Forward genetics

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Forward genetics is a molecular genetics approach of determining the genetic basis responsible for a phenotype. Forward genetics provides an unbiased approach because it relies heavily on identifying the genes or genetic factors that cause a particular phenotype or trait of interest. [1]

Contents

This was initially done by using naturally occurring mutations or inducing mutants with radiation, chemicals, or insertional mutagenesis (e.g. transposable elements). Subsequent breeding takes place, mutant individuals are isolated, and then the gene is mapped. Forward genetics can be thought of as a counter to reverse genetics, which determines the function of a gene by analyzing the phenotypic effects of altered DNA sequences. [2] Mutant phenotypes are often observed long before having any idea which gene is responsible, which can lead to genes being named after their mutant phenotype (e.g. Drosophila rosy gene which is named after the eye colour in mutants). [3]

Techniques used in Forward Genetics

Forward genetics provides researchers with the ability to identify genetic changes caused by mutations that are responsible for individual phenotypes in organisms. [1] There are three major steps involved with the process of forward genetics which includes: making random mutations, selecting the phenotype or trait of interest, and identifying the gene and its function. [4] Forward genetics involves the use of several mutagenesis processes to induce DNA mutations at random which may include:

Chemical mutagenesis

Chemical mutagenesis is an easy tool that is used to generate a broad spectrum of mutant alleles. Chemicals like ethyl methanesulfonate (EMS) cause random point mutations particularly in G/C to A/T transitions due to guanine alkylation. [3] These point mutations are typically loss-of-function or null alleles because they generate stop codons in the DNA sequence. [5] These types of mutagens can be useful because they are easily applied to any organism but they were traditionally very difficult to map, although the advent of next-generation sequencing has made this process considerably easier.

Another chemical such as ENU, also known as N-ethyl-N-nitrosourea works similarly to EMS. ENU also induces random point mutations where all codons are equally liable to change. These point mutations modify gene function by inducing different alleles, including gain or loss of function mutations in protein-coding or noncoding regions in the genome. [6]

The figure shows the chemical compounds ethyl methansulfonate (shown on the left) and N-ethyl-N-nitrosourea (shown on the right). Chemchemicals.png
The figure shows the chemical compounds ethyl methansulfonate (shown on the left) and N-ethyl-N-nitrosourea (shown on the right).

Radiation mutagenesis

Other methods such as using radiation to cause large deletions and chromosomal rearrangements can be used to generate mutants as well. [3] Ionizing radiation can be used to induce genome-wide mutations as well as chromosomal duplications, inversions, and translocations.

Similarly, short wave UV light works in the same way as ionizing radiation which can also induce mutations generating chromosomal rearrangements. When DNA absorbs short wave UV light, dimerizing and oxidative mutations can occur which can cause severe damage to the DNA sequence of an organism.

Insertional mutagenesis

Mutations can also be generated by insertional mutagenesis. Most often, insertional mutagenesis involves the use of transposons, which introduces dramatic changes in the genome of an organism. Transposon movements can create random mutations in the DNA sequence by changing its position within a genome, therefore modifying gene function, and altering the organism’s genetic information. For example, transposable elements containing a marker are mobilized into the genome at random. These transposons are often modified to transpose only once, and once inserted into the genome a selectable marker can be used to identify the mutagenized individuals. Since a known fragment of DNA was inserted this can make mapping and cloning the gene much easier. [3] [7]

Post mutagenesis

Once mutagenized and screened, typically a complementation test is done to ensure that mutant phenotypes arise from the same genes if the mutations are recessive. [3] [8] If the progeny after a cross between two recessive mutants have a wild-type phenotype, then it can be inferred that the phenotype is determined by more than one gene. Typically, the allele exhibiting the strongest phenotype is further analyzed. A genetic map can then be created using linkage and genetic markers, and then the gene of interest can be cloned and sequenced. If many alleles of the same genes are found, the screen is said to be saturated and it is likely that all of the genes involved producing the phenotype were found. [8]

Flowchart of basic steps involved in forward genetics approach. Forward genetics steps.png
Flowchart of basic steps involved in forward genetics approach.

