mutagenesis

basic information about mutagenesis

1. Presentation on MUTAGENESIS Submitted by : Desai Vruddhi K. M.Sc.( Agri ), 3 rd sem. Reg.No:04-AGRMA-01581-17 Dept. of GPB, C.P.C.A, SDAU GP-505 :- MUTagenesis and mutation breeding

2. Introduction Types of mutagenesis Application of mutagenesis. Handling of segregating population Screening/selection Methods for Validation of mutants Advantages and l imitations of mutation breeding Contents..... . 2

3. MUTAGENESIS Mutagenesis  :- is a process by which the genetic information of an  organism is changed, resulting in a  mutation . It may occur spontaneously in nature, or as a result of exposure to  mutagens. It can also be achieved experimentally using laboratory procedures. In nature mutagenesis can lead to cancer and various  heritable diseases, but it is also a driving force of evolution.

4. Types of mutagenesis Directed mutagenesis Site-directed mutagenesis PCR mutagenesis Insertional mutagenesis Signature tagged mutagenesis Transposon mutagenesis Sequence saturation mutagenesis

5. Directed mutagenesis Directed mutagenesis , also known as  directed mutation , was a hypothesis proposing that organisms can respond to environmental stresses by orthogenetically directing mutations to certain genes or areas of the genome. Different types of directed mutagenesis are a ) oligonucleotide directed mutagenesis with M13 DNA b) oligonucleotide directed mutagenesis with plasmid DNA c) PCR Amplified oligonucleotide directed mutagenesis. advantages :- Mutation rates is high All mutations can be induced Systematic and detailed Investigation of the targeted mutation can be made

6. Site-directed mutagenesis Site-directed mutagenesis  is a molecular biology method that is used to make specific and intentional changes to the DNA sequence of a gene and any gene products. Also called  site-specific mutagenesis  or  oligonucleotide -directed mutagenesis , it is used for investigating the structure and biological activity of DNA, RNA, and protein molecules, and for protein engineering

7. Conti… In molecular biology, insertional mutagenesis is the creation of mutations of DNA by the addition of one or more base pairs. Such insertional mutations can occur naturally, mediated by viruses or transposons , or can be artificially created for research purposes in the lag. Signature-tagged mutagenesis  ( STM ) is a genetic technique used to study gene function. Recent advances in genome sequencing have allowed us to catalogue a large variety of organisms' genomes, but the function of the genes they contain is still largely unknown. Using STM, the function of the product of a particular gene can be inferred by disabling it and observing the effect on the organism. The original and most common use of STM is to discover which genes in a pathogen are involved in virulence in its host, to aid the development of new medical therapies/drugs.

8. Transposon mutagenesis Transposon mutagenesis , or  transposition mutagenesis , is a biological process that allows genes to be transferred to a host organism's chromosome, interrupting or modifying the function of an extant gene on the chromosome and causing mutation.

9. APPLICATION Site directed mutagenesis is used to study changes in protein activity that occurs as a result of the DNA manipulation. It is used to assess the activity of proteins containing known amino acid substitutions. It is used to select or screen for mutations (at the DNA, RNA or protein level) that have a desired property; and which can be manipulated further for enhance biological activity. It is used to change specific amino acid in an enzyme. In this case, the modified enzyme molecule is assayed and compared to the wild-type enzyme molecule. It is used to introduce or remove restriction endonuclease sites or tags. Site-directed mutagenesis helps scientists to study the mechanism of action behind biological reactions catalyzed by enzymes  in vivo .

10. APPLICATION Recombinant DNA technology and the use of synthetic DNA molecules make it possible for molecular biology scientists to induce specific mutations in specific genes.   In vitro  mutagenesis is used to purposefully change genetic information. The analysis of the subsequent changes in gene expression and gene products helps elucidate the functional effect of the mutation. There are various types of mutagenesis. Site-directed mutagenesis, cassette mutagenesis and random mutagenesis are some examples of mutagenesis. It is noteworthy that the particular mutagenesis method you choose to use will depend on the goal of the project and the information you have about the target gene sequence to be altered

11. Handling of segregating population

12. Conti…

13. SCREENING/SELECTION A  genetic screen  or  mutagenesis screen  is an experimental technique used to identify and select for individuals who possess a phenotype of interest in a mutagenized population. Hence a genetic screen is a type of phenotypic screen. 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.

