Exam 15: Analysis of Gene Function by Forward Genetics and Reverse Genetics

In creating a recombinant DNA molecule, what are two methods that prevent re- ligation of the cloning vector that contains no insert DNA?

In producing a recombinant plasmid to be used to clone a given donor insert, it is possible to cut both the donor and plasmid with the same restriction enzyme, resulting in complementary sticky ends. Assuming plenty of plasmid DNA is available, why is further selection necessary before the introduction of the plasmid into a cellular system?

Suppose you have engineered a plasmid vector in order to produce a functional animal protein in E. coli. But the product you collect, while it has the correct amino acid sequence, is in fact nonfunctional. What might you try that would have a greater chance of success?

Which of the following is true of the sequences found in DNA libraries from human tissues?

Which type of DNA library (genomic or cDNA) would include introns and promoters?

Discuss the ethical and societal reasons for and against germ- line gene therapy.

Which lists the potential steps for genetic therapy for mice with sickle cell disease (fiS/fiS) in the correct order? I. Differentiation of iPS cells into hematopoietic progenitor (HP) cells by infecting with retrovirus expressing HoxB4. II. Reprogramming tail fibroblasts into induced pluripotent stem (iPS) cells by infecting them with vectors containing transcription factors. III. Infection with a viral vector expressing Cre recombinase to remove transcription factor(s) from f iPS cells. IV. Transplantation of corrected (þA/þA) hematopoietic cells back into irradiated mice. V. Homologous recombination with fiS and wild- type fiA allele via CRISPR- Cas9 genome editing

In the case of Jesse Gelsinger, a young man who died during gene therapy, the therapy involved delivery via a viral vector. Was this germ- line or somatic therapy?

The binary approach uses both a transformation vector that contains the T- region flanked by border sequences and a disarmed ________with the genes required for virulence and conjugative transfer.

Which of the following lists the steps for cDNA library preparation in the correct order? I. Partially degrade the mRNA with RNase H II. Isolate mRNA from a specific cell/ tissue with an oligo- dT primer that hybridizes to poly- A tail III. Synthesize a strand of cDNA using DNA polymerase with mRNA fragments as primers IV. Ligate cDNAs into a cloning vector V. Create a single- stranded DNA molecule complementary to the mRNA using reverse transcriptase