Lecture RR3. PCR and DNA sequencing Flashcards
What are the components needed for a PCR reaction (Polymerase Chain Reaction)
- DNA template
- a single DNA polymerase
- Primers
- dNTPs
what are the steps to PCR (Polymerase Chain Reaction)
- separation of the 2 strands, done by temperature (denaturation)
- (Annealing) The temperature is lowered to allow primers to base pair to complementary DNA template
- (extension) Polymerase extends primer to form a nascent strand
These steps are repeated 20 - 40 times
What does the Tm of DNA depend on
the sequence (the more G-C, the higher the Tm)
the length
of the DNA
why is it preferable to use DNA in labs instead of RNA
Because RNA is very easily degradable and it is less chemically stable than DNA
why do DNA molecules migrate on electrophoresis gel?
Because it is negatively charged due to the phosphate groups in the backbones
The molecules are separated by length
What are some applications of PCR (Polymerase Chain Reaction)
- Sequencing (some approaches)
- DNA cloning (isolating a particular gene or over-expression of proteins)
- Detection of pathogens (COVID)
- Gene editing (CRISPR, etc)
What are some applications for DNA sequencing?
- some applications of sequencing:
- Phylogenetic relations between species
- Human (animal) ancestry
- Catching criminals
- Diagnosis and predisposition to disease
What did Frederick Sanger invent?
The classical Sanger sequencing, the most used form of sequencing
also called: Dideoxy Chain-Termination Method of DNA sequencing
what is the difference between dNTP and ddNTP?
ddNTP has NO hydroxyl group whereas dNTP still has one
ddNTP stops the growth, since there is no hydroxyl group to bind to and continue
When separating DNA with a gel are the shorter fragments at the bottom or at the top of the gel?
bottom (closer to the 5’ end)
