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| ===Suppressor Screen=== | ===Suppressor Screen=== | ||
| A '''genetic suppressor screen''' is used to identify suppressor mutations which alleviate or revert the phenotype of the original mutation. 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 <ref name= WormS> Hodgkin J. Genetic suppression. 2005 Dec 27. In: WormBook: The Online Review of C. elegans Biology . Pasadena (CA): WormBook; 2005-.</ref>. | A '''genetic suppressor screen''' is used to identify suppressor mutations which alleviate or revert the phenotype of the original mutation. 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 <ref name= WormS> Hodgkin J. Genetic suppression. 2005 Dec 27. In: WormBook: The Online Review of C. elegans Biology . Pasadena (CA): WormBook; 2005-.</ref>. 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 <ref name= Mcgraw>. | ||
| ===Temperature Sensitive Screen=== | ===Temperature Sensitive Screen=== | ||
Revision as of 16:22, 3 May 2012
This article is about a method to identify the functions of genes. For screening or testing for genetic diseases, see genetic testing.A genetic screen (also known as a phenotypic screen), is an experimental technique or procedure used to identify and select for individuals who possess a phenotype of interest in a mutagenized population . 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 .
Basic Screening
Forward genetics (or a forward genetic screen) is an approach used to identify genes (or set of genes) responsible for a particular phenotype of an organism. Reverse genetics (or a reverse genetic screen) on the other hand, analyzes the phenotype of an organism following the disruption of a known gene. In short, forward genetics starts with a phenotype and moves towards identifying the gene(s) responsible, whereas reverse genetics starts with a known gene and assays the effect of its disruption by analyzing the resultant phenotypes. Both forward and reverse genetic screens aim to determine gene function .
Successful forward genetic screens often have two key components. The first is a defined genetic background of the organism being used and the second is a simple yet constant experimental procedure to identify mutants of interest. Defined genetic backgrounds allow researchers to identify and locate affected genes in mutant individuals with greater efficiency. A simplified screening method is beneficial because it 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 . 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 transposons 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.
Screening Variations
Many variations have been cleverly devised to elucidate a gene that leads to a mutant phenotype of interest.
Enhancer Screen
An enhancer screen begins with a mutant individual that has an affected process of interest with a known gene disruption/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 which exacerbate (or 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 much stronger 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 Screen
A genetic suppressor screen is used to identify suppressor mutations which alleviate or revert the phenotype of the original mutation. 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 <ref name= Mcgraw>.
Temperature Sensitive Screen
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.
Mapping mutants
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. 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.
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 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.
External links
References
- ^ Hartwell, L. H., Hood, L., Goldberg, M. L., Reynolds, A. E., Silver, L. M., & Veres, R. C. (2008). Genetics: From Genes to Genomes. New York: McGraw-Hill
- Nature. (2002). The Art and Design of Genetic Screens. Nature Reviews.
- Page, Damian (2002). "The Art and Design of Genetic Screens: Arabidopsis thaliana" (PDF). Nature. 3: 124–136. Retrieved 2 May 2012.
{{cite journal}}: Unknown parameter|coauthors=ignored (|author=suggested) (help); Unknown parameter|month=ignored (help) - Herman RK, Yochem J. Genetic enhancers. 2005 Sep 16. In: WormBook: The Online Review of C. elegans Biology . Pasadena (CA): WormBook; 2005-.
- Hodgkin J. Genetic suppression. 2005 Dec 27. In: WormBook: The Online Review of C. elegans Biology . Pasadena (CA): WormBook; 2005-.