Single-nucleotide polymorphisms (SNPs) are point mutations, found in every living organism, that occur at greater than one percent frequency in a genomic population. SNPs are biologically important as markers and potential contributors to disease risk factors and drug treatment response variations. They constitute the fundamental data foundation of many heritable disease studies, as well as tumor profiling, bacterial strain differentiation, and myriad other genomic analysis applications.
Knowing, on average, that a SNP occurs once every 1,000 base pairs enables the estimation of approximately 3 million SNPs in the 3 billion base-pair human genome. Most SNPs are binary, meaning that the process of genotyping a single SNP typically consists of determining which one of two nucleotide bases is present at the SNP locus. Methods for making that determination are diverse, and include array-based hybridization, PCR, and sequencing.
Array-based hybridization
Array-based hybridization is carried out using oligonucleotide probes, of known primary sequence, arrayed on a solid support such as a glass slide, silicon chip, or microbead. The immobilized probes are used to interrogate a fragmented DNA sample for complementary sequences. The primary sequence of the sample, including the nucleotide present at the SNP locus, is inferred from the pattern of hybridization.
Though limited-throughput arrays can be constructed in research laboratories, the design and manufacture of commercial arrays is an industrial undertaking, and this method of SNP genotyping ranks low in assay flexibility. Throughput is the strength of commercial arrays, which can rapidly genotype hundreds of thousands of SNPs from tens to hundreds of individuals.
In addition to a genome-wide human SNP array with probes for the detection of nearly one million SNPs, Thermo Fisher offers a variety of additional genotyping arrays covering biologically important SNPs found in humans, research animals, agricultural crops and livestock, and other organisms. Arrays from Thermo Fisher are prepared using a photolithographic process to create probe arrays on microplates or cartridges. High-capacity arrays from Illumina cover numerous species and areas of research focus. Illumina’s arrays are composed of silica microbeads bearing oligonucleotides. The microbeads self assemble on planar silica slides or fiber optic bundles to form the array.
PCR-based methods
The dual-probe TaqMan assay is highly sensitive and therefore of short-list consideration when sample size is limiting. In TaqMan SNP genotyping, allele-specific probes labeled with different FRET report dyes assay both SNP alleles simultaneously. Taking advantage of the 5’ nuclease activity of Taq polymerase, amplicon production releases the specific reporter dye for the amplified allele, freeing it from the effect of the quenching dye and enabling fluorescent signal to be emitted and quantified. To improve specificity, a minor groove binder at the 3’ ends of probes stabilizes the complementary association between probes that are a perfect match for their DNA targets. While TaqMan SNP genotyping assay probe design is a specialized endeavor, Thermo Fisher maintains a library containing 17 million SNP assays, and researchers may find among them one or more that fits their needs.
A variety of artful molecular techniques distinguish other PCR-based SNP assays optimized for specific genotyping needs. BHQplus probes, manufactured by LGC Biosearch Technologies, incorporate a duplex-stabilizing chemistry, which makes them suitable for the design of shorter probes and challenging AT-rich targets.
RNA-DNA hybrid primers, a feature of the rhAMP SNP Genotyping System from Integrated DNA Technologies, improve assay precision by requiring a perfect complement to target DNA before allele-specific primer extension can proceed. This mechanism, combined with a novel Taq polymerase, is designed to improve the ability to genotype SNPs embedded in difficult regions.
Of particular interest to researchers on a tight budget, the Melt Analysis of Mismatch Amplification Mutation Assay (Melt-MAMA) is a SNP genotyping method that does not require fluorescent labels or special enzymes. In Melt-MAMA, two different primers are designed corresponding to the SNP alleles. A third, common, opposite-strand primer enables PCR and amplicon production. Unlike other PCR-based approaches, which use specialized molecular techniques to stabilize complementary oligonucleotide associations, the Melt-MAMA approach deliberately destabilizes the primer-template complex by adding a mismatch near the 3’ end of the primer. Therefore, two non-complementary base pairs exist in the non-matching complex, making the matching primer far more stable by comparison and minimizing amplification of the non-complementary primer. At the PCR endpoint, amplicons generated from the allele-complementary primer greatly outnumber amplicons generated from the non-complementary primer. A GC clamp designed into one of the primers enables correct structural assignment of the prevailing amplicon by temperature-association (melt) curve analysis.
Sequencing
In cases when research study requirements include genotyping of a sample type or SNP category for which no suitable off-the-shelf assays exist, and it doesn’t make sense to develop and validate a new assay, targeted Sanger sequencing, which can provide conclusive allele identification data, may become an attractive option. Genomic services provider GENEWIZ describes their SNP Genotyping service as a PCR and Sanger sequencing based solution, and suggests that the service is appropriate for SNP analysis and detection as well as comparing results obtained from NGS- or microarray-based approaches.
Whole-genome sequencing (WGS) is an efficient way to produce data for SNP discovery. For SNP genotyping, though, WGS may be overkill, both in terms of cost and the amount of data generated. Budget allowing, an exception might be made in the case of highly valuable samples, such as rare tissue samples with rich, high-quality auxiliary information attached, such as clinical histories relating to a research study. In that case, WGS can be used to determine the genotypes of immediately relevant SNPs, while the same data can also be used to carry out an extended genome-wide association study.
Next-generation sequencing (NGS) panels, available from NGS instrumentation manufacturers as well as many other commercial suppliers, enable targeted sequence analysis of a defined set of genes. Analysis of the sequence data produced by these panels will yield SNP genotypes along with the other data for which the panel was designed.
How to Choose
One can easily make some initial decisions about which SNP genotyping method to choose based on the scale of the research project. For instance, a TaqMan assay approach will not meet the needs of a project requiring rapid genotyping of hundreds of thousands of SNPs, and a high-throughput commercial array would be a poor match for a project with a weeks-long average time between sample acquisitions.
A second consideration is whether the needs of the study can be met with an existing, validated, commercial solution that matches study requirements and can be delivered on short notice. This would enable research to get underway sooner and at lower cost. If no ready-made solution is available, a variety of customization options can be explored, even as add-ons to the high-throughput commercial arrays. Manufacturers of solutions based on PCR-based methods offer on-line access to probe-design software and other tools that researchers can use to create and order purpose-made SNP genotyping kits.
Last, but not least, is the consideration of cost. Any customization required will require investments of both time and capital resources. Researchers seeking to reduce the disadvantages of limited capital may wish to consider the capabilities of the Melt-MAMA method, which requires no special dyes, enzymes, or equipment, and is now used as an investigative tool to genotype pathogenic strains of Y. pestis in a resource-constrained environment.