Biochemistry Questions: DNA

1. Attach a page to your test that incorporates a diagram of just the plasmid map and describe all of the important freatures of the plasmid that are important for the cloning and screening of your DNA segment.

2. Describe how you would select for positive E.coli clones containing the plasmid of interest with the inserted DNA segment - see Wikipedia- cloning vector.

3. Describe how DNA fingerprinting works with amplification of STRs for three different loci - use tpox, csf1po, and d7s820 as examples from STR-base (google this site)

4. Using only the common alleles for each locus described above, what are the exact repetitive sequences for each locus and describe the results you are looking for in the fingerprinting analysis?

5. Describe how PCR is used to distinguish between male and female DNA using the amelogenin STR - be specific in what investigators are trying to determine from the results and give exact base sequence difference between male and female.

6. Also, 1 in 7,000 men have deletion of the amelogenin STR in the X chromosome (called X-null). Could the FBI still determine sex with a blood sample from an X-null individual? Explain.

7. Describe all of the experiments that were used to determine the binding site and catalytic active site of the enzyme lysozyme.

8. Using molecular diagrams, give a details description of the catalytic mechanism or the hydrolysis of NAM-NAG and indicate how the binding of the ES complex facilitates the chemistry of the catalytic mechanism.

9. Describe how a cocktail of these and other HIV drugs (AZT, ddCTP) might be effective in slowing the progresssion of the AIDS disease. What are some of the side or adverse affects of these therapies?

1.

Different types of cloning vectors are used for different types of cloning experiments. The vector is chosen according to the size and type of DNA to be cloned. One type of vectors used for cloning are Plasmid vectors. Every vector contains features that allow for the removal or insertion of DNA fragment in or out of the vector. This can be performed by exposing the vector and the foreign DNA with a restriction enzyme.

Plasmids with a phage origin, the pBluescript II phagemids, are used for cloning and sequencing (construction of nested deletions for DNA sequencing, site-specific mutagenesis and gene-mapping). These phagemids have an extensive polylinker with 21 restriction enzyme recognition sites. Side polylinker T7 and T3 RNA polymerase promoters, which are used for synthesizing RNA in vitro....

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