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Laboratory #7 PCR

PCR• Polymerase chain reaction (PCR)• PCR is DNA replication in a test tube.

– In vitro enzymatic amplification of a specific segment of DNA.• Many Applications.

– direct cloning from DNA or cDNA.– Mutagenesis and engineering of DNA– Genetic fingerprinting of DNA samples– Assays for the presence of infectious agents– Prenatal diagnosis of genetic diseases– Analysis of allelic sequence variations– Analysis of RNA transcript structure– Genomic footprinting– Nucleotide sequencing of genomic and cDNA.

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PCR

PCR

• A single cycle of PCRconsists of three steps

• Denature DNA annealprimers extend primers.

• First, the reaction mixture isheated to 95˚C to denature(separate the strands of thedouble helix) the target DNA.

• Problem - DNA polymerasebreaks down at hightemperatures.

Denature DNA

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PCR• Thermus aquaticus -

bacteria which naturally livein hot springs (50 to 80ºC).

• The DNA polymerase usedby Thermus aquaticus isstable at high temperatures.

• Taq polymerase is used inthe PCR reaction.

• The PCR reaction wasdeveloped by Kary Mullis in1983.

• Kary Mullis won the Nobelprize in chemistry for thisdiscovery

PCR• Taq polymerase adds the appropriate nucleotide to the

replicating DNA sequence.• Can complete no more than 30 amplification cycles.• Taq polymerase adds an ‘A’ to the 3´ end of a linear vector.• Has the ability to anneal primers at room temperature to

regions of low complimentary.– May result in numerous unwanted PCR products.– ‘Hot start’ Taq polymerase

• Reversible inhibition of Taq polymerase by an antibody prevents Taq activityat room temperature.

• A five minute denaturation of the antibody at 94°C eliminates the antibodyand initiates Taq activity.

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PCR• Second, the temperature is lowered (generally to

around 50-55˚C), to allow the primers to anneal (bind)to the denatured strands of target DNA.

Anneal Primers

PCR• Primers are necessary in order for DNA replication to begin• Tm (Annealing Temperature) =Temperature at which the

primers stick to the correct sequence on the DNA strands.• Tm = 4(G+C) + 2(A+T)

– Notice that the annealing temperature increases with length andG+C content.

• The DNA strands will separate above the annealing temperature.• Too low of an annealing temperature the primers will anneal to

sequences other than the true target.• Too high of an annealing temperature and too little product is

made.• The annealing temperature should be as close to 50°C as

possible.• The design of the primer should be complex enough so that the

likelihood of annealing to sequences other than the chosentarget is low.

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PCR• Third, the temperature is raised to 72˚C,

which is the optimal temperature for Taqpolymerase to synthesize DNA extended fromthe annealed primers.

Extend Primers

PCR

• Elongation temperature = The temperatureat which the dNTPs bind to the DNA strands.

• Normally between 70-72°C.• Taq polymerase activity is optimal around

70°C• 1 minute is sufficient for amplification of 2kb

sequences• 3 minutes is good for 3kb and above.

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Overview

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PCR• It is only when we reach the end of the third

cycle do we get DNA of the desired length.

PCR• It is only when we reach the end of the third cycle do

we get DNA of the desired length.

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PCR• It is only when we reach the end of the third cycle do

we get DNA of the desired length.

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PCR• I Initial Denaturation

– 94°C for 5 minutes (This insures that the DNA isthoroughly denatured.)

• II Amplification (The PCR thermocycler will repeat thesethree steps in order 30 times.)– 94°C for 1 minute (Denaturation of target DNA)– 50°C for 1 minute (Annealing of primer to template DNA)– 72°C for 1 minute (Elongation of primer to produce new

DNA strand)• III Additional Elongation (This step insures all DNA

strands are full length)– 72°C for 5 minutes

• After step III, the PCR machine has been programmed todrop the temperature of the heating block to 4°C. Yoursamples will be very stable if left at 4°C.

PCR

• The maximum number of cycles is 30.• The reaction reaches a plateau.

• Degradation of the reactants (dNTPs, Taqpolymerase)

• Reaction depletion (primers, dNTPs)• End product inhibition - The concentration of

the product inhibits the formation of newproducts.

• Competition for reactants by other products.• Competition for primer binding.

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WolffiacDNAInsert

What size is the Wolffia cDNA insert?

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Grow an overnight (ON) culture of thedesired bacteria in 2 ml of LB mediumcontaining the appropriate antibiotic forplasmid selection. Incubate the cultures at37°C with vigorous shaking.

Grow the bacteria

Protocol

• Dilute aliquots of your overnight cultureof your plasmid DNA samples 40-fold.Label four fresh microfuge tubes andcombine 200 µl of H2O with 5 µl ofovernight culture. Mix each sample tubeby tapping the side

• The mini-prep DNA is too concentrated and mustbe diluted in order for the PCR reaction to work.

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Prepare the PCR Reaction.Final conc. (or amt.) Stock 1X Rx1X Taq Buffer 10X

0.2 mM dNTP 10 mM Mix PCR Bead1 Unit Taq Polymerase 1 U/µlsterile ddH2O ----- 18.0 µl1 pM/µl M13 For Primer 10 pM/µl 2.5 µl1 pM/µl M13 Rev Primer 10 pM/µl 2.5 µl4 ng Plasmid DNA ~2 ng/µl (1-50 dil) 2.0 µl

=25 µl =

Prepare the 5 Rxn. PCR Mix for 4 Samples:

1 Rxn. 5 Rxns. PCR MixSterile ddH2O 18.0 µl 90.0 µlFor Primer (10 pmole/µl) 2.5 µl 12.5 µlRev Primer (10 pmole/µl) 2.5 µl 12.5 µlDiluted culture (~2 ng) 2.0 µl ** (DO NOT ADD DNA!)

=25.0 µl

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Protocol• Obtain a strip of 4 Tubes of “PCR Beads.” Label

each with the plasmid sample that you will add to it.• To each tube containing a PCR Bead add 23 ul of

the “5 Rxn. PCR Mix.• Then add 2 ul of the appropriate diluted culture that

you prepared in Step 1. Mix each by gently tappingthe tube.

Protocol• Add your tubes to the PCR machine.

MAKE SURE THAT YOU HAVELABELED THE TUBES WITH YOURCLONE NAME.