5.2 Reaction Particulars

Reaction conditions

Specific conditions for maximal yields of a given DNA sequence usually have to be worked out . As a starting point, the general reaction mixture (in 0.1 ml) may contain:

The concentration of Mg ions is quite critical, and may have to be determined by testing various concentrations.

Typically, the thermal cycler will be set to denature at 94° for 20 sec, anneal the primers to the DNA at 55° for 20 sec, and allow the polymerase to synthesize DNA at 72° for 30 sec. The machines may heat at about 0.3° per sec and cool at about 1° per sec, so that a complete cycle takes 3.75 min. The automated machine might run this program for 20 to 30 cycles to amplify the desired sequence.

Reference: Ausubel, F. M., et al., Short protocols in molecular biology, 2nd edit., J. Wiley & Sons, N. Y. 1992.

Primer Selection

Select primers (if possible) that do not have repeat stretches of one base, or primers that might fold over on themselves to form internal hairpin structures, or that might hydrogen bond with each other. Primers should be between 20 and 30 nucleotides in length and be of typical G plus C content. If only shorter primers are available (12 to 15 nucleotides) the polymerase reaction temperature will have to be lowered to perhaps 50-60° so that the primers do not dissociate from the template DNA before polymerase can act. Unrelated nucleotide sequences may be added onto the 5' end of primers to provide other functions in the product. Different sequences could be placed at each end. An example might be the addition of an endonuclease restriction site]

 

The DNA polymerase

The PCR method first used DNA polymerase I from E. coli (the Klenow fragment). This enzyme has an ordinary temperature stability, and so fresh enzyme had to be added after each denaturation step. The Taq polymerase was isolated from Thermus aquaticus which is was found in a hot spring in Yellowstone National Park. The microorganism grows optimally at 70-75° and the isolated enzyme catalyzes DNA synthesis optimally at 75-80°. It is quite stable at still higher temperatures and retains about 50% of its activity after one hr at 94°. Thus, the enzyme needs to be added only once at the beginning of the procedure. In contrast to the E. coli enzyme, Taq polymerase has no "editing" function, and may make more errors in synthesizing DNA. Usually this is not a serious problem, but if the product is used to determine the base sequence of a gene, several cloned PCR products should be sequenced to be sure no errors have crept in.

The DNA sample

Often, whole cells may be added to the reaction mixture and the high temperature and alkaline conditions of the denaturation step will lyse the cells and liberate DNA. This procedure works with isolated white blood cells, but not with whole blood. Hematin and other porphyrin breakdown products of hemoglobin were found to inhibit PCR. In some cases, it may be preferable to isolate and quantitate the DNA before using it in PCR. A procedure for plucked hairs is as follows:

The product

Usually the approximate size of the gene being isolated is known or suspected. Gel electrophoresis should be used to verify that PCR yielded predominately one product in the correct size range. If impurities are detected, often the DNA in the band of the correct size may be eluted from the gel and used. The DNA may be precipitated with ethanol, redissolved and used in DNA sequencing reactions (see above reference for details). PCR also may be used to produce mostly single-stranded product by using one primer in 100-fold excess over the other.

Reaction plateau

Amplification usually slows or stops after 20-30 cycles. The reason for this is believed to be that the Taq polymerase cannot keep up with the many strands to be copied after their concentration reaches high levels. Another cause might be that the new strands will anneal with one another before the primers can bind and be extended. If more product is needed, it is simplest to run as many duplicate tubes as required.]