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A genetic screen or mutagenesis screen is an experimental technique used to identify and select individuals who possess a
phenotype In genetics, the phenotype () is the set of observable characteristics or traits of an organism. The term covers the organism's morphology or physical form and structure, its developmental processes, its biochemical and physiological pr ...
of interest in a mutagenized population. Hence a genetic screen is a type of
phenotypic screen In genetics, the phenotype () is the set of observable characteristics or traits of an organism. The term covers the organism's morphology or physical form and structure, its developmental processes, its biochemical and physiological proper ...
. Genetic screens can provide important information on
gene In biology, the word gene (from , ; "... Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a b ...
function as well as the molecular events that underlie a biological process or pathway. While
genome project Genome projects are scientific endeavours that ultimately aim to determine the complete genome sequence of an organism (be it an animal, a plant, a fungus, a bacterium, an archaean, a protist or a virus) and to annotate protein-coding genes and o ...
s have identified an extensive inventory of genes in many different organisms, genetic screens can provide valuable insight as to how those genes function.


Basic screening

Forward genetics 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 partic ...
(or a forward genetic screen) starts with a phenotype and then attempts to identify the causative mutation and thus gene(s) responsible for the phenotype. For instance, the famous screen by
Christiane Nüsslein-Volhard Christiane (Janni) Nüsslein-Volhard (; born 20 October 1942) is a German developmental biologist and a 1995 Nobel Prize in Physiology or Medicine laureate. She is the only woman from Germany to have received a Nobel Prize in the sciences. Nà ...
and
Eric Wieschaus Eric Francis Wieschaus (born June 8, 1947 in South Bend, Indiana) is an American evolutionary developmental biologist and 1995 Nobel Prize-winner. Early life Born in South Bend, Indiana, he attended John Carroll Catholic High School in Birming ...
mutagenized fruit flies and then set out to find the genes causing the observed mutant phenotypes. Successful forward genetic screens often require a defined genetic background and a simple experimental procedure. That is, when multiple individuals are mutagenized they should be genetically identical so that their wild-type phenotype is identical too and mutant phenotypes are easier to identify. A simple screening method allows for a larger number of individuals to be screened, thereby increasing the probability of generating and identifying mutants of interest. Since natural allelic mutations are rare prior to screening geneticists often mutagenize a population of individuals by exposing them to a known mutagen, such as a chemical or radiation, thereby generating a much higher frequency of chromosomal mutations. In some organisms mutagens are used to perform saturation screens, that is, a screen used to uncover ''all'' genes involved in a particular phenotype.
Christiane Nüsslein-Volhard Christiane (Janni) Nüsslein-Volhard (; born 20 October 1942) is a German developmental biologist and a 1995 Nobel Prize in Physiology or Medicine laureate. She is the only woman from Germany to have received a Nobel Prize in the sciences. Nà ...
and
Eric Wieschaus Eric Francis Wieschaus (born June 8, 1947 in South Bend, Indiana) is an American evolutionary developmental biologist and 1995 Nobel Prize-winner. Early life Born in South Bend, Indiana, he attended John Carroll Catholic High School in Birming ...
were the first individuals to perform this type of screening procedure in animals.
Reverse 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 pr ...
(or a reverse genetic screen), starts with a known gene and assays the effect of its disruption by analyzing the resultant phenotypes. For example, in a knock-out screen, one or more genes are completely deleted and the deletion mutants are tested for phenotypes. Such screens have been done for all genes in many bacteria and even complex organisms, such as ''C. elegans''. A reverse genetic screen typically begins with a gene sequence followed by targeted inactivation. Moreover, it induces mutations in model organisms to learn their role in disease. Reverse genetics is also used to provide extremely accurate statistics on mutations that occur in specific genes. From these screens you are able to determine how fortuitous the mutations are, and how often the mutations occur.


Screening variations

Many screening variations have been devised to elucidate a gene that leads to a mutant phenotype of interest.


