Project
Leader: Dr
Dave Bunyan
Company website: www.twt.com (Third Wave™ Technologies)
Invader® assay
technology:
The Invader® DNA assay enables simultaneous detection of
two different alleles in a bi-allelic system in a non-PCR based
assay. Two oligonucleotide probes (an allele specific primary
probe and an Invader® probe) hybridise in tandem to the target
DNA to form a specific overlapping structure. The 5’-end
of the primary probe contains a 5’-Flap that is non-complementary
to the target DNA and so is unable to hybridise to the target
sequence. The 3’-end of the bound Invader® probe overlaps
the primary probe by a single base this is the site of the allelic
variant or SNP.
A Cleavase® enzyme (Flap endonuclease I) recognises the overlapping structure
and cleaves the 5’-Flap on the primary probe at the base of the overlap
releasing it as a target specific product. If the probe does not hybridise
perfectly at the site of interest no overlapping structure is formed and no
cleavage occurs and the target specific product is not released. The reaction
consists of two primary probes and two invader probes that are specific to
either allele 1 or allele 2 of a bi-allelic system meaning two target specific
products are generated.
The target specific 5’-Flap oligos’s are involved
in a secondary reaction where they act as Invader® probes
on a Fluorescent resonance energy transfer (FRET™) cassette
leading to the formation of an overlapping structure that is
recognised by the Cleavase® enzyme. When the FRET™ cassette
is cleaved a fluorophore is released form a quencher on the FRET™ generating
a fluorescence signal. There are two signal fluorophores attached
to two different FRET™ cassettes (FRET™ 1 and FRET™ 2)
that are spectrally distinct and specific to either allele of
the bi-allelic system. The ratios of the two fluorescent signals
then allows a genotype to be assigned.
The reaction
conditions (63oC) allow cycling of the primary probes and Invader® probes
meaning multiple rounds of primary probe cleavage occurs per
DNA target and
multiple fluorescent
signals are generated per DNA target.
Detection of Factor V Leiden (G1691A), Factor II (G20210A
) & MTHFR
(C677T) variants:
The Invader® assay was evaluated in conjunction with other
SNP detection technologies (Pyrosequencing™ and NanoChip®)
by testing 100 anonymised thrombophilia patients samples for
the Factor V Leiden (G1691A), Factor II (G20210A ) & MTHFR
(C677T) variants that had previously been genotyped using restriction
fragment length polymorphism (PCR based assay). The FVL and FII
results display 100% concordance to the RFLP method and testing
of the MTHFR variant is currently being undertaken.
Evaluation
of the Invader® assay platform
for molecular analysis of the Factor V (G1691A) and Factor
II (G20210A) mutations.
Presentation to the CMGS Spring Meeting in 2004 available for
download here.
Other applications of the Invader® assay platform:
Genetics:
- Factor
V leiden
- Factor
II
- MTHFR
- PLA1/A2
- ApoE
- PAI-1
- Cystic
fibrosis screen
- Connexin
26
- Tay-
sachs
- Rett
syndrome
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Pharmacogenetics:
- CYPP450
2D6
- CYPP450
2D9, 2C19
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Chromosomal
analysis (CA):
- Prenatal
aneuploidy
- Additional CA applications
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