Gene amplification- PCR Flashcards
what is PCR
polymerase chain reaction
an artificial way of replicating DNA, rapidly producing billions of identical copies of DNA fragments
what components are needed for PCR to take place
1) PCR primers
2) TAQ polymerase
3) free nucleotides
4) buffer solutions
what are PCR primers
short single-stranded sequences of DNA which are complementary to the start of a sequence- the 3’
why are PCR primers needed
they are needed to bind to a section of DNA as DNA polymerase is unable to bind free nucleotides to form a new single strand if there is no existing DNA backbone
what is TAQ polymerase and why is it used
a polymerase enzyme from a thermophilic bacteria so it doesn’t denature when exposed to the high temperatures used in the PCR
outline the method of PCR
1) DNA is denatured- heated to 95 degrees operating 2 strands of DNA by breaking hydrogen bonds
2) it is cooled to 55 degrees triggering PCR primers to anneal (join) to their complementary base sequence at the end of the single-stranded DNA
3) chain is extended- heated to 72 degrees causing TAQ polymerase to catalyze the synthesis of a complementary strand for each single-stranded DNA producing 2 identical strands
4) repeated doubling the quantity of DNA each time
what is the difference between PCR and normal DNA replication
1) can only replicate short sequences of DNA not the entire DNA
2) requires primers
3) uses a cycle of heating and cooling to separate and bind strands whilst DNA replication uses helicase
what are the uses of PCR
1) classification of organisms
2) mutation detection
3) cancer research
4) genetic engineering
what are the limitations of PCR
1) contamination could affect amplification
2) error rate- sometimes DNA polymerase inserts wrong nucleotide
3) after a while the process slows
what are the limitations of PCR
1) contamination could affect amplification
2) error rate- sometimes DNA polymerase inserts wrong nucleotide
3) after a while the process slows
why after 20 cycles does the pcr reaction slow
1) reagent conc becomes limiting
2) enzymes denature after repeated heating
3) DNA conc increases so single strands bind to each other
