How TaqMan SNP Genotyping Assays Work

What are SNPs?

Single nucleotide polymorphisms (SNPs) are the most common type of genetic variation in humans. There are more than 10 million SNPs in the human genome, and they underlie traits such as height, response to drugs, and risk of developing diseases. Some diseases, such as sickle cell, stem from a single mutation. Others, including most neurodegenerative diseases, are much more genetically complex. Genotyping can help us understand the mechanisms of disease, predict an individual's risk of developing certain diseases, and even help to design personalized therapies.

Applied Biosystems TaqMan SNP genotyping assays have been used to identify variants associated with blood disorders [1] and many other diseases. We know, for instance, that SNPs in the gene encoding the cholesterol carrier Apolipoprotein E (ApoE) influence an individual’s lifetime risk of developing Alzheimer’s disease. Recently, researchers used TaqMan SNP genotyping assays for accurate high-throughput ApoE genotyping, evaluating almost 1,200 human DNA samples [2].

TaqMan assays can be used to genotype transgenic mice [3], helping to create pure lines in animals and reduce variability in experimental results. The assays have also been applied to evaluate the origin of human and animal parasites which is valuable for medicine and agriculture [4].

These studies and many others demonstrate the speed, specificity, sensitivity, ease of use, and reproducibility of TaqMan assays, as well as their ability to detect sequences using tiny concentrations of DNA, in the nanogram range. TaqMan assays are an ideal and cost-effective method for investigating SNPs in large populations.

How do TaqMan SNP Genotyping Assays work?

Like all TaqMan Assays, TaqMan SNP genotyping assays require a double-stranded DNA template, the Taq polymerase enzyme, and two primers—forward and reverse—which are specific to the sequence to be amplified. Unlike gene expression qPCR, SNP detection requires two probes with different fluorescent reporters. This allows us to differentiate homozygous from heterozygous samples [5].

The first probe—labeled with VIC fluorescent dye—detects the first allele sequence, while the second probe—labeled with FAM fluorescent dye—detects the second. If the sample is homozygous for allele 1, the fluorescence readout or ‘allelic discrimination plot’ will show mostly VIC fluorescence, while if the sample is homozygous for allele 2, the plot will mainly show signal from the FAM dye. If the sample is heterozygous, however, there should be roughly equal signal for each dye. Across an entire plate, the data should resolve into three discrete clusters. Exceptions can arise if the minor allele frequency is very low, if there is a copy number variation (CNV) of the gene in question, or if it lies on the X chromosome.

For our TaqMan SNP genotyping assays, we always list the context sequence of the assay in the forward orientation, regardless of the strand on which the SNP is typically reported. It is important to compare the context sequence of the SNP assay to the SNP sequence in the NCBI’s dbSNP database to determine which dye is associated with the minor allele.

Discrimination of a single base

Three factors contribute to discrimination based on a single mismatch:

1. The mismatch has a disruptive effect on hybridization. A mismatched probe will have a lower melting temperature (Tm) than a perfectly matched probe. Choosing the correct annealing/extension temperature in the PCR will favor hybridization of an exact-match probe over a mismatched probe.
   
   
2. The assay is performed under competitive conditions with both probes present in the same reaction tube. Mismatched probes are therefore prevented from binding due to stable binding of exact-match probes.
   
   
3. The 5’ end of the probe must start to be displaced before cleavage occurs. The 5’ nuclease activity of Taq DNA polymerase actually recognizes a forked structure with a displaced 5’strand of at least 1 to 3 nucleotides [6]. Once a probe starts to be displaced, complete dissociation occurs faster with a mismatch than with an exact match. This allows less time for cleavage to occur with a mismatched probe. Thus, the presence of a mismatch promotes dissociation rather than probe cleavage.

The lower Tm of the mismatched probe creates a window between the Tm of the perfectly matched probe and the Tm of the mismatched probe. You can achieve allele discrimination using an annealing/extension temperature within the Tm window. As probes get longer, a single mismatch becomes less disruptive. For longer probes, this reduces the difference in Tm between a matched and mismatched probe, leading to a smaller Tm window. Thus, shorter probes display better mismatch discrimination.

