PCR
The Polymerase Chain Reaction
Teresa Lewis Ph. D.
Assistant Researcher
Hawaii Institute of Marine Biology
Polymerase Chain Reaction
PCR first described in mid 1980s, Mullis Nobel prize in
1993
An in vitro method for the enzymatic synthesis of specific
DNA sequences
Selective amplification of target DNA from a heterogeneous,
complex DNC/cDNA population
Requires
Two specific oligonucleotide primers
Thermostable DNA polymerase
dNTPs
Template DNA
Sequential cycles of (generally) three steps (temperatures)
Initially PCR used the Klenow fragment of E. coli DNA
polymerase - inactivated by high temperatures
Kleppe, Ohtsuka, Kleppe, Molineux, Khorana. 1971. J. Mol. Biol. 56:341.
Required a thermostable DNA polymerase - Taq
DNA polymerase from Thermus aquaticus
a thermophilic eubacterial microorganism
isolated from a hot spring in Yellowstone
National Park
Kcat = 150 nucleotides/sec/enzyme (at Topt)
Taq1/2 =
92.5 oC
130 min
95.0 oC
40 min
97.5 oC
5 min
PCR - before the thermocycler
95 C
5 min
55 C
3 min
35 times
8 BORING hours per PCR!
72 C
5 min
Thermocyclers
heated lids
adjustable ramping times
single/multiple blocks
gradient thermocycler blocks
standard tube, volume, cost
evaporation & heat transfer concerns
thin walled tube, volume, cost
evaporation & heat transfer concerns
Directional Synthesis
2 copies
Cycle 2
4 copies
Cy
cle
1 copy
PL
sa US
d
pr lts,T NTP
im aq
ers p s, b
ol, uf
fer
,
Xeroxing DNA
Cycle 3
8 copies
Cycle 35
n36 = 68,719,476,736 copies in ~ 2 hrs
A simple thermocycling protocol
1X
35X
1X
94C 94C
72C
3 min 1 min
1 min
45 sec
extension
annealing
denaturation
Initial denaturation
of DNA
55C
4C
hold
Step 1:
Denaturation
dsDNA to ssDNA
Step 2:
Annealing
Primers onto template
Step 3:
Extension
dNTPs extend 2nd strand
Vierstraete 1999
extension products in one cycle serve as template in the next
Basic Components of PCR
Template DNA (0.5 - 50 ng)
< 0.1 ng plasmid DNA, 50 ng to 1 g gDNA for single copy genes
Oligonucleotide primers (0.1 2.0 M)
dNTPs (20 250 M)
Thermostable DNA pol (0.5 2.5 U/rxn)
MgCl2 (1 5 mM) affects primer annealing and Taq activity
Buffer (usually supplied as 10X)
Working concentrations
KCL (10 50 mM)
Tris-HCl (10 mM, pH 8.3)
NaCl2 (sometimes)
Buffer
Primers
AC TG
dNTPs
Taq polymerase
DNA template
+ + MgCl
2
MgCl2 (mM)
1.5 2
3 4
Magnesium Chloride
(MgCl2 - usually 0.5-5.0mM)
Magnesium ions have a variety of effects
Mg2+ acts as cofactor for Taq polymerase
Required for Taq to function
Mg2+ binds DNA - affects primer/template interactions
Mg2+ influences the ability of Taq pol to interact with
primer/template sequences
More magnesium leads to less stringency in
binding
PCR Problems
Taq is active at low temperatures
At low temperatures mis-priming is likely
Temp
Extension Rate
55o C
37o C
24 nt/sec
1.5 nt/sec
22o C
0.25 nt/sec
150 nucleotides in 10 min
Cures for mis-priming
Cheap fixes
Physical separation DNA-in-the-cap
Set up reactions on ice
Hot-start PCR holding one or more of the PCR components
until the first heat denaturation
Manually - delay adding polymerase
Wax beads
Polymerase antibodies
Touch-down PCR set stringency of initial annealing
temperature high, incrementally lower with continued cycling
PCR additives
0.5% Tween 20
5% polyethylene glycol 400
betaine
DMSO
Primer Design
1. Typically 20 to 30 bases in length
2. Annealing temperature dependent upon
primer sequence (~ 50% GC content)
3. Avoid secondary structure, particularly 3
4. Avoid primer complementarity (primer dimer)
