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| Homework 5 | |||
Answer Key in PDF format
Bioc471a/571a Homework 2 - Due at the start of class September 25
1. (2 pts) Why is it not a good idea to have RNase H activity present in the first strand cDNA reaction, but it is required for the second strand reaction in the replacement strategy? Does the replacement strategy result in synthesis of full-length cDNA products that include the first nucleotide of the mRNA transcript? Explain.
2. (2 pts.) Based on the flow scheme for oligonucleotide adaptors in figure 5.4, and use of the lZapII cDNA cloning vector, would blue-white screening of plagues distinguish between recombinants that contain EcoRI adapted cDNA inserts, and those that only have EcoRI adaptor dimers (false-positive)? Explain. If after isolating a recombinant cDNA clone from this library you discover that the insert contains an internal Eco RI site, what two explanations could account for this and how would you distinguish between these possibilities?
3. (2 pts.) Based on the flow scheme for oligonucleotide screening of a cDNA library in figure 5.6, how many different oligonucleotide sequences are represented in the NT probe pool? What is the rationale for using the two probes in successive screenings, i.e., screen first with the NT probe and then the CT probe? Will any bona fide cDNAs be missed using this strategy? Explain.
4. (2 pts.) What three criteria must be met for a functional cDNA screening strategy to work using the plasmid sib selection approach? Describe the functional screening assay initially used by Caterina et al. (Nature 389:816-824, 1997) to identify the cDNA insert encoding VR1. Describe the assay they used to quantitate the binding specificity of VR1 for capsaicin relative to compounds in the poblano verde chili.
5. (2 pts.) What are three key functional components required for functional cDNA screening using the yeast two-hybrid strategy? What function in the bait protein would give a high background signal, i.e., show a positive signal even in the absence of a target protein? What is the advantage of having both the Gal4-dependent His3 and lacZ reporter genes in the host yeast strain?
