When first developed, multiple cycles of the PCR process were clumsy to use. First, the DNA polymerases available at the time were inactivated at the temperature of the denaturation and fresh aliquot of the enzyme had to be added at each PCR cycle. Second, three water baths at three different temperatures were necessary. In the evolution of the PCR, solving these problems have greatly contributed to the success of the procedure. The first development was the purification of a heat - stable DNA polymerase. This enzyme (Taq polymerase) was isolated from a bacterium Thermus aqaticus in 1988. Thermus aquaticus grows at high temperature in natural hot springs. The second, also in 1988, was the invention of a thermal cycler, which allows the automation of temperature cycling.

All components of a polymerase chain reaction are readily available from commercial suppliers. A PCR test tube should contain:

- DNA polymerase

- reaction buffer with magnesium chloride

- template (a piece of DNA)

- large quantities of the four nucleotides (dNTPs)

- large quantities of the primers

DNA polymerase

DNA polymerases are enzymes which catalyze the synthesis of long polynucleotide chains from monomer deoxynucleoside triphosphates (dNTPs) using one of the oryginal parental strands as a template for the synthesis of a new complementary strands. The synthesis always preceeds in the 5' to 3' direction.

In the PCR thermostable DNA polymerases are used. The most commonly used is Taq polymerase, but alternative heat stable enzymes are available, too.

Reaction buffer

There are several reaction buffer available for PCR. The most common, 10 times concentrated buffer contain:

-100mM Tris-HCl, pH 8.3 (at room temperature)

-500mM KCl

-15mM MgCl

-0.1% (w/v) gelatin

Most suppliers usually provide a 10 x buffer for use with the respective enzyme.

The concentration of MgCl

The magnesium chloride concentration can be varied usually within the range 0.5 - 5.0 mM. Generally, insufficient Mg ions lead to low yields and excess of these ions will result in the accumulation of nonspecific products.

dNTPs (deoxynucleoside triphosphate)

PCR is performed with dNTP concentrations around 200 M. The concentration of dNTPs depends on:

- the MgCl concentration

- the reaction stringency

- the primer concentration

- the length of the amplified products

Taq DNA polymerase has a higher fidelity at lower dNTP concentration.

Some dNTPs analogs can be use in the PCR. Some applications of these modified nucleotides are listed in Table.

Used in dideoxy DNA sequencing reactions


labeled ddNTPs

Used in automated fluorescent DNA sequencers
[- P]dNTPs
Radiolabeling of PCR products
[- S]dATP
Radiolabeling of PCR products
Nonradioactive labeling of PCR products
Nonradioactive labeling of PCR products


The primers used in PCR have between 20 and 30 nucleotides. This length allows use of a reasonably high annealing temperature. The primers should have similar G+C content, minimal secondary structure and low complementarity to each other, particularly in the 3' region. This precaution will reduce the incidence of primer-dimer formation which is an amplification artifact caused when one primer es extended by the polymerase using the other primer or itself as a template, resulting in a short incorrect product.


The sample may be single- or double-stranded DNA or RNA. It is important to ensure that inhibitors of the reaction:

- detergent (some non-ionic detergents are beneficial to PCR)


- traces of phenol

are not present.

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Dominika Borek
Department of Crystallography
Faculty of Chemistry
Adam Mickiewicz University
Grunwaldzka 6
Poznan, Poland