Applications of Luminex xMAP technology for rapid, high-throughput multiplexed nucleic acid detection

Abstract

Background: As we enter the post-genome sequencing era and begin to sift through the enormous amount of genetic information now available, the need for technologies that allow rapid, cost-effective, high-throughput detection of specific nucleic acid sequences becomes apparent. Multiplexing technologies, which allow for simultaneous detection of multiple nucleic acid sequences in a single reaction, can greatly reduce the time, cost and labor associated with single reaction detection technologies.

Methods: The Luminex xMAP system is a multiplexed microsphere-based suspension array platform capable of analyzing and reporting up to 100 different reactions in a single reaction vessel. This technology provides a new platform for high-throughput nucleic acid detection and is being utilized with increasing frequency. Here we review specific applications of xMAP technology for nucleic acid detection in the areas of single nucleotide polymorphism (SNP) genotyping, genetic disease screening, gene expression profiling, HLA DNA typing and microbial detection.

Conclusions: These studies demonstrate the speed, efficiency and utility of xMAP technology for simultaneous, rapid, sensitive and specific nucleic acid detection, and its capability to meet the current and future requirements of the molecular laboratory for high-throughput nucleic acid detection.

Fig. 1

Luminex xMAP system and components. The four main components of the xMAP system are shown, clockwise from top-right: biomolecular reactants; fluorescently color-coded microspheres; fluidics and optics; and high-speed digital signal processing. The Luminex 100 analyzer with XY platform and sheath delivery system is pictured in the center.

Fig. 2

Diagram of the microsphere-based direct hybridization assay format. Target DNA is PCR-amplified. One of the primers is biotinylated. The amplified products are denatured, hybridized to allele-specific probe-coupled microsphere sets and labeled for detection with streptavidin-R-phycoerythrin.

Fig. 3

Diagram of the microsphere-based competitive hybridization assay format. Left: In the absence of target DNA, the biotinylated competitor oligonucleotides hybridize to the allele-specific probe-coupled microsphere sets. The hybridized microsphere sets are labeled with streptavidin-R-phycoerythrin, resulting in 100% signal. Right: When target DNA is present, the biotinylated competitor oligonucleotides hybridize to the target DNA instead of the allele-specific probe-coupled microsphere sets. The target DNA/competitor oligonucleotide hybrids are labeled with streptavidin-R-phycoerythrin, resulting in a reduction of signal on the allele-specific probe-coupled microsphere sets.

Fig. 4

Diagram of assay format using solution-based genotyping with microsphere capture. A. Solution-based enzymatic genotyping assay is performed, incorporating the unique capture sequences into the products. B. Address probe-coupled microsphere sets. C. Products are captured onto the microspheres through hybridization of the capture and address sequences, and labeled with streptavidin-R-phycoerythrin.

Fig. 5

Diagram of ASPE, OLA and SBCE procedures used for addressed microsphere capture assays. ASPE: 1. Target DNA is combined with capture sequence-tagged allele specific primers and denatured; 2. Target DNA and primers are annealed in a reaction containing a DNA polymerase and dNTPs (one of which is biotinylated); 3. Primer extension; and 4. Capture sequence-tagged ASPE products. OLA: 1. Target DNA is combined with capture sequence-tagged allele specific probes and denatured; 2. Target DNA and probes are annealed in a reaction containing a DNA ligase and biotinylated reporter probe; 3. Oligonucleotide ligation; and 4. Capture sequence-tagged OLA products. SBCE: 1. Target DNA is combined with a capture sequence-tagged primer (in separate reactions for each allele) and denatured; 2. Target DNA and primer is annealed in a reaction containing a DNA polymerase and a biotinylated ddNTP; 3. Single base primer extension; and 4. Capture sequence-tagged SBCE products are multiplexed for detection.