If there is d there is a crossover between col and

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linked to d at marker 4. If there is D, there is a crossover between col and Leu? (combination events) - Ex. Double mutant of XY, Y mutant looks like X mutant, then we can say X is masking Y. (LOOK AT EPISTASIS ANALYSIS) - Ex. Tyrp1, enzyme that stimulates the pigment precursor. MC1R, enzyme that helps to deposit the colour on fur. If dnt have this protein, then no pigment, appears yellow. - Positional cloning: AG between n and d. which gene is actually agamous? Look at gene closer to to AG. Can see how close are x and y to agamous, then we can see what genes are on the moleulcar markers, and clone the genes to further test what gene causes the phenotype. - Design sequencing primers, determine what are the open reading frames, determine the sequences on the genes. - Sequencing: ull know what mutation is exactly involved. Also called Sanger (chain- termination) method. - 3’ has OH, need OH for synthesis!! And 5’ has the phosphates . that’s why we say synthesize from 5’ tpp 3’ - ddNTP will termninate dna polymerization. (no OH present) - add ddATP, when it gets thrown in at random, it generates fragments that terminates Everytime when theres ddATP. Different fragments, all terminating with A. - Run on gel, sensitive enough to detect a nucleotide. Can detect which ends at A, T, C, G. - Lane 1, all bands end with A, lane 2 all bands terminate with T. largest size ends with A, 2 nd is A and the next is T (all separated by a single bp) through ths, we can tell what the sequence is!! Need to do 4 separate reactions. - Use colour, to determine which ends wit A, which ends with C etc. fluorescently labelled. - Contains lots of introns that don’t code the protein? So they get spliced out. Genome is quite large. mRNA 14000 nucleotides…too large to fit in and to clone it. - poly a tail at 3’ end. Then primer needs to have lots of t nucleotieds, to anneal to poly a tailRetroviruses converts RNA TO DNA. - Once we sequenced the location, we can make pcr primers. Will get a cDNA copy of all the transcripts. - Once weve cloned our gene of interest, we can use BLAST (pull out everything that looks similar to it, see what other genes are similar to it kind of like turnitin.). and we can manipulate the clone gene further. LECTURE 4: GENE REGULATION - Methods to detect transcript distribution: RNA hybridization, RT-PCR, microarrays Next- Gen sequencing. - Q: what makes cells carry out diff functions? Different red blood cells. Ex. Differentiate into being antibodies to help protect against infections/pathogens, and skin cell will produce keratin etc.
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- All diff cells express diff proteins all from genes. Have to turn on diff genes, diff regulation for all the genes, for them to become a certain type. Ex. Phosphorylation of protein can turn on or of the protein (type of regulation) - GOAL; Know the types of methods and which method to use? Best method to use for different studies.
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