Genotyping

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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.

Contents

Techniques

Current methods of genotyping include restriction fragment length polymorphism identification (RFLPI) of genomic DNA, random amplified polymorphic detection (RAPD) of genomic DNA, amplified fragment length polymorphism detection (AFLPD), polymerase chain reaction (PCR), DNA sequencing, allele specific oligonucleotide (ASO) probes, and hybridization to DNA microarrays or beads. Genotyping is important in research of genes and gene variants associated with disease. Due to current technological limitations, almost all genotyping is partial. That is, only a small fraction of an individual's genotype is determined, such as with (epi)GBS (Genotyping by sequencing) or RADseq. New [2] mass-sequencing technologies promise to provide whole-genome genotyping (or whole genome sequencing) in the future.

Applications

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.

Human genotyping

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.

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.

Ethical concerns

The ethical concerns 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. [3] The benefits of population wide genotyping have been contended by ethical concerns on consent and general benefit of wide span screening. [3] 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. [4] 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. [5] [4] Much of the ethical concerns surrounding genotyping arise from information availability, as in who can access the genotype of an individual in various contexts. [4]

Tuberculosis

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. [6] 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. [6] 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. [6]

Agricultural Usage

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. [7] 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. [7] 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. [7]

See also

Related Research Articles

In molecular biology, restriction fragment length polymorphism (RFLP) is a technique that exploits variations in homologous DNA sequences, known as polymorphisms, populations, or species or to pinpoint the locations of genes within a sequence. The term may refer to a polymorphism itself, as detected through the differing locations of restriction enzyme sites, or to a related laboratory technique by which such differences can be illustrated. In RFLP analysis, a DNA sample is digested into fragments by one or more restriction enzymes, and the resulting restriction fragments are then separated by gel electrophoresis according to their size.

<span class="mw-page-title-main">Single-nucleotide polymorphism</span> Single nucleotide in genomic DNA at which different sequence alternatives exist

In genetics and bioinformatics, a single-nucleotide polymorphism is a germline substitution of a single nucleotide at a specific position in the genome that is present in a sufficiently large fraction of considered population.

<span class="mw-page-title-main">DNA sequencing</span> Process of determining the nucleic acid sequence

DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. The advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery.

A genetic marker is a gene or DNA sequence with a known location on a chromosome that can be used to identify individuals or species. It can be described as a variation that can be observed. A genetic marker may be a short DNA sequence, such as a sequence surrounding a single base-pair change, or a long one, like minisatellites.

A molecular marker is a molecule, sampled from some source, that gives information about its source. For example, DNA is a molecular marker that gives information about the organism from which it was taken. For another example, some proteins can be molecular markers of Alzheimer's disease in a person from which they are taken. Molecular markers may be non-biological. Non-biological markers are often used in environmental studies.

In molecular biology, SNP array is a type of DNA microarray which is used to detect polymorphisms within a population. A single nucleotide polymorphism (SNP), a variation at a single site in DNA, is the most frequent type of variation in the genome. Around 335 million SNPs have been identified in the human genome, 15 million of which are present at frequencies of 1% or higher across different populations worldwide.

Public health genomics is the use of genomics information to benefit public health. This is visualized as more effective preventive care and disease treatments with better specificity, tailored to the genetic makeup of each patient. According to the Centers for Disease Control and Prevention (U.S.), Public Health genomics is an emerging field of study that assesses the impact of genes and their interaction with behavior, diet and the environment on the population's health.

SNP genotyping is the measurement of genetic variations of single nucleotide polymorphisms (SNPs) between members of a species. It is a form of genotyping, which is the measurement of more general genetic variation. SNPs are one of the most common types of genetic variation. An SNP is a single base pair mutation at a specific locus, usually consisting of two alleles. SNPs are found to be involved in the etiology of many human diseases and are becoming of particular interest in pharmacogenetics. Because SNPs are conserved during evolution, they have been proposed as markers for use in quantitative trait loci (QTL) analysis and in association studies in place of microsatellites. The use of SNPs is being extended in the HapMap project, which aims to provide the minimal set of SNPs needed to genotype the human genome. SNPs can also provide a genetic fingerprint for use in identity testing. The increase of interest in SNPs has been reflected by the furious development of a diverse range of SNP genotyping methods.

An allele-specific oligonucleotide (ASO) is a short piece of synthetic DNA complementary to the sequence of a variable target DNA. It acts as a probe for the presence of the target in a Southern blot assay or, more commonly, in the simpler dot blot assay. It is a common tool used in genetic testing, forensics, and molecular biology research.

Marker assisted selection or marker aided selection (MAS) is an indirect selection process where a trait of interest is selected based on a marker linked to a trait of interest, rather than on the trait itself. This process has been extensively researched and proposed for plant- and animal- breeding.

Personal genomics or consumer genetics is the branch of genomics concerned with the sequencing, analysis and interpretation of the genome of an individual. The genotyping stage employs different techniques, including single-nucleotide polymorphism (SNP) analysis chips, or partial or full genome sequencing. Once the genotypes are known, the individual's variations can be compared with the published literature to determine likelihood of trait expression, ancestry inference and disease risk.

Population genomics is the large-scale comparison of DNA sequences of populations. Population genomics is a neologism that is associated with population genetics. Population genomics studies genome-wide effects to improve our understanding of microevolution so that we may learn the phylogenetic history and demography of a population.

