User:KKnowles2022/Forward genetics

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. 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. 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. These point mutations are typically loss-of-function or null alleles because they generate stop codons in the DNA sequence. 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.

Radiation mutagenesis
Other methods such as using radiation to cause large deletions and chromosomal rearrangements can be used to generate mutants as well. 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.

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. 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.