The polymerase chain

Reaction (PCR)

07/03/09 Abraham Mikru, University of Helsinki, 281108 1

The polymerase chain Reaction (PCR)
• Selective amplification of Target segment
• Sequence of flanks must be known
• Flanks hybridize with primers
• Primers delimit the target sequence
• Amplification by thermo-stable Taq
polymerase
• Three temperature cycles
 Denaturation usually 94 0C
 Annealing 52-65 0C
 Extension 72-74 0C
• The cycle repeated 25 to 30 times
• Result in hundred million copies of target
segment
07/03/09 Abraham Mikru, University of Helsinki, 281108 2
 Analyzing the PCR Product
• Reaction mixture agarose gel
electrophoresed
• Stained with ethidium bromide
• Amplified target visible as discrete band
• May by itself suffice as useful info
• As in symbiotic gene targeted PCR
• PCR product can also be cloned into vector
• Sequence determined
• Compared with data base for identity

07/03/09 Abraham Mikru, University of Helsinki, 281108 3

 PCR in More Detail
• Experimental set up is easy
• Requires careful planning
Critical issues
 Sequence of primers
 Temperatures
What can be done with the amplified DNA?

07/03/09 Abraham Mikru, University of Helsinki, 281108 4

Primer Design
• Key to the success of PCR
• Appropriately designed primer
• Should result in only the target DNA
• Inappropriate primer may result in
 None target fragment
 Truncated bands with more than one fragment
• Must correspond with flanks
• Complementary to their template strand
• 3’ ends of fd and rvs primers point to each other
• Target segment shouldn’t exceed 3kbp
• Best between 1kb and 3 kbp
• The longer the fragment the less efficient
• More difficult to obtain consistent result

07/03/09 Abraham Mikru, University of Helsinki, 281108 5

Primer Design cont.
• Up to 10 kb can be amplified by standard PCR
• But less reproducible and inefficient
• Targets more than 40 kbp requires special method
• Primer length is the most critical issue
• Too short primer lead to random hybridization
• Result in undesired product
• If a pair of 8mers used on human genomic DNA
• Hybridization sites for the primers expected
• On average once every 48= 65,536 bp ≈46000 possible
sites in the 3 million kbp nucleotide sequ
• Thus very unlikely 8mers will give single target
• If 17mers used, number of possible priming sites once
every 417= 1.18 million bp. This figure is over 5 times
greater than the human genomic DNA
• So 17mer primer expected to give single target

07/03/09 Abraham Mikru, University of Helsinki, 281108 6

Primer Design & Annealing Temp.
• Too long primer reduce efficiency of PCR
• Longer time for annealing
• Primer longer than 3omer rarely used

The annealing temperature is key

• DNA-DNA hybridization is temperature dependent
• If too high no annealing
• If too low mismatched hybridization
• If mismatches are tolerated truncated bands
• Ideal annealing temperature:
• Low enough to allow hybridization
• High enough to prevent mismatch hybrids
• Estimated from Tm=4x(G+C)+2x(A+T) 0C of primer-
template hybrid
• Annealing Temp is best 1 to 2 0C lower than Tm
• The Tms for the fd and rvs primers should be equal

07/03/09 Abraham Mikru, University of Helsinki, 281108 7

 PCR mix
1. Buffer optimized with Mg ions
2. Sterile milli Q water
3. Fd and Rvs primers
4. DNTPs
5. Thermostable Taq polymerase
6. Dispense into PCR tubes in 50 µl
amounts
7. Add 1 µl of sample and load into
the thermocycler
07/03/09 Abraham Mikru, University of Helsinki, 281108 8
 Studying the PCR Product
• PCR is only the start of a story
• Target amplicon is studied in various ways
• Wide range of procedures
Three important techniques
 Gel electrophoresis
 Cloning
 Sequencing
• Portion (5 µl ) of the PCR reaction mixture is
run
• On 1.5% agarose in 0.5% TAE for 1 hr
• pGEM included as size marker (ladder)
• If expected band is absent or truncated
• Something has gone wrong- repeat indicated

07/03/09 Abraham Mikru, University of Helsinki, 281108 9

 Agarose gel electrophoresis

• The PCR product mix may be restricted

• To get info on presence of rest sites
• The AGE in this case is RFLP
• Important in constructing genome map
• Also in the study of genetic diseases
• Exact size can be used to see
• Deletion or insertion mutation
• This length mutation profiling is used
• In forensic DNA profiling
• In some cases mere presence of target
sequence is diagnostic
07/03/09 Abraham Mikru, University of Helsinki, 281108 10
Measurement of DNA concentration
• Concentration data crucial
• In cloning experiment
• Accurate measurement by UV spectrophotometry
(Nano drop)
• Absorbance directly proportional to the amount of
DNA
• Absorbance of 1 measured at A260 wave length
corresponds to 50µg of dsDNA/ml
• UV absorbance can also be used to check purity of
DNA
• With pure sample of DNA the ratio of
A260 / A280 is 1.8
• Ratios <1.8 indicate contamination (protein/Phenol)

07/03/09 Abraham Mikru, University of Helsinki, 281108 11