xTAG Technology

The xTAG technology developed by Luminex provides a method for the simultaneous detection of 100 different nucleotide sequences (or other analytes) in a single reaction. An xTAG microsphere is a labeled 5.6 micron diameter polystyrene sphere. The labeling is done using two spectrally distinct fluorochromes. The beads are pre-coupled with an xTAG oligonucleotide sequence. The xTAG oligonucleotide sequences, also known as anti-tag sequences, are 24 bp long and are complementary to the tags attached to the ASPE primers. Since each fluorochrome has a different intensity, 100 microsphere sets with specific spectral addresses are created for multiplex detection.

xTAG Challenges

Despite the target specificity of the xTAG oligonucleotide sequences, chances are, that some of them may cross-react with custom designed oligos. Also, it is critical that no matter which beads are selected, if they are being multiplexed in one assay, they need to be from the same MFI group. Software that assists researchers in choosing the best TAG for each designed ASPE oligo can automate this process.

xTAG Mechanism

xTAG microspheres are interrogated individually in a fluid stream as they pass by two separate lasers in the Luminex 100, Luminex 200 or Bio-Plex 200 analyzer. A 635-nm 10-mW red diode laser excites the two fluorochromes contained within the microspheres and a 532-nm, 13-mW yttrium aluminum garnet (YAG) laser excites the reporter fluorochrome (R-phycoerythrin, Alexa 532, or Cy3) bound to the microsphere surface. High-speed digital signal processing classifies the microspheres based on its spectral address and quantifies the reaction on the surface. Thousands of microspheres are interrogated per second by the signal detection platform, resulting in an analysis system capable of analyzing and reporting up to 100 different reactions in a single reaction vessel in just a few seconds per sample.

xTAG Chemistry
1. Multiplex PCR: The region of interest is amplified using Multiplex PCR. Design multiplex PCR primers using PrimerPlex.
2. Exo/SAP treatment of PCR Product: To remove the excess primers and oligonucleotide, the PCR reaction is treated.
3. Multiplex ASPE: In this step, the PCR reaction is now subjected to a primer extension assay. It should be specific to the allele that is being analyzed (Allele Specific Primer Extension). The 5’ end of the ASPE primer is attached to an xTAG universal tag sequence. ASPE primer design using PrimerPlex.
4. Universal Array Sorting: The hybridization of the 5' universal tag to the complementary anti-tag sequence takes place. The anti-tag sequence is coupled to a particular xTAG bead set.
5. Detection: After hybridization, the xTAG beads are read by the analyzer. Then the data analysis software analyzes the results. Applications 1. SNP genotyping 2. Genetic disease screening 3. Gene expression profiling 4. Microbial detection