The 3’ end of all TaqMan MGB probes contains a specific binding element that binds to the minor groove of the DNA helix (MGB or minor groove binder), which improves hybridization and stabilizes binding, allowing smaller probes to be used. Smaller probes improve mismatch discrimination and allow you to design assays for more complex sequences, including multiple nucleotide polymorphisms (MNPs) and insertions or deletions [7]. In addition, using a nonfluorescent quencher molecule, which suppresses fluorescence when the probes are unbound, improves the signal-to-noise ratio.

Applied Biosystems conducted a study where we measured matching and mismatching Tms for a set of 60 MGB probes ranging from 13 to 18 nucleotides in size. The average delta Tm (matching-mismatching) was 9.7°C. This broad Tm window makes it easy to design probes with a matching Tm above the annealing/extension temperature of PCR (nominally 60°C) and a mismatching Tm below this temperature.

A qualitative assay

As in all PCR, temperature and template sequence are key considerations. Unlike in a TaqMan gene expression assay, where the probe should have a melting temperature (Tm) at least 10°C above the primer, in a TaqMan SNP genotyping assay the Tm of the probe should only be 5–6°C above that of the primer.

This means the Tm for the mismatched probe will be lower than the annealing Tm for the primers ensuring that if a probe is present, it will more likely be the matched probe. This implies that, while promoting specificity, genotyping assays cannot be used for quantification. Genotyping assays aim to discriminate between sequences, rather than measure the level of a particular DNA sequence.

If the scatter plot could show the trajectory taken by each data point over the course of the experiment, it would look like a clam shell. This clam-shelling effect only occurs in SNP genotyping assays of homozygous samples.

At the beginning of your experiment, the probes will be in a 1:1 ratio. As the experiment progresses, the probe specific to your allele will start to bind and become depleted in the solution, leaving an excess of the mismatched probe in the solution. As it only has a single base difference, it will continue to bind to the mismatched sequence. When that probe gets cleaved, you may observe fluorescent contribution from the dye on the mismatched probe. The kinetics of the reaction therefore depends on the concentration and binding energy of both the matched and mismatched probes.

We recommend using TaqMan Genotyper or the Genotyping Module on the Thermo Fisher Cloud to analyse your genotyping data. Both programs make genotyping calls based on dye ratios and cluster orientation, not threshold cycle (Ct) values. The algorithms in these programs can differentiate between clusters even if there is mismatch binding.

Getting started

To get started, search the TaqMan SNP genotyping assay search tool. You can narrow down your search from over 7 million available assays based on species, assay type, target, and chromosomal location.

The Applied Biosystems assay library includes almost 7 million genome-wide human assays and 10,000 mouse assays, as well as over 2,500 drug metabolism genotyping assays designed to detect polymorphisms in drug metabolism enzyme (DME) markers. You can also submit target SNP sequences for any genome using the Custom TaqMan Design Tool. TaqMan SNP Genotyping Assays offer the world’s biggest collection of single-tube, ready-to-use SNP assays.

References

1. Teh L-K, Lee T-Y, Tan JAMA et al. (2015) The use of Taqman genotyping assays for rapid confirmation of β-thalassaemia mutations in the Malays: Accurate diagnosis with low DNA concentrations. Int J Lab Hematol 37(1):79–89. doi: 10.1111/ijlh.12240.
2. Zhong L, Xie Y-Z, Cao T-T et al. (2016) A rapid and cost-effective method for genotyping apolipoprotein E gene polymorphism. Mol Neurodegener 11(1):2. doi: 10.1186/s13024-016-0069-4.
3. Vaisman BL (2013) Genotyping of transgenic animals by real-time quantitative PCR with TaqMan probes. Methods Mol Biol 1027:233–251. doi: 10.1007/978-1-60327-369-5_11.
4. Burnet JB, Ogorzaly L, Tissier A et al. (2013) Novel quantitative TaqMan real-time PCR assays for detection of Cryptosporidium at the genus level and genotyping of major human and cattle-infecting species. J Appl Microbiol 114(4):1211–1222. doi: 10.1111/jam.12103.
5. Woodward J (2014) Bi-allelic SNP genotyping using the TaqMan assay. Methods Mol Biol 1145:67–74. doi: 10.1007/978-1-4939-0446-4_6.
6. Lyamichev V, Brow, MAD, Dahlberg JE (1993) Structure-specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases. Science 260:778-783.
7. Fedick A, Su J, Treff NR (2012) Development of TaqMan allelic discrimination based genotyping of large DNA deletions. Genomics 99(3):127–131. doi: 10.1016/j.ygeno.2012.01.003.