PCR Flashcards
What does this stand for?
Principles of polymerase chain reaction
What type of method is this?
In vitro (in glass) outside of the body
What does this do?
Amplify the DNA fragments
All 3 temperatures
95 degree C
55 degree C
72 degree C
All the things in the test tube
DNA sample
DNA polymerase
Free DNA nucleotides
DNA primers
Step 1
Heat test tube to 95 degree C to break hydrogen bonds between DNA strands so the DNA separates
Step 2
Cool to 55 degree C to allow DNA primers to hydrogen bond by complementary base pairs
What are DNA primers?
Short, single-stranded DNA molecules that are used to mark the start and end of the specific DNA base sequence to be amplified
Step 3
Heat to 72 degree C and nucleotides are then added to each primer via complementary base pairing with the template strand.
DNA polymerase joins the nucleotides together via phosphodiester bonds
When does the process stop?
Mixture runs out of primers or free DNA nucleotides
How does the number of copies of DNA molecules increase each time?
Doubles
Why do we want to amplify DNA fragments?
Requires a lot of same DNA for meaningful research and development
Gene cloning
Amplification of a specific gene
Why are primers needed?
Mark the start and end of base sequence to be copied
DNA polymerase will only start adding nucleotides if it can bind to a primer first
primers also prevent original DNA strands re-joining
Why is DNA helicase not needed to break hydrogen bonds between DNA?
Step 1 heats to 95 degrees which breaks hydrogen bonds between DNA strands
Why is PCR popular method of amplification?
Fast
Does not require culturing cells
What type of DNA polymerase is used and why?
Thermostable so can withstand high temperatures without denaturing = no change in tertiary structure so active site of enzyme still complementary = still functional