14. 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.

15. enhancer screen 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 which 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.

16. Temperature sensitive A  temperature sensitive screen  involves performing temperature shifts to enhance a 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.

17. suppressor screen A  suppressor screen  is used to identify suppressor mutations which 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.

18. Screening/selection Mainly three types screening/selection techniques in M2 and subsequent generation Visual Mechanical/Physical Other methods

19. i ) Visual screening most effective and efficient method for identifying mutant phenotypes Visual selection often is the prime basis for selecting for disease resistance , earliness, plant height, colour changes, ion-shattering, adaptation to soil, climate, growing period etc.

20. ii) Mechanical/Physical Very efficient for seed size, shape, weight, density, etc., using appropriate sieving machinery chemical, biochemical, physiological etc. E.g.- Low alkaloid content mutants can be selected using colorimetric tests chromatographic or electrophoresis techniques may be used to select isolate protein variants

21. Methods for validation of mutants Genome-wide chips Difference screening Microarray PCR screening TILLING and ECO-TILLING

22. DNA chip technology utilizes microscopic arrays ( microarrays ) of molecules immobilized on solid surfaces for biochemical analysis. Microarrays can be used for expression analysis, polymorphism detection, DNA resequencing , and genotyping on a genomic scale. Advanced arraying technologies such as photolithograpy , micro-spotting and ink jetting, coupled with sophisticated fluorescence detection systems and bioinformatics, permit molecular data gathering at an unprecedented rate. Microarray -based characterization of plant genomes has the potential to revolutionize plant breeding and agricultural biotechnology. This review provides an overview of DNA chip technology, focusing on manufacturing approaches and biological applications.

23. TILLING and Eco-TILLING TILLING is a general reverse genetic technique that combines chemical mutagenesis with PCR based screening to identify point mutations in regions of interest TILLING is powerful technology that employed heteroduplex analysis to detect which organism in a population carry single nucleotide mutation in specific genes. Eco-TILLING is similar to TILLING, except that is objective is to identify natural genetic variation as opposed to induced mutations.

24. Methods for generating mutant varieties mutagenesis Forward genetics -chemicals -radiation Reverse genetics Insertional mutagenesis Agrobacterium mediated transformation Virus induced gene silencing RNA mediated interference transposon tagging TILLING Next generation sequencing

25. Advantages Induced mutagenesis is used for the induction of cytoplasmic male sterility. Ethidium bromide has been used for induction of cytoplasmic male sterility in perlmillet and barley mutation breeding is cheap and repid method developing new variety as compair to backcross, pedigree and bulk method Mutation breeding is more effective for the improvement of oligogenic characters such as disease resistance than polygenic traits Mutation breeding is the simple, quick and best way when a new character is to be include in vegetatively propagated crops.

26. Disadvantages Most of the mutation are deleterious and undesirable. Identification of micro mutations, which are more useful to plant breeder is usually very defficult . Since useful mutations are produced at a very low frequency, a very large plant population has to be screened to identify and isolate desirable mutants. Mutation breeding has limited scope for the genetic improvement of quantitative or polygenic characters.

27. Future prospects of mutation The mutants which have no breeding value are generally thrown away by the researchers, are now becoming important tool in genome research. Damage to the DNA of particular gene sequence is now possible in pooled samples taken from large mutated populations using novel mutation detection technique. This technique is known as ‘targets included local lesions in genomes’ or ‘TILLING’ (McCallum et al., 2000a ) which is gaining popularity in these days. ‘Denaturing high performance liquid chromatography’ or ‘DHPLC’ In this approach point mutation of high density are required for which highly efficient chemical mutagens and ionizing radiations are generally used to develop of mutated generations. McCallum et al. (2000b) though demonstrated this technique in Arabidopsis for the first time, yet it is adopted for other plant species as well. If the sequences of the targeted gene is known and the methodology for detection of single nucleotide substitutions is available, TILLING can be applied successfully.