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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.
