Nucleic Acid Amplification in Molecular Genetics Clinical Lab

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.