BPS 4101 Lecture Notes - Lecture 12: Taqman, Myf5, Reference Genome
23 Jan 2018
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How would you define the term transcriptome: entire set of rna transcripts in an organism, complete set of rna transcripts (mrna, ncrna) that are produced by the genome, under specific circumstances or in a specific cell. Isolate mrna fraction from total mrna and convert it to cdna: can use oligodt primer. Avoids possible contamination of genomic dna - will only get product if primer binds to cdna. Fluorescent signal increases1000 x when bound to dsdna. Disadvantage: lack of specificity (binds to all dsdna - primer dimers); cannot multiplex (multiple genes). Seq specific probe (fluo quenched when not bound) Exploits 5"-3" exonuclease activity of pol cleaves the bond between nts of probe. Diff reporters emitting light at diff wave lengths for ea. gene (multiplex) How can we get a larger snapshot of mrna expression in the cell: thousands of mrnas simultaneously, microarrays, rna seq.
