Medium Throughput SNP Genotyping: 
The Luminex Microsphere Sorter  

Principle of Operation:

The Luminex fluorescent microsphere sorter can be used for low to medium throughput genotyping projects, capable of analyzing anywhere from one SNP in one individual up to 50 SNPs in unlimited numbers of individuals. This device analyzes beads labeled with varying ratios of two fluorescent dyes, and can resolve 100 different dye sets. The fluorescent microspheres used in the SNP assay are coupled to oligonucleotides (the "TAG sequence") such that each particular bead's fluorescent address is associated with a known 20 nucleotide sequence. User-provided oligos, comprised of a SNP specific sequence coupled to the complement of the bead-associated oligo sequence, are used in an allele specific extension reaction with biotinylated nucleotides. These products are combined with selected fluorescent bead-oligo combinations, then incubated with strepavidin-phycoerythrin (SA-PE).  As a result, SNP allele-specific extension products are labeled with PE.   Reaction products are then analyzed on the Luminex instrument, which quantifies the amount of PE signal associated with a particular fluorescent bead address. Each address is linked to a particular oligo which in turn is linked to a particular SNP based on the experimental design. Thus, differences in signal intensity determine which alleles are present in a given input PCR fragment. Additional documentation describing the use of this assay can be found on the Luminex web site, and on our downloads page.

Beginning the Project:

The assay input material is a PCR fragment encompassing the SNP of interest (PCR fragment generation is an associated service carried out in the facility). Specific guidelines for the PCR fragment characteristics used in the assay have not been formulated; we have successfully assayed SNPs in fragments ranging from 150 bps to several kb in length.  Multiple SNPs can also be assayed simultaneously on the same fragment.  One consideration is that the 3' end of the PCR fragment should be far enough away from the SNP such that the allele specific extension product generated later will incorporate enough biotinylated C nucleotide to yield a good signal following incubation with SA-PE. 

Users also need to design the allele specific primer extension (ASPE) primers.   Some general guidelines for Luminex ASPE primer design are:

* ASPE primers should be synthesized for all sequence variants and be from the same DNA strand (per target sequence).
* ASPE primers should be matched for melting temperature at 51-56 °C.
* ASPE primers should extend out to and include the SNP as the 3' nucleotide.
* The primer is synthesized with the TAG sequence incorporated at the 5' end.

Two ASPE primers are needed in order to resolve each possible allele pair. There is a preferred order for selection of fluorescent microspheres used in the assay; not all of the 100 available dye possibilities are equally detectable, so it is recommended to start with the TAG sequences on the most favorable dye sets and proceed from there.  A csv file summarizing this information is viewable here. The table lists the TAG sequence for each bead type, in their recommended order of use.  For example, bead types 33 and 34 are the most preferred for the first SNP, followed by 35 and 36 for the second, and so on. The table shows the exact sequence to be added to the 5' end of your (approximately) 20 base pair allele specific primer.  Please contact us if this is not clear.

Running the samples:

Several options are currently available for the SNP analysis service utilizing Luminex. These range from a full service option, where genomic DNA is delivered in return for data files, to the user participating in instrument operation (at a reduced cost). In the full service option, the researcher provides quantified genomic DNA ready for amplification at a concentration of 20 ng/ul, together with PCR cycling parameters and evidence of parameter efficacy (e.g., a gel photo) from previous experiments. In addition to genomic DNA, the researcher provides both the amplifying and the ASPE oligonucleotides. We will carry out all subsequent steps and provide you with the data files. The most cost effective option involves the user providing the SNP-containing PCR fragments to be assayed. Cleanup of the fragments to remove excess primers and nucleotides is built into the cost of the assay. Multiplex reactions are recommended (see below). User also orders and provides the ASPE oligos at 100 uM concentration. We recommend that the user has positive and negative controls present in each assay.

Other Considerations:

Before scaling up the project we recommend pilot experiments be run with PCR fragments containing previously characterized SNPs, particularly in the case of multiplexed assays. This will allow the user to assess SNP "conversion," that is, how clear cut the allele calls are in this assay. Sometimes one allele of a SNP will amplify disproportionately and obscure the presence of the other allele. Running the assay on a known heterozygote will indicate whether this will be a problem. The amount of input PCR fragment needed to give adequate signal will also be determined empirically at this point. Since much of the expense of the assay is on a per tube basis, the assay is most economical when multiple SNPs are assayed in each tube. Theoretically it is possible to multiplex 50 different SNPs with the 100 different bead addresses. While prices can vary with project parameters, some idea of the relationship between cost/SNP and multiplex level can be seen here (these figures are based on running a 96 well plate, the most cost effective option).

Output:

Output data from the instrument is provided in csv spread sheet format (Excel compatible); recommended values to use for downstream graphing or analysis are the median fluorescent (PE) intensities for each bead type in each sample. Signal intensities correlate with the presence of the different alleles. Our primary responsibility will be to make sure the assay is performed correctly and that the instrument is functioning within specifications. Further analyses and conclusions are the responsibility of the researcher.

Other Luminex Applications:

In addition to genotyping, multiplexed bead technology serves as the basis for numerous other assays involving simultaneous detection of different proteins and/or certain post-translational modifications of these proteins.  The UC Davis Clinical Proteomics facility can provide information and service for some of these applications.  Check out their web site for more details and contact information.