Genotyping
Genotyping is the process of determining differences in the genetic make-up (genotype) of an individual by examining the individual's DNA sequence using biological assays and comparing it to another individual's sequence or a reference sequence. It reveals the alleles an individual has inherited from their parents.[1] Traditionally genotyping is the use of DNA sequences to define biological populations by use of molecular tools. It does not usually involve defining the genes of an individual.
Techniques
[edit]Restriction Fragment Length Polymorphisms
[edit]A restriction fragment length polymorphism (RFLP) is a variation between different people at sites of the genome recognized by restriction enzymes. DNA containing different restriction sites will be cut by bacterial restriction enzymes differently and this can be seen using gel electrophoresis. When running the sample through, a successfully cleaved sample will contain two bands, while the sample with a different restriction site polymorphism will have one band as it had not been cleaved. A small change is enough to cause that restriction site to deny the restriction enzyme. This method is often used to trace the inheritance of DNA through families.[2]
Random Amplified Polymorphic Detection
[edit]The random amplified polymorphic detection (RAPD) method relies on PCR methods to amplify and isolate lengths of DNA fragments. Oligonucleotide primers are used which bind to denatured DNA fragments which have been produced through heat treatment. Two primers, one to define the starting point and ending point of PCR DNA synthesis, are used in this process. The fragments of DNA will range from two to three kbp and different primers are tried until the desired trait is isolated from the genome. This method is useful in locating small differences to differentiate between species.[3]
Amplified Fragment Length Polymorphisms
[edit]The amplified fragment length polymorphism (AFLP) detection method is much like RAPD as it also relies on PCR amplification of DNA, with the difference being that this process is more precise but also more time consuming than the RAPD counterpart.[4] It also does not require random primers, instead the DNA is digested by restriction enzymes and the ends are then ligated to adaptors which allow for specification of strands when performing PCR amplification, this is where the improved precision of this method comes from.[5]
DNA Microarrays/Beads
[edit]This process uses specific oligonucleotides which are placed on a DNA microarray which bind to complementary strands of DNA. This method is optimal for detecting single nucleotide polymorphisms in the DNA. The DNA will bind to the oligonucleotide bead up until one base pair before the SNP, where a single labeled nucleotide will be incorporated. This will be seen through dyes and fluorescently labeled proteins which indicate which SNP can be found at the locus of interest.[6][7]
Applications
[edit]Microbial
[edit]Genotyping applies to a broad range of individuals, including microorganisms. For example, viruses and bacteria can be genotyped. Genotyping in this context may help in controlling the spreading of pathogens, by tracing the origin of outbreaks. This area is often referred to as molecular epidemiology or forensic microbiology.[citation needed]
Human
[edit]Humans can also be genotyped. For example, when testing fatherhood or motherhood, scientists typically only need to examine 10 or 20 genomic regions (like single-nucleotide polymorphism (SNPs)), which represent a tiny fraction of the human genome.[citation needed]
When genotyping transgenic organisms, a single genomic region may be all that needs to be examined to determine the genotype. A single PCR assay is typically enough to genotype a transgenic mouse; the mouse is the mammalian model of choice for much of medical research today.[citation needed]
Tuberculosis
[edit]Genotyping is used in the medical field to identify and control the spread of tuberculosis (TB). Originally, genotyping was only used to confirm outbreaks of tuberculosis; but with the evolution of genotyping technology it is now able to do far more. Advances in genotyping technology led to the realization that many cases of tuberculosis, including infected individuals living in the same household, were not actually linked.[8] This caused the formation of universal genotyping in an attempt to understand transmission dynamics. Universal genotyping revealed complex transmission dynamics based on things like socio-epidemiological factors. This led to the use of polymerase chain reactions (PCR) which allowed for faster detection of tuberculosis. This rapid detection method is used to prevent TB.[8] The addition of whole genome sequencing (WGS) allowed for identification of strains of TB which could then be put in a chronological cluster map. These cluster maps show the origin of cases and the time in which those cases arose. This gives a much clearer picture of transmission dynamics and allows for better control and prevention of transmission. All of these different forms of genotyping are used together to detect TB, prevent its spread and trace the origin of infections. This has helped to reduce the number of TB cases.[8]
Agricultural Usage
[edit]Many types of genotyping are used in agriculture. One type that is used is genotyping by sequencing because it aids agriculture with crop breeding. For this purpose, single nucleotide polymorphisms (SNPs) are used as markers and RNA sequencing is used to look at gene expression in crops.[9] The knowledge gained from this type of genotyping allows for selective breeding of crops in ways which benefit agriculture. In the case of alfalfa, the cell wall was improved through selective breeding that was made possible by this type of genotyping.[9] These techniques have also resulted in the discovery of genes that provide resistance to diseases. A gene called Yr15 was discovered in wheat, which protects against a disease called yellow wheat rust. Selective breeding for the Yr15 gene then prevented yellow wheat rust, benefiting agriculture.[9]
Ethical concerns
[edit]The ethics of genotyping humans have been a topic of discussion. The rise of genotyping technologies will make it possible to screen large populations of people for genetic diseases and predispositions for disease.[10] The benefits of population wide genotyping have been contended by ethical concerns on consent and general benefit of wide span screening.[10]
Psychological
[edit]Genotyping identifies mutations that increase susceptibility of a person to develop a disease, but disease development is not guaranteed in most cases, which can cause psychological damage.[11]
Discrimination
[edit]Discrimination can arise from various genetic markers identified by genotyping, such as athletic advantages or disadvantages in professional sports or risk of disease development later in life.[12][11]
Eugenics
[edit]Availability
[edit]Much of the ethical concerns surrounding genotyping arise from information availability, as in who can access the genotype of an individual in various contexts.[11]
See also
[edit]- Mendelian error – Error in the mendelian inheritance
- Quantitative trait locus – DNA locus associated with variation in a quantitative trait
- SNP genotyping – Measurement of genetic variations
References
[edit]- ^ "Genotyping definition". NIH. 2011-09-21. Retrieved 2011-09-21.
