In Vitro Gene Cloning Flashcards
What does PCR stand for?
polymerase chain reaction
What happens at 95C in PCR?
the hydrogen bonds between the complementary bases in DNA are broken.
What happens at 55C in PCR?
the primers can anneal to the exposed complementary bases (because of complementary base pairing)
What happens at 72C in PCR?
Taq/DNA polymerase catalyses the reaction forming phosphodiester bonds between adjacent nucleotides
What is the importance of primers in PCR?
- they provide a binding site for DNA polymerase
- they prevent strands reforming hydrogen bonds
What are the 3 stages in a PCR reaction?
separation of DNA strands
addition of primers
synthesis of DNA
What is required for PCR?
thermocycler
DNA fragment
supply of nucleotides
(heat-tolerant) DNA polymerases (taq polymerase)
complementary primers
What does anneal mean?
bind to
What is a primer?
a short sequence of single stranded DNA, these are deliberately created to be complementary to the start/end of the DNA fragment
Why does PCR stop DNA replication?
limited number of primers/free nucleotides
Why might PCR require various types of primers?
as base sequences differ and primers must be complementary so they can bind
Why is a graph showing DNA increase with PCR increase a curve?
DNA doubles with each cycle, so increases exponentially
Why does a graph showing DNA increase with PCR plateau?
as nucleotides can be all used up
