PCR Lecture
PCR Lecture
PCR Lecture
Requires
Two specific oligonucleotide primers
Thermostable DNA polymerase
dNTPs
Template DNA
Sequential cycles of (generally) three steps (temperatures)
92.5 oC
130 min
95.0 oC
40 min
97.5 oC
5 min
95 C
5 min
55 C
3 min
35 times
72 C
5 min
Thermocyclers
heated lids
adjustable ramping times
single/multiple blocks
gradient thermocycler blocks
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
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
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
Cheap fixes
Physical separation DNA-in-the-cap
Set up reactions on ice
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 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
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
(tetramethylammonium chloride)
dC7GTP
(7-deaza-2'-deoxyguanosine)
BSA
(bovine serum albumin)
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
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
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
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?