Biology 3594A Lecture Notes - Lecture 19: Ethidium Bromide, Agarose Gel Electrophoresis, Beta Thalassemia
13 May 2018
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Negative result in PCR –is that evidence that the target was not present?
In a previous lecture: PCR result was negative, therefore the Y chromosome didn’t amplify
True Negative –biologically, the thing you assayed is not there.
False Negative –it’s there but you failed to detect it
1. Is it sufficient evidence that the target was not present? No! Had to demonstrate by other means
The paper about the Y chromosome had this problem –they used a low p value and a reconstruction experiment
Should: use more negative controls, more positive controls, diff technique -> to increase evidence that indicates that whatever you’re looking for is not there
Can sequence stuff to check for inversions and mutations
Reproducibility (until it can be done by other people it’s not a marketable assay, can’t just have one person who can do it), diff assays being used, diff laboratories ->
increase confidence level
When designing a clinical test you want a high resolution product, reproducible
Often publications don’t pass the review because there is insufficient reproducibility – need to have a large sample size and the results were reproduced
This slide is a good short answer to short essay question
Would be published as important because there are many diff fields and ways we can use it
If we were looking for a small amount of foreign DNA it would be great technology
Also for cell free DNA, fetal DNA in mother’s plasma – learn about disease or sex of fetus in first trimester, this technique would make it possible. Can sequence fetal
DNA in the first trimester
Tumour DNA –individual who had cancer and used chemo. Minimal residual disease –what if there is one tumour genome, one cell still there? People want to know,
beyond a PET scan
Fetal DNA –lots of cell apoptosis, means DNA is floating around
Women who have had male children, Y dna is in their brain and can be detected
ctDNA –chemo destroys the cells. Could gather the DNA and assay for mutations
Individualized medicine –specifically looking in an individual for a diff form of DNA
Problem with all of these that you are looking for a mutant form of DNA in very low abundance
Current assays aren’t very good at finding it
Look at a few assays that help with low abundance DNA detection, in the paper they call it mutant DNA
Chop up WT DNA so all that is left is the mutant
Mutant enrichment protocol
CRISPR Cas 9 is used in DNA editing because its specific and it cuts –specific to the WT and allows for mutant enrichment
Assay is not about discovering new mutations –for a mutation that you know about. Cut up WT and leave the mutant there
Can do this for known disorders and diseases
Mutant fragment has diff form than the WT
Refer to it as MU and “mutant DNA” – a form of DNA that differs from the WT by a known mutation
Might be in vitro (fragment in solutions), cell cultures (in vivo)
Can test how far you can go with the technology to find the mutant, when there is more and more WT present
Typically the WT does not cause cancer or a blood disorder
Mutant will be a part of lung cancer or beta thalassemia
Can’t use agarose gel, need smaller -> acrylamide gel
Sequencing used to be gels, PAGE
Could tell single nucleotide differences
Need better stain than ethidium bromide
Silver staining –more sensitive than ethidium bromide
High percentage gel
More deposit of silver than ethidium bromide –higher sensitivity in detecting ds DNA
Depositing silver metal, happening in the presence of nucleic acids
high sensitivity to detect length diffs
Capillary electrophoresis
After PAGE gels the next wave of technology
Tiny tubes containing compounds that can further slow the migration and tell single nucleotide diffs
Diff between peaks can be as little as a single nucleotide diff in length
Instead of a big slab of gel, have small tubing that is the size of a pin, can electrophoresce DNA through that to detect sin gle nucleotide diffs in length
Size differences detection: duplication and deletion, doesn’t help with base substitution
If mixture of WT and mutant, might be able to tell 5% of mutant but can’t get to 1% or 0.1% (where we want to be for cell fre e DNA)
Could eliminate WT by not amplifying it and use allele specific primer to amplify the mutation
Hypothesis: mutant exists in Wt and want to amplify the mutant
Lec 19 DNA editing for mutation detection
May 13, 2018
2:57 PM
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Document Summary
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