Updated: May 27, 2022
Genotyping is a technology that detects small genetic differences that can lead to major changes in phenotype including physical differences and pathologic changes underlying diseases. It is used across many fields but especially in medicine and agriculture. It does this by comparing a DNA sequence to that of another sample or a reference sequence. It identifies small variations in genetic sequence within populations, such as single-nucleotide polymorphisms (SNPs). SNPs (often pronounced “snips”) are single base-pair changes in DNA that occur at specific places in the genome. There are over 660 million SNPs in the human genome, which makes them the most common type of genetic variation in humans. They can explain traits such as eye color and inherited diseases such as cystic fibrosis and sickle cell anemia, as well as act as markers indicating a risk of developing complex common diseases like diabetes and Alzheimer's disease. SNP genotyping can accelerate the era of personalized medicine by predicting an individual's risk of developing certain diseases or designing targeted therapies specific to the genetic basis of the disease.
There are many methods of detecting novel and known SNPs. These include DNA sequencing, mass spectrometry, molecular beacons, SNP microarrays, and PCR-based methods (ever heard of PCR tests? :D). SNP detection can be broken down into two sub-groups: SNP discovery and SNP screening. SNP discovery includes SNPs that are not yet known. Researchers are looking for new SNPs in targeted areas and on a genome-wide scale. SNP screening limits you to known SNPs and researchers are typically looking to genotype individuals or determine if a particular SNP is involved in producing a certain characteristic. Real time PCR is the most common and easiest method of screening known SNPs. The benefits of real-time PCR are that it is easy, accurate, and the bioinformatic analysis is also not as complex as sequencing and microarrays.
You might be wondering why researchers perform SNP genotyping… well, one reason is disease association. Genome-wide association studies (GWAS) can identify connections between SNPs and common disease risk by comparing the polymorphisms across two different populations (one healthy and one diseased). This can also be used in terms of population genomics. SNPs have many implications for evolutionary biology, so GWAS can be useful in identifying forms of genetic variation that show phenotypic differences between healthy individuals. Understanding this normal genetic variation across different populations helps us to understand how different groups have evolved, and may have implications for protecting certain species against future environmental challenges. Also, understanding genetic variation benefits the agricultural industry, where trait selection in plants and livestock is used to increase quantity and quality. While traditional selective breeding involved purely observational methods (selecting only plants or animals with superior phenotypic traits, such as size or strength, for breeding), modern selective breeding relies heavily on molecular biology techniques, including SNP genotyping. Selective breeding generates breeds with more desirable phenotypes and detecting these genetic changes helps us understand which genes and sequences are associated with particular phenotypic traits. This is useful for designing more intelligent breeding programs. Single-celled organisms, such as bacteria, also have SNPs. SNP genotyping can show the difference between bacterial isolates and can also be used to characterize strains of antibiotic resistance (see my previous blog: What is antibiotic resistance?). SNP-based strain detection is relevant in both clinical and agricultural research and has been used to study a range of infectious diseases in both humans and plants.
A haplotype is a series of adjacent SNPs from an individual and can serve as a signature for a specific phenotypic trait. The haplotype group, or block, is inherited together from a single parent since they are close together in the chromosome, which also means that haplotypes can be used to trace lineage, as well as valuable traits for crop production. Signature haplotypes may also indicate a predisposition to a specific disease or drug sensitivities, which are key for developing personalized medical diagnoses or treatments.
There are certain modern technologies that are used to study SNP genotyping. There are varied approaches to SNP genotyping depending on the number of samples, the number of genotypes to be tested, and the amount of sample material available, but below are some of the most common ones.
For approaches that need more high index technologies, SNP analysis using microarrays and targeted sequencing methods such as amplicon sequencing and hybridization capture technology are very helpful. Simply put, amplicon sequencing uses PCR to create sequences of DNA called amplicons. Amplicons from different samples can be multiplexed, also called indexed or pooled, which involves adding a barcode (index) to samples so they can be identified. Hybridization capture uses probes to hybridize to the regions of interest. It is particularly helpful when trying to detect rare variants.
For approaches that do not need as high of an index, multiplex quantitative PCR and multiplex digital PCR are used. Multiplex quantitative PCR, also called multiplex qPCR, allows you to have multiple targets that are amplified in a single reaction tube. Each target is amplified by a different set of primers, and a uniquely-labeled probe distinguishes each PCR amplicon. This way, you can measure the expression levels of several targets or genes of interest quickly. Multiplex digital PCR, also called multiplex dPCR, provides a highly sensitive measure of the absolute amount of a target DNA. Because of the sensitivity, properly designed assays can detect rare alleles, copy number variations, low abundance transcripts, rare microRNAs, and very low viral loads.
Except some more news in the future about all the ways that genotyping is advancing our knowledge of the medical and agricultural industries!
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