Lecture 6 - PLB 113 Lecture II. Genome Org and Gene...

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PLB 113 – Lecture II. Genome Org and Gene Expression F. Differential Gene Expression III. Gene Transfer A. Gene Transfer Systems RNA excess mRNA-single copy DNA saturation hybridization experiment • Purpose: to determine the RNA complexity of a population - number of mRNAs in a population • Starting material: — mRNA from a plant organ - leaf — Single copy DNA of known complexity • Experiment — Hybridize an excess of mRNA with labeled scDNA to saturation — Determine the fraction of scDNA that hybridizes • Analysis — Based on the fraction of scDNA that hybridizes, RNA complexity can be calculated * The amount of copies of each mRNA doesn’t matter. Why do you want excess m RNA? Want the hybridization between any single copy DNA and the corresponding mRNA to occur. When hybridization occurs, one strand of the scDNA will hybridize with the complementary mRNA. A-a’, B-b’, and C-c’. Other stuff isn’t hybridized. (doesn’t matter since they are not labeled)
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Single strand scDNA and mRNA are degraded (the ones got crossed out) by the applied enzyme. Why do we need to the factor of 2? We start the hybridization experiment with double stranded single copy DNA as a probe . One strand of the scDNA will hybridize with the corresponding mRNA. Another strand doesn’t hybridize with any mRNA. But, when we are looking for the fraction of scDNA hybridized with the mRNA, we use the number of single strand single copy DNA as the denominator. Thus, to find the complexity of mRNA from the fraction of scDNA hybridized and the complexity of scDNA, we need the factor of 2 to get the correct complexity of mRNA. **will introns be a problem in this experiment?
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Lecture 6 - PLB 113 Lecture II. Genome Org and Gene...

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