Overview - Applied Biosystems 7900HT User Manual

Chapter 5 Analyzing End-Point Data

Overview

Allelic
Discrimination
on the 7900HT
Instrument
Employing the
5´ Nuclease
Assay for Allelic
Discrimination
5-6 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
September 1, 2004 11:39 am, CH_End-Point.fm
The Applied Biosystems 7900HT Fast Real-Time PCR System supports allelic
discrimination using TaqMan
two variants of a single nucleic acid sequence are detected in a prepared sample.
Allelic discrimination chemistry can be used for single-nucleotide polymorphism
(SNP) detection.
Allelic discrimination on the 7900HT instrument is made possible through the use of
the fluorogenic 5´ nuclease assay (see
probes anneal specifically to complementary sequences between the forward and
reverse primer sites on the template DNA. Then during extension, AmpliTaq Gold
DNA polymerase cleaves the probes hybridized to the matching allele sequence(s)
present in each sample. The cleavage of each matched probe separates the reporter dye
from the quencher dye, which results in increased fluorescence by the reporter. After
thermal cycling, the plate is run on the 7900HT instrument, which reads the
fluorescence generated during the PCR amplification. By quantifying and comparing
the fluorescent signals using the SDS software, it is possible to determine the allelic
content of each sample on the plate.
Mismatches between a probe and target reduce the efficiency of probe hybridization.
Furthermore, AmpliTaq Gold
mismatched probe than to cleave it, releasing the reporter dye. By running the extension
phase of the PCR at the optimal annealing temperature for the probes, the lower melting
temperatures (T ) for mismatched probes minimizes their cleavage and consequently
m
their fluorescent contribution.
between target and probe sequences in TaqMan
et al., 1995; Livak et al., 1999).
Allele
V
X
Probe-target sequence
Match
Allele
F
Y
Probe-target sequence
Match
Figure 5-3 Target and Probe Sequence Interaction
Table 5-1 shows the correlation between fluorescence signals and sequences present
in the sample.
Table 5-1 Signal and Sequence Correlation
A substantial increase in...
VIC ® dye fluorescence only
FAM ™ dye fluorescence only
DRAFT
® probes. Allelic discrimination is the process by which
page D-2). During the PCR, the fluorogenic
® DNA polymerase is more likely to displace the
Figure 5-3 illustrates results matches and mismatches
Q
Probe-target sequence
Mismatch
mismatch (higher T )
m
Q
Probe-target sequence
Mismatch
mismatch (higher T )
m
Indicates...
homozygosity for Allele X.
homozygosity for Allele Y.
® PDARs for AD assays (Livak
Legend
V
VIC
F
FAM
Q
Quencher
AmpliTaq
Gold DNA
Polymerase
GR1556
®