PCR Flashcards
what does pcr stand for?
polymerase chain reaction
what is unique about PCR?
it doesn’t use the aid of vectors or host cells
what is PCR used for?
making large amounts of DNA that is located within a defined region between two primers
what primers flank the region that PCR targets?
oligonucleotides
what are oligonucleotides?
short segments of DNA, usually about 15-20 nucleotides in length
what are the three steps of PCR?
1) denaturation
2) primer annealing
3) primer extension
denaturation
DNA is firs denatured by heat treatment, causing the strands to separate
primer annealing
as the temperature is lowered, oligonucleotide primers bind to the DNA
primer extension
once the primers have annealed, the temperature is raised slightly, and taq polymerase catalyzes synthesis of complementary DNA strands - this doubles the amount of DNA
what is the chain reaction of PCR?
the three step process repeating itself many times to amplify DNA, and the products of each reaction are used as templates for the next reaction
thermocycler
the machine that carries out PCR
- automates the timing of the temperature changes in each cycle
- each cycle lasts about 2-3 minutes
how many cycles are usually run in PCR?
20-30- cycles
can PCR be used on RNA?
PCR can be used to detect the amount of specific RNAs living in cells.
how is pcr used with RNA?
- RNA is isolated and mixed with reverse transcriptase
- generates single strand cDNA that is used as template DNA
- RNA has been amplified to produce many copies of DNA
what is the primer used with RNA in PCR?
reverse transcriptase
