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How can you separate DNA in terms of size?
You can separate things as small as 1000 nucleotides or DNA chromosomes at 2.5 million nucleotides
Characteristics of gel electrophoresis
- agarose gel separates DNA fragments based on size, but not with good precision
- stained with EtBr; gets stuck between base pairs
Why can't we use PAGE?
because acrylamide gel spaces that DNA would move through are much smaller
How do you make a probe?
Fragment of DNA or RNA is radioactively or fluorescently labeled to detect the presence of nucleotide sequences
How would you obtain a lot of genes?
grow fragment in a plasmid to get a lot of genes
Explain labeling DNA in vitro.
A purified DNA polymerase enzyme labels all the nucleotides in a DNA molecule adn can produce highly radioactive DNA probes
Hexanucleotides are used. They bind. DNA polymerase then elongates, incorporating labeled nucleotides, resulting in a population ofDNA molecules that contain labeled examples of all sequences on both strands
What is another way to label?
Polynucleotide kinase labels only the 5' ends of DNA strands; therefore, when labeling is followed by rrestriction nuclease cleavage, DNA molecules containing a single 5' end labeled strand are readily obtained
- introduced chemically via conjugation into biomolecules to be detected in further assays
- Anti-DIG antibodies with high affinities can find it.
It is an all-purpose, nonradioactive labeling, immuno-tag
The methyl group of Thymine on DIG is replaced by a spacer arm linked to digoxigenin, which is easily detected
- 1) isolate RNA from tissues and purify
- 2) run a gel
- 3) Take RNA out of gel and put a stack of paper towels on top.
- 4) Sit the gel on a sponge in the buffer and allow the buffer to migrate up from the gel to the paper and towels. RNA will follow.
- 5) RNA now on paper. Put in ziplock with probes and wait for hybridization.
- 6) Wash away= view bands where hybridization occurred with autoradiography
Purpose of southern blotting?
Purpose of Northern blotting?
Purpose of southern blotting is to detect the presence of a particular DNA fragment in a Sample
purpose of northern blotting is to detect mRNAspresent in tissues and thus measure gene expression
Explain southern blotting
Big giant pieces of DNA that were cut with restriction enzyme to get shorter pieces of DNA. Melt the DNA to get ssDNA. Run on gel and probe it.
Differences between southern and northern blotting
- Southern: DNA
- Northern: TNA
- Southern: requires denaturation of strands
- northern: no denaturation needed
- southern: treated with restriction enzymes
- northern: not treated with restriction
What happens after a genomic library is made?
you have a tube full of clones. You need to screen.
- Clones spread out on plate. Bacteria grow on agar plate. Each spot corresponds to a different clone. Plaques/ colonies form.
- Use a disc of absorbant paper, place on petri dish and peel the paper from the dish to produce a replica of colonies.
- Lyse the bacteria to denature the DNA and incubate with probe.
- Wash. The sequence we're looking for binds to the colonies. Once you isolate the clone, you can freeze them so they can be reused any time
Difference between dideoxyribonucleotides and deoxyribonucleotides
One has a 3'-OH and the other doesn't
Explain the dideoxy method?
- We take groups of fragments of known nucleotide sequences and put them in plasmids.
- Take fragment.
- Get single strad
- Add the hexonucleotide label.
- Let it build
- Throw in dideoxyribonucleotide, causing elongation to stop
- Do this for all four nucleotides in separate tubes.
- Run a gel. Start at bottom and read to the top to get sequence
How is a Southern Blot run?
we need a probe for a gene we believe to be involved in the tissue being studied, called Tumor, in this example.
- 1) The two strands will be cut by a restriction enzyme. The two strands are melted to allow running of the gel.
- 2) A nitrocellulose membrane is then placed on the gel. Buffer transfer then occurs from the bottom up to the membrane, with the DNA following
- 3) The membrane is then baked in a vacuum or regular oven or exposed to UV to permanently keep the DNA there
- 4) The membrane is then placed in a bag with buffer and the DNA probe.
- 5) Await hybridization
- 6) probe is washed off and viewed
For a Southern blot, what must be the case with the mutation?
It has to either create or remove a restriction site. This will cause different bands to appear and thus an awareness of where the error is
How was the dideoxy method improved?
Label each nucleotide with different fluorescent tags. use DNA polymerase and cloned template to get another strand and mix it with four different colors. Run the gel in the same lane. The UV spec will read it and separate the sequences
What are the characteristics of a plasmid?
several promoters (ex: T7 and T3: little stretches of nucleotides that are known promoters)
needs an origin of replication
features can be modified in other plasmids)
What is the case with bacteria and plasmids?
only a small fraction of the bacteria will take up a plasmid. So, we kill them with antibiotic. We want to keep the ones with our genes alive. They are resistant.
Select for plasmids with the genes by growing them in ampicillin
What are the steps for cloning?
- 1) isolate and purify the gene of interest
- 2) Use a restriction enzyme to cut the DNA
- 3) use the same restriction enzyme to cut the plasmid to open it up. This will be at the Multiple Cloning Site (MCS)
- 4) Allow the DNA fragment and plasmids to be ligated together by DNA ligase
- 5) Plasmids are incorporated into host bacterial cell via transfection
- 6) Each cell has a different segment of DNA from the organism, making up a DNA library
- 7) They can be plated and the colony with the desired gene is isolated