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Introduction 1-9

Preventing Contamination and Nonspecific Amplification

Overview

The DNA amplification capability of the PCR process makes special

laboratory practices necessary. Potential contamination can be

introduced by samples with high DNA concentrations, from the DNA

Template Controls, or from PCR carryover contamination. In addition,

due to the nonspecific nature of SYBR Green I Detection, any double

stranded DNA will be detected. Therefore, it is recommended to check

for nonspecific product formation by dissociation curve or gel analysis.

For more information on the polymerase chain reaction, refer to Kwok

and Higuchi, 1989. For more information on the prevention of

unintended products, refer to Mullis and Faloona, 1987.

Hot Start PCR

To improve PCR specificity and sensitivity by controlling mispriming

events, the Hot Start technique was introduced (Faloona et al., 1990).

Hot Start PCR is a simple modification of the original PCR process in

which the amplification reaction is started at an elevated temperature.

This was initially performed manually, by adding an essential

component of the reaction to the reaction mixture only after that mixture

had been heated to an elevated temperature. However, this approach

was often cumbersome and time consuming, especially when using

large numbers of samples.

AmpliTaq Gold

DNA Polymerase

Applied Biosystems introduced AmpliTaq Gold

DNA Polymerase to

perform an automated, convenient, and efficient Hot Start. AmpliTaq

Gold DNA Polymerase is a chemically modified form of AmpliTaq

DNA

Polymerase. The modification renders the enzyme inactive.

Upon thermal activation, the modifier is released, resulting in active

enzyme. The high-temperature incubation step required for activation

ensures that active enzyme is generated only at temperatures where

the DNA is fully denatured.

When AmpliTaq Gold DNA Polymerase is added to the reaction mixture

at room temperature, the inactive enzyme is not capable of primer

extension. Any low-stringency mispriming events that may have

occurred will not be enzymatically extended and subsequently

amplified.

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Master GREEN 200 Manual de usuario Pagina 15