Genetic screen
Encyclopedia
A genetic screen is a procedure or test to identify and select individuals who possess a phenotype
of interest. A genetic screen for new genes is often referred to as forward genetics as opposed to reverse genetics
, the term for identifying mutant
alleles in genes that are already known. Mutant alleles that are not tagged for rapid cloning
are mapped and cloned by positional cloning.
Since unusual allele
s and phenotypes are rare, geneticists expose the individuals that are to be screened to a mutagen
, such as a chemical or radiation, which generates mutations in their chromosomes. The use of mutagens enables "saturation screens" one of the first of which was performed by Nobel laureates Christiane Nüsslein-Volhard
and Eric Wieschaus
. A saturation screen is performed to uncover every gene
that is involved in a particular phenotype in a given species
. This is done by screening and mapping genes until no new genes are found. Mutagens such as random DNA insertions by transformation
or active transposon
s 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.
A phenotypic screen involves looking for a phenotype of interest in a mutated population. Examples include screening fruit flies
for mutants with no wings, Arabidopsis
flowers with unusually coloured petals or for mice that are deaf.
Many variations have been cleverly devised to elucidate a gene that leads to the mutant phenotype of interest. One type of screen is a temperature sensitive screen that involves temperature shifts to enhance the mutant phenotype. A population grown at low temperature 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
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
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 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 and Paul Nurse
to identify mutants defective in cell cycle in S. cerevisiae and S. pombe, respectively.
An enhancer/suppressor screen is a type of modifier genetic screen. In this case a mutagenised population has an allele of a gene that leads to a weak mutant phenotype in the biological process of interest. For example, with regard to fruit fly wing development, a weak allele may have small abnormal wings whereas a strong/null allele would have no wings. In this sensitised background it is possible to discover new mutants that either enhance the phenotype (small wings to no wings) or suppress the phenotype (small wings to normal wings). Such a screen has two advantages. First, new genes identified in the screen are often involved in the same biological process as the weak allele in the genetic background, in this case wing formation. Second, due to genetic redundancy
, the mutant genes discovered may not have a visible phenotype of their own. In a more basic screen these would not be discovered, however, in the sensitised genetic background a visible phenotype is clear.
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 allele
s. With the advent of genomic sequences for model systems such as Drosophila, Arabidopsis and C. elegans
many SNPs
have now been identified that can be used as traits for mapping. 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.
Tests used for this purpose include cross-species hybridization, identification of unmethylated CpG islands, exon trapping, 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
and is known as chromosome walking. 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
and mutant
DNA in that region is then required to locate the DNA mutation
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, Huntington's and Cystic Fibrosis. However, complications in the analysis arise if the disease exhibits locus heterogeneity.
Phenotype
A phenotype is an organism's observable characteristics or traits: such as its morphology, development, biochemical or physiological properties, behavior, and products of behavior...
of interest. A genetic screen for new genes is often referred to as forward genetics as opposed to reverse genetics
Reverse genetics
Reverse genetics is an approach to discovering the function of a gene by analyzing the phenotypic effects of specific gene sequences obtained by DNA sequencing. This investigative process proceeds in the opposite direction of so-called forward genetic screens of classical genetics...
, the term for identifying mutant
Mutation
In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...
alleles in genes that are already known. Mutant alleles that are not tagged for rapid cloning
Cloning
Cloning in biology is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments , cells , or...
are mapped and cloned by positional cloning.
Since unusual allele
Allele
An allele is one of two or more forms of a gene or a genetic locus . "Allel" is an abbreviation of allelomorph. Sometimes, different alleles can result in different observable phenotypic traits, such as different pigmentation...
s and phenotypes are rare, geneticists expose the individuals that are to be screened to a mutagen
Mutagen
In genetics, a mutagen is a physical or chemical agent that changes the genetic material, usually DNA, of an organism and thus increases the frequency of mutations above the natural background level. As many mutations cause cancer, mutagens are therefore also likely to be carcinogens...
, such as a chemical or radiation, which generates mutations in their chromosomes. The use of mutagens enables "saturation screens" one of the first of which was performed by Nobel laureates Christiane Nüsslein-Volhard
Christiane Nüsslein-Volhard
Christiane Nüsslein-Volhard is a German biologist who won the Albert Lasker Award for Basic Medical Research in 1991 and the Nobel Prize in Physiology or Medicine in 1995, together with Eric Wieschaus and Edward B...
and Eric Wieschaus
Eric F. Wieschaus
-External links:***, excellent profile**...
. A saturation screen is performed to uncover every gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
that is involved in a particular phenotype in a given species
Species
In biology, a species is one of the basic units of biological classification and a taxonomic rank. A species is often defined as a group of organisms capable of interbreeding and producing fertile offspring. While in many cases this definition is adequate, more precise or differing measures are...
. This is done by screening and mapping genes until no new genes are found. Mutagens such as random DNA insertions by transformation
Transformation (genetics)
In molecular biology transformation is the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material from its surroundings and taken up through the cell membrane. Transformation occurs naturally in some species of bacteria, but it can...
or active transposon
Transposon
Transposable elements are sequences of DNA that can move or transpose themselves to new positions within the genome of a single cell. The mechanism of transposition can be either "copy and paste" or "cut and paste". Transposition can create phenotypically significant mutations and alter the cell's...
s 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.
