The Polymerase Chain Reaction (pcr)

The polymerase chain Reaction (PCR)

07/03/09

Abraham Mikru, University of Helsinki, 281108

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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

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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

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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

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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

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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

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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 primertemplate 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

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PCR mix 1. 2. 3. 4. 5. 6.

Buffer optimized with Mg ions Sterile milli Q water Fd and Rvs primers DNTPs Thermostable Taq polymerase 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

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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

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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

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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

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