Enhancer

An enhancer screen begins with a mutant individual that has an affected process of interest with a known gene mutation. The screen can then be used to identify additional genes or gene mutations that play a role in that biological or physiological process. A genetic enhancer screen identifies mutations that enhance a phenotype of interest in an already mutant individual. The phenotype of the double mutant (individual with both the enhancer and original background mutation) is more prominent than either of the single mutant phenotypes. The enhancement must surpass the expected phenotypes of the two mutations on their own, and therefore each mutation may be considered an enhancer of the other. Isolating enhancer mutants can lead to the identification of interacting genes or genes which act redundantly with respect to one another.


Suppressor

A suppressor screen is used to identify suppressor mutations that alleviate or revert the phenotype of the original mutation, in a process defined as synthetic viability. Suppressor mutations can be described as second mutations at a site on the chromosome distinct from the mutation under study, which suppress the phenotype of the original mutation. If the mutation is in the same gene as the original mutation it is known as intragenic suppression, whereas a mutation located in a different gene is known as extragenic suppression or intergenic suppression. Suppressor mutations are extremely useful to define the functions of biochemical pathways within a cell and the relationships between different biochemical pathways.


Temperature sensitive

A temperature-sensitive screen involves performing temperature shifts to enhance a mutant phenotype. A population grown at low temperatures would have a normal phenotype; however, the mutation in the particular gene would make it unstable at a higher temperature. A screen for temperature sensitivity in fruit flies, for example, might involve raising the
temperature Temperature is a physical quantity that expresses quantitatively the perceptions of hotness and coldness. Temperature is measurement, measured with a thermometer. Thermometers are calibrated in various Conversion of units of temperature, temp ...
in the cage until some flies faint, then opening a portal to let the others escape. Individuals selected in a screen are liable to carry an unusual version of a
gene In biology, the word gene (from , ; "... Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a b ...
involved in the phenotype of interest. An advantage of alleles found in this type of screen is that the mutant phenotype is conditional and can be activated by simply raising the temperature. A
null mutation A null allele is a nonfunctional allele (a variant of a gene) caused by a genetic mutation. Such mutations can cause a complete lack of production of the associated gene product or a product that does not function properly; in either case, the all ...
in such a gene may be lethal to the embryo and such mutants would be missed in a basic screen. A famous temperature-sensitive screen was carried out independently by
Lee Hartwell Leland Harrison (Lee) Hartwell (born October 30, 1939 in Los Angeles, California) is former president and director of the Fred Hutchinson Cancer Research Center in Seattle, Washington. He shared the 2001 Nobel Prize in Physiology or Medicine wit ...
and
Paul Nurse Sir Paul Maxime Nurse (born 25 January 1949) is an English geneticist, former President of the Royal Society and Chief Executive and Director of the Francis Crick Institute. He was awarded the 2001 Nobel Prize in Physiology or Medicine along ...
to identify mutants defective in the cell cycle in ''
S. cerevisiae ''Saccharomyces cerevisiae'' () (brewer's yeast or baker's yeast) is a species of yeast (single-celled fungus microorganisms). The species has been instrumental in winemaking, baking, and brewing since ancient times. It is believed to have bee ...
'' and ''
S. pombe ''Schizosaccharomyces pombe'', also called "fission yeast", is a species of yeast used in traditional brewing and as a model organism in molecular and cell biology. It is a unicellular eukaryote, whose cells are rod-shaped. Cells typically measur ...
'', respectively.


RNAi

RNA interference (RNAi) screen is essentially a forward genetics screen using a reverse genetics technique. Similar to classical genetic screens in the past, large-scale RNAi surveys success depends on a careful development of phenotypic assays and their interpretation. In
Drosophila ''Drosophila'' () is a genus of flies, belonging to the family Drosophilidae, whose members are often called "small fruit flies" or (less frequently) pomace flies, vinegar flies, or wine flies, a reference to the characteristic of many speci ...
, RNAi has been applied in cultured cells or in vivo to investigate gene functions and to effect the function of single genes on a genome-wide scale. RNAi is used to silence gene expression in Drosophila by injecting dsRNA into early embryos, and interfering with
Frizzled Frizzled is a family of atypical G protein-coupled receptors that serve as receptors in the Wnt signaling pathway and other signaling pathways. When activated, Frizzled leads to activation of Dishevelled in the cytosol. Species distribution Fr ...
and Frizzled2 genes creating defects in embryonic patterning that mimic loss of wingless function.


