Tools Of Molecular Genetics Flashcards
Why do we usually have to denature RNA before running on a gel?
RNA has a tendency to BP with itself forming a secondary molecular structure. (Add formaldehyde to denature during a run)
How do we visualize DNA bands on a GE?
Soak the gel in a dye that binds DNA and fluoresces
Ex. Ethidium Bromide
Apart from dye-fluorescence, how else can we visualize DNA bands on a gel?
Autoradiography
Expose xray film to the gel and see what gets darker
How are samples denatured when preparing them for hydridization?
High temperature High pH (NaOH)
How do we increase our chances that probe-target heteroduplexes will be in our final assay?
Stringency of conditions
How do we manipulate stringency of conditions to optimize probe-target heteroduplexes?
If we want very specific probe-target sequences, then during mixing use high temperature and/or a higher concentration of NaOH.
Shorter probes => more stringent
Lower temp, allows for less strigent BP to be stable
Can also decrease stringency via increasing length of probes
What are RFLP good at visualizing?
Inherited difference in the pattern of restriction enzyme digestion.
Length polymorphisms:
- Loss of restriction site
- New restriction site
- Insertion of DNA
- Deletion of DNA
Use southern blotting or PCR to enhance
What probes would you use in the distinct blotting techniques?
Southern/Northern: cDNA
Western: Antibody
What do we use Southern blotting for? What is a typical process?
Used to detect specific DNA fragments
- Unlabeled DNA cut with restriction nucleases
- GE separates strands by size
- Blotted onto nitrocellulose paper
- Labeled DNA probe hybridized to separated DNA
- Visualized by autoradiography
What are we visualizing during a Northern blot?
RNA. Probe is still going to be ssDNA
What are DNA microarrays used for?
Expression of many thousands of genes simultaneously
Comparing two samples.
One color- expression of one sample
Another color- expression of another sample
Hybrid color - both expressing
No color - no expression
What are ASOs? How are they useful?
Allele-specific oligonucleotides
AKA Allele-specific dot blotting
Very stringent hybridization technique.
What directs the PCR amplification of a desired piece of DNA?
PCR primers. Can produce 10^9 amount of DNA in a few hours.
What three steps repeat over and over in PCR?
- Heat to separate strands
- Hybridization of primers
+Heat stable DNA polymerase (+necleotide analogs) - DNA syn. From primers.
Describe allele-specific PCR?
At least one primer is chosen to specific a mutation near the 3’-OH end. .
Under stringnet conditions a mismatched primer will not initiate replication.
The appearance of a product indicated the genotype.
