Exam 8: Recombinant Dna Technology and Molecular Cloning

In the context of recombinant DNA technology, plasmids are used as vectors. This means That plasmids

How does a genomic library differ from a gene clone?

Reverse transcriptase synthesizes

If you had an antibody to the protein encoded by an unknown gene of interest, what type of library could you screen to identify the sequence of the gene? Using a diagram, explain how this would work.

The following DNA ends were generated by some common restriction enzymes. The dashes indicate the presence of additional nucleotides. Which of these ends could be labeled using Klenow? (There may be more than one.) Explain your answer.

To analyze the results of a DNA sequencing reaction, which type of gel should you prepare?

The following is a physical map of a region you are mapping for RFLP analysis: ‪ The numbered vertical lines represent restriction sites recognized by SmaI. The red sites (2 and 3) are polymorphic, the others are not. You cut the DNA with SmaI, electrophorese the fragments, and Southern blot them to a membrane. You have a choice of two probes that recognize the DNA regions shown above: probe A, green line; probe B, blue line. (a) Explain which probe you would use for analysis and why the other choice would be unsuitable. (b) Give the sizes of bands you will detect in individuals homozygous for the following genotypes with respect to sites 2 and 3.

What does it mean to say that a genetic marker and a disease gene are closely linked?

Compare and contrast degenerate probes and EST-based probes.

Starting with the nucleotide sequence of the human DNA ligase I gene, describe how you would search for a homologous gene in another organism whose genome has been sequenced, such as the pufferfish Tetraodon nigroviridis. Then, describe how you would obtain the protein and test it for ligase activity.

It is common to use ddNTPs (dideoxynucleoside triphosphates) for sequencing DNA because, when incorporated into a growing DNA polymer, the ddNTP acts as a chain terminator, allowing the researcher an opportunity to determine the last-added base to a given chain. From your knowledge of nucleotide structure and DNA synthesis, what is the most likely attribute of ddNTPs for DNA sequencing?

Describe the principle of antibody-affinity chromatography. For one protein that is recognized by the antibody and a second protein that is not, illustrate at which point each protein will be released from the column.

In red-white selection for recombinant yeast artificial chromosome (YAC) vectors in yeast cells, a red colony contains

Which of the following enzymes is involved in the PCR process?

A chromotography column in which oligo-dT is linked to an inert substance is useful in separating eukaryotic mRNA from other RNA molecules. On what principle does this column operate?

Describe the process of cloning a DNA fragment into a YAC. How would you screen for clones that contain an insert?

Which of the following statements regarding 454 pyrosequencing is not true?

Compare and contrast genomic libraries with cDNA libraries.

Automated DNA sequencing differs from manual DNA sequence in which way?

Compare and contrast the principles of autoradiography and phosphorimaging.