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Understanding genotyping


Genotyping is the process of determining the DNA sequence, called a genotype, at specific positions within the genome of an individual. Sequence variations can be used as markers in linkage and association studies to determine genes relevant to specific traits or disease.

pipette tips used in genotyping analysis

Identifying sequence variation

Each species is defined by a distinct set of common characteristics, but even within one species, there are subtle differences among individuals. For example, we can easily spot physical differences among people, but every species has individual differences, even those considered non-living, such as viruses. The differences are not drastic enough to be separated into a different species, but instead, an individual of a species with a slightly different characteristic is called a variant. 

Variation is the term used to describe the differences either among individuals or populations within a single species.  For example, some populations of soybean plants will have resistance to a common fungal pathogen such as phytophthora root rot; whereas, other populations will be susceptible. In simplistic terms, this is a population variation. The phytophthora root rot resistant population has some sort of change enabling the plants to grow in the presence of the fungus without being infected.

Environmental and genetic differences are the root of these visible or phenotypic changes. Since environmental changes are not heritable, most are interested in the genetic variation that results in the physical differences. Genetic variation can be passed onto the next generation, and increase the fitness for the species.

Genotyping is the experimental procedure that identifies the differences in DNA sequence among individuals or populations, which is used to understand the connection between genotypes and phenotypes. An individual genome is identified as a distinct variant when compared to a reference sequence, which is derived from the general population or a defined subgroup. A variant sequence can differ from the reference sequence in numerous ways. Types of variation include single nucleotide variants (SNV), single nucleotide polymorphisms (SNPs), insertions and deletions (indels), and copy number variation (CNV).

SNPs are the most common type of sequence variant investigated by researchers; they are typically defined as SNVs that occur at >1% in the population. Based on the number of SNPs cataloged in Build 149 of the SNP database, dbSNP, maintained by the National Center for Biotechnology Information (NCBI) [1] and a genome size of 3.4 x 109 bp [2], the human genome should contain a SNP approximately once every 22 bases! Other common model systems show a similarly high frequency of SNPs [3]

What is a haplotype?

A series of adjacent SNPs from an individual represent a haplotype and can serve as a signature for a specific phenotypic trait (Figure 1). The haplotype group or block is inherited together from a single parent since they are close together in the chromosome, which means the groups can trace lineage also. Haplotypes are used to trace valuable traits for crop production such as yield or disease resistance. Signature haplotypes may also indicate a predisposition to a specific disease or drug sensitivities, which are key for developing personalized medical diagnoses or treatments.

Figure 1. Haplotypes are groups of SNPs that are inherited as a single block. An example of SNPs (A or G, G or T, and A or T) found in close proximity in the genome. During reproduction, these three SNPs are inherited as a group. Therefore, an individual can inherit one of three distinct haplotypes (AGT, GTA, AGA) depending on the SNP profile of their parents. 

Associating sequence variation to specific traits

By comparing genetic variations among individuals of a species, researchers can identify heritable genetic signatures or markers relevant to specific traits. These unique differences can be used as markers in linkage and association studies.

Genome-wide association studies or GWAS compares genetic differences across entire genomes from two individuals or populations. For example, the genomes of a group of people that have a disease can be compared to a genomic sequences from a similar group of people without the disease. Any SNP or haplotypes that is more prevalent in those with the disease is called an associated genetic marker.

The association is just the beginning, and many studies are needed to confirm if the genetic variation is truly the underlying cause of the disease. NCBI maintains a registry of human genomic variations and their relationship to health called ClinVar. The website archives variation in human genes and the evidence supporting the association to a particular phenotype. Besides human health, finding the relationship of genotype to phenotype has many applications:

  • Match effective medical treatments to populations with specific genotypes (personalized medicine)
  • Facilitate animal breeding and plant breeding, such as the selection of a desired genotype from a cross hybrid
  • Trace ancestry or the origins of disease; map evolution, such as phylogenetic relationships between species; or test for family relationship for inheritance or paternity
  • Perform forensics, using genotyping to identify a specific individual
  • Do pathogen typing and resistance screening
  • Monitor biodiversity

Technologies used to study genotyping

There are different approaches to SNP genotyping with the number of samples, the number of genotypes to be tested, and the amount of sample material available, all factoring into choice of technology.