Human diseases

Human diseases and disorders can be the result of mutations. [9] Forward genetics methods are employed in studying heritable diseases to determine the genes that are accountable. [10] With single-gene or mendelian disorders a missense mutation can be significant; single nucleotide polymorphisms (SNPs) can be analyzed to identify gene mutations that are associated with the disorder phenotype. Before 1980 very few human genes had been identified as disease loci until advances in DNA technology gave rise to positional cloning and reverse genetics. In the 1980s and 1990s, positional cloning consisted of genetic mapping, physical mapping, and discerning the gene mutation. [11] Discovering disease loci using old forward genetic techniques was a very long and difficult process and much of the work went into mapping and cloning the gene through association studies and chromosome walking. [3] [12] Despite being laborious and costly, forward genetics provides a way to obtain objective information regarding a mutation's connection to a disease. [13] Another advantage of forward genetics is that it requires no prior knowledge about the gene being studied. [10] Cystic fibrosis however demonstrates how the process of forward genetics can elucidate a human genetic disorder. Genetic-linkage studies were able to map the disease loci in cystic fibrosis to chromosome 7 by using protein markers. Afterward, chromosome walking and jumping techniques were used to identify the gene and sequence it. [14] Forward genetics can work for single-gene-single phenotype situations but in more complicated diseases like cancer, reverse genetics is often used instead. [12] This is usually because complex diseases tend to have multiple genes, mutations, or other factors that cause or may influence it. [9] Forward and reverse genetics operate with opposite approaches, but both are useful for genetics research. [10] They can be coupled together to see if similar results are found. [10]

Classical forward genetics

By the classical genetics approach, a researcher would then locate (map) the gene on its chromosome by crossbreeding with individuals that carry other unusual traits and collecting statistics on how frequently the two traits are inherited together. Classical geneticists would have used phenotypic traits to map the new mutant alleles. Eventually the hope is that such screens would reach a large enough scale that most or all newly generated mutations would represent a second hit of a locus, essentially saturating the genome with mutations. This type of saturation mutagenesis within classical experiments was used to define sets of genes that were a bare minimum for the appearance of specific phenotypes. [15] However, such initial screens were either incomplete as they were missing redundant loci and epigenetic effects, and such screens were difficult to undertake for certain phenotypes that lack directly measurable phenotypes. Additionally, a classical genetics approach takes significantly longer.

History

Gregor Mendel experimented with pea plant phenotypes and published his conclusions about genes and inheritance in 1865. [10] Around the early 1900s Thomas Hunt Morgan was mutating Drosophila using radium and attempting to find heritable mutations. [16] Alfred Sturtevant later began mapping genes of Drosophila with mutations they had been following. [17] In the 1990s forward genetics methods were utilized to better understand Drosophila genes significant to development from embryo to adult fly. [18] In 1995 the Nobel Prize went to Christiane Nüsslein, Edward Lewis, and Eris Wieschaus for their work in developmental genetics. [18] The human genome was mapped and the sequence was published in 2003. [19] The ability to identify genes that contribute to Mendelian disorders has improved since 1990 as a result of advances in genetics and technology. [9]

See also

Related Research Articles

An allele, or allelomorph, is a variant of the sequence of nucleotides at a particular location, or locus, on a DNA molecule.

<span class="mw-page-title-main">Genetics</span> Science of genes, heredity, and variation in living organisms

Genetics is the study of genes, genetic variation, and heredity in organisms. It is an important branch in biology because heredity is vital to organisms' evolution. Gregor Mendel, a Moravian Augustinian friar working in the 19th century in Brno, was the first to study genetics scientifically. Mendel studied "trait inheritance", patterns in the way traits are handed down from parents to offspring over time. He observed that organisms inherit traits by way of discrete "units of inheritance". This term, still used today, is a somewhat ambiguous definition of what is referred to as a gene.