5. The last 3 nucleotides at the 3` end is the
substrate for DNA polymerase - G or C
6. Many good freeware programs available
Primer Design Software
Many free programs available online
OLIGO
PRIMER
PrimerQuest
Primer Dimers
Pair of Primers
5-ACGGATACGTTACGCTGAT-3
5-TCCAGATGTACCTTATCAG-3
Complementarity of primer 3 ends
5-ACGGATACGTTACGCTGAT-3
3-GACTATTCCATGTAGACCT-5
Results in PCR product
Primer 1
5-ACGGATACGTTACGCTGATAAGGTACATCTGGA-3
3-TGCCTATGCAATGCGACTATTCCATGTAGACCT-5
Primer 2
Rules of thumb for PCR conditions
Add an extra 3-5 minute (longer for Hot-start Taq) to your cycle
profile to ensure everything is denatured prior to starting the PCR
reaction
Approximate melting temperature (Tm) = [(2 x (A+T)) +(4 x
(G+C))]C
If GC content is < 50% start 5C beneath Tm for annealing
temperature
If GC content 50% start at Tm for annealing temperature
Extension @ 72C: rule of thumb is ~500 nucleotide per minute.
Use 3 minutes as an upper limit without special enzymes
Special PCR cycling protocols
Touchdown PCR
Step-up PCR
Gradient cycling
Common PCR additives
BSA (usually at 0.1 to 0.8 g/L final concentration)
Stabilize Taq polymerase & overcome PCR inhibitors
DMSO (usually at 2-5% v/v, inhibitory at 10% v/v)
Denaturant - good at keeping GC rich template/primer strands
from forming secondary structures.
Glycerol (usually at 5-10% v/v)
Increases apparent concentration of primer/template mix, and
often increases PCR efficiency at high temperatures.
Stringency enhancers (Formamide, Betaine, TMAC)
Concentrations used vary by type
Enhances yield and reduces non-specific priming
Non-ionic detergents (Triton X, Tween 20 or Nonidet P-40) (0.11%)
NOT SDS (0.01% SDS cuts Taq activity to ~10% of normal)
Stabilize Taq polymerase & suppress formation of 2 structure
PCR additives - Literature
Additive
References
DMSO
Amplifications 5: 16
Gene 140: 1
Nucleic Acids Research 18: 1666
(dimethyl sulfoxide)
Betaine
(N,N,N-trimethylglycine
= [carboxymethyl]
trimethylammonium)
Formamide
Non-ionic detergents
e.g. Triton X-100, Tween 20 or
Nonidet P-40 (NP-40)
TMAC
Biochemistry 32: 137
BioTechniques 21: 1102
Genome Research 6: 633
Nucleic Acids Research 25: 3957
Proceedings of the National Academy of Sciences of the United States of America
70: 298
Trends in Biochemical Science 22: 225
Nucleic Acids Research 18: 7465
Biotechniques 12: 332
Nucleic Acids Research 18: 1309
(tetramethylammonium chloride)
Nucleic Acids Research 18: 4953
Nucleic Acids Research 23: 3343
dC7GTP
Nucleic Acids Research 16: 3360
(7-deaza-2'-deoxyguanosine)
BSA
(bovine serum albumin)
Applied and environmental microbiology 62:1102-1106
BioTechniques 23:504
BioTechniques 25:564
Nucleic Acids Research 16: 9775
Typical PCR Temps/Times
Initial
denaturation
90o 95o C
13
min
Denature
90o 95o C
0.5 1
min
Primer
annealing
45 65 C
0.5 1
min
Primer
extension
70o 75o C
0.5 2
min
Final
extension
70o 75o C
5 10
min
Stop reaction
4o C or 10 mM
EDTA
hold
25 40
cycles
Gel electrophoresis
Workhorse method of the
biotech laboratory
Separation through a matrix
(agarose or acrylamide)
Analytical or preparative method
Separates fragments with large
range of molecular weights
Driven by simple current and the
fact nucleic acids are uniformly negatively charged
Sample buffer (SB) or tracking dye (TD) or loading buffer used to keep sample in the well and visualize run
An aside: Ohms law
V = IR
voltage = current x resistance
(electric field) (milliamps) (ohms)
What does this REALLY mean to you?
