PCR

Polymerase Chain Reaction

 Polymerase Chain Reaction
PCR – first described in mid 1980’s, 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
dNTP’s
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
 Short History of PCR
• 1983: Dr. Kary Mullis developed PCR
• 1985: First publication of PCR by Cetus Corporation
appears in Science.
• 1986: Purified Taq polymerase is first used in PCR
• 1988: PerkinElmer introduces the automated
thermal cycler.
• 1989: Science declares Taq polymerase "molecule of
the year.
 Short History of PCR
• 1990: amplification and detection of specific DNA
sequences using a fluorescent DNA-binding dye,
laying the foundation for future "real-time" or
"kinetic" PCR.
• 1991: RT-PCR is developed using a single
thermostable polymerase, rTth, facilitating
diagnostic tests for RNA viruses.
• 1993:Dr. Kary Mullis shares Nobel Prize in
Chemistry for conceiving PCR technology.
 PCR - before the thermocycler

95º C 55º C 72º C

5 min 3 min 5 min

35 times

8 BORING hours per PCR!

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
 “Xeroxing” DNA
1 copy

2 copies

Cycle 2

4 copies

Cycle 3

8 copies

Cycle 35

n36 = 68,719,476,736 copies in ~ 2 hrs

A simple thermocycling protocol

1X 35X 1X
94ºC 94ºC
3 min 1 min 72ºC
1 min
55ºC
Initial denaturation
of DNA 45 sec

denaturation

extension
4ºC

annealing
∞ hold
PCR Steps

1. Denaturation of ds DNA template

2. Annealing of primers

3. Extension of ds DNA molecules

 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

 Denaturation
• Temperature: 92-94C
• Double stranded DNA melts single stranded
DNA
5’ 3’

3’ 5’
92C

5’ 3’

+
3’ 5’
 Annealing
• Temperature: ~50-70C (dependant on the melting
temperature of the expected duplex)
• Primers bind to their complementary sequences

5’ 3’

Forward primer Reverse primer

3’ 5’
 Extension
• Temperature: ~72C
• Time: 0.5-3min
• DNA polymerase binds to the annealed primers and
extends DNA at the 3’ end of the chain

Taq

5’ Taq

3’
5’
Cycling
 Products of Extension
5’ 3’

Taq
3’ 5’

5’ 3’

3’ 5’

Taq
 Example
PCR reaction condition
Effect number of cycles
Basic Components of PCR
• Template DNA (0.5 - 50 ng)
< 0.1 ng plasmid DNA, 50 ng to 1 μg gDNA for single copy
genes
- contains the sequence to be amplified

• Oligonucleotide primers (0.1 – 2.0 μM)

oligonucleotides that define the sequence to be amplified.
Size: 18-25 bp
• dNTP’s (20 –250 μM)
DNA building blocks
As subtrat for enzyme

• Thermostable DNA pol (0.5 – 2.5 U/rxn)

enzyme that catalyzes the reaction
• Buffer (usually supplied as 10X)
Working concentrations
KCl (10 – 50 mM)
Tris-HCl (10 mM, pH 8.3)
NaCl2 (sometimes)

Buffer
Primers
ACTG dNTPs
Taq polymerase

DNA template
+ + MgCl
2
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

MgCl2 (mM)
1.5 2 3 4 5
Example
PCR Reaction Composition for 20ul final volume
PCR Master Mix
Contain all component of PCR reaction
• Taq DNA Polymerase
• MgCl
• Buffer
• Enhancer (DMSO, Betain)

Except / must add to PCR

• DNA template
• Oligonucleotide primer
• dH2O to final volume

Usually supplied 2X concentration

How to calculate and prepare reagent
 Basic requirements for PCR
reaction
• 1) DNA sequence of target region must be
known.

2) Primers - typically 20-30 bases in size.

These can be readily produced by commercial
companies. Can also be prepared using a DNA
synthesizer
 Basic requirements for PCR
reaction
• 3) Thermo-stable DNA polymerase - eg Taq
polymerase which is not inactivated by
heating to 95C

4) DNA thermal cycler - machine which can be

programmed to carry out heating and cooling
of samples over a number of cycles.
PCR Enhancer and Inhibitor
PCR Problems
Taq is active at low temperatures
At low temperatures mis-priming is likely

Temp Extension Rate

55o C 24 nt/sec
37o C 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))]ºC
• If GC content is < 50% start 5ºC beneath Tm for annealing
temperature
• If GC content ≥ 50% start at Tm for annealing temperature

• Extension @ 72ºC: 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 µg/µL 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) (0.1–1%)

NOT SDS (0.01% SDS cuts Taq activity to ~10% of normal)
Stabilize Taq polymerase & suppress formation of 2º structure
PCR additives - Literature
Additive References
Amplifications 5: 16
DMSO Gene 140: 1
(dimethyl sulfoxide) Nucleic Acids Research 18: 1666
Biochemistry 32: 137
BioTechniques 21: 1102
Betaine Genome Research 6: 633
(N,N,N-trimethylglycine
Nucleic Acids Research 25: 3957
= [carboxymethyl] Proceedings of the National Academy of Sciences of the United States of America
trimethylammonium) 70: 298
Trends in Biochemical Science 22: 225
Formamide Nucleic Acids Research 18: 7465

Non-ionic detergents Biotechniques 12: 332

e.g. Triton X-100, Tween 20 or Nucleic Acids Research 18: 1309
Nonidet P-40 (NP-40)
TMAC Nucleic Acids Research 18: 4953
(tetramethylammonium chloride) Nucleic Acids Research 23: 3343

dC7GTP Nucleic Acids Research 16: 3360

(7-deaza-2'-deoxyguanosine)
Applied and environmental microbiology 62:1102-1106
BSA BioTechniques 23:504
(bovine serum albumin) BioTechniques 25:564
Nucleic Acids Research 16: 9775
Typical PCR Temps/Times
Initial 90o – 95o C 1–3
denaturation min

Denature 90o – 95o C 0.5 – 1

min
25 – 40
Primer 45o – 65o C 0.5 – 1
cycles
annealing min
Primer 70o – 75o C 0.5 – 2
extension min

Final 70o – 75o C 5 – 10

extension min
Stop reaction 4o C or 10 mM hold
EDTA
Troubles in PCR
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 (don’t believe it!)

Don’t get discouraged…validating PCRs can be tricky

Any Questions?