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Mutagenesis and Genetic Screens

Mutagenesis and Genetic Screens. General pathway for mutational dissection of a biological process “Forward Genetics”. Fig. 12-39. General pathway for mutational dissection of a biological process “Forward Genetics”. Fig. 12-39. Classification of Mutant Type.

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Mutagenesis and Genetic Screens

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  1. Mutagenesis and Genetic Screens

  2. General pathway for mutational dissection of a biological process “Forward Genetics” Fig. 12-39

  3. General pathway for mutational dissection of a biological process “Forward Genetics” Fig. 12-39

  4. Classification of Mutant Type

  5. Test for type of mutation recovered: haplo-insufficient null Fig. 12-38

  6. Test for type of mutation recovered: haplo-insufficient null Fig. 12-38

  7. Test for type of mutation recovered: haplo-insufficient leaky Fig. 12-38

  8. Test for type of mutation recovered: gain-of-function mutation Fig. 12-38

  9. Test for type of mutation recovered: neomorphic mutation Fig. 12-38

  10. General pathway for mutational dissection of a biological process “Forward Genetics” Fig. 12-39

  11. From phenotype to gene • Once an interesting mutant is found and characterized, we want to find the gene in which the mutant occurred • Positional cloning • First use genetic mapping • Then use chromosome walking chromosome contig candidate genes mutation

  12. Candidate-gene approach • If the mutated gene is localized to a sequenced region of the chromosome, then look for genes that could be involved in the process under study • Last step: confirm gene identification • Rescue of phenotype • Mutations in same gene in different alleles

  13. Insertional mutagenesis • Alternative to chromosome walking • To reduce time and effort required to identify mutant gene • Insert piece of DNA that disrupts genes • Inserts randomly in chromosomes • Make collection of individuals • Each with insertion in different place • Screen collection for phenotypes • Use inserted DNA to identify mutated gene

  14. Insertional mutagens • Transposable elements • Mobile elements jump from introduced DNA • e.g., P elements in Drosophila • Or start with a small number of nonautonomous elements • Mobilize by introducing active element • e.g., AC/DS elements in plants • Single-insertion elements • e.g., T-DNA in plants • Once insert, can’t move again

  15. Genome-Wide Phenotypic Analysis:“Phenomics”

  16. High-Throughput Genetics Applications of genomics approaches to genetics

  17. High-throughput genetic screens • Some genetic screens are relatively straightforward • e.g., For a visible phenotype like eye color • If phenotype is subtle or needs to be measured, the screen is more time consuming • Examples • Seed weight • Behavioral traits

  18. Industrial setting for screens

  19. High-throughput genetic screen • Paradigm Genetics, Inc. performs “phenotypic profiling” • Take measurements of mutants’ physical and chemical parameters • e.g., plant height, leaf size, root density, and nutrient utilization • Different developmental times: compare to wild type

  20. Finding random mutations in your gene of interest (or every gene in the genome) • Random insertion of transposons • Random point mutations/indels

  21. Screening an insertion library PCR primers insert • PCR used to find insertion • One primer complementary to insert • Other primer complementary to gene • If get an amplification product then you have insertion • Sequence product for exact location gene Z PCR amplification insert gene Z + – amplification product on gel indicates presence of insert near gene

  22. P element piggyBac

  23. Summary of P element Gene Disruption Project

  24. TILLING • Method for finding mutations produced by chemical mutagens in specific genes • Chemical mutagenesis • Usually produces point mutations • Very high mutagenic efficiency • Generally gives more subtle phenotypes than insertions • e.g., hypomorphs, temperature sensitive mutants

  25. gene Z WT gene Z mutant PCR amplification from wild type and mutant WT mutant TILLING in Arabidopsis I EMS mutagenize seed • EMS used to mutagenize Arabidopsis • Grow individual mutagenized lines • Make primers flanking gene of interest • Amplify using PCR

  26. TILLING in Arabidopsis II • Denature DNA from pools of mutant lines • Allow to hybridize to wild-type DNA • Detect mismatches in hybridized DNA • Denaturing HPLC • Cel I enzyme cuts at mismatches • Sequence to identify site of mutation ATGCGGACTG |||||| ||| TACGCCGGAC + Cel 1 ATGCGG CTG |||||| ||| TACGCC GAC

  27. Arabidopsis TILLING Project

  28. Summary I • Forward genetics • Mutation to gene function • Genetic screens • Cloning genes identified in screens • Genomics approaches to forward genetics • High-throughput genetic screens • Insertional mutagenesis • Activation tagging • Enhancer trapping and gene trapping

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