polymerase chain reaction (pcr) lab 2. today’s lab principles of pcr the actual reaction primer...

Polymerase Chain Reaction (PCR)

Lab 2

Today’s lab

• Principles of PCR

• The actual reaction

• Primer design

• Limitations, troubleshooting and uses

• Variations

DNA Structure

Sugar-Phosphate backbone

What Is PCR?

• In vitro technique

• Amplification of specific DNA sequence between two regions of known sequence

• Invented 1985

Overall Principle of PCR

DNA – 1 copy

Known sequenceSequence of interestKnown sequence

PCR

Lots of copies

Why PCR?

• To ‘pull the needle out of the haystack’

• Rapid & easy

• Sensitive

• Robust

• Widespread applications

The Cycling Reactions

• Denature: ‘separate D.S DNA’

• Anneal: ‘stick primers to S.S DNA’

• Extend: ‘make new DNA from template’

Components of Reaction

• Template DNA

Taq

dTTP

dCTP dGTP

dATP

Primers

DNA Polymerase

dNTPs

Denaturation

• Temperature: 92-94C

• Double stranded DNA melts single stranded DNA

92C

3’5’

3’ 5’

+

5’3’

5’ 3’

Annealing

• Temperature: ~50-70C (dependant on the melting temperature of the expected duplex)

• Primers bind to their complementary sequences

5’3’

5’ 3’

Forward primer Reverse primer

Extension

• Temperature: ~72C• Time: 0.5-3min• DNA polymerase binds to the annealed primers and

extends DNA at the 3’ end of the chain5’ 3’

Taq5’

5’3’

Taq5’

Extension (2)

5’ 3’

5’3’

Taq

Taq5’

5’

Properties of Polymerase

• Taq polymerase originally isolated from from Thermus aquaticus

• Heat stable (half life of ~ 30min at 95C)• Taq DNA polymerase has no proof-

reading function in 3’―5’ direction• Primer extension occurs at up to 100

bases/sec• Plateau is reached eventually

Products of Extension

3’5’

3’ 5’

3’5’

3’ 5’

Taq

Taq

Cycle Begins Again………

3’5’

3’ 5’

3’5’

3’ 5’

Products after 2 cycles

3’5’

3’ 5’3’5’

3’ 5’

3’5’

3’ 5’

3’5’

3’ 5’

Products after 3 cycles…..

Geometric Amplification

1 2 4 8

16 32 64 128

256 512 1024 2048

4096 8192 16384 32768

Timescale: e.g. 93C: 5 min

93C: 45 sec

53C: 45 sec

72C: 1 min

72C: 5 min

X 30

Polymorphisms

Single base change at 3’ - primer won’t bind fully

3’

5’ 3’

5’

Normal Sequence

Sequence containing polymorphism

5’3’

5’3’

• Dolan DNA learning centre

• http://www.dnai.org/b/index.html

Primer Design

• Base composition: GC content 40-60%

• Length: ~18-24 bases

• Melting temp (Tm) of 2 primers: shouldn’t differ by more than ~5C

• Tm of amplification product: shouldn’t differ from Tm of primers by those of primers by more than ~10C

Primer Design (cont)• 3’ terminal sequences critical• Cycling and buffer conditions adjusted for

each primer pair• Complimentary sequences on pairs of

primers to be avoided

Primer 1

Primer 23’

3’

Primer Dimer

Primer Design (cont)

• Self complimentary sequences on primers also to be avoided

Primer

2 new primers generated

Primer secondary structure

Not complimentary to DNA sequence – cannot bind

PCR Primer Design - Summary

• PRIMER LENGTH - usually 18-30 bases is optimal. A primer of 18 bases should, in theory, only hybridize in one position in a eukaryotic genome.

• SELF-COMPLEMENTARITY -primer pairs should always be checked for complementarity. Complementarity at the 3’end can often lead to primer dimer formation. Complementary sequences within a single primer - especially if GC rich - can lead to hairpin loops or other secondary structures being formed. Commercially available computer programmes can be used to search for complementary sequences in primers so that these can be avoided.

• SPECIFICITY - the 3’ terminal sequence of a primer is critical for PCR specificity and sensitivity. A run of 3 or more G or C bases at this position should be avoided ( can cause non-specific annealing ). Also T should not be used at the 3’ end as it is more prone to mis-priming than the other nucleotides. Bases at the 5’ end are less critical for primer annealing, so if a restriction enzyme site is to be introduced, it should be closer to this end.

• G + C CONTENT - aim for 40% - 60%.

Things to try if PCR does not work

• A) If no product ( of correct size ) produced:– 1 Check DNA quality

– 2 Reduce annealing temperature

– 3 Increase magnesium concentration

– 4 Add dimethylsulphoxide ( DMSO ) to assay ( at around 10% )

– 5 Use different thermostable enzyme

– 6 Throw out primers - make new stocks

• B) If extra spurious product bands present– 1 Increase annealing temperature

– 2 Reduce magnesium concentration

– 3 Reduce number of cycles

– 4 Try different enzyme

Basic requirements for PCR reaction

1) DNA sequence of target region must be known.

2) Primers - typically 20-30 bases in size.These can be readily produced by commercial companies. Can also be prepared using a DNA synthesizer

3) Thermo-stable DNA polymerase - eg Taq polymerase which is not inactivated by heating to 95C

4) DNA thermal cycler - machine which can be programmed to carry out heating and cooling of samples over a number of cycles.

STANDARD PCR REACTION

• Genomic DNA - 100ng ( approx )• Forward & reverse primers at 0.1-0.5μM concentration.• dATP, dCTP, dGTP, dTTP - each at 0.2mM conc.• Appropriate buffer containing magnesium.• Taq polymerase - 0.5 - 1 unit• Sterile H2O - to give required total reaction volume

STANDARD PCR METHOD

• Prepare mix containing primers, dNTP’s, buffer and water sufficient for all reaction tubes or microtitre plate wells

• Aliquot appropriate volume to each 0.2ml thin walled tube or plate well. (robotic system can be used with plates)

• Add DNA to each tube - using a new tip for each sample• Add Taq polymerase - load tubes on PCR machine• If PCR machine does not have heated lid, 1 drop of

mineral oil should be added to each tube before adding Taq. The Taq can be added with the tubes in the PCR machine block at 80C = hot start.

• Start PCR programme

Example of PCR programme

• Initial denaturation 95C for 5 mins

• Thermo-cycle file - 30 cycles of• Denaturation : 95C for 30 secs• Annealing : 55C for 30 secs• Extension : 72C for 45 secs

• Final extension 72C for 5 mins• Holding ( soak ) file usually 4C

Advantages of PCR

• Small amount of DNA is required per test - 100-200ng for PCR; 5-10μg for Southern blotting.

• Result obtained more quickly - usually within 1 day for PCR; 5-10 days for Southern.

• Usually not necessary to use radioactive material (32P) for PCR.

• PCR is much more precise in determining the sizes of alleles - essential for some disorders.

• PCR can be used to detect point mutations.

Checklist

• Is there a product formed?

• Is the product of the correct size?

• Is only one product formed?

1800bp

What can you do with PCR products?

• Gel electrophoresis – presence/absence of product, size determination

• Restriction endonuclease digestion• Sequencing

Applications

• Amplify DNA for genetics applications, cloning, sequencing, mapping DNA segments, infectious disease diagnosis, sex determination, forensics, evolutionary biology, archaeology…….