CRISPR

CRISPR/Cas is primarily used for reverse genetic screens. CRISPR has the ability to create libraries of thousands of precise genetic mutations and can identify new tumors as well as validate older tumors in cancer research. Genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences identify genes essential for cell viability in cancer. Bacterial CRISPR–Cas9 system for engineering both loss of function (LOF) and gain of function (GOF) mutations in untransformed human intestinal organoids in order to demonstrate a model of Colorectal cancer (CRC). It can also be used to study functional consequences of mutations in vivo by enabling direct genome editing in somatic cells.


Mapping mutants

By the
classical genetics Classical genetics is the branch of genetics based solely on visible results of reproductive acts. It is the oldest discipline in the field of genetics, going back to the experiments on Mendelian inheritance by Gregor Mendel who made it possible ...
approach, a researcher would then locate (map) the gene on its
chromosome A chromosome is a long DNA molecule with part or all of the genetic material of an organism. In most chromosomes the very long thin DNA fibers are coated with packaging proteins; in eukaryotic cells the most important of these proteins are ...
by
crossbreeding A crossbreed is an organism with purebred parents of two different breeds, varieties, or populations. ''Crossbreeding'', sometimes called "designer crossbreeding", is the process of breeding such an organism, While crossbreeding is used to main ...
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
allele An allele (, ; ; modern formation from Greek ἄλλος ''állos'', "other") is a variation of the same sequence of nucleotides at the same place on a long DNA molecule, as described in leading textbooks on genetics and evolution. ::"The chro ...
s. With the advent of genomic sequences for model systems such as ''
Drosophila melanogaster ''Drosophila melanogaster'' is a species of fly (the taxonomic order Diptera) in the family Drosophilidae. The species is often referred to as the fruit fly or lesser fruit fly, or less commonly the " vinegar fly" or "pomace fly". Starting with ...
,'' '' Arabidopsis thaliana'' and ''
C. elegans ''Caenorhabditis elegans'' () is a free-living transparent nematode about 1 mm in length that lives in temperate soil environments. It is the type species of its genus. The name is a blend of the Greek ''caeno-'' (recent), ''rhabditis'' (r ...
'' many single nucleotide polymorphisms (SNPs) have now been identified that can be used as traits for mapping. In fact, the Heidelberg screen, allowing mass testing of mutants and developed in 1980 by Nüsslein-Volhard and Wieschaus, cleared the way for future scientists in this field. SNPs are the preferred traits for mapping since they are very frequent, on the order of one difference per 1000 base pairs, between different varieties of organism. Mutagens such as random DNA insertions by
transformation Transformation may refer to: Science and mathematics In biology and medicine * Metamorphosis, the biological process of changing physical form after birth or hatching * Malignant transformation, the process of cells becoming cancerous * Tran ...
or active
transposons A transposable element (TE, transposon, or jumping gene) is a nucleic acid sequence in DNA that can change its position within a genome, sometimes creating or reversing mutations and altering the cell's genetic identity and genome size. Tran ...
can also be used to generate new mutants. These techniques have the advantage of tagging the new alleles with a known molecular (DNA) marker that can facilitate the rapid identification of the gene.


