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Introduction 1-13

Amplicon Independent Amplification (Including Primer-Dimer)

Introduction

This section discusses the use of dissociation curves to detect

nonspecific amplification.

Dissociation Curve

Defined

A dissociation curve is a graph that displays dissociation data from the

amplicons of quantitative PCR runs. Change in fluorescence is plotted

against temperature. The change in fluorescence is due to a dye or

probe interacting with double-stranded DNA.

Using Dissociation

Curves

General Information

The GeneAmp

5700 Sequence Detection System (SDS), the

ABI P

RISM

7900HT SDS,

ABI PRISM

7700 SDS, and the

ABI PRISM

7000 SDS

enable dissociation curves to be run to detect nonspecific

amplification through computer software. Nonspecific amplification,

including primer-dimers, may affect the quality of amplification data.

The dissociation curves above show typical primer-dimer formation.

The specific product is shown with a melting temperature (T

) of

80.5 °C, while the primer-dimer has a characteristically lower T

75 °C.

Primer-dimer will be most prevalent in No Template Control (NTC) wells

and sample wells containing low concentrations of template.

Temperature (°C)

Dissociation

curve of

specific primer

Dissociation

curve of a

primer dimer

1 2 ... 14 15 16 17 18 19 20 21 22 23 24 ... 55 56

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Master GREEN 200 Manual de usuario Pagina 19