DNA-Based Tissue Typing
Rapid · Sensitive · Specific

Introduction

Luminex has developed a multiplexed DNA hybridization system which simultaneously analyzes up to sixty-four unique DNA sequences in the same reaction with point mutant discrimination. Oligonucleotides complementary to each DNA sequence to be analyzed are coupled individually to unique FlowMetrix microsphere sets as the "target" sequences; complementary reporter oligonucleotides for each target are fluorescently labeled. After combining all of the microsphere-bound targets and fluorescent reporters, the multiplexed mixture is hybridized in the presence of amplified sample DNA, such as PCR product, and then analyzed with the FlowMetrix system. The presence or absence of each DNA sequence in the test sample is indicated by competitive inhibition of target-reporter hybridization for each microsphere set. The entire hybridization and analysis can be performed in less than an hour. The PCR resequencing described here employs multiplexed competitive hybridization of sixteen distinct oligonucleotide probes with a 229 bp PCR product derived from exon 2 of the HLA-DQA1 gene. These probes represent allelic DNA sequences that define the eight major alleles of the HLA-DQA1 gene. The results indicate that the FlowMetrix system is a rapid and sensitive method for resequencing analysis of nucleic acids using competitive hybridization in a multiplexed format.

To establish the specificity of the multiplexed analysis, each of the sixteen oligonucleotides was used as a model competitor in the multiplexed assay. The data shown in Figure 1 demonstrate that each of the oligonucleotide sequences preferentially inhibits hybridization of its homologous target microsphere and fluorescent reporter.

Figure 1. Specificity of the Multiplexed Competetitive Hybridization Assay

 

 

 

Materials

Oligonucleotide Conjugation to Microspheres: Each target oligonucleotide was covalently coupled to a unique FlowMetrix microsphere set using a one-step EDC coupling method. Each coupling reaction contained 0.1 mM of amino-substituted oligonucleotide and 1 x 10⁸ microspheres/ml in 0.1 M MES, pH 4.5; EDC was added at 0.5 mg/ml and the reaction was incubated for thirty minutes at room temperature, followed by a second EDC addition and incubation. The coupled microspheres were washed once and stored at 4^oC in the same buffer.

Multiplex assay: Reactions were performed in 50 ul hybridization buffer containing the sixteen oligonucletide target microspheres, forty femtomoles of each fluorescent reporter oligonucleotide, and ten to twenty microliters of unlabeled, double-stranded PCR product or oligonucleotide as competitor. The competitive hybridization assay was performed by mixing competitor DNA (PCR product) with the mixed fluorescent reporters; the mixture was heat denatured at 95° C for five to ten minutes and then hybridized at 55° C for ten to fifteen minutes in hybridization buffer (2.5 M tetramethylammonium chloride + 0.15% SDS + 3 mM EDTA + 75 mM TrisHCl, pH 8.0). The mixture of target set microspheres was added to the hybridization mixture and after an additional ten to fifteen minutes at 55° C, the hybridization mixture was analyzed in fifteen to twenty seconds using the FlowMetrix system.

Results

Specificity of the Multiplexed System: As shown in Figure 1 each oligonucleotide sequence preferentially inhibits hybridization of its homologous target microsphere and fluorescent reporter.

Multiplexed PCR Analysis: Figure 2 depicts the HLA-DQA1 typing of PCR products derived from human genomic DNA from one homozygous and one heterozygous individual. Each DQA1 allele contains only one sequence from each of the four separate regions (25, 34, 41, and 55) of the PCR product; in many cases these alternate sequences represent point mutations. The pattern of alternate sequences present at each of these four regions defines the DQA1 allele. As shown in Figure 2, the homozygous sample (0101/0101) contains the sequences 2501, 3401, 4101 and 5501. The heterozygous sample (0101/0401) contains two alternate sequences at each region except region 25, since sequence 2501 is shared by these two DQA1 alleles. IC1 and IC2 are internal control sequences which are not used for allele designation. In a double-blind study, the system correctly typed 34 of 34 samples containing both homozygous and heterozygous individuals (data not shown).

Figure 2. Multiplexed Analysis of HLA-DQA1 PCR Product

Conclusions

These studies have demonstrated that the FlowMetrix system can perform rapid and accurate resequencing analysis of PCR products in a multiplexed, competitive hybridization assay. The model system used here required the analysis of fourteen allelic DNA sequences to perform genetic tissue typing of the eight major alleles of HLA-DQA1. The FlowMetrix system was able to perform this analysis in a multiplexed format, using a single sample of a single PCR product in a single reaction tube. The entire assay, including hybridization and analysis can be accomplished in less than an hour after PCR amplification of the sample.

Luminex Corporation provides the system including microspheres in a format ready to apply to your specific application. Using this Assay Development Kit, your research group can develop multiplexed assays of interest in a matter of days. For further information in the US call toll free 1-888-219-8020, outside the US call 1-512-219-8020 or email scombs@luminexcorp.com.

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