PCR Flashcards
What is PCR?
a polymerase chain reaction, a method of amplifying dna
What can PCR do? Why might we find it useful?
Can use PCR to make billions of replications of DNA supa fast, applications in forensics- can replicate burglar hair for ex
Whats the three purposes of PCR?
-Detect presence or absence of a gene
- Getting enough dna copies to perform sequencing
- cloning and genetic engineering
What is the first thing needed in pcr in DNA pcr?
extract genomic dna (all the dna in the genome)
What is the 2 thing needed in PCR?
design primers which bind to either side of your region of interest
What are key features of primers?
Are written in 5’ to 3’ direction, have a free OH group and are aprrox 20 nucleotides long, are complimentary to the sequence they bind to
What is the third thing needed in PCR?
dNTPS
What is the fourth thing neede din PCR?
taq polymerase, special kind of polymerase which withstands heating and cooling
What do all components of pcr go into?
A thermocycler
What is the first step of PCR? In what conditions (time and temp)?
Denaturation (break strands of dna template) around 30 sec at 95
What is the second step of PCR? In what conditions (time and temp)?
Annealing (1 min at 55-66 degrees)
have primers bind to locations on template
What is the third step of PCR? In what conditions (time and temp)?
Synthesis: taq polymerase adds nuceletide to 3’ end.
How much DNA do you get after every round of PCR?
double the amount
How many copies of dna would you get after say, 27 rounds of pcr?
2^27
What determines the temperature at which primers anneal in PCR?
the melting temp of the primers we’re using, is when 50% are bound and 50% are not, varibale on GC richness and length of primers
