Recitation 15 Mutant Hunt By complementation Example Imagine a mutant yeast that unlike all other yeast cannot synthesize arginine To nd the gene we make a plasmid library in which each plasmid has a wild type copy of a different gene We then transform a different mutant yeast colony with a different plasmid We then see which one rescues turn the mutant into a wild type the mutant phenotype The conclusion we can draw from this is that the plasmid transformed in that rescued colonies contains the gene of interest Caveats to this method You must be working with recessive mutations usually a loss of function Adding wild type to a gain of function will not give us a rescued phenotype Must be in something that you can transform genetically Mutational mapping with SNPs Example ATTGC and ATCGC SNPs can be causative of a phenotype If it does not cause a different phenotype it could be a silent mutation it could not be translated i e it would be in an intron it might not be transcribed SNPs single nucleotide polymorphisms meaning we are looking at a single base pair difference at a given position among a population Transcription starts at the promoter and stops at the transcriptional termination site Translation starts at AUG the start codon and stops at the stop codon Regions that are transcribed but not translated are a part of the untranslated region UTR Find a SNP close to your mutation by testing recombination frequencies We then map our disease mutation more exactly by chromosome walking walking from the SNP to nd a mutation that everyone with the disease has and then we know we have found the diseased allele Sequencing the Human Genome What do we nd Microarrays Genes Transposons inserted repeats of DNA Non coding regions i e promoters Used for expression analysis which is testing transcriptional level control Isolate mRNA from a given tissue Use reverse transcriptase to get cDNA Let the cDNA hybridize to a chip Each spot on the chip has a different gene sequence Conjugate stick on each mRNA molecule with a orescent molecule If orescent more expression
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