- ^ "Restriction Fragment Length Polymorphism (RFLP)". www.genome.gov. Retrieved 2025-03-28.
- ^ Heldt, Hans-Walter; Piechulla, Birgit (2021-01-01), Heldt, Hans-Walter; Piechulla, Birgit (eds.), "Chapter 22 - Biotechnology Alters Plants to Meet the Requirements of Agriculture, Nutrition, and Industry", Plant Biochemistry (Fifth Edition), Academic Press, pp. 533–569, ISBN 978-0-12-818631-2, retrieved 2025-03-28
- ^ Garcia, Antonio A. F.; Benchimol, Luciana L.; Barbosa, Antônia M. M.; Geraldi, Isaias O.; Souza Jr., Cláudio L.; Souza, Anete P. de (2004). "Comparison of RAPD, RFLP, AFLP and SSR markers for diversity studies in tropical maize inbred lines". Genetics and Molecular Biology. 27: 579–588. doi:10.1590/S1415-47572004000400019. ISSN 1415-4757.
- ^ Paun, Ovidiu; Schönswetter, Peter (2012). "Amplified fragment length polymorphism: an invaluable fingerprinting technique for genomic, transcriptomic, and epigenetic studies". Methods in Molecular Biology (Clifton, N.J.). 862: 75–87. doi:10.1007/978-1-61779-609-8_7. ISSN 1940-6029. PMC 3513352. PMID 22419490.
- ^ Bumgarner, Roger (January 2013). "Overview of DNA microarrays: types, applications, and their future". Current Protocols in Molecular Biology. Chapter 22: Unit 22.1. doi:10.1002/0471142727.mb2201s101. ISSN 1934-3647. PMC 4011503. PMID 23288464.
- ^ Kockum, Ingrid; Huang, Jesse; Stridh, Pernilla (2023). "Overview of Genotyping Technologies and Methods". Current Protocols. 3 (4): e727. doi:10.1002/cpz1.727. ISSN 2691-1299.
- ^ a b c García De Viedma, Darío; Pérez-Lago, Laura (2018-09-07). Baquero, Fernando; Bouza, Emilio; Gutiérrez-Fuentes, J.A.; Coque, Teresa M. (eds.). "The Evolution of Genotyping Strategies To Detect, Analyze, and Control Transmission of Tuberculosis". Microbiology Spectrum. 6 (5). doi:10.1128/microbiolspec.MTBP-0002-2016. ISSN 2165-0497. PMC 11633623. PMID 30338753. S2CID 53016602.
- ^ a b c Scheben, Armin; Batley, Jacqueline; Edwards, David (2017). "Genotyping-by-sequencing approaches to characterize crop genomes: choosing the right tool for the right application". Plant Biotechnology Journal. 15 (2): 149–161. doi:10.1111/pbi.12645. ISSN 1467-7652. PMC 5258866. PMID 27696619.
- ^ a b Hall, Alison Elizabeth (2013). "What ethical and legal principles should guide the genotyping of children as part of a personalised screening programme for common cancer?". Journal of Medical Ethics.
- ^ a b c Mathaiyan, Jayanthi; Chandrasekaran, Adithan; Davis, Sanish (2013). "Ethics of genomic research". Perspectives in Clinical Research. 4 (1): 100–104. doi:10.4103/2229-3485.106405. ISSN 2229-3485. PMC 3601693. PMID 23533991.
- ^ Lippi, Giuseppe (2004). "Athletes Genotyping: Ethical and Legal Issues". International Journal of Sports Medicine. 25 (2): 159, author reply 160–1. doi:10.1055/s-2004-819956. PMID 14986202.