PCRFISH 543 / OCEAN 575

Molecular Techniques

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

PCR

• How does it work:– Separate the two strands (94oC)

– Anneal primers (55oC)• Replication start

– Extension (72oC)• = replication

– Repeat 20 – 30 times

94°

55°

72°

94°

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 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

• 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

PyrimidinesPurines

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’

3’

5’ 3’

5’

5’

5’

3’

3’

DNA sequences are always written 5’ 3’

5’-5’-GGCCCCAATTAAGGAATTGGCCAAGGCCCCTTGGAAGGAATTCCAAGGCCAATTGGCCAA-3’-3’

5’-5’-GGCCCCAATTAAGGAATTGGCCAAGGCCCCTTGGAAGGAATTCCAAGGCCAATTGGCCAA-3’-3’3’-3’-AACCGGTT-5’-5’

3’-3’-CCGGGGTTAATTCCTTAACCGGTTCCGGGGAACCTTCCTTAAGGTTCCGGTTAACCGGTT-5’-5’

3’-3’-CCGGGGTTAATTCCTTAACCGGTTCCGGGGAACCTTCCTTAAGGTTCCGGTTAACCGGTT-5’-5’5’-5’-GGCCCCAA-3’-3’

5’-5’-TTGGCCAA-3’-3’5’-5’-GGCCCCAA-3’-3’ and

So the Primers are

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• 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.

• Minimal (<3bp) between-primer-complementarity

• Primers should have no self complementarity

5’-A5’-ACCTTGGTTGGCCCCAATTAAGGAATTGGCCAAGG-3’-3’ |||| |||| 3’-3’-CCAAAACCTTGGCCAACCCCGGTTAATTGGCCAATT-5’-5’

5’-5’-AACCTTGGTT AAGGAATT-3-3

GGCCCC

AATTAAGG

GGCC

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

PCR - in practice

Sample Single Reaction

Template DNA 1-2 µg genomic1-2 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 >1 µl

Total 25 µl

Buffer is polymerase-specific, purchased with the enzyme,

Caution: some buffers are Mg++ free, others are not

Primers, dNTPS and Mg are often made up as 10X stocks for ease of setting up

reactions

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

Number of PCR cycles

Am

ou

nt

of

PC

R p

rod

uct

Desired product

Non-specific product

Non-specific product with

higher amplification

efficiency than desired

product

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