PCR Flashcards
What is PCR?
Polymerase Chain Reaction
A rapid procedure for in vitro(test tube) enzymatic amplification of a specific segment of DNA
What are the applications of PCR?
- Covid Testing
- Assays for presence of infectious disease
- Quantifying RNA transcripts
- Genetic Fingerprinting of Forensic Samples
and more
4 steps of PCR testing
- Reverse transcription PCR of naso-pharyngeal swabs
- RNA extracted from samples is reverse transcribed to cDNA
- Primers to the ORF 1ab gene of SARS-CoV-2
- Real time PCR to quantify the RNA and therefore virus
What is the bond between bases?
H bonding
How do DNA strands run?
anti-parallel
What group is at the 5’ end?
Phosphate group
What group is at the 3’ end?
OH sugar
G and C are _____ than A and T because…
stronger, they have 3 hydrogen bonds vs 2
What does DNA polymerase need? Why?
Primer, because it needs to attach a nucleotide to the 3’OH
Where does DNA polymerase add a nucleotide?
DNA POL adds a nucleotide to the 3’OH of the primer
Where do the primers need to be tightly bound to?
The 3’ end of the primer because that is where the nucleotide is being layed
If the 3’ end of the primer needs to be tightly bound to the template what nucleotides will be there? Why?
G and C because they have strong (3) H bonds
Forward Primer
Anneals to the lower strand
Reverse Primer
Anneals to the upper strand
PCR Protocol 6 steps
- Initial Melting at 94C for 5 min
- Denaturation of the DNA strands (94-94C) (High temp)
- Annealing of oligonucleotides primers (55-65C) (Lower temp for primers to find their spots)
4.Extension/polymerization with polymerase (72C) - Repeat steps 2-4 for 25-30 cycles
- Final extension at 72C for 5-10min
