1. Northern blotting is a technique used to detect specific RNA sequences in a sample. It involves separating RNA fragments by gel electrophoresis, transferring them to a membrane, then using a complementary DNA or RNA probe to identify the target sequence through hybridization. 2. Key steps include isolating RNA from cells, separating fragments by size using gel electrophoresis, blotting onto a membrane, incubating with a labeled probe, washing unbound probe, and detecting the bound probe to identify the target RNA sequence. 3. Northern blots are useful for determining gene expression levels in different tissues and cell types by detecting the presence and amount of specific mRNA transcripts. They allow investigation of how genes are regulated at the transcriptional level.

1. Ministry of Higher education of Iraq
University of Kerbala
Faculty of veterinary medicine
blotting type and uses
By
Adil atyeh
Ali Kadhim
Juman Khalil

2. What is blotting?
• Blots are techniques for transferring DNA,
RNA and proteins onto a solid support (carrier)
generally nylon or nitrocellulose membranes.
so they can be separated, and often follows the
use of a gel electrophoresis.

3. • Blotting of nucleic acid is the central technique
for hybridization studies. Nucleic acid labeling
and hybridization on membranes have formed
the basis for a range of experimental
techniques involving understanding of gene
expression, organization, etc.

4. • Identifying and measuring specific proteins in
complex biological mixtures, such as blood,
have long been important goals in scientific
and diagnostic practice.

5. • More recently the identification of abnormal
genes in genomic DNA has become
increasingly important in clinical research and
genetic counseling. Blotting techniques are
used to identify unique proteins and nucleic
acid sequences.

6. • They have been developed to be highly
specific and sensitive and have become
important tools in both molecular biology and
clinical research.

8. Southern Blotting

9. • A Southern blot is a method used in molecular
biology for detection of a specific DNA
sequence in DNA samples. Southern blotting
combines transfer of electrophoresis -separated
DNA fragments to a filter membrane and
subsequent fragment detection by probe
hybridization.

10. The method is named after its inventor, the British
biologist Edwin Mellor Southern.

11. • The key to this method is hybridization.
• Hybridization is a process of forming a double-
stranded DNA molecule between a single-
stranded DNA probe and a single-stranded
target patient DNA.

12. 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.

13. 1. Extract and purify DNA from cells;
2. DNA is restricted with enzymes;;
3. Denature DNA;
4Separated by electrophoresis;
5. Transfer to nitrocellulose paper;
6. Add labeled probe for hybridization to take place;
7. Wash off unbound probe;
8. Autoradiograph.
Steps for hybridization

15. 3.The restriction fragments present in the gel are
denatured with alkali and transferred onto
4. a nitrocellulose filter or nylon membrane by
blotting.
This procedure preserves the distribution of the
fragments in the gel, creating a replica of the
gel on the filter.

16. 5. The filter is incubated under hybridization
conditions with a specific radiolabeled DNA
probe.
The probe hybridizes to the complementary
DNA restriction fragment.

17.  This procedure preserves the distribution of
the fragments in the gel, creating a replica of
the gel on the filter.

18. • Excess probe is washed away and the probe
bound to the filter is detected by
autoradiography, which reveals the DNA
fragment to which the probe hybridized.

23. Southern blots are used in gene discovery , mapping,
To identify specific DNA in a DNA sample.
To Isolate desired DNA for construction of rDNA.
Identify mutations, deletions, and gene
rearrangements.
 Used in prognosis of cancer and in prenatal
diagnosis of genetic diseases.

24.  In RFLP (in molecular biology; Southern blot
analysis is used for the detection of DNA or
gene sequence in circular or large DNA. ...
Where as RFLP (Restriction Fragment Length
Ploymorphism) is used for compare the DNA
fragments after reaction with restriction
enzymes.

25.  Diagnosis of HIV-1 and infectious disease.
In DNA fingerprinting:
Paternity and Maternity Testing
Criminal Identification and Forensics
Personal Identification

26. Effective way to detect a specific DNA
sequence in a large, complex sample of
DNA. Can be used to quantify the amount
of the present DNA.
Cheaper than DNA sequencing

27. • Southern blots allow investigators to determine the
molecular weight of a restriction fragment and to
measure relative amounts in different samples.

28. More expansive than most other tests.
Complex and labor-intensive.
Time consuming and cumbersome.

29. WESTERN BLOTTING
1

30. INTRODUCTION
 Named after scientist: Western Neal Burnette.
 Western blotting is a technique used to detect the
presence of a specific protein in a complex protein
mixture.

31. PRINCIPLE OF WESTERN BLOTTING
 Western blotting is an Immunoblotting technique
which rely on the specificity of binding between a
molecule of interest and a probe to allow detection
of the molecule of interest in a mixture of many
other similar molecules.
 In Western blotting, the molecule of interest is a
protein and the probe is typically an antibody raised
against that particular protein.
 The SDS PAGE technique is a prerequisite for
Western blotting .

32. STEPS IN WESTERN BLOTTING
1. A protein sample is subjected
to electrophoresis on an SDS-
Polyacrylamide gel.
2. Electroblotting transfers the
separated proteins from the gel
to the surface of a nitrocellulose
membrane.
4

33. 3. The blot is incubated with a generic protein (such
as milk proteins or BSA) which binds to any
remaining sticky places on the nitrocellulose.
4. An antibody that is specific for the protein of
interest (the primary antibody-Ab1) is added to the
nitrocellulose sheet and reacts with the antigen.
•Only the band containing the protein of interest
binds the antibody , forming a layer of antibody
molecules.

