11.2 Flashcards
what are the minimum requirements for DNA synthesis in vitro?
DNA strand for template
DNA primer
DNA polymerase
dNTPs
Mg+ (for dna poly)
who invented PCR?
kary mullis in 1983
what is PCR?
enzymatic copying of double-stranded DNA using two primers, complementary to opposite stands, that lead to exponential increase in amount of target sequence
how many temperatures are needed for PCR?
three
what are the three temperature stages of PCR?
denaturation - 94-96c
annealing - 50-65c (depends on Tm)
elongation/extension - 72c
what “ingredients” are needed for PCR?
dNTPs
Mg2+
Primers (usually two)
Template DNA
Taq polymerase
what are primers?
short moleucles of ssDNA
what gives the growth in PCR?
primers anneal to opposite strands
what determines the size of PCR product?
how far apart the annealing sites of the two primers are
how big are most PCR products?
2 kb or less
how long are primers usually?
18-25 b long
why are primers usually 18-25 b long?
shorter ones would bind too often (not specific enough)
longer ones would take more time to make, but not be any more specific
what are some applications of pcr?
amplifying target sequences for further study
detection of rare DNA sequences
what are the stages of pcr?
exponential phase
linear phase
platueua phase
what happens in the exponential phase of PCR?
only limited by the amount in the previous cycle, exponential growth
what happens in the linear phase of PCR?
dNTPs are less abundant and DNA poly may wear out, leading to slower growth of product
what happens in the plateau phase in PCR?
growth slows down and then stops as dNPTs run out and DNA poly becomes exhausted
what is qPCR?
PCR where DNA is quantified each cycle using reporter dye and fluorescence reader
what dye is used for qPCR?
SYBR green
why do we use SYBR green for qPCR?
is simplest and cheapest
is much brighter when bound to dsDNA
what does SYBR green do?
binds to minor groove in dsDNA
what are some applications of qPCR?
quantify amount of starting DNA
measuring rate at which gene is transcribed