High-throughput genotyping methods include whole genome analysis by NGS, SNP analysis using microarrays, and targeted sequencing methods such as amplicon sequencing or hybridization capture technology.

Low-throughput analyses include using multiplex qPCR, PACE (PCR Allele Competitive Extension) SNP genotyping, and multiplex dPCR to identify the genotype of a specific SNP.


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Overview of genotyping terms

Learn more about the language used in genotyping studies.

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IDT genotyping solutions

Genotyping by qPCR

Quantitative polymerase chain reaction (qPCR) is a commonly used genotyping technique. Often employing a primer-pair and target-specific fluorescent probe, qPCR can be a sensitive and specific way to detect SNPs. IDT offers a complete SNP genotyping solution with predesigned assays, as well as complementary easy- and ready-to-use reagent mixes. For other applications, modified probes are available that can be incorporated into custom qPCR assays.

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Targeted sequencing

Targeted sequencing uses deep sequencing to detect known and novel variants within your region of interest. Thus, it can be used as a method of gene expression analysis, mutation detection, and gene structure analysis.

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PACE™ SNP Genotyping Assays

PACE™ SNP genotyping uses competitive, allele-specific PCR and a simple, easily detected fluorescent readout for bi-allelic genotyping. Obtain primer sets from IDT for cost-effective, high-throughput assays.

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Genotyping by digital PCR (dPCR)

Digital PCR (dPCR) includes the same reagents found in a typical qPCR assay and amplified in a similar manner, but dPCR divides the reaction into nano-sized droplets or wells prior to amplification. The partitions are so small that either 1 or 0 templates are in each. After amplification, the fluorescence in the well or drop represents the genotype.

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Get started with genotyping

For user-defined methods, order custom probes with modifications, such as Affinity Plus™ qPCR locked nucleic acid (LNA) Probes, that increase probe stability and enable designs within difficult sequences and with selective target identification.

rhAmpSeq amplicon sequencing system

The rhAmpSeq™ system enables highly accurate amplicon sequencing on Illumina® next generation sequencing (NGS) platforms. Whether you are investigating thousands of targets or a few, the fast and easy rhAmpSeq workflow generates NGS-ready amplicon libraries for deep, targeted resequencing.

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Affinity Plus qPCR Probes

Use Affinity Plus™ qPCR Probes for SNP genotyping, transcript variant identification, and more sensitive target detection in challenging samples (FFPE tissue, biofluids). The Affinity Plus bases used in these qPCR probes are locked nucleic acid monomers. When incorporated into a probe, they impart heightened structural stability, leading to increased hybridization melt temperature (Tm).

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PACE™ SNP Genotyping Assays

PACE™ SNP genotyping uses competitive, allele-specific PCR and a simple, easily detected fluorescent readout for bi-allelic genotyping. Obtain primer sets from IDT for cost-effective, high-throughput assays.

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rhAmp SNP Genotyping System

The rhAmp SNP Genotyping System is a fully integrated genotyping solution that includes an extensive predesigned assay collection, a custom design tool, optimized reagent mixes, and optional synthetic control templates. SNP detection may be performed on any commonly available qPCR instrument.

Precise and easy-to-use, the rhAmp SNP Genotyping System offers an out-of-the-box solution for SNP genotyping studies for small discovery or large screening projects.

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  1. NCBI dbSNP Build 149 (Nov 7, 2016) [Accessed Dec 19, 2016].
  2. Gregory TR. (2005) Animal genome size database. [Accessed Dec 19, 2016].
  3. Prediger E. (2017) Consider SNPs when designing PCR and qPCR assays, [Online] Coralville, Integrated DNA Technologies. [Accessed Jan 6, 2020].