Southern Blotting, CGH And FISH Flashcards
What factors is DNA strand denaturation dependant upon?
Temperature, Salt concentration, Organic solvents, Nucleic acid concentration, Complexity of nucleic acids.
Complete reannealing is difficult as DNA if very complex.
How can we use the understand of the requirements for DNA denaturing and annealing in hybridisation?
By understanding the requirements to denature and reanneal DNA we can control how well a piece of DNA will hybridise to a complimentary sequence.
A high stringency protocol will identify exact matches in DNA. A low stringency protocol will enable us to identify sequences that are similar but not exactly the same.
Describe the southern/northern blotting process.
1) . Take genomic DNA from a patient and fragment it using restriction enzymes. The whole genome will be separated into different sized fragments.
2) . Run fragments on agarose gel along with a size ladder.
3) . Transfer the gel onto a nitrocellulose sheet. Buffer, sponge, gel membrane, paper stack (from bottom to top) encourges a flow of liquid through the gel and results in the DNA binding to the nitrocellulose membrane.
4) . The nitrocellulose membrane will now have an image of the gel on it. If the DNA is single stranded (denatured) it can be permanently bound on to the nitrocellulose. The nitrocellulose membrane can be treated at a specific stringency to only allow certain levels of hybridisation to remain.
5) . We can then introduce a probe DNA and the labelled probe will hybridise to complimentary DNA or RNA.
6) . Bands can be visualised using auto radiography.
What can FISH analysis support the identification of?
1) . Chromosomal alterations (deletions, duplications, translocation, inversions).
2) . Gene duplications.
Gene-specific or even chromosome translocation specific probes can be designed to show us if there are a wrong number of gene copies or a specific translocation is present. Can be done on interphase nuclei.
True or false. FISH can be applied to metaphase or interphase cell preparations.
True.
List some of the variations of FISH.
multitarget FISH (mFISH), Spectral Karyotype Analysis (SKY).
mFISH can target different parts of the same chromosome to detect inversions etc.
What is the main use of CGH?
Molecular comparison of copy number (loss or gain of DNA) in tumour cells.
Describe how CGH was carried out historically. How is it usually carried out now?
Historically you would label tumour and control DNA with different markers, mix them in equal parts and then carry out a simultaneous hybridisation of the two samples to normal metaphase spreads. The probes then hybridised to the relevant targets and could be detected as different colours depending on the quantity of each label that hybridise to the chromosomes.
If you are using a green probe for the tumour sample and a red probe for the control balanced DNA content would appear orange, over-expression of the whole of a chromosome in the tumour sample would make the whole chromosome appear green, under-representation of the whole of a chromosome in the tumour sample would make the chromosome appear red and amplification of a specific region of a chromosome in the tumour sample will make that region of the chromosome specifically appear green.
This is an old method however and it requires the identification of each chromosome in turn. Now aCGH is used.
Describe aCGH.
You can effectively use aCGH to carry out a virtual karyotype.
Test and control DNA are hybridised to a series of sequential chromosome probes arrayed onto a slide. Many thousands of probes a an cover all of the chromosomes.
The hybridisation of equal quantities of sample to probe DNA will give us a series of probe-specific fluorescences.
It is a competitive hybridisation process so if there are equal amounts of both sample and control DNA you should get equal hybridisation to the probe and equal amounts of each type of fluorescence. The fluorescence levels should only vary if there is a difference in copy number in the test sample compared to the control.
Differences in fluorescence show differences on copy number.
The log2 of the ratio between the two signals should be around 1 if there is no change in copy number.
We can use this method to identify the areas at which abnormalities are occurring.
This is a much easier CGH method to report than historical methods as the data is numerical.
DNA strands are covalently associated. True or false?
False. DNA strands are non-covalently associated. This means we can denature and reanneal the strands by changing the temperature.
