Molecular Inversion Probe Technology for SNP Genotyping
BackgroundThe availability of large collections of single nucleotide polymorphisms (SNPs), along with the recent large-scale linkage disequilibrium mapping efforts, have brought the promise of whole genome association studies to the forefront of current thinking in human genetics.
ParAllele has developed a novel technology based on the concept of Molecular Inversion Probes that enables up to 20,000 SNPs to be scored in a single assay. This unprecedented level of multiplexing is made possible through exquisite enzymological specificity using a unimolecular interaction that is insensitive to cross-reactivity amongst multiple probe molecules. The technology has been demonstrated to exhibit high accuracy while enabling a high rate of conversion of individual SNPs into working multiplexed assays. For more details about Molecular Inversion Probes, please visit our publications page.
Molecular Inversion Probes are so named because the oligonucleotide probe central to the process undergoes a unimolecular rearrangement from a molecule that cannot be amplified (step 1), into a molecule that can be amplified (step 6). This rearrangement is mediated by hybridization to genomic DNA (step 2) and an enzymatic "gap fill" process that occurs in an allele-specific manner (step 3). The resulting circularized probe can be separated from cross-reacted or unreacted probes by a simple exonuclease reaction (step 4). Figure 1 shows these steps.
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| Figure 1: Schematic of the Molecular Inversion Probe |
SNP Genotyping Using Molecular Inversion ProbesThe SNP genotyping process using molecular inversion probes is outlined diagrammatically in Figure 2a below.
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| Figure 2a: 10,000 multiplex MIP assay detected on Tag Microarray |
Molecular Inversion probe detection is possible using multiple different detection platforms. Four-color data from a microarray platform is shown below.
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| Figure 2b |
Applications of Molecular Inversion ProbesMolecular Inversion Probe technology is invaluable as a high-throughput SNP genotyping method for both targeted and whole genome SNP analysis projects as well as allele quantitation.
Quality Control and Performance MetricsThe MIP assay has been extensively validated over the past two years as part of the International HapMap project. Every step of the genotyping process is rigorously quality controlled by designated tags built-in to each assay, leading to high-quality unambiguous data, an example of which is shown in Figure 3.
For more details on the performance metrics as well as use of the Molecular Inversion Probe technology, please visit Products and Services.
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Figure 3: The ParAllele basecaller and browser. This view shows a single marker in a 6000plex assay called in 100 patients. Three clusters are clearly visible, distinguishing the homozygotes and heterozygotes. The software can automatically call the genotypes as well as provide a rich environment for browsing and troubleshooting the data |
The Power of Molecular Inversion Probe TechnologyMolecular Inversion Probes represent one of the most scalable methods to perform targeted whole-genome association studies. The technology has a number of advantages over competing methods, including the following:
- No Pre-PCR
No PCR amplification is required at the point of mutation detection, obviating an enormous amount of labor and expense.
- Multiplexing
The unimolecular design of this assay affords a unique ability to multiplex without backgrounds from cross-reactions among probes. The intermolecular interactions between probes are erased by exonuclease treatment, which does not affect circularized probes. PCR is applied after mutation detection and when each molecular inversion probe has been converted to a standard length with similar sequence composition and common primers. These features enable the high degree of multiplexing of this method.
- Scalability
Genotyping up to 10,000 SNPs with this method currently requires a single assay and a single microarray. A modest infrastructure is therefore needed to use this approach.
- Conversion Rate
Several levels of intrinsic specificity are built into this assay: the constraint of dual-recognition sequences within a single probe allowing only local interaction with DNA, error checking activity of the dual-gap fill enzymes, and the tag sequences chosen for high hybridization specificity eliminating cross-talk at the detection level. It is the product of all of these individual specificities that allows this assay's unprecedented level of multiplexing at the genomic level. The result is that a large fraction of all SNPs can be converted to working multiplexed assays in a single pass.
- Robust Genotype Calling
Uniformity of signal and robust algorithms allows the simple, highly accurate calling of genotypes.
- Integrated Quality Control and Tracking
A very powerful and unique aspect of this approach is the ability to have an ongoing check on the signal to noise through the monitoring of the unexpected allele channels and built-in quality controls.
- Single Molecular Probe
Molecular inversion requires a single probe per marker so that any damage or loss of performance of that probe will affect both alleles equally and not lead to spurious genotypes as is the case with allele-specific probes.
Taken together, these advantages amount to an enabling advances in genotyping technology that allow the power of large-scale SNP analysis to be realized.
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