PCR

1
PCR

• FISH 543 / OCEAN 575
• Molecular Techniques

2
DNA Replication in the TubePCR

• 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

3
PCR

• How does it work
• Separate the two strands (94oC)
• Anneal primers (55oC)
• Replication start
• Extension (72oC)
• replication
• Repeat 20 30 times

94
94
72
55
4
PCR
5
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

6
DNA Replication in the TubePCR

• 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?

7
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

Pyrimidines
Purines
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The structure of DNA

• Complimentary binding
• Hydrogen bonds
• Purine with Pyrimidine
• A T
• G C
• Chain is antiparallel

9
Action of DNA polymerase is always 5? 3

5
3
5
3
5
3
3
5
10
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-TGCA-3
5-GCCA-3
and
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PCR primers

• Annealing temperature
• Optimal temperature for primers to attach to the
  template DNA
• Too high
• Bonds dont work
• Primer doesnt 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 dependson GC content

12
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

13
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

• Minimal (lt3bp) between-primer-complementarity

5-ACTGTGCCATAGATGCAG-3
3-CAACTGCACCGTATGCAT-5

• Programs on the web to design primers
• Links on webpage

14
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 20mM 2.5 µl 10X
buffer 2.5 µl H2O 11.5 µl Taq 0.5 U gt1
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
15
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
  samples worth) extra to allow for pipetting
  errors
• Negative control
• No template DNA
• Check for contamination
• Positive control
• Something you know works

16
Common PCR Problems

• Contamination
• No or weak product
• Primer dimers
• Non-specific products

17
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

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If it happens

• Try somebody elses ingredients
• Change ingredients
• chemicals
• water
• Clean gear
• pipettes
• bench (bleach)
• Be more careful
• Pipetting
• Use of contaminated tips
• Causes chemical contamination

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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

20
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

21
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

22
Desired product

Non-specific product
Amount of PCR product
Non-specific product with higher amplification
efficiency than desired product
Number of PCR cycles
23
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

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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