In this report, we describe a new method for multiplexed SNP genotyping using the Qbead system. The Qbead system uses spectral signatures or ‘barcodes’ to identify individual microspheres. Just as traditional barcodes use black and white stripes of various positions and widths to track and identify items, the Qbead system uses combinations of different Qdot nanocrystals with various emission wavelengths and intensities to create unique codes for microsphere identification. To our knowledge, this is the first study to demonstrate a practical biological application for nanocrystal-encoded bead technology. Earlier studies used water-dispersible nanocrystals covalently linked to biomolecules to investigate the suitability of Qdot nanocrystals for ultrasensitive labeling of molecules in a biological context (26
). Recently, nanocrystals covalently linked to streptavidin have been used to visualize molecular targets at the subcellular level (30
). In addition, a proof-of-concept study used nanocrystals conjugated to microspheres as a model to detect DNA hybridization of oligonucleotide probes (31
). Our work both confirms and extends the findings of these earlier studies in that we have validated the Qbead system as an accurate, sensitive and robust method for multiplexed genetic analyses. Our study represents a significant advance in that this is the first demonstration of multiplexed genotype analysis of actual patient samples using nanocrystal-encoded bead technology.
We wished to demonstrate a practical working system for real-world genotyping applications rather than modeling individual components of such a system. For this reason, we chose genes from the cytochrome P450 family. This family is particularly relevant to modern drug discovery due to the critical metabolic role played by many of these genes (32
). Furthermore, the cytochrome P450 family is a notoriously difficult gene family due to the high degree of homology among its members that impacts not only multiplexed detection (e.g. because of cross-reactivities) but also the ability to multiplex PCR amplification of targets. In addition, we made use of only authentic genomic samples rather than relying upon clean model oligonucleotides.
The Qbead system offers a number of advantages for highly multiplexed biological and genetic applications. The broad excitation spectra and narrow emission spectra of the nanocrystals make it possible to excite many different nanocrystals as well as the fluorophore of the hybridized target with a single wavelength of light, resulting in emission colors that can be detected simultaneously. Moreover, it is possible to produce nanocrystals that emit light at any desired wavelength with a narrow 20–30 nm FWHM (full-width at half maximum) band width by controlling the mean size and size distribution of nanocrystals during synthesis. Nanocrystals also have superior stability and a reduced photo-bleaching rate as compared with organic fluorophores (29
). One of the key features of nanocrystal-encoded bead technology is the potential encoding capacity that enables the high level of multiplexing necessary for genetic analysis. In theory, N
intensity levels with m
colors will produce Nm
– 1 unique codes. For example, a combination of five colors and six intensity levels theoretically would produce 7776 unique codes. In practice, however, fewer unique codes may be produced due to spectral overlapping, fluorescence intensity variations and signal-to-noise requirements. Nonetheless, a realistic scheme using 5–6 colors with six intensity levels would be expected to yield at least 10 000–40 000 recognizable codes (31
). When compared with other bead-based technologies such as the Luminex platform (33
), the Qbead system provides much more potential upside in terms of multiplexing since thousands more spectral codes are possible using nanocrystals instead of traditional dyes.
Our study demonstrates several key features of the Qbead system that are important for SNP genotyping. First, the Qbead system is highly accurate. In analyzing 86 SNP genotypes from GSK genomic DNA samples and 200 SNP genotypes from Coriell genomic DNA samples, results of the Qbead system were 100% concordant with those of direct DNA sequencing. In fact, the call rate of the Qbead system was higher than that of DNA sequencing. Whereas sequencing discerned 280 of the 286 genotypes tested (97.9% call rate), the Qbead system identified 286 of the 286 SNP genotypes (100% call rate). One possible explanation for the relatively low call rate by DNA sequencing is that the PCR products may not have been sufficiently clean for DNA sequencing, despite purification of products with the QIAquick™ PCR purification kit. Nonetheless, using the same samples without QIAquick™ PCR purification, the Qbead system was able to discern SNP genotypes for all samples tested. In determining the 940 SNP genotypes of the GSK genomic DNA samples, results of the Qbead system were 100% concordant between operators and 100% concordant with results of the GSK 5′ nuclease TaqMan® in-house assays.