A phenotypic screen involves looking for a phenotype of interest in a mutated population. Examples include screening fruit flies
Drosophila melanogaster
Drosophila melanogaster is a species of Diptera, or the order of flies, in the family Drosophilidae. The species is known generally as the common fruit fly or vinegar fly. Starting from Charles W...
for mutants with no wings, Arabidopsis
Arabidopsis thaliana
Arabidopsis thaliana is a small flowering plant native to Europe, Asia, and northwestern Africa. A spring annual with a relatively short life cycle, arabidopsis is popular as a model organism in plant biology and genetics...
flowers with unusually coloured petals or for mice that are deaf.
Many variations have been cleverly devised to elucidate a gene that leads to the mutant phenotype of interest. One type of screen is a temperature sensitive screen that involves temperature shifts to enhance the mutant phenotype. A population grown at low temperature 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
Temperature is a physical property of matter that quantitatively expresses the common notions of hot and cold. Objects of low temperature are cold, while various degrees of higher temperatures are referred to as warm or hot...
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
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
involved in the phenotype of interest. An advantage of alleles found in this type of screen is that the mutant phenotype is conditional
Conditional
Conditional may refer to:*Causal conditional, if X then Y, where X is a cause of Y*Conditional mood , a verb form in many languages*Conditional probability, the probability of an event A given that another event B has occurred...
and can be activated by simply raising the temperature. A null mutation 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 and Paul Nurse
Paul Nurse
Sir Paul Maxime Nurse, PRS is a British geneticist and cell biologist. He was awarded the 2001 Nobel Prize in Physiology or Medicine with Leland H. Hartwell and R...
to identify mutants defective in cell cycle in S. cerevisiae and S. pombe, respectively.
An enhancer/suppressor screen is a type of modifier genetic screen. In this case a mutagenised population has an allele of a gene that leads to a weak mutant phenotype in the biological process of interest. For example, with regard to fruit fly wing development, a weak allele may have small abnormal wings whereas a strong/null allele would have no wings. In this sensitised background it is possible to discover new mutants that either enhance the phenotype (small wings to no wings) or suppress the phenotype (small wings to normal wings). Such a screen has two advantages. First, new genes identified in the screen are often involved in the same biological process as the weak allele in the genetic background, in this case wing formation. Second, due to genetic redundancy
Genetic redundancy
Genetic redundancy is a term typically used to describe situations where a given biochemical function is redundantly encoded by two or more genes. In these cases, mutations in one of these genes will have a smaller effect on the fitness of the organism than expected from the genes’ function....
, the mutant genes discovered may not have a visible phenotype of their own. In a more basic screen these would not be discovered, however, in the sensitised genetic background a visible phenotype is clear.
Mapping mutants
By the classical geneticsClassical genetics
Classical genetics consists of the technique and methodologies of genetics that predate the advent of molecular biology. A key discovery of classical genetics in eukaryotes was genetic linkage...
approach, a researcher would then locate (map) the gene on its chromosome
Chromosome
A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.Chromosomes...
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 allele
Allele
An allele is one of two or more forms of a gene or a genetic locus . "Allel" is an abbreviation of allelomorph. Sometimes, different alleles can result in different observable phenotypic traits, such as different pigmentation...
s. With the advent of genomic sequences for model systems such as Drosophila, Arabidopsis and C. elegans
Caenorhabditis elegans
Caenorhabditis elegans is a free-living, transparent nematode , about 1 mm in length, which lives in temperate soil environments. Research into the molecular and developmental biology of C. elegans was begun in 1974 by Sydney Brenner and it has since been used extensively as a model...
many SNPs
Single nucleotide polymorphism
A single-nucleotide polymorphism is a DNA sequence variation occurring when a single nucleotide — A, T, C or G — in the genome differs between members of a biological species or paired chromosomes in an individual...
have now been identified that can be used as traits for mapping. 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.
Positional cloning
Positional cloning is a method of gene identification in which a gene for a specific phenotype is identified, with only its approximate chromosomal location (but not the function) known, also known as the candidate region. Initially, the candidate region can be defined using techniques such as linkage analysis, 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, exon trapping, 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 (genetics)
In the fields of genetics and genetic computation, a locus is the specific location of a gene or DNA sequence on a chromosome. A variant of the DNA sequence at a given locus is called an allele. The ordered list of loci known for a particular genome is called a genetic map...
and is known as chromosome walking. With the completion of genome sequencing projects such as the Human Genome Project
Human Genome Project
The Human Genome Project is an international scientific research project with a primary goal of determining the sequence of chemical base pairs which make up DNA, and of identifying and mapping the approximately 20,000–25,000 genes of the human genome from both a physical and functional...
, 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
Genetic recombination
Genetic recombination is a process by which a molecule of nucleic acid is broken and then joined to a different one. Recombination can occur between similar molecules of DNA, as in homologous recombination, or dissimilar molecules, as in non-homologous end joining. Recombination is a common method...
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
Wild type
Wild type refers to 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, "mutant" allele...
and mutant
Mutant
In biology and especially genetics, a mutant is an individual, organism, or new genetic character, arising or resulting from an instance of mutation, which is a base-pair sequence change within the DNA of a gene or chromosome of an organism resulting in the creation of a new character or trait not...
DNA in that region is then required to locate the DNA mutation
Mutation
In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...
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, Huntington's and Cystic Fibrosis. However, complications in the analysis arise if the disease exhibits locus heterogeneity.