1. FISH 543 / OCEAN 575 Molecular Techniques
PCR
FISH 543 / OCEAN 575
Molecular Techniques

2. 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

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 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?

7. The structure of DNA Sugar-phosphate backbone Nitrogenous base
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

8. 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’

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’-GCCA-3’
and
5’-TGCA-3’

11. PCR primers Annealing temperature Remember:
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

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
5’-ACTGT
AGAT-3
GCC
ATA
GGC
Minimal (<3bp) 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 µl
Forward Primer 10 mM µl
Reverse Primer 10 mM µl
dNTPS 8mM µl
Mg++ 20mM µl
10X buffer µl
H2O µl
Taq 0.5 U >1 µl
Total 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 sample’s 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

18. 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

19. No or weak product Missing ingredient Wrong concentrations
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 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. Amount of PCR product Number of PCR cycles
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

24. 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