Overview - Applied Biosystems 7900HT User Manual

Chapter 6 Analyzing Real-Time Data

Overview

About
Dissociation
Curve Analysis
Employing the
SYBR Green 1
Dye
Mathematical
Transformations
6-38 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
September 1, 2004 11:39 am, CH_Real-Time.fm
The Applied Biosystems 7900HT Fast Real-Time PCR System supports dissociation
curve analysis of nucleic acids using SYBR
dye chemistry. The objective of dissociation curve analysis is to accurately determine
the melting temperature (T
unknown PCR sample. Typical uses of dissociation curves include detection of
non-specific products and primer concentration optimization.
Dissociation curve analysis on the 7900HT instrument is made possible through the
use of the fluorogenic SYBR Green 1 double-stranded DNA binding dye chemistry
(see page D-3). Dissociation curves are commonly performed following the PCR
stage of a SYBR Green dye experiment to screen for non-specific products. To
generate the data needed to create a curve, the 7900HT instrument performs a
programmed temperature 'ramp' in which it slowly elevates the temperature of the
plate over several minutes. The specific binding characteristic of the SYBR Green 1
Dye permits the 7900HT instrument to monitor the hybridization activity of the
nucleic acids present in the sample. During the run, the instrument records the
decrease in SYBR Green fluorescence resulting from the dissociation of dsDNA.
After the run, the SDS software processes the raw fluorescence data from the SYBR
Green 1 Dye to generate a more meaningful representation of the relationship
between spectral change and temperature for the dissociation curve run.
Multicomponenting and Normalization
The first mathematical transformation involves the conversion of the raw data,
expressed in terms of Fluorescent Signal vs. Wavelength, to pure dye components
using the extracted pure dye standards. At the same time, the software determines the
contribution of each dye in the raw data using the multicomponent algorithm.
Afterwards, the software normalizes the data using the component of the passive
reference dye as shown below.
R
( )
SYBR
R = --------------------------------------------- -
n
R
( )
PassiveReference
Derivation of Dissociation Curve Data
The SDS software then computes the first derivative of the normalized data (R
each reading taken by the 7900HT instrument during the temperature ramp. The
resulting derivative data (R
temperature (see below).
dR
' n
R = ---------
n
dT
The software plots the negative of the resulting derivative data on graph of -R
versus temperature (T) that visualizes the change in fluorescence at each temperature
interval. The T for the target nucleic acid can be determined from the graph by
m
identifying the maximum for the rate of change (displayed as a peak) for the
appropriate amplification curve.
DRAFT
®
Green 1 double-stranded DNA binding
) of a single target nucleic acid sequence within an
m
´) is the rate of change in fluorescence as a function of
n
) for
n
´
n