PCR- nucleic acid amplification Flashcards
PCR
polymerase chain reaction
primer-directed in vitro enzymatic reaction for the massive amplification of a specific DNA fragment
PCR uses
screen for organism/mutation
viral titers
DNA sequencing
etc
standard PCR reaction mix
0.2 mM dNTPs 50 mM KCl 10 mM tris ph 8.4 1.5 mM MgCl2 2.5 units of themostable DNA polymerase (Taq) 0.25 mM each primer DNA for use as a template
Template for PCR
must be DNA (ss or ds)
if starting w/ RNA must use reverse transcriptase
should be as high quality as possible
usually 1-200 ng of genomic DNA
PCR primers
single stranded 18-30 bp fragments complementary to sequences flanking the region to be amplified
primers determine the specificity of the PCR product
DNA polymerase
heat stable DNA polymerase from Thermus aquaticus (Taq)
cloned & purified
MgCl2
cofactor for DNA polymerase
chelated by EDTA
step 1 (denaturation)
initial denaturation
95C for 5-15 minutes
split template DNA strands
step 2 (anneal)
annealing
anneal primer to template
50-68C ( 5C lower than the Tm of the primer) for 20-30 seconds
step 3 (extend)
extension @ 72C
optimal temperature for taq
1kb/minute
amplicon
PCR product
number of amplicons= 2^n
n= number of cycles
misprimes
may occur due to nonspecific hybridization of primers
primer dimers
may occur due to hybridization of primers to each other
negative control
contamination control
most dangerous contaminant is PCR product from a previous reaction
contamination control
physical separation: air locks, positive air flow, PCR hoods w/ UV
dUTP + uracil-N-glycosylase
10% bleach
avoiding misprimes
use proper annealing temperature
design primers carefully
use a sequestered enzyme
sequestered enzyme
requires an initial heat activation (hot start)
has an inactivating antibody bound to the enzyme that is only blasted off at high temperatures
primer design
goal: target specificity
avoid sequence homologies
G/C content less than 80%
product size as small as possible
PCR product cleanup
removes all reaction components from PCR product as well as misprimes & primer dimers
required before many subsequent steps
solid-phase isolation of PCR product
small sephadex G-50columns for the microfuge
real time / quantitative PCR
standard PCR w/ an added probe or dye to generate a fluorescent signal from the prroduct
threshold cycle
input (starting DNA amount) is inversely proportional to threshold cycle (cycle at which fluorescence crosses the threshold level
higher amount of DNA starting material = crosses threshold faster
viral copy number qantification
take difference of control threshold cycle & sample threshold cycle
log of the difference
times the control concentration
thermal melt curve
each DNA fragment has a unique melting profile
take the derivative of the change in fluorescence/derivative of the temperature = melt curve
used in BRAF mutated in cancers
