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1-10 Introduction

False Positives

Special laboratory practices are necessary in order to avoid false

positive amplifications (Higuchi, et al., 1989). This is because of the

capability for single DNA molecule amplification provided by the PCR

process (Saiki et al., 1985; Mullis et al., 1987; Saiki et al., 1988).

Because of the enormous amplification possible with PCR, amplicon

carryover can result in sample contamination. Other sources of

contamination could be from samples with high DNA levels or from

positive control templates.

When dUTP replaces dTTP as a dNTP substrate in PCR and the

method described below is used, AmpErase UNG treatment can

prevent the reamplification of carryover PCR products in subsequent

experiments Sninsky and Gelfand, pers. comm.) This method uses

enzymatic and chemical reactions analogous to the

restriction-modification and excision-repair systems of cells to degrade

specifically PCR products from previous PCR amplifications or to

degrade mis-primed, non-specific products produced prior to specific

amplifications, but not degrade native nucleic acid templates.

The method used to make PCR products susceptible to degradation

involves substituting dUTP for dTTP in the PCR mix and treating

subsequent PCR mixes with the enzyme uracil N-glycosylase (UNG,

EC 3.2.2-) prior to amplification (Longo et al., 1990).

The AmpErase UNG provided in this product is a pure, nuclease-free,

26-kDa enzyme encoded by the Escherichia coli uracil N-glycosylase

gene which has been inserted into an E. coli host to direct the

expression of the native form of the enzyme (Higuchi et al., 1989).

Although the protocol and reagents described here are capable of

degrading or eliminating large numbers of carried over PCR products,

we encourage users to continue using the specific devices and

suggestions described in this protocol booklet and in Kwok (1990) and

Higuchi(1989) to minimize cross-contamination from non-dU-containing

PCR products or other samples.

Optional Use of

AmpErase UNG

AmpErase

uracil-N-glycosylase (UNG) treatment can be useful in

preventing the reamplification of carryover PCR products. Although the

SYBR Green PCR Master Mix does not contain UNG, dTTP has been

replaced with dUTP, thus making the SYBR Green Master Mix

compatible with the use of UNG. If PCR carryover contamination is

suspected, UNG should be used to troubleshoot the problem. UNG can

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Master GREEN 200 Manual de usuario Pagina 16