@#. Fjc. southern and northen.pdf

Biotechnology

1. Southernblotting
and Northen blotting
techniques

2. CONTENTS
Blotting
Typesofblotting
Southernblotting
•Principle
•Apparatus
•Steps involved in southernblotting
•Application
•Advantages andDisadvantages
Northernblotting
•Steps involved in northernblotting
•Applications
•disadvantages

3. BLOTTING
A blot, in molecular biology and genetics, is a method of
transferring proteins, DNA or RNA, onto acarrier.
The term "blotting" refers to the transfer of biological
samples from a gel to a membrane and their subsequent
detection on the surface of themembrane.
TechniquefortransferringDNA,RNAandProteinsonto
acarriersotheycanbeseparated,andoftenfollowsthe
useofagelelectrophoresis.

4. TYPES OFBLOTTING

5. 1.SOUTHERNBLOTTING
A Southern blot is a method used
in molecular biology for detection ofa
specific DNA sequence in DNAsamples.
Southern blotting combinestransfer
of electrophoresis -separated DNA
fragments to a filter membraneand
subsequent fragment detection byprobe
hybridization.
The method is named after its inventor,
the British biologist Edwin Mellor
Southern.

6. •
PRINCIPLE
The key to this method is hybridization. Hybridization:
It is the process of forming a double-stranded DNA molecule
between a single-stranded DNA probe and a single-stranded
targetDNA.
•There are 2 important features of hybridization:
•The reactions are specific-the probes will only bind to
targets with a complementarysequence.
•The probe can find one molecule of target in a
mixture of millions of related but non-complementary
molecules.

7. STEPS INVOLVED IN SOUTHERN
BLOTTING
1.Extract and purify DNA fromcells;
2.DNA is restricted withenzymes;;
3.Separated byelectrophoresis;
4.DenatureDNA;
5.Transfer to nitrocellulosepaper;
6.Add labeled probe for hybridization to take
place;
7.Wash off unboundprobe;
8.Autoradiograph.

8. APPARATUS

9. 1.Extract and purify DNA fromcells
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 andDNA.
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 inbuffer.
2.DNA is restricted withenzymes.

10. 3.Separated byelectrophorosis.
•The complex mixture of fragments is subjected to gel
electrophoresis to separate the fragments according tosize.

11. 4. DenatureDNA.
•The restriction fragments present in the gel are denatured
withalkali.
•This causes the double stranded to becomesingle-stranded.
•DNA is then neutralizedwithNaClto preventre-hybridization
before adding theprobe.

12. 5.Transfer to nitrocellulosepaper.
•Transfer the DNA from the gel to a solid support, ie,
blotting.
•The blot is made permanentby:
–Drying at~80°C
–Exposing to UV irradiation
6. Add labeled probe forhybridization.
•The filter is incubated under hybridization conditions with
a specific radiolabeled DNAprobe.

13. •The probe hybridizes to the complementary DNA
restrictionfragment.
7. Wash off unboundprobe.
•Blot is incubated with wash buffers containing NaCl and
detergent to wash away excess probe and reduce
background.

14. 8.Autoradiograph.
•If the probe is radioactive, the particles emits when
expose to X-rayfilm.
•There will be dark spots on the film wherever the probe
bound.

16. APPLICATIONS







To identify specific DNA in a DNAsample.
To Isolate desired DNA for construction of rDNA.
Identify mutations, deletions, and generearrangements.
Used in prognosis of cancer and in prenatal diagnosis of
genetic diseases.
InRFLP.
Diagnosis of HIV-1 and infectious disease.
In DNAfingerprinting:
Paternity and MaternityTesting
Criminal Identification andForensics
PersonalIdentification

17. ADVANTAGES
•Effective way to detect a specific
DNA sequencein a large, complex
sample of DNA.
•Can be used to quantify the
amount of the present DNA.
•Cheaper thanDNA
sequencing.
•More expansive than most other
tests.
•Complex andlabor-intensive.
•Time consumingand
cumbersome.
DISADVANTAGES

18. 2.NorthernBlotting
•Northern blotting is a technique for detection of
specific RNA sequences. Northern blotting was
developed by James Alwine and George Stark
at Stanford University (1979) and was named
such by analogy to Southernblotting.

19. StepsinvolvedinNorthern blotting
1. RNA is isolated from several
biological samples (e.g.various
tissues, various developmental
stages of same tissueetc.)
* RNA is more susceptible to
degradation than DNA.

20. 2. Sample’s are loaded on gel
and the RNA samples
are separated according to
their sizeon an agarose gel
.
•The resulting gel
following after the
electrophoresisrun.

21. 3. The gel is then blotted on a
nylon membrane or a
nitrocellulose filter paper
by creating the sandwich
arrangement.

22. 4. The membrane is placed
in a dish containing
hybridization buffer with a
labeledprobe.
•Thus, it will hybridize to
the RNA on the blot that
corresponds to the
sequence ofinterest.
5. The membrane is washed
to remove unbound
probe.

23. 6. The labeled probe is detected via
autoradiography or via a
chemiluminescence reaction (if a
chemically labeled probeisused).Inboth
cases this results in the formation of a
dark band on an X-rayfilm.
•Nowtheexpressionpatternsofthe
sequenceofinterestinthedifferent
samplescanbecompared.

24. APPLICATIONS
•A standard for the study of gene expression at the
level of mRNA (messenger RNAtranscripts).
•Detection of mRNA transcript size.
•Study RNA degradation.
•Study RNA splicing.
•Study RNAhalf-life.
•Often used to confirm and check transgenic /
knockout mice (animals).

25. Disadvantage of Nourthern Blotting
1.The standard northern blot method is relatively
less sensitive than nuclease protection assays
andRT-PCR.
2.Detection with multiple probes is aproblem.
3.If RNA samples are even slightly degraded by
RNAses, the quality of the data and
quantitation of expression is quite negatively
affected.