Nucleic Acid Amplification
Target Amplification Increasing the number of target fragments, most used in molecular diagnostics
Probe amplification Increasing the number of target-specific probes, relies on ligation
Signal amplification Increasing the production of signal on probe
PCR
Source DNA + dNTPs + 2 primers + reaction buffer + dIH2O + Taq
3 steps Denaturation (95C, 15 sec) + Annealing (primer binding, 50-65C, 30 sec) + extension (72C,
1 min) + final extension (72C, 5 min) + 4C hold
Hot Start Reduces background non-specific bands
Heat too long will reduce Taq activity
Annealing temp is based on melting temp (Tm), determined by the physical properties of primers
Too high temp primers cannot bind
Too low temp primers bind non-specifically
Shorter fragments amplify better than longer ones
Too many cycles can see increasing primer dimer, should between 25-35 cycles
PCR buffer
Increase [Mg2+] decrease reaction stringency
[Mg2+] influences enzyme activity, increases Tm of dsDNA, forms soluble complexes with
dNTP
EDTA binds to Mg2+
o Nucleic acid amplification & detection occur in the same step = homogenous reaction
o Nucleic acid amplification & detection occur in separate steps = heterogeneous reaction
o Stringency Ability of hybridization reaction to tolerate mismatches
o High stringency = Better chance of only seeing expected products
o Low stringency = Nonspecific amplification
o Increasing annealing temp Increase stringency up to a point
o Annealing temp > Tm, primers won’t bind, no product
o Increase Mg conc. Decrease stringency
o PCR Additives:
DMSO (1-10%)
Disrupt base pairing lower Tm
Used to improve amplification of GC rich regions & long DNA fragments
Can inhibit Taq activity & disrupt antibody binding used in Hot Start
Glycerol (5-20%)
Stabilize Taq
Lower Tm
PCR Enhancer (higher yield)
Betaine (1M)
Enzyme stabilizing agent
Lowers Tm
Reduces 2nd structure formation (most common additive for GC rich DNA amplification)
DMSO + betaine is preferred over formamide
Formamide (1.25 – 10%)
Binds major/minor helix in DNA = Disrupt base pairing = Lowers Tm (same effect as DMSO)
Can inhibit Taq activity at >5% conc.
Target Amplification Increasing the number of target fragments, most used in molecular diagnostics
Probe amplification Increasing the number of target-specific probes, relies on ligation
Signal amplification Increasing the production of signal on probe
PCR
Source DNA + dNTPs + 2 primers + reaction buffer + dIH2O + Taq
3 steps Denaturation (95C, 15 sec) + Annealing (primer binding, 50-65C, 30 sec) + extension (72C,
1 min) + final extension (72C, 5 min) + 4C hold
Hot Start Reduces background non-specific bands
Heat too long will reduce Taq activity
Annealing temp is based on melting temp (Tm), determined by the physical properties of primers
Too high temp primers cannot bind
Too low temp primers bind non-specifically
Shorter fragments amplify better than longer ones
Too many cycles can see increasing primer dimer, should between 25-35 cycles
PCR buffer
Increase [Mg2+] decrease reaction stringency
[Mg2+] influences enzyme activity, increases Tm of dsDNA, forms soluble complexes with
dNTP
EDTA binds to Mg2+
o Nucleic acid amplification & detection occur in the same step = homogenous reaction
o Nucleic acid amplification & detection occur in separate steps = heterogeneous reaction
o Stringency Ability of hybridization reaction to tolerate mismatches
o High stringency = Better chance of only seeing expected products
o Low stringency = Nonspecific amplification
o Increasing annealing temp Increase stringency up to a point
o Annealing temp > Tm, primers won’t bind, no product
o Increase Mg conc. Decrease stringency
o PCR Additives:
DMSO (1-10%)
Disrupt base pairing lower Tm
Used to improve amplification of GC rich regions & long DNA fragments
Can inhibit Taq activity & disrupt antibody binding used in Hot Start
Glycerol (5-20%)
Stabilize Taq
Lower Tm
PCR Enhancer (higher yield)
Betaine (1M)
Enzyme stabilizing agent
Lowers Tm
Reduces 2nd structure formation (most common additive for GC rich DNA amplification)
DMSO + betaine is preferred over formamide
Formamide (1.25 – 10%)
Binds major/minor helix in DNA = Disrupt base pairing = Lowers Tm (same effect as DMSO)
Can inhibit Taq activity at >5% conc.
