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Blotting technique
A blot, in molecular biology and genetics, is a method
of transferring proteins, DNA or RNA, onto a carrier.
The term "blotting" refers to the transfer
of biological samples from a gel to a membrane and their subsequent detection
on the surface of the membrane.
Southern blot
Used to study how genes are organized within genomes
by mapping restriction sites in and around segments of genomic DNA for which
specific probes are available.
Combines transfer of electrophoresis -separated DNA
fragments to a membrane and subsequent fragment detection by probe
hybridization.
Named after its inventor, the British biologist Edwin
Mellor Southern.
Edwin Mellor Southern
The
technique is based on HYBRIDIZATION.
Hybridization is the process of forming a complementary
base-pairing between a single-stranded DNA probe and a single-stranded
target DNA.
The reactions are highly specific i.e. probes
will only bind to targets with a complementary sequence.
Principle of hybridization
Transfer methods
The transfer of electrophoretically separated DNA from
gels to two-dimensional solid supports is a key step in Southern hybridization.
Upward
Capillary Transfer: DNA
fragments carried from the gel in an upward flow of liquid and deposited on the surface of the solid support
Downward
Capillary Transfer: DNA fragments
carried in a downward flow of alkaline buffer and deposited on the surface of a
solid support
Simultaneous Transfer to Two Membranes: For a high concentration of target DNA, the capillary method can transfer DNA simultaneously to two solid
supports
Electrophoretic
Transfer: Used for analysis of
small fragments of DNA separated by PAGE. Only for nylon membranes
Vacuum
Transfer: DNA and RNA can be
transferred rapidly and quantitatively from gels under vacuum
Upward Capillary
Transfer
The liquid is drawn upward through the gel by capillary
action.
Rate of transfer depends on:
◦
size of
the DNA fragments
◦
concentration
of agarose in the gel
Small fragments of DNA (<1 kb) are transferred
within 1 hour
Larger fragments are transferred more slowly and less
efficiently.
Capillary transfer of DNAs > 15 kb requires at least
18 hours
Set-up for upward capillary transfer
How we do it!
STEP-I:
Isolate genomic DNA (10 μg per lane required)
STEP-II: Restriction digestion with one or more
enzymes and agarose gel electrophoresis
STEP-III: The digested DNA
fragments are denatured in the presence of alkali in situ (in gel)
STEP-IV: The fragments are then neutralized with NaCl
to prevent renaturation before the addition of probe
STEP-V: Upward capillary transfer of DNA fragments to the membrane (solid support). Usually, a nitrocellulose or nylon membrane is used.
STEP-VI: UV cross-linking of the transferred DNA
fragments to the membrane
STEP-VII:
Preparation of radiolabelled probe
usually with α-P32
labelled dCTP or dATP. Fluorescence labeling with Digoxigenin (DIG) is
also possible.
STEP-VIII: Hybridization of the
membrane with the labeled probe under specific conditions. The probe hybridizes with
the complementary DNA fragment.
Before hybridization blocking is done with salmon
sperm DNA to eliminate non-specific probe binding
STEP-IX: Wash the unbound probe.
STEP-X: Detection by autoradiography
A typical Southern blot
Application of Southern Blot
·
Primary
usage is to identify a specific DNA in a DNA sample.
To confirm integration of a transgene in the
host genome.
To identify copy number of the transgene
integrated in the host genome.
Other applications:
Identify mutations, deletions, and gene
rearrangements.
In RFLP
Used in the prognosis of cancer and in prenatal diagnosis
of genetic diseases.
Diagnosis of HIV-1 and infectious disease.
In DNA
fingerprinting:
◦
Paternity
and Maternity Testing
◦
Criminal
Identification and Forensics
◦
Personal
Identification
Northern Blot
Analysis
• Northern blotting is a technique for the detection of
specific RNA sequences in the transcriptome.
• Northern blotting was developed by James Alwine and
George Stark (1979) and was named such by analogy to Southern blotting.
Application of Northern Blot
Used for the study of gene expression at the level of
mRNA (messenger RNA transcripts).
Detection of mRNA transcript size
Study RNA degradation
Study RNA splicing
Study RNA half-life
Often used to confirm and check transgene expression.
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