PCR
FISH 543 / OCEAN 575
Molecular Techniques
DNA Replication in the Tube
PCR
• Polymerase Chain Reaction
– Most important recent discovery (1985)
– Patented – all PCR reactions pay royalty
• Repeated replication of specific DNA sections
– Small quantities
• Feathers, hair etc.
– Specific regions of DNA
• Target specific sequences
• Logarithmic replication
– 2  4  8  16  32  64 128  256  512  1028
PCR
• How does it work:
94°
94°
– Separate the two strands (94oC)
– Anneal primers (55oC)
72°
• Replication start
– Extension (72oC)
• = replication
– Repeat 20 – 30 times
55°
PCR
PCR in practice
• Reaction ingredients
– Buffer
• Keep pH constant
– Template DNA
– Primers
• As a starting point
• Forward and reverse
– Nucleotides
• To synthesize DNA
– Polymerase
• Taq polymerase
– MgCl2
• Aids enzyme activity
• Needs accurate temperature
control
– PCR machines
– Automatic cycling of
temperature
DNA Replication in the Tube
PCR
• Need PCR primers
– Polymerase can only start synthesizing from double stranded
DNA
• Start where primer anneal
• What are primers?
– Short artificial DNA sequences
•
•
•
•
15-20 bp
Match template DNA
Can pick where we want to start PCR
Which direction?
The structure of DNA
• Sugar-phosphate
backbone
– 5 C-atoms in the sugar
• Chain is directional
– #3 on one side
– #5 on the other
• Nitrogenous base
– Purines: A, G
– Pyrimidines: C, T
Purines
Pyrimidines
The structure of DNA
• Complimentary binding
– Hydrogen bonds
– Purine with Pyrimidine
• A–T
• G–C
– Chain is antiparallel
Action of DNA polymerase is always
5’ 3’
5’
3’
3’
5’
3’
5’
3’
5’
DNA sequences are always written 5’ 3’
5’-GCCATAGATGCAGCCTGAGATCAGCATGCA-3’
3’-CGGTATCTACGTCGGACTCTAGTCGTACGT-5’
5’-GCCATAGATGCAGCCTGAGATCAGCATGCA-3’
3’-ACGT-5’
5’-GCCA-3’
3’-CGGTATCTACGTCGGACTCTAGTCGTACGT-5’
So the Primers are
5’-GCCA-3’
and
5’-TGCA-3’
PCR primers
• Annealing temperature
– Optimal temperature for primers to attach to the template
DNA
• Too high
– Bonds don’t work
– Primer doesn’t anneal
• Too low
– Primer may attach anywhere
– ‘Non-specific amplification’
– Depends on strength of bonds
• Remember:
– G-C – three hydrogen bonds
– A-T – two hydrogen bonds
– Annealing temperature depends
on GC content
Primers
• Where do we get primer sequences from?
– Somebody may have isolated them
• Check databases
• Freely available on internet (GenBank)
– Results not publishable without primer information
– Heterologous primers
• Isolated from related species
• Very useful for many applications
• Problem
– may not exactly match
– PCR does not always work
– Primer design from published sequences
• Align related species
• Design primers in conserved regions
• Amplify variable regions
– Primer isolation
• Very lengthy and expensive procedure
• several months work
Primer design
• Primer pairs should have similar annealing temp
–
–
length, %GC content
Tm = 4(G + C) + 2(A + T) oC.