Positional cloning

Positional cloning is a method of gene identification in which a gene for a specific phenotype is identified only by its approximate chromosomal location (but not the function); this is known as the candidate region. Initially, the candidate region can be defined using techniques such as
linkage analysis 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 separ ...
, and positional cloning is then used to narrow the candidate region until the gene and its mutations are found. Positional cloning typically involves the isolation of partially overlapping DNA segments from genomic libraries to progress along the chromosome toward a specific gene. During the course of positional cloning, one needs to determine whether the DNA segment currently under consideration is part of the gene. Tests used for this purpose include cross-species hybridization, identification of unmethylated
CpG islands The CpG sites or CG sites are regions of DNA where a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases along its 5' → 3' direction. CpG sites occur with high frequency in genomic regions called CpG isl ...
,
exon trapping Exon trapping is a molecular biology technique to identify potential exons in a fragment of eukaryote DNA of unknown intron- exon structure.Duyk, G. M, S. W. Kim, R. M Myers, and D. R Cox. 1990. “Exon Trapping: a Genetic Screen to Identify Ca ...
, direct cDNA selection, computer analysis of DNA sequence, mutation screening in affected individuals, and tests of gene expression. For genomes in which the regions of genetic polymorphisms are known, positional cloning involves identifying polymorphisms that flank the mutation. This process requires that DNA fragments from the closest known genetic marker are progressively cloned and sequenced, getting closer to the mutant allele with each new clone. This process produces a contig map of the
locus Locus (plural loci) is Latin for "place". It may refer to: Entertainment * Locus (comics), a Marvel Comics mutant villainess, a member of the Mutant Liberation Front * ''Locus'' (magazine), science fiction and fantasy magazine ** ''Locus Award' ...
and is known as
chromosome walking Primer walking is a technique used to clone a gene (e.g., disease gene) from its known closest markers (e.g., known gene). As a result, it is employed in cloning and sequencing efforts in plants, fungi, and mammals with minor alterations. This te ...
. With the completion of genome sequencing projects such as the Human Genome Project, modern positional cloning can use ready-made contigs from the genome sequence databases directly. For each new DNA clone a polymorphism is identified and tested in the mapping population for its recombination frequency compared to the mutant phenotype. When the DNA clone is at or close to the mutant allele, the recombination frequency should be close to zero. If the chromosome walk proceeds through the mutant allele, the new polymorphisms will start to show increase in recombination frequency compared to the mutant phenotype. Depending on the size of the mapping population, the mutant allele can be narrowed down to a small region (<30 Kb). Sequence comparison between
wild type The wild type (WT) is the phenotype of the typical form of a species as it occurs in nature. Originally, the wild type was conceptualized as a product of the standard "normal" allele at a locus, in contrast to that produced by a non-standard, "m ...
and
mutant In biology, and especially in genetics, a mutant is an organism or a new genetic character arising or resulting from an instance of mutation, which is generally an alteration of the DNA sequence of the genome or chromosome of an organism. It ...
DNA in that region is then required to locate the DNA
mutation 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 replication, DNA or viral repl ...
that causes the phenotypic difference. Modern positional cloning can more directly extract information from genomic sequencing projects and existing data by analyzing the genes in the candidate region. Potential disease genes from the candidate region can then be prioritized, potentially reducing the amount of work involved. Genes with expression patterns consistent with the disease phenotype, showing a (putative) function related to the phenotype, or homologous to another gene linked to the phenotype are all priority candidates. Generalization of positional cloning techniques in this manner is also known as positional gene discovery. Positional cloning is an effective method to isolate disease genes in an unbiased manner and has been used to identify disease genes for
Duchenne muscular dystrophy Duchenne muscular dystrophy (DMD) is a severe type of muscular dystrophy that primarily affects boys. Muscle weakness usually begins around the age of four, and worsens quickly. Muscle loss typically occurs first in the thighs and pelvis follow ...
,
Huntington's disease Huntington's disease (HD), also known as Huntington's chorea, is a neurodegenerative disease that is mostly inherited. The earliest symptoms are often subtle problems with mood or mental abilities. A general lack of coordination and an uns ...
, and cystic fibrosis. However, complications in the analysis arise if the disease exhibits locus heterogeneity.


References

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External links


Principles of Map-based or Positional Cloning of Plant Genes


Classical genetics Molecular genetics Medical genetics Genetics experiments