PCR- nucleic acid amplification

Question 1

PCR

Answer 1

polymerase chain reaction

primer-directed in vitro enzymatic reaction for the massive amplification of a specific DNA fragment

Question 2

PCR uses

Answer 2

screen for organism/mutation
viral titers
DNA sequencing
etc

Question 3

standard PCR reaction mix

Answer 3

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

Question 4

Template for PCR

Answer 4

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

Question 5

PCR primers

Answer 5

single stranded 18-30 bp fragments complementary to sequences flanking the region to be amplified
primers determine the specificity of the PCR product

Question 6

DNA polymerase

Answer 6

heat stable DNA polymerase from Thermus aquaticus (Taq)

cloned & purified

Question 7

MgCl2

Answer 7

cofactor for DNA polymerase

chelated by EDTA

Question 8

step 1 (denaturation)

Answer 8

initial denaturation
95C for 5-15 minutes
split template DNA strands

Question 9

step 2 (anneal)

Answer 9

annealing
anneal primer to template
50-68C ( 5C lower than the Tm of the primer) for 20-30 seconds

Question 10

step 3 (extend)

Answer 10

extension @ 72C
optimal temperature for taq
1kb/minute

Question 11

amplicon

Answer 11

PCR product
number of amplicons= 2^n
n= number of cycles

Question 12

misprimes

Answer 12

may occur due to nonspecific hybridization of primers

Question 13

primer dimers

Answer 13

may occur due to hybridization of primers to each other

Question 14

negative control

Answer 14

contamination control

most dangerous contaminant is PCR product from a previous reaction

Question 15

contamination control

Answer 15

physical separation: air locks, positive air flow, PCR hoods w/ UV
dUTP + uracil-N-glycosylase
10% bleach

Question 16

avoiding misprimes

Answer 16

use proper annealing temperature
design primers carefully
use a sequestered enzyme

Question 17

sequestered enzyme

Answer 17

requires an initial heat activation (hot start)

has an inactivating antibody bound to the enzyme that is only blasted off at high temperatures

Question 18

primer design

Answer 18

goal: target specificity
avoid sequence homologies
G/C content less than 80%
product size as small as possible

Question 19

PCR product cleanup

Answer 19

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

Question 20

real time / quantitative PCR

Answer 20

standard PCR w/ an added probe or dye to generate a fluorescent signal from the prroduct

Question 21

threshold cycle

Answer 21

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

Question 22

viral copy number qantification

Answer 22

take difference of control threshold cycle & sample threshold cycle
log of the difference
times the control concentration

Question 23

thermal melt curve

Answer 23

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