Secondly, the Qbead system is able to function accurately with very low quantities of DNA. In the experiments shown here, we used 1 ng of genomic DNA for 10-plex PCR, and the resulting PCR products were used for multiplex SNP genotype determinations (0.1 ng per SNP genotype). We have since found that we obtain comparable results using 0.2 ng of genomic DNA for 10 SNP genotype determinations (0.02 ng per SNP genotype). The amount of genomic DNA used for each SNP determination by the Qbead system is substantially less than that required by non-multiplexed assays or multiplexed assays without direct multiplexed amplification of genomic DNA.
A third feature of the Qbead system is its flexibility. It is relatively straightforward to customize the Qbead system by selecting allele-specific oligonucleotides according to the SNPs of interest, and different multiplexed assays can be created simply by mixing various combinations of encoded microspheres. In addition, the Qbead system can, in principle, be adapted to other assay chemistries for SNP genotyping such as allele-specific primer extension (ASPE), oligonucleotide ligation assay (OLA), PCR combined with ligase detection reaction (PCR/LDR), single base extension assay and molecular beacon probes. Also, by modifying the types of probes conjugated to microspheres, the Qbead system can easily be adapted to other applications such as analysis of gene expression and protein–protein interactions. The ease with which oligonucleotides and spectral codes can be added or changed renders the Qbead system far more flexible while simultaneously less expensive than methods such as DNA microarrays in which all probes are located on a single platform. Moreover, because each custom-encoded microsphere population can be tested individually and checked for quality control, the Qbead system does not suffer from the chip-to-chip variation problems common with microarrays.
Efficiency is a fourth feature of the Qbead system. Because the PCR amplification of genomic DNA is performed in multiplex, only one amplification step is performed. As compared with systems in which genomic DNA is first amplified in singleplex PCRs and the products then applied to other amplification steps, the multiplex PCR of the Qbead system reduces the amount of not only the genomic DNA required but also the enzymes and reagents needed for PCR amplification. In addition, because the reaction products from the muliplexed PCR amplification are used directly, the Qbead system saves the time and expense associated with enzymatic or column purification of PCR products. The rapid reaction kinetics and reduced hybridization times of the Qbead system also enhance efficiency. As compared with microarrays, suspended bead-based systems such as the Qbead system have the advantage of fast solution-like reaction kinetics instead of the comparatively sluggish kinetics characteristic of two-dimensional chip technologies. This is advantageous from the standpoint of high throughput and, more importantly, potentially critical to the ultimate accuracy of the genotyping systems.
Finally, the Qbead system has high-throughput capability. In the experiments shown here, 10 SNP alleles were determined in multiplexed reactions using combinations of nanocrystals with two emission colors and different intensity levels. To date, more than 110 unique codes have been created and decoded successfully using Qbead technology (manuscript in preparation). The multiplexed PCR amplification and hybridization reactions can be performed in a 96-well plate, and high throughput can be facilitated by adapting the Qbead system to automated sample handling and transfer devices. Also, although data for this study were generated using a BDIS FACScan, assays can now be analyzed with a fluorescence-based imaging and data analysis instrument that has been developed for the Qbead system (35
). Information about the microscope-based detection platform as well as other information about quantum dot technology can be viewed at http://www.qdots.com
In summary, we have developed an accurate and sensitive method for multiplexed SNP genotyping using the Qbead system. Furthermore, we have demonstrated the actual use of the Qbead system for SNP genotyping of the cytochrome P450 family and produced extremely accurate parallel results in spite of the known difficulties often encountered with this gene family. We have done so while simultaneously reducing the amount of genomic DNA typically required for SNP genotype analysis. Bead-based genetic analysis in general, and Qbead microsphere-based analysis in particular, promise flexibility, efficiency and easy automation in a robust and cost-effective platform for a number of biological and genetic applications.