<span class="mw-page-title-main">Mutation</span> Alteration in the nucleotide sequence of a genome

In biology, a mutation is an alteration in the nucleic acid sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA or viral replication, mitosis, or meiosis or other types of damage to DNA, which then may undergo error-prone repair, cause an error during other forms of repair, or cause an error during replication. Mutations may also result from insertion or deletion of segments of DNA due to mobile genetic elements.

<span class="mw-page-title-main">Dominance (genetics)</span> One gene variant masking the effect of another in the other copy of the gene

In genetics, dominance is the phenomenon of one variant (allele) of a gene on a chromosome masking or overriding the effect of a different variant of the same gene on the other copy of the chromosome. The first variant is termed dominant and the second is called recessive. This state of having two different variants of the same gene on each chromosome is originally caused by a mutation in one of the genes, either new or inherited. The terms autosomal dominant or autosomal recessive are used to describe gene variants on non-sex chromosomes (autosomes) and their associated traits, while those on sex chromosomes (allosomes) are termed X-linked dominant, X-linked recessive or Y-linked; these have an inheritance and presentation pattern that depends on the sex of both the parent and the child. Since there is only one copy of the Y chromosome, Y-linked traits cannot be dominant or recessive. Additionally, there are other forms of dominance, such as incomplete dominance, in which a gene variant has a partial effect compared to when it is present on both chromosomes and co-dominance, in which different variants on each chromosome both show their associated traits.

Genetic linkage is the tendency of DNA sequences that are close together on a chromosome to be inherited together during the meiosis phase of sexual reproduction. Two genetic markers that are physically near to each other are unlikely to be separated onto different chromatids during chromosomal crossover, and are therefore said to be more linked than markers that are far apart. In other words, the nearer two genes are on a chromosome, the lower the chance of recombination between them, and the more likely they are to be inherited together. Markers on different chromosomes are perfectly unlinked, although the penetrance of potentially deleterious alleles may be influenced by the presence of other alleles, and these other alleles may be located on other chromosomes than that on which a particular potentially deleterious allele is located.

<span class="mw-page-title-main">Molecular genetics</span> Scientific study of genes at the molecular level

Molecular genetics is a branch of biology that addresses how differences in the structures or expression of DNA molecules manifests as variation among organisms. Molecular genetics often applies an "investigative approach" to determine the structure and/or function of genes in an organism's genome using genetic screens. 

A genetic screen or mutagenesis screen is an experimental technique used to identify and select individuals who possess a phenotype of interest in a mutagenized population. Hence a genetic screen is a type of phenotypic screen. Genetic screens can provide important information on gene function as well as the molecular events that underlie a biological process or pathway. While genome projects have identified an extensive inventory of genes in many different organisms, genetic screens can provide valuable insight as to how those genes function.

<span class="mw-page-title-main">ENU</span> Chemical compound

ENU, also known as N-ethyl-N-nitrosourea (chemical formula C3H7N3O2), is a highly potent mutagen. For a given gene in mice, ENU can induce 1 new mutation in every 700 loci. It is also toxic at high doses.

<span class="mw-page-title-main">Mosaic (genetics)</span> Condition in multi-cellular organisms

Mosaicism or genetic mosaicism is a condition in which a multicellular organism possesses more than one genetic line as the result of genetic mutation. This means that various genetic lines resulted from a single fertilized egg. Mosaicism is one of several possible causes of chimerism, wherein a single organism is composed of cells with more than one distinct genotype.

<span class="mw-page-title-main">Non-Mendelian inheritance</span> Type of pattern of inheritance

Non-Mendelian inheritance is any pattern in which traits do not segregate in accordance with Mendel's laws. These laws describe the inheritance of traits linked to single genes on chromosomes in the nucleus. In Mendelian inheritance, each parent contributes one of two possible alleles for a trait. If the genotypes of both parents in a genetic cross are known, Mendel's laws can be used to determine the distribution of phenotypes expected for the population of offspring. There are several situations in which the proportions of phenotypes observed in the progeny do not match the predicted values.

