RR1 Flashcards
How are molecular probes made?
1) Stick macromolecular mixture to a nylon or nitrocellulose membrane
2) Create a probe that is reverse complement of target, it will stick to its target
3) Remove all non-specifics, now have specific target
Use molecular weight to keep record
What do we use to label sequences?
Use PNKs and oligonucleotides
1) Have known sequence
2) Synthesize complementary oligonucleotide
3) Polynucleotide Kinase transfers alpha phosphates of ATP to 5’ hydroxyl end of synthetic oligonucleotide (Alpha phosphate acts as label)
How can we label DNA probes using PCR?
1) Denature/unwind DNA
2) dNTP with isotopic radiolabel on alphaphosphate is incorporated into amplified DNA
3) Primers are elongated, now radiolablled
4) Product is rendered single stranded (remove impurities)
There will now be a labelled C where nucleotides were added
Nucleic acid creation and solid state supports
- DNA and RNA are separated using agarose gel
- Requires molecules to be denatured
- Smaller DNA pieces moce further up
- Using transfer buffer, with a nitrocellulose membrane, we create a permanent copy
What is UV crosslinking?
The permanent binding of nucleic acids to a membrane
- Records abundance and position of molecules followeing their separation on a gel
Can come down to specific number of mols
What are different ways we can detect polymorphisms?
Normally, we use EcoRI to cut DNA into 2 fragments - we run these fragments on a gel and the probe will interact with both.
With a mutation on the EcoRI site (a polymorphism), ECORI WILL NOT cut. This means there is a polymorphism
*Polymorphism changes DNA so EcoRI can no longer identify fragment site”
Southern analysis
1) Separation of DNA fragements based on size with electrophoresis, transfer to a membrane
2) Label sequence with a probe and put on gel
Allows the creation of a pedigree *can see heterozygote or homozygote
For DNA
Northern analysis
Can quantify gene expression
1) Run RNA on gel, transfer to a membrane to see RNA fragemnts
2) Allows us to see which genes are more active in which stages of embryonic development
for RNA
Why does RNA bind to nylon and nitrocellulose membranes?
RNA has a negative charge and membrane has a positive charge
Describe the steps of cDNA library creation using reverse transcriptase
1) mRNA gets 3’ poly(A) tail, hybridized by an oligo-dT primer
2) Reverse transcribe RNA into cDNA
3) Use alkali to remove RNA strand
4) PolydG initiates synthesis of 2nd DNA strand using oligoDC
5) OligoDC is extended by E-coli DNA pol I
now have cDNA that becomes double stranded and incorporated into library
Creation of DNA copy of RNA
What is RT-qPCR
“How much of a given gene product is present in a sample I have?”
Use reverse transcriptase of mRNA and cDNA
- Subjected to PCR that puts fluorescent dye into polymers
The more DNA produced, the more fluorescent signal detectable
How is mRNA abundance related to time to reach plateau in fluorescence?
The fluorescent plateau is reached faster with more starting material
We can uses the amount of cycles it takes to reach plateau using the initial mRNA abundance
What is RNA sequencing?
- Allows the veiwing of all RNA transcripts at the same time
- Can run all RNA over column collection at all RNA present over initial population
Describe the steps of RNA sequencing
1) Extract RNA from population, isolate for poly-A tail and size
2) Reverse transcriptase, creating a cDNA library
3) Add adaptors for next generation sequencing
4) Add adaptors for NGS (needed for binding of primers)
5) Use PCR to amplify
