattern.
 Restriction Fragment Length Polymorphisms (RFLPs)
In this method, unequal lengths of DNA fragments are obtained by cutting Variable
Number of Tandem Repeat (VNTRs) sequences up to 30 sequences long with
restriction enzymes at specific sites.

 There are different VNTRs, as there are different plant species, number and location
of restriction enzyme-recognition sites.

 PCR amplification of DNA is not required for this method. The routine southern blot
experiment can be used.

 The complimentary DNA sequences are radiolabeled on agarose gel for visualization
in this method. This method is used to identify the origins of a particular plant
species.
 This method is not much favored for DNA fingerprinting, as it has many drawbacks.
The results cannot indicate the chance of match between two organisms.
 The other drawback of RFLPs is a costly process which involves lot of labor and
money.
 Randomly Amplified Polymorphic DNAs (RAPDs)
This method is most commonly used for primary assay.
 This method helps in screening the differences in DNA
sequences of two species of plants.
 This method is used to search the sequences required for random
amplification. In this method, using short single primers at low
annealing tempratures, DNA is cut and amplified.
 Using electrophoresis and superimposing the gels, a banding
pattern is identified. The gel is cut where the target band is found
and the DNA is isolated and sequenced.
 This target is used to assess DNA from other cultivars. This
technique is more cost-effective than RFLPs. The drawback for
this method is that RAPDs lack specificity due to low annealing
temperatures and easier reaction conditions.
Simple Sequence Repeats (SSR)
Simple sequence repeats are microsarellites. They
show high degree of polymorphism.
They are isolated using hybridized probes followed by
their sequences. They are detected by gel
electrophoresis using specific dyes or radiolabelling.
The advantage of SSRs is that the amount of DNA
required is less than RFLPs. The assays involving SSRs
are more robust, making them more efficient than
RAPDs.
The drawback of this method is that seperate SSR
primers are needed for each species.
Amplified Fragment Length Polymorphism (AFLP)
This method is a PCR based derivative of RFLP. Here
sequences are selectively amplified using the
primers. This method is more useful than RFLP or
RAPD as more loci can be evaluated. AFLP helps in
determining a large number of polymorphism. This
method is also cost effective.
 ADVANTAGES OF DNA FINGERPRINTING IN PLANTS ARE AS
FOLLOWS:
DNA fingerprinting is used for the identification of genetic diversity
within a breeding population. It is used to identify a gene of interest.
In the United States, it is also used to detect a genetically modified
organisms in agriculture.
 RFLP markers are used to detect the genetic distance in wheat.
 RAPD markers are used for characterization, estimation of genetic
relatedness and determination of genetic diversity of tea
germplasm. It is also used to find genetic relatedness and difference
in figs.
 AFLP markers help in assessing genetic diversity among cultivars
such as wheat. It also helps detect higher level of polymorphism.
 DNA fingerprinting of herbal drugs can be useful in authenticating
the various claims of medical uses related to the plants.