Diversity Arrays Technology (DArT) is a high-throughput genetic marker technique that can detect allelic variations to provides comprehensive genome coverage without any DNA sequence information for genotyping and other genetic analysis. The general steps involve reducing the complexity of the genomic DNA with specific restriction enzymes, choosing diverse fragments to serve as representations for the parent genomes, amplify via polymerase chain reaction (PCR), insert fragments into a vector to be placed as probes within a microarray, then fluorescent targets from a reference sequence will be allowed to hybridize with probes and put through an imaging system. The objective is to identify and quantify various forms of DNA polymorphism within genomic DNA of sampled species.

Molecular Inversion Probe (MIP) belongs to the class of Capture by Circularization molecular techniques for performing genomic partitioning, a process through which one captures and enriches specific regions of the genome. Probes used in this technique are single stranded DNA molecules and, similar to other genomic partitioning techniques, contain sequences that are complementary to the target in the genome; these probes hybridize to and capture the genomic target. MIP stands unique from other genomic partitioning strategies in that MIP probes share the common design of two genomic target complementary segments separated by a linker region. With this design, when the probe hybridizes to the target, it undergoes an inversion in configuration and circularizes. Specifically, the two target complementary regions at the 5’ and 3’ ends of the probe become adjacent to one another while the internal linker region forms a free hanging loop. The technology has been used extensively in the HapMap project for large-scale SNP genotyping as well as for studying gene copy alterations and characteristics of specific genomic loci to identify biomarkers for different diseases such as cancer. Key strengths of the MIP technology include its high specificity to the target and its scalability for high-throughput, multiplexed analyses where tens of thousands of genomic loci are assayed simultaneously.

<span class="mw-page-title-main">Exome sequencing</span> Sequencing of all the exons of a genome

Exome sequencing, also known as whole exome sequencing (WES), is a genomic technique for sequencing all of the protein-coding regions of genes in a genome. It consists of two steps: the first step is to select only the subset of DNA that encodes proteins. These regions are known as exons—humans have about 180,000 exons, constituting about 1% of the human genome, or approximately 30 million base pairs. The second step is to sequence the exonic DNA using any high-throughput DNA sequencing technology.

<span class="mw-page-title-main">Restriction site associated DNA markers</span> Type of genetic marker

Restriction site associated DNA (RAD) markers are a type of genetic marker which are useful for association mapping, QTL-mapping, population genetics, ecological genetics and evolutionary genetics. The use of RAD markers for genetic mapping is often called RAD mapping. An important aspect of RAD markers and mapping is the process of isolating RAD tags, which are the DNA sequences that immediately flank each instance of a particular restriction site of a restriction enzyme throughout the genome. Once RAD tags have been isolated, they can be used to identify and genotype DNA sequence polymorphisms mainly in form of single nucleotide polymorphisms (SNPs). Polymorphisms that are identified and genotyped by isolating and analyzing RAD tags are referred to as RAD markers. Although genotyping by sequencing presents an approach similar to the RAD-seq method, they differ in some substantial ways.

Molecular breeding is the application of molecular biology tools, often in plant breeding and animal breeding. In the broad sense, molecular breeding can be defined as the use of genetic manipulation performed at the level of DNA to improve traits of interest in plants and animals, and it may also include genetic engineering or gene manipulation, molecular marker-assisted selection, and genomic selection. More often, however, molecular breeding implies molecular marker-assisted breeding (MAB) and is defined as the application of molecular biotechnologies, specifically molecular markers, in combination with linkage maps and genomics, to alter and improve plant or animal traits on the basis of genotypic assays.

Multiple Annealing and Looping Based Amplification Cycles (MALBAC) is a quasilinear whole genome amplification method. Unlike conventional DNA amplification methods that are non-linear or exponential, MALBAC utilizes special primers that allow amplicons to have complementary ends and therefore to loop, preventing DNA from being copied exponentially. This results in amplification of only the original genomic DNA and therefore reduces amplification bias. MALBAC is “used to create overlapped shotgun amplicons covering most of the genome”. For next generation sequencing, MALBAC is followed by regular PCR which is used to further amplify amplicons.

<span class="mw-page-title-main">Kompetitive allele specific PCR</span>

Kompetitive allele specific PCR (KASP) is a homogenous, fluorescence-based genotyping variant of polymerase chain reaction. It is based on allele-specific oligo extension and fluorescence resonance energy transfer for signal generation.

In the field of genetic sequencing, genotyping by sequencing, also called GBS, is a method to discover single nucleotide polymorphisms (SNP) in order to perform genotyping studies, such as genome-wide association studies (GWAS). GBS uses restriction enzymes to reduce genome complexity and genotype multiple DNA samples. After digestion, PCR is performed to increase fragments pool and then GBS libraries are sequenced using next generation sequencing technologies, usually resulting in about 100bp single-end reads. It is relatively inexpensive and has been used in plant breeding. Although GBS presents an approach similar to restriction-site-associated DNA sequencing (RAD-seq) method, they differ in some substantial ways.

References

  1. ↑ "Genotyping definition". NIH. 2011-09-21. Retrieved 2011-09-21.
  2. ↑ "Genotyping at Illumina, Inc". Illumina.com. Archived from the original on 2011-04-16. Retrieved 2010-12-04.
  3. 1 2 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.
  4. 1 2 3 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.
  5. ↑ 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.
  6. 1 2 3 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. PMID   30338753. S2CID   53016602.
  7. 1 2 3 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.
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