Laboratory #7 PCR PCR

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Dale Bond
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1 1 Laboratory #7 Polymerase chain reaction () 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.

First, the reaction mixture is heated to 95 C to denature (separate

2 2 A single cycle of consists of three steps Denature DNA anneal primers extend primers. First, the reaction mixture is heated to 95 C to denature (separate the strands of the double helix) the target DNA. Problem - DNA polymerase breaks down at high temperatures. Denature DNA

3 Thermus aquaticus - bacteria which naturally live in hot springs (50 to 80ºC). The DNA polymerase used by Thermus aquaticus is stable at high temperatures. Taq polymerase is used in the reaction.

3 3 Thermus aquaticus - bacteria which naturally live in hot springs (50 to 80ºC). The DNA polymerase used by Thermus aquaticus is stable at high temperatures. Taq polymerase is used in the reaction. The reaction was developed by Kary Mullis in Kary Mullis won the Nobel prize in chemistry for this discovery 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 products. Hot start Taq polymerase Reversible inhibition of Taq polymerase by an antibody prevents Taq activity at room temperature. A five minute denaturation of the antibody at 94 C eliminates the antibody and initiates Taq activity.

4 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.

4 4 Second, the temperature is lowered (generally to around C), to allow the primers to anneal (bind) to the denatured strands of target DNA. Anneal Primers Primers are necessary in order for DNA replication to begin T m (Annealing Temperature) =Temperature at which the primers stick to the correct sequence on the DNA strands. T m = 4(G+C) + 2(A+T) Notice that the annealing temperature increases with length and G+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 chosen target is low.

5 Third, the temperature is raised to 72 C, which is the optimal temperature for Taq polymerase to synthesize DNA extended from the annealed primers.

5 5 Third, the temperature is raised to 72 C, which is the optimal temperature for Taq polymerase to synthesize DNA extended from the annealed primers. Extend Primers Elongation temperature = The temperature at which the dntps bind to the DNA strands. Normally between 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.

6 Overview 6

7 7 It is only when we reach the end of the third cycle do we get DNA of the desired length. It is only when we reach the end of the third cycle do we get DNA of the desired length.

8 8 It is only when we reach the end of the third cycle do we get DNA of the desired length.

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12 12 I Initial Denaturation 94 C for 5 minutes (This insures that the DNA is thoroughly denatured.) II Amplification (The thermocycler will repeat these three 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 machine has been programmed to drop the temperature of the heating block to 4 C. Your samples will be very stable if left at 4 C. The maximum number of cycles is 30. The reaction reaches a plateau. Degradation of the reactants (dntps, Taq polymerase) Reaction depletion (primers, dntps) End product inhibition - The concentration of the product inhibits the formation of new products. Competition for reactants by other products. Competition for primer binding.

13 13 What size is the Wolffia cdna insert? Wolffia cdna Insert

14 Grow the bacteria Grow an overnight (ON) culture of the desired bacteria in 2 ml of LB medium containing the appropriate antibiotic for plasmid selection.

14 14 Grow the bacteria Grow an overnight (ON) culture of the desired bacteria in 2 ml of LB medium containing the appropriate antibiotic for plasmid selection. Incubate the cultures at 37 C with vigorous shaking. Protocol Dilute aliquots of your overnight culture of your plasmid DNA samples 40-fold. Label four fresh microfuge tubes and combine 200 µl of H 2 O with 5 µl of overnight culture. Mix each sample tube by tapping the side The mini-prep DNA is too concentrated and must be diluted in order for the reaction to work.

15 15 Prepare the Reaction. Final conc. (or amt.) Stock 1X Rx 1X Taq Buffer 10X 0.2 mm dntp 10 mm Mix Bead 1 Unit Taq Polymerase 1 U/µl sterile ddh 2 O µl 1 pm/µl M13 For Primer 10 pm/µl 2.5 µl 1 pm/µl M13 Rev Primer 10 pm/µl 2.5 µl 4 ng Plasmid DNA ~2 ng/µl (1-50 dil) 2.0 µl =25 µl = Prepare the 5 Rxn. Mix for 4 Samples: 1 Rxn. 5 Rxns. Mix Sterile ddh 2 O 18.0 µl 90.0 µl For Primer (10 pmole/µl) 2.5 µl 12.5 µl Rev Primer (10 pmole/µl) 2.5 µl 12.5 µl Diluted culture (~2 ng) 2.0 µl ** (DO NOT ADD DNA!) =25.0 µl

16 Protocol Obtain a strip of 4 Tubes of Beads. Label each with the plasmid sample that you will add to it. To each tube containing a Bead add 23 ul of the 5 Rxn. Mix.

16 16 Protocol Obtain a strip of 4 Tubes of Beads. Label each with the plasmid sample that you will add to it. To each tube containing a Bead add 23 ul of the 5 Rxn. Mix. Then add 2 ul of the appropriate diluted culture that you prepared in Step 1. Mix each by gently tapping the tube. Protocol Add your tubes to the machine. MAKE SURE THAT YOU HAVE LABELED THE TUBES WITH YOUR CLONE NAME.

Polymerase Chain Reaction

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