Genetics, a discipline of biology, is the science of heredity and variation in living organisms.

<span class="mw-page-title-main">Gene mapping</span> Process of locating specific genes

Gene mapping or genome mapping describes the methods used to identify the location of a gene on a chromosome and the distances between genes. Gene mapping can also describe the distances between different sites within a gene.

P elements are transposable elements that were discovered in Drosophila as the causative agents of genetic traits called hybrid dysgenesis. The transposon is responsible for the P trait of the P element and it is found only in wild flies. They are also found in many other eukaryotes.

In molecular biology, insertional mutagenesis is the creation of mutations in DNA by the addition of one or more base pairs. Such insertional mutations can occur naturally, mediated by viruses or transposons, or can be artificially created for research purposes in the lab.

A phene is an individual genetically determined characteristic or trait which can be possessed by an organism, such as eye colour, height, behavior, tooth shape or any other observable characteristic.

Balancer chromosomes are a type of genetically engineered chromosome used in laboratory biology for the maintenance of recessive lethal mutations within living organisms without interference from natural selection. Since such mutations are viable only in heterozygotes, they cannot be stably maintained through successive generations and therefore continually lead to production of wild-type organisms, which can be prevented by replacing the homologous wild-type chromosome with a balancer. In this capacity, balancers are crucial for genetics research on model organisms such as Drosophila melanogaster, the common fruit fly, for which stocks cannot be archived. They can also be used in forward genetics screens to specifically identify recessive lethal mutations. For that reason, balancers are also used in other model organisms, most notably the nematode worm Caenorhabditis elegans and the mouse.

Transposons are semi-parasitic DNA sequences which can replicate and spread through the host's genome. They can be harnessed as a genetic tool for analysis of gene and protein function. The use of transposons is well-developed in Drosophila and in Thale cress and bacteria such as Escherichia coli.

<span class="mw-page-title-main">Reverse genetics</span> Method in molecular genetics

Reverse genetics is a method in molecular genetics that is used to help understand the function(s) of a gene by analysing the phenotypic effects caused by genetically engineering specific nucleic acid sequences within the gene. The process proceeds in the opposite direction to forward genetic screens of classical genetics. While forward genetics seeks to find the genetic basis of a phenotype or trait, reverse genetics seeks to find what phenotypes are controlled by particular genetic sequences.

<span class="mw-page-title-main">Mutagenesis (molecular biology technique)</span>

In molecular biology, mutagenesis is an important laboratory technique whereby DNA mutations are deliberately engineered to produce libraries of mutant genes, proteins, strains of bacteria, or other genetically modified organisms. The various constituents of a gene, as well as its regulatory elements and its gene products, may be mutated so that the functioning of a genetic locus, process, or product can be examined in detail. The mutation may produce mutant proteins with interesting properties or enhanced or novel functions that may be of commercial use. Mutant strains may also be produced that have practical application or allow the molecular basis of a particular cell function to be investigated.

This glossary of genetics and evolutionary biology is a list of definitions of terms and concepts used in the study of genetics and evolutionary biology, as well as sub-disciplines and related fields, with an emphasis on classical genetics, quantitative genetics, population biology, phylogenetics, speciation, and systematics. Overlapping and related terms can be found in Glossary of cellular and molecular biology, Glossary of ecology, and Glossary of biology.

References

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  15. ↑ Gibson G, Muse SV (2009). A Primer of Genome Science (Third ed.). Sinauer Press.
  16. ↑ Hamilton V (2016-07-19). "The Secrets of Life". Science History Institute. Retrieved 2018-09-25.
  17. ↑ "An Overview of the Human Genome Project". National Human Genome Research Institute (NHGRI). Retrieved 2018-09-25.
  18. 1 2 Gilbert S (2014). Developmental Biology. Sutherland, MA: Sinauer Associates Inc. ISBN   978-0-87893-978-7.
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