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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.

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slide1

PCR

FISH 543 / OCEAN 575

Molecular Techniques

dna replication in the tube pcr
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
slide3
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°

pcr in practice
PCR in practice
  • Needs accurate temperature control
    • PCR machines
    • Automatic cycling of temperature
  • 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
dna replication in the tube pcr1
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?
the structure of dna
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 dna1
The structure of DNA
  • Complimentary binding
    • Hydrogen bonds
    • Purine with Pyrimidine
      • A – T
      • G – C
    • Chain is antiparallel
slide9

Action of DNA polymerase is always 5’ 3’

5’

3’

5’

3’

5’

3’

3’

5’

slide10

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’

5’-TGCA-3’

and

pcr primers
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 dependson GC content
primers
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
slide13

Primer design

ATA

GGC

GCC

5’-ACTGT

AGAT-3

  • 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 (<3bp) between-primer-complementarity

5’-ACTGTGCCATAGATGCAG-3’ |||| 3’-CAACTGCACCGTATGCAT-5’

  • Programs on the web to design primers
    • Links on webpage
slide14

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++ 20mM2.5 µl

10X buffer 2.5 µl

H2O 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

slide15

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
slide16

Common PCR Problems

  • Contamination
  • No or weak product
  • Primer dimers
  • Non-specific products
the worst problem contamination
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
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

No or weak product
primer dimers
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
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
slide22

Desired product

Non-specific product

Amount of PCR 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
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

PCR optimization - rules