Lesson 4 - Southern Blotting + Hybridization Method Flashcards
- devised by Edward Southern in 1975
- DNA fragments separated on a gel
- blotted to a charged surface/membrane
- hybridized with a DNA probe
souther blotting + hybridization
devised southern blotting
Edward Southern (1975)
what is done to the DNA in southern blotting
separated on a gel
how is the DNA fragments separated
blotting to a charged surface/ membrane
example of charged surface/ membrane
nitrocellulose membrane
used to find gene of interest
DNA probe
how is the location of DNA+probe on the filter detected
by autoradiography
autoradiography can detect ~5 µg single copy sequence
high sensitivity
what can the autoradiography detect
~5µg single copy sequence
applications of souther blotting/hybridization
- RFLP mapping
- forensic investigation
- genetic screening
RFLP
restriction fragment length polymorphism
Steps in Southern Blotting + Hybridization
- restriction digestion
- electrophoresis
- blotting and denaturation
- hybridization with radioactive probe
- autoradiography
- DNA samples to be tested are prepared from the approprate sources
- restriction enzyme is added to the samples of DNA to produce restriction fragments
restriction fragment preparation
what is produced when a restriction enzyme is added to the samples of DNA
restriction fragments
- the mixtures of restriction fragments from each sample are separated by this
- each sample forms a characteristic pattern of bands
electrophoresis
- capillary action pulls an alkaline solution upward through the gel and through a sheet of nitrocellulose paper laid on top of it
- transfers the DNA to the paper and denaturing it in the process
- single strands of DNA stick to the paper, positioned in bands exactly as on the gel
blotting
pulls the alkaline solution upward through the gel and through a sheet of nitrocellulose paper
capillary action
after blotting, what is done
DNA is fixed to the membrane
how is DNA fixed to the membrane
- bake at 80°C or
- expose to UV
- the paper is exposed to a solution containing a radioactively labeled probe
- probe is single-stranded DNA complementary to the DNA sequence of interest, and it attaches by base pairing to restriction fragments of complementary sequence
hybridization with radioactive probe
where is the paper blot exposed to afterward
solution containing radioactively labeled probe
what is a probe
single-stranded DNA complementary to DNA sequence of interest
how is unhybridized probe removed
washing (then dry)
- sheet of photographic film is laid over the paper
- the radioactivity in the bound probe exposes the film to form an image corresponding to specific DNA bands – the bands containing DNA that base pairs with the probe
autoradiography
Other blotting methods
- northern blotting
- western blotting
- eastern blotting
- RNA blotted on membrane
- DNA probe
northern blotting
what is blotted in northern blotting?
RNA
probe in northern blotting
DNA probe
- proteins blotted
- labeled antibody specific for the protein
western blotting
what is blotted in western blotting
proteins
how is detection in western blotting done
labeled antibody specific for the protein
used to detect glycoproteins and lipoproteins
eastern blotting
what is detected in eastern blotting
- glycoproteins
- lipoproteins
western blotting is used to verify what?
protein expression
verification of ZmCA1 gene insertion
PCR
verification of protein expression
western blot analysis
quantification of carbonic anhydrase activity
enzyme assay
what is quantified in enzyme assay
carbonic anhydrase activity
detection of copy number of gene insertion
southern blot analysis
what is detected in southern blot analysis
copy number of gene insertion
