PCR Flashcards
PCR Concepts and Types of PCR
Who invented the PCR? When?
Kary Mullis, 1985
What does PCR stand for?
DNA Polymerase Chain Reaction
Stages of PCR?
denaturation, annealing, extension
purpose and temperature of denaturation?
unwind DNA to become single stranded, 92-95
purpose and temperature of annealing?
annealing of forward and reverse primers to single stranded target DNA so DNA polymerase can bind, 50-68
purpose and temperature of extension?
extension of primers using dNTPs to complement the target DNA, 70-72
which strand does the reverse primer bind to?
sense strand
which way does DNA polymerase extend?
3’ end
how long are primers?
15-40 oligonucleotides
what is the melting temperature (Tm)?
temperature at which half of the DNA strands become melt apart and become single stranded
how many hydrogen bonds do guanine and cytosine form?
three
what does it mean when a primer has a specific melting temperature?
temperature at which half of primer molecules are bound to DNA template
which dyes intercalate DNA and are UV active (when visualising PCR product)?
ethidium bromide or SYBR safe
which PCR troubleshooting technique involves setting a temperature gradient in the machine?
running PCR at different annealing temperatures
how can you enhance the activity of DNA polymerase?
change concentration of magnesium chloride