34. 5. After washing for
removal of non-specifically
bound Ab1, secondary
antibody (Ab2) is added.
•Ab2 specifically recognizes the
primary antibody and binds.
•Ab2 is radioactively labeled, or is
covalently linked to a reporter enzyme,
which allows to visualize the protein-Ab1-
Ab2 complex.

35. Column 1: positive control; column 2: negative control; column 3-4: positive anti-HIV-1 serum;
column 5: positive anti-HIV-1 serum group O; column 6: positive anti-HIV-2 serum; columns 7-
10:sequential results from serum testing of a patient newly infected with HIV-1.

36. APPLICATIONS
 The confirmatory HIV test .
 Used as the definitive test for Bovine spongiform
encephalopathy ( Mad cow disease).
 Some forms of Lyme disease testing employ
Western blotting.
 Western blot can also be used as a confirmatory test for
Hepatitis B infection.
 In veterinary medicine, western blot is sometimes used to
confirm FIV+ status in cats.

37. Northern Blotting

38. What is Blot?
In molecular biology blots is a technique for transferring DNA
, RNA and proteins onto a carrier so they can be separated,
and often follows the use of gel electrophoresis.
Types OF Bolting:
Southern Blotting: Used For DNA Detection in a sample
Northern Blotting : Used For RNA Detection in a sample
Western Blotting : Used For Proteins in a sample

39. Northern Blotting

40. The northern blot is a technique used in molecular
biology to study gene expression by detection
of RNA (or isolated mRNA) in a sample.
Developed by Alwnie and his colleagues in 1979.
This method was named for its similarity to the
technique known as a Southern blot.

41. Northern Blotting

42. Applications
 Northern blots are particularly useful for determining the conditions
under which specific genes are being expressed, including which tissues
in a complex organism express which of its genes at the mRNA level.
 When trying to learn about the function of a certain protein, it is
sometimes useful to purify mRNA from many different tissues or cell
types and then prepare a northern blot of those mRNAs, using a cDNA
clone of the protein of interest as the probe.
 Only mRNA from the cell types that are synthesizing the protein will
hybridize to the probe.

43. Procedure
STEP 1 : ISOLATAION OF RNA :
All the target RNA’s molecules should be extracted from the
sample.
Isolate RNA from cells or tissue samples using the TRI
reagent or genelute™ kit for mammalian cells or tissues.
Step 2 : Agarose Gel Electrophoresis :
In gel electrophoresis the mixture of mRNA molecules
separated/denatured to small fragments/molecules according to their size
using an electric field.

44. Agarose Gel Electrophoresis
The sample should be pushed to electrical field
through a gel that containing small pores at a
speed that are inversely proportional to the size.
As we know that DNA/RNA have negative
charge due to the sugar-phosphate backbone
will move toward positive end of the field.

45. Agarose Gel Electrophoresis
Agarose & Buffer : Agarose & buffer are used is a gel to conduct
electrical current.
Formaldehyde :
Formaldehyde is used to unfragment the branched RNA molecule to
simple linear one and to prevent it form coiling again.

47. Gel Electrophoresis

48. Blotting/ Transfer
The transfer or blotting is the step in
which the mRNA from the
electrophoresis gel will be transferred
onto a nylon membrane so it may be
accessible to a probe for hybridization
and detection. The separated mRNA
bands are then blotted on chemically
reactive filter paper.

49. Blotting/ Transfer
Traditionally, a nitrocellulose membrane is used, although
nylon or a positively charged nylon membrane may be used.
Nitrocellulose typically has a binding capacity of about
100µg/cm, while nylon has a binding capacity of about 500
µg/cm. Many scientists feel nylon is better since it binds more
and is less fragile.
RNA,s will be covalently link to membrane.

50. Blotting/ Transfer

51. Hybridization/ Probe
In molecular biology hybridization means the process of
forming a double stranded nucleic acid from joining two
complementary strands of DNA (or RNA).
Pre-hybridization before hybridization blocks non-specific
sites to prevent the single-stranded probe from binding just
anywhere on the membrane.
Incubate membrane with labeled DNA or RNA probe with
target sequence.

52. Hybridization/ Probe
Incubate for several hours at suitable renaturation
temperature that will permit probe to anneal to its
target sequence(s).
Probe sequence is complementary to the RNA of
interest.

53. Hybridization/ Probe

54. Washing And Visualization
Wash Nylon from excess of probe & dry.
Place Nylon sheet over x-ray film.
X-ray film darkens where the fragments are complementary to
the radioactive probes.

56. Advantages/Application
 A standard for direct study of the gene expression at the level of
mRNA.
 Detection of mRNA transcript size
DISADVANTAGE :
 Time consuming procedure .
 Use of radioactive probes.
 Detection with multiple probes is a problem.

57. Northern And Southern Blotting Difference
Bothe are same in protocol but some difference as below:
Character Southern Northern
Introduction Southern blotting was
developed for the very
first time by the E.M.
Southern in 1975.
This method was
developed by the
and his colleagues in
1979.
Function For DNA For RNA
Denaturation Need of Denaturation No Need
Hybridization DNA-DNA hybridization RNA-DNA hybridization
Name Southern name of
Inventor
Northern is a misnomer