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The enzyme DNA polymerase is needed to manufacture the DNA copies. The PCR procedure involves several high temperature steps so a heat resistant DNA polymerase is required. This came originally from heat resistant bacteria living in hot springs at temperatures up to 90°C. Taq polymerase from Thermus aquaticus is most widely used.

A supply of nucleotides is needed by the polymerase to make the new DNA. These are supplied as the nuceoside triphosphates.

Finally we need a PCR machine to keep changing the temperature (Fig. 23.02). The PCR process requires cycling through several different temperatures. Because of this, PCR machines are sometimes called thermocyclers.

PCR needs primers to start DNA synthesis which means that we must know some DNA sequence in or close to the region of interest.

The requirement for primers means that some knowledge of the sequence of the DNA template is needed. As described in Chapter 24, ever-increasing quantity of genomic DNA sequences is now available. Unknown sequences are dealt with in a variety of ways (for some specialized approaches see below). However, since binding of a primer need not be perfect, related sequences can often be used successfully, especially if longer primers are used.

When Kary Mullis invented PCR in 1987, he used normal DNA polymerase. Since the temperature needed to separate DNA into single strands destroys this enzyme, he had to add a fresh dose of polymerase to each tube every cycle! Luckily, heat resistant DNA polymerase was purified from Thermus aquaticus just a year or two later. Taq polymerase can be added to the reaction mixture at the beginning and survives all of the heating steps. It actually requires a high temperature to manufacture new DNA.

PCR machine See thermocycler

Taq polymerase Heat resistant DNA polymerase from Thermus aquaticus that is used for PCR

thermocycler Machine used to rapidly shift samples between several temperatures in a pre-set order (for PCR)

Thermus aquaticus Thermophilic bacterium found in hot springs and used as a source of thermostable DNA polymerase

Kary Mullis Invents PCR after a Vision

Science, like nothing else among the institutions of mankind, grows like a weed every year. Art is subject to arbitrary fashion, religion is inwardly focused and driven only to sustain itself, law shuttles between freeing us and enslaving us."—Kary Mullis

Kary Mullis won the Nobel Prize in Chemistry in 1993 for developing the polymerase chain reaction (PCR). The PCR is one of modern biology's most useful techniques and has been used in virtually every area of molecular biology and biotechnology. Kary Mullis is one of sciences true eccentrics. In addition to molecular biology he has also contributed to other areas of science.While a doctoral candidate working on bacterial iron transport, he published an article entitled "The Cosmological Significance of Time Reversal" (Nature 218:663 (1968)), which deals with his notion that about half of the mass in the universe is going backward in time.

Kary Mullis invented PCR while working as a scientist for the Cetus Corporation. He conceived the idea while cruising in a Honda Civic on Highway 128 from San Francisco to Mendocino in April 1983. Mullis recalls seeing the polymerase chain reaction as clear as if it were up on a blackboard in his head. In lurid pink and blue. He pulled over and started scribbling. One basic ingredient of the PCR is that it amplifies DNA by constant repetition—rather like the computer programs Mullis was then involved in writing. Kary Mullis was given a $10,000 bonus by Cetus, who at first failed to realize the significance of the discovery. Later they sold the technology to Roche for $300,000,000.

In 1999, Kary Mullis mentioned the computer DNA connection again, "It is interesting that biochemistry developed alongside computers. If computers had not come along at about the same time as the structure of DNA was discovered, there would be no biochemistry. You always needed the computer to process the information. Without it we would have rooms and rooms full of monks writing out the sequences."

FIGURE 23.03 Kary Mullis Sees PCR in a Vision

FIGURE 23.04 Denaturing the Template and Binding the Primers

In the steps of PCR, a very small amount of template DNA is heated to 90°C, which separates the two strands of the double helix. When the temperature is lowered to 50-60°C, the primers can anneal to the ends of the target sequence. Since the primer is present in large excess over the template DNA, essentially all template strands will bind to primers rather than re-annealing to each other.

5' 3'

-Target sequence CGTCAATGGT'-

-ATTCCAGAGC—Target sequence

Heat to 90°c STRANDS SEPARATE

3' 5'

5'

-Target sequence CGTCAATGGT'-

3'

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