polymerase chain reaction (PCR) Animation

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A sample of chromosomal DNA, also called genomic DNA, can be used as the starting material for the polymerase chain reaction (PCR). With PCR, an investigator can amplify a single copy of a DNA segment into billions of identical copies. The DNA segment of interest, or target DNA, is indicated in red.

In addition to the target DNA, a PCR reaction contains several other ingredients. These include free nucleotides, DNA primers, and the enzyme Taq polymerase. The primers are typically about 20 nucleotides long and are complementary in sequence to the ends of the target DNA. Taq polymerase is derived from hot springs bacteria and can tolerate the intense heat of a PCR reaction.

A PCR reaction lasts several hours and typically consists of 20 to 35 repeating cycles. A cycle begins by heating the reaction mixture to 95 degrees Celsius. The heat denatures the DNA, breaking the hydrogen bonds that hold the strands together.

After denaturing the DNA, the temperature is reduced to around 60 degrees so that the primers can form hydrogen bonds, or anneal, with their complementary sequences in the target DNA. Note that the primers and the target DNA follow base-pairing rules. An adenine (A) pairs with a thymine (T), and a cytosine (C) pairs with a guanine (G).

In the next phase, the temperature is raised to 72 degrees Celsius. Taq polymerase functions optimally at this temperature and begins polymerization, adding nucleotides to the 3' end of each primer attached to a DNA strand. After one complete cycle, there are two double-stranded copies of the target DNA.

The PCR reaction mixture contains many copies of the primers and an abundant supply of nucleotides to perform many addition cycles. After a second cycle, there are four copies of the target DNÁ.

After cycle 3 is finished, there are eight copies of the double-stranded target DNA sequence. Note that only two of the double-stranded copies consist of just the target fragment. The others also include flanking DNA regions.

As the number of cycles increases, the products consist of a greater proportion of fragments with just the target DNA. After four cycles, half of the fragments consist of just target DNA, and half of the fragments also contain flanking DNA.

With each additional cycle, the number of copies of our target sequence doubles. At the end of cycle 25 there are more than 33 million copies of this double stranded target region.