Genetic screen 340952 218270037 2008-06-09T22:06:52Z Ciar 2026926 rv spam {{Otheruses4|a method to identify the functions of [[gene]]s|screening or testing for genetic diseases|genetic testing}} A '''genetic screen''' (often shortened to '''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 [[Genetic screen#Mapping mutants|mapped]] and cloned by '''positional cloning'''. ==Creating a mutant population== 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 F. Wieschaus|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 [[genetic map|mapping]] genes until no new genes are found. Mutagens such as random DNA insertions by [[Transformation (genetics)|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. ==Types of screen== A '''basic screen''' involves looking for a phenotype of interest in the mutated population. One might screen for obvious phenotypes such as [[Drosophila melanogaster|fruit flies]] with no wings or an ''[[Arabidopsis thaliana|Arabidopsis]]'' flower with no petals. More subtle 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. An '''enhancer/suppressor screen''' is the most sophisticated type of 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. ==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 [[Trait (biological)|trait]]s 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 ''[[Caenorhabditis elegans|C. elegans]]'' many [[Single nucleotide polymorphism|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 (genetics)|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== * [http://www.cc.ndsu.nodak.edu/instruct/mcclean/plsc731/map-based/map-based1.htm Principles of Map-based or Positional Cloning of Plant Genes [[Category:Classical genetics]] [[Category:Molecular genetics]] [[Category:Medical genetics]] [[Category:Genetics experiments]]