For a given current, decreasing the thickness of the gel or
ionic strength of the running buffer increases the mobility
of the nucleic acid fragments
Manipulate this when possible to speed up the pay-off
Did your PCR work?!!
Electrophoresis
Variations on a Theme
DGGE
Denaturing gradient gel electro4
TGGE
Temperature gradient gel electro4
Allows separation of single base
polymorphisms
Pulsed field gel electrophoresis (PFGE)
Schwartz and Cantor (1984) Cell 37:67-75
DNA (from cells or DNA, undigested or digested)
Embedded into agarose plugs
Electrical field constantly changes direction (non-continuous)
allows for increased resolution of very h.m.w. gDNA
(Kb to Mb ranges)
chiller, pump, flatbed electro4 box
can use digested or undigested
RNA electrophoresis
Requires special solution
treatment to protect RNA
from degradation or
from folding in on itself
RNA is denatured and run on agarose gels containing
formaldehyde. Formaldehyde forms unstable Schiff bases
with the single imino group of guanine bases. This maintains
RNA in a denatured state so it electrophoreses properly
according to its molecular weight.
Uses same gel box and power supply as traditional DNA
electrophoresis
Houston, we have a PCR product
BUT what if you dont
Troubleshooting PCR
Non-specific bands on your gel
Reagents, set-up
Run negative control
Template concentration inappropriate
Review guidelines
Annealing temp too low
Optimize by gradient PCR
Extension time too short
time for longer products
Cycle number too high
Review guidelines
Primer design not appropriate
specificity
Primer concentration too high
Optimize by titration
Non-specific priming
specificity, Hot Start
MgCl2 concentration too high
Optimize by titration
GC-rich template, 2 structure
PCR additives
Contaminating DNA
Decontaminate work area:
use ARTs, wear gloves,
pipettor, reagents,
UV treat plastics
Troubleshooting PCR
Diffuse smearing on your gel
Template concentration inappropriate
Review guidelines
Taq concentration too high
Optimize by titration
Extension time inappropriate
Review guidelines
Cycle number too high
Reduce, review guidelines
Primer design not appropriate
specificity
Primer concentration too high
Optimize by titration
Non-specific priming
use Hot Start
MgCl2 concentration too high
Optimize by titration
GC-rich template, 2 structure
PCR additives
Contaminating DNA
Decontaminate work area:
use ARTs, wear gloves,
pipettor, reagents,
UV treat plastics
Troubleshooting PCR
Poor or no amplification of bands
Problem with thermocycler, set-up,
Run positive control
reagents
Enzyme concentration low
Concentration
Annealing temp too low
Optimize by gradient PCR
Extension time too short
Time for longer products
Cycle number too low
Review guidelines
Primer design not appropriate
Specificity
Primer concentration too high
Optimize by titration
Non-specific priming
Specificity, Hot Start
MgCl2 concentration too low
Optimize by titration
GC-rich template, 2 structure
PCR additives
Troubleshooting PCR
Prioritizing Approaches
Pilot error ( set-up errors common in the interim between
training with someone and working independently)
Template dilution error (concentration matters!)
Thermocycling parameter errors (temps/times)
Bad reagents (1. dNTPs, 2. primers, 3. Taq)
Unique template or template structure issues
BAD KARMA (dont believe it!)
Dont get discouragedvalidating PCRs can be tricky
Questions?
