The Polymerase Chain Reaction

The Polymerase Chain Reaction (PCR) provides an extremely sensitive means of amplifying small quantities of DNA. The development of this technique resulted in an explosion of new techniques in molecular biology (and a Nobel Prize for Kary Mullins in 1993) as more and more applications of the method were published. The technique was made possible by the discovery of Taq polymerase, the DNA polymerase that is used by the bacterium Thermus auquaticus that was discovered in hot springs. This DNA polymerase is stable at the high temperatures need to perform the amplification, whereas other DNA polymerases become denatured.
Since this technique involves amplification of DNA, the most obvious application of the method is in the detection of minuscule amounts of specific DNAs. This important in the detection of low level bacterial infections or rapid changes in transcription at the single cell level, as well as the detection of a specific individual's DNA in forensic science (like in the O.J. trial). It can also be used in DNA sequencing, screening for genetic disorders, site specific mutation of DNA, or cloning or subcloning of cDNAs.

The Reaction


PCR, like DNA sequencing, is based on the DNA polymerization reaction. A primer and dNTPs are added along with a DNA template and the DNA polymerase (in this case, Taq). The main difference with PCR is that, in addition to using a primer that sits on the 5' end of the gene and makes a new strand in that direction, a primer is made to the opposite strand to go in the other direction. The original template is melted (at 94^oC), the primers anneal (@ 45-55^oC) and the polymerase makes two new strands (@ 72^oC), doubling the amount of DNA present. This provides 2 new templates for the next cycle. The DNA is again melted, primers anneal, and the Taq makes 4 new strands:
Click here to download a short movie of the PCR reaction.
OR . . .
Click here to download a static figure of the PCR reaction (if the movie doesn't work with your web browser).


Figure 1
Notice:

• Every cycle results in a doubling of the number of strands DNA present.
• After the first few cycles, most of the product DNA strands made are the same length as the distance between the primers.

The result is a dramatic amplification of a the DNA that exists between the primers. These cycles are repeated 20 to 40 times, each cycle providing 2 new templates for the next cycle. The amount of amplification is 2 raised to the n power; n represents the number of cycles that are performed. After 20 cycles, this would give approximately 1 million fold amplification. After 40 cycles the amplification would be 1 X 10¹². The reaction is performed in a thermocycler, which is programmable heating block that will cycle between melting, annealing and polymerization temperatures.

Limitations/Difficulties


While a very powerful technique, PCR can also be very tricky. The polymerase reaction is very sensitive to the levels of divalent cations (especially Mg²⁺) and nucleotides, and the conditions for each particular application must be worked out. Primer design is extremely important for effective amplification. The primers for the reaction must be very specific for the template to be amplified. Cross reactivity with non-target DNA sequences results in non-specific amplification of DNA. Also, the primers must not be capable of annealing to themselves or each other, as this will result in the very efficient amplification of short nonsense DNAs. The reaction is limited in the size of the DNAs to be amplified (i.e., the distance apart that the primers can be placed). The most efficient amplification is in the 300 - 1000 bp range, however amplification of products up to 4 Kb has been reported. Also, Taq polymerase has been reported to make frequent mismatch mistakes when incorperating new bases into a strand.
The most important consideration in PCR is contamination. If the sample that is being tested has even the smallest contamination with DNA from the target, the reaction could amplify this DNA and report a falsely positive identification. For example, if a technician in a crime lab set up a test reaction (with blood from the crime scene) after setting up a positive control reaction (with blood from the suspect) cross contamination between the samples could result in an erroneous incrimination, even if the technician changed pipette tips between samples. A few blood cells could volitilize in the pipette, stick to the plastic of the pipette, and then get ejected into the test sample. The powerful amplification of PCR may be able to detect this cross contamination of samples. Modern labs take account of this fact and devote tremendous effort to avoiding this problem.

Procedure:


Primers
As stated above, the selection of primers is very important to the efficiency of the reaction. Usually the primers are custom synthesized based on the sequence of the DNA that is being amplified. In your reactions, two primers would have to be made for each of the inserts and the primers that you use would be based on which insert you have in your plasmid. However, since all of the inserts are in the pBluescript plasmid, we can take advantage of the vector sequences that are common to all of the plasmids. For this reason you will all be using the same primers; one primer from the vector sequences at the 5' end of your insert and one from vector sequences at the 3' end of your insert. When the products are run on agarose gel they should each be the size of insert that you predicted from your restriction mapping.

Dilutions
This lab involves doing a serial dilution (see the lambda phage lab) of your isolated plasmid (from lab # 4), setting up 2 PCR reactions with this diluted template, running the PCR in the thermocycler, and then sizing the resultant fragments by agarose gel electrophoresis. This whole procedure should take about 6 hrs., so it will be done over two weeks. The first week, you will do the serial dilutions, set up the reactions and put the reactions in the thermocycler. The next week you will run the reactions out on an agarose gel.
Note: If your insert is greater than 2.0 Kb, tell me and I will give you a different plasmid because this is too large for efficient amplification.

Setting up the Reactions
1. Take three tubes and mark them 10^-2, 10^-4 and 10^-6. Put 199 ul of H₂O in the 10^-2 tube and 990 ul the 10^-4 and 10^-6 tubes. Put 2 ul of your original plasmid in the 10-2 tube and mix well. Put 10 ul of the solution from the 10^-2 tube in the 10^-4 tube and mix well. Put 10 ul of the solution from the 10^-4 tube in the 10^-6 tube and mix well. Each of these is a 100 fold dilution.
2. Label 2 PCR tubes (the 0.65 ml tubes provided) 10^-4 and 10^-6.
3. Set up the following reactions in each of the tubes (I will be adding the buffer mix and Taq polymerase):

       34.5 ul   H2O
        1.0 ul   diluted DNA solution
       14.0 ul   Buffer mix (buffer, primers, MgCl2, and dNTPs)
        0.5 ul   Taq polymerase

       50.0 ul   Total

4. Place these three tubes in the thermocycler.

After you have run the agarose gel, note the extent to which you diluted the DNA in the reactions. Assuming that your original plasmid stock was approximately 1 ug / ul (which is probably a good estimate for most of you), the 10^-6 dilution had a concentration of 1 femtogram / ul. This means that your 10^-6 PCR reaction only has about 1 picogram of DNA template. (Imagine - one picogram!) For this reason, on the agarose gel you should only see the amplified fragment and not the DNA from the original template.