Lecture 18 Flashcards
Job of restriction endonucleases
cleave the DNA by locating palindrome
How can we obtain mRNA from cells?
mRNA can be isolated from cell extracts using a poly-T tail immobilized on a bead which will bind to poly-A tail of mRNA
What hybridization mean?
After denaturing ds DNA strands probe or primer can be added which will hybridize to target DNA strand and anneal back.
What is the significance of using dideoxynucleotides in DNA sequencing technique?
Labeled Dideoxynucleotides does not have free 3’OH, thus stops the DNA strand elongation, so when dideoxy is incorporated instead of normal dNTP, shorter fragments of DNA are produced. These fragments with various length are run on gel which shows the sequence in order.
What is the purpose of allele specific oligonucleotides (ASOs)
single stranded pieces of DNA which will anneal to specific sequence of DNA, is formulated to discriminate between mutations
What is the purpose of Restriction Fragment Length Polymorphism (RFLP)
Any changes in DNA sequence that alter restriction sites can be detected using this technique as insertion or deletion can affect the length of DNA between a set of restriction site.
How is RFLP carried out?
Probe binds to target site, and if there is mutation in restriction site, it leads to longer fragment while if there is no mutation, there is shorter fragments. Then, the cells are loaded onto gel for southern blotting.
Mechanism of southern blot
DNA is digested by restriction enzyme, the run on gel. The gel is pressed onto nitrocellulose with alkaline solution transferrring DNA from gel to nitrocellulose, Then labeled DNA or RNA probe is inserted for hybridization.
What is northern blotting used for?
RNA analysis
What is western blotting used for?
Protein analysis
How is western blotting done?
protein is coated with negative charges by SDS solution.
Then, so that all proteins migrate to the anode and are separated on the basis of size.
For protein that has sulfide bond, how is it treated for western blotting done?
heating with SDS and mercaptoethanol to break sulfide bond
Why RFLP and western blotting used together?
to see if mutation in restriction site cause any problem in stability of protein
a quality-control mechanism that selectively degrades mRNAs harboring premature termination (nonsense) codons at the ribosomes
Nonsense-mediated mRNA decay
