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-Amplifying DNA: The Polymerase Chain ReactionOften, DNA samples are in quantities that are too small to work with. Luckily, a technique invented in the 1980s called the Polymerase Chain Reaction (or PCR ), can be used to "amplify" the amounts of DNA in these samples. The PCR machine is actually nothing more than a very precise heating and cooling device. The machine has small slots which hold small tubes containing the DNA sample and other necessary reaction ingredients. These additional ingredients include a generous supply of individual nucleotides (A's, T's C's and G's), short single-stranded DNA molecules called primers and an enzyme called Thermus aquaticus polymerase (Taq polymerase, for short). Taq polymerase is derived from bacteria that live in hot springs and are among the few enzymes that can function at very high temperatures. The cycle begins when the machine heats the tube to a temperature somewhere around 90-95 C, pausing each doublestranded DNA molecule in the original sample to separate into two single stranded pieces. (Recall that the hydrogen bonds that connect the two complementary strands of the DNA double helix are much weaker than the covalent bonds that connect the nucleotides together that make up each chain. The heating causes the hydrogen bonds to be broken, thus unravelling and separating the two strands, while the covalent bonds remain unaffected.) Next, the temperature is lowered slightly, which allows the DNA primers to bind to the separated strands. The primers bind because they are complementary to certain sequences of each DNA strand which "flank" the DNA that is to be replicated in the middle. Once the primers have attached themselves to the single strands, Taq polymerase synthesizes, using the individual nucleotides floating around in the tube, a complementary strand for each original single strand. This completes the first cycle, and doubles the amount of sample DNA present in the tube. In the next cycle, the PCR machine heats and cools as before, causing the new double-stranded DNA molecules to separate, and for new complementary

strands to be synthesized by Taq polymerase.

Every time one cycle is performed, the amount of copies of the desired DNA sequence (located between the two primers) theoretically doubles. After about 30 cycles (which will typically take about 3 hours), enough DNA copies of this DNA sequence will have been made for the application of further biotechnology techniques. The DNA Learning Centre, has developed an online video that gives a very good explanation of the PCR technique.

Isolating A Gene

Introducing A Gene To A Cell

Reference List 1. 2.
L.A. Moran, K.G. Scrimgeour, H.R. Horton, R.S. Ochs and J.D. Rawn, Biochemistry, (New Jersey: Neil Patterson, 1994) 33-24. E.S. Grace, Biotechnology Unzipped: Promises & Realities, (Toronto: Trifolium, 1997) 52-53.

Publication Date: 2000-10-16 Author:Industry Canada - Life Sciences Branch

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