• Primers should have no self complementarity
5’-ACTGT
GGC
GCC
ATA
AGAT-3
• Minimal (<3bp) between-primer-complementarity
5’-ACTGTGCCATAGATGCAG-3’
||||
3’-CAACTGCACCGTATGCAT-5’
• Programs on the web to design primers
– Links on webpage
PCR - in practice
Sample Single Reaction
Template DNA
1-2 µg genomic
1-2 µg mtDNA
1µl
Forward Primer 10 mM
2.5 µl
Reverse Primer 10 mM
2.5 µl
dNTPS 8mM
2.5 µl
Mg++
2.5 µl
20mM
10X buffer
2.5 µl
H2 O
11.5 µl
Taq 0.5 U
>1 µl
Total
25 µl
Primers, dNTPS and Mg are often made up as
10X stocks for ease of setting up reactions
Buffer is polymerase-specific, purchased
with the enzyme,
Caution: some buffers are Mg++ free,
others are not
Use high quality nuclease free water
PCR - in practice
• You are never setting up only a single PCR
reaction
– Make up master mix
• Buffer, primers, MgCl2, water, dNTPs, Taq
– When calculating master mix volume, add a bit (~1
sample’s worth) extra to allow for pipetting errors
• Negative control
– No template DNA
• Check for contamination
• Positive control
– Something you know works
Common PCR Problems
• Contamination
• No or weak product
• Primer dimers
• Non-specific products
The worst problem – Contamination
• Exponential copying of template
– Very sensitive
– Tiny amounts of contaminant can cause problems
• Main culprit
– PCR products
• Perfectly matching short sequences
• Massive amounts
• Can swamp new template DNA
• You are your own worst enemy!
• Solutions
–
–
–
–
Use ultra-clean chemicals
Separate pre- and post PCR
Always use negative control
Aliquot reagents in small batches
• Can be discarded if problem
– Use filtertips
– Pipet carefully
If it happens…
• Try somebody else’s ingredients
• Change ingredients
– chemicals
– water
• Clean gear
– pipettes
– bench (bleach)
• Be more careful
– Pipetting
– Use of contaminated tips
• Causes chemical contamination
No or weak product
• Missing ingredient
– Check your lab book
– Do it again
• Wrong concentrations
–
–
–
–
Template
Primer
Taq
MgCl2
• Wrong primers
– Check sequence
– Try alternatives
– Use positive control
• Bad template
– Check template on
agarose gel
• Fragmentation
– PCR inhibitors
• Add to working PCR
– Too much
• Wrong conditions
– Reduce stringency
• Reduce annealing temp
• Increase MgCl2
• Failed staining
– Check visualization
– Use standard
Primer dimers
• Primers annealing to each other
– Small products 50-100 bp
• Usually because of template problems
– Primers try to anneal to something
• Solution
– Positive control
– Redesign primers
– Hot Start
Non-specific products
• Detection
– Electrophoresis on a gel
• Wrong product size
– Always use a standard
• Know your size
• Solution
– Increase stringency
• Increase annealing temperature
• Reduce MgCl2
– Change program
• Extension times
– Different primers
– Reduce number of cycles
Amount of PCR product
Desired
product
Non-specific
product
Non-specific
product with
higher
amplification
efficiency
than desired
product
Number of PCR cycles
PCR optimization
• Very sensitive procedure
– Each primer pair needs to
be optimized
– Can vary between PCR
machines
• Usually need to be
optimized
– Concentrations
• MgCl2 conc
• Primer & template
concentration
– Template can inhibit
PCR - dilute
– Ratio often important
• dNTP conc
– Cycling parameters
• Annealing temp
– Based on primer Tm
• Extension times
• Potentially lots of variables
• Ways to make it easier
– Gradient cycles
• Allow annealing temp gradient
across the block
• Can vary MgCl2 at same time
– Touch-down PCR
• Start with high annealing temp
– Produce few very specific
copies
• Lower annealing temp
– More efficient replication
– Touch-up PCR
• Start with low annealing temp
– Make sure there are some
copies
• Increase annealing temp
– Primers prefer PCR products
– Prevents non-specific
amplification after many cycles
PCR optimization - rules
• Maximize stringency
– Highest annealing temp
– Lowest MgCl2
• Minimize number of cycles
– Taq degradation
– Production of non-specifics
– Taq errors
• Most significant parameters
– Annealing temperature
– MgCl2