RR1 Flashcards
How are nucleic acids analyzed and quantified?
There are molecular probes that are used to “find a needle in a haystack.”
1) Stick macromolecular mixture to a nylon or nitrocellulose membrane
2) Create a probe that is a reverse complement of a target so that it will stick to its target
3) Remove all the non-specifics, so now it has a specific target.
Can now get a record from molecular weight.
What is a oligonucleotide?
Oligonucleotides, or oligos, are short single strands of synthetic DNA or RNA that serve as the starting point for many molecular biology and synthetic biology applications
How do you label segments?
1) Have a known sequence
2) Synthesize complementary oligonucleotide
3) Polynucleotide kinase will transfer phosphates of ATP to 5’ hydroxyl end of synthetic oligonucleotide. This will then label complementary oligonucleotide which is bound to a gene/sequence of interest.
How do you make labelled DNA probes using PCR? (amplified)
1) Denature/unwind DNA
2) dNTP with isotopic radio-labeled on (alpha)phosphate is incorporated into amplified DNA (the ones that do not incorporate are removed)
3) Primers are elongated and now radio-labelled
4) Product is rendered (melted) before use, because you need sticky ends.
There is a labelled C where the polymerase was added.
How do you create records of nucleic acids using solid state supports?
- DNA and RNA can be separated using agarose gel
- required molecules to be denatures
- Smaller DNA pieces will move further up
- Using a transfer buffer (RNA) and Alkaline solution (DNA) with a nitrocellulose membrane creates a permanent copy (abundance and size of molecules)
- This can now be hybridized to a sequence of interest
How do you detect polymorphisms using probes?
1) Use EcoRI to cut DNA into z fragments
2) Run 2 fragments on a gel, hybridize, making 2 bonds, creating a ‘signature’
- With a mutation impacting the EcoRI site, it will not cut. Running this on a gel will only make 1 large fragment. So you will know that there is a Polymorphism. Polymorphism changes DNA so EcoRI can no longer identify fragment site.
What is Southern Analysis? How does the process work?
It is the seperation of DNA fragments based on size via electrophoresis, transfer them to membrane. You have to label a sequence with a probe and put on a gel. You can then make a pedigree of where elements are added to the pedigree, for example if you see 2 different fragments, Heterozygotes. Can compare DNA fragments and see amplification, deletion etc.
If you do not have any probes, how would you complete southern analysis?
You could use specific primers and cut with any enzyme and then you would have 2 fragments.
What is Northern Analysis? How does the process work?
With northern analysis, you can see whether a gene is over or under expressed. You need to run RNA on a gel, transfer to nylon, to see how the RNA fragments differ. This allows us to see which genes are more active in different stages of embryonic development. Allows gene expression in different cells and tissues.
1) RNA is run on agarose gel then transferred to Nylon membrane.
2) Filter paper and weight placed on top of nylon and gel - RNA binds to membrane.
3) (-) RNA binds easily due to (+) charge of nylon membrane.
4) RNA is immobilized on membrane using heat.
5) Probe has complementary sequence and will bind if on nylon membrane.
What are the steps in making cDNA libraries? What is a cDNA libary?
A cDNA library is a DNA copy of RNA.
1) mRNA gets 3’ poly(A) tail is hybridized by an oligo-dT primer (TTT)
2) Reverse transcribe RNA into cDNA
3) Use alkali to remove RNA strand
4) Poly dG initiates synthesis of 2nd DNA strand with Oligo Dc
5) Oligo Dc is extended by Ecoli DNA pol I
What method would you use if you were trying to determine how much of a given gene product is present in a sample you have?
RT-qPCR will determine mRNA levels
How does RT-qPCR work?
It is reverse transcriptase mRNA to cDNA. It is subjected to PCR that puts fluorescent dye that is incorporated into polymers. The more DNA produced, the more fluorescence signal is detectable.
In RT-qPCR, what is the starting material directional proportional too?
The amount of starting material is directly proportional to mRNA abundance in the original sample. You can determine from the amount of cycles it takes to reach the plateau phase.
What is the purpose of RNA sequencing? What are the steps?
RNA sequencing allows viewing of all RNA transcripts at the same time. You can run all RNA over column collection of all RNA present over initial population.
1) Extract RNA from population, isolate for poly-A and size
2) Reverse transcriptase, create cDNA library
3) Add adaptors for Next Generation Sequencing
4) Use PCR to amplify
Can now look at everything at the same time in 1 experiment!
