Detection of Single Nucleotide
Polymorphism (SNP) by flowmetrix multiplex hybridization assay using fluorescent
microbeads as solid support. Experience with the LabMAP (Luminex) system
Mads V. Hollegaard1,2,
Bent Nørgaard-Pedersen1, Poul Thorsen2 and
1Dept. of Clinical
Biochemistry, Statens Serum Institut, Copenhagen, and 2NANEA, Aarhus
University, Aarhus
The
Luminex Multi-Analyte Profiling
technology (LabMAP) (Austin, Texas, USA, www.luminexcorp.com) is based on the
use of polystyrene microspheres as solid support for multiplex assays. By
mixing two flourochromes at different ratios a set of 100 different colourcoded
beads has been developed. By varying the classification laser emission ratios
the Luminex 100 system classifies each microsphere according to its predefined
flouroscent emission ratio. This, in theory, allows the user to do 100
different analyses at one time. To be able to quantify the extent of the
biomolecular interaction between the sample and the microsphere a third
flourochrome is coupled to a reporter molecule and excited by a reporter laser.
The high multiplex capacity combined with a relatively short read-out time and
low cost makes this a interesting high throughput technology.
The
association between genetic polymorphisms and various diseases has lately
gained a great deal of interest. In our laboratory we are especially interested
in associations between SNP’s in gene regulatory regions for pro-inflammatory
cytokines and certain diseases . For the purpose of running large case-control
studies we have applied different SNP detection methods to the LabMAP system.
Until now our preferred method has been the multiplex hybridisation assay,
where bead-coupled oligonucleotides complementary to wild type and mutant DNA
regions are hybridized to the relevant biotinylated PCR amplicons. Technical
aspects, strengths and shortcomings will be discussed.