Exam 8: Recombinant Dna Technology and Molecular Cloning

You plan to use the polymerase chain reaction to amplify part of the DNA sequence shown below, using oligonucleotide primers that are hexamers matching the regions shown in red. (In practice, hexamers are too short for most purposes). State the sequence of the primer oligonucleotides that should be used, including their polarity (5′3′), and give the sequence of the DNA molecule that results from amplification. 5′-TAGGCATGCAATGGTAATTTTTCAGGAACCAGGGCCCTTAAGCCGTAGGCAT-3′ 3′-ATCGGTACGTTACCATTAAAAACTCCTTGGTCCCGGGAATTCGGCATCGGTA-5′

You want to join together two pieces of DNA that have been cut with SmaI. Outline a strategy you could use to increase the efficiency of blunt end ligation.

You want to clone a 1.5 kb cDNA. Which vectors discussed would be appropriate to use? Which would be inappropriate? Explain your answer.

Which step in Southern blotting allows an investigator to identify a particular DNA fragment that is present within a complex mixture of fragments?

What is suggested by the fact that type II restriction endonuclease recognition sequences are usually palindromic?

How can an amino acid sequence be used to design a gene-specific hybridization probe?

Following the injection of a linear phage DNA into E. coli host cells, the phage genome circularizes by

Describe the principle of ion-exchange chromatography. Describe the behavior of a positively charged protein and a negatively charged protein in a column containing positively charged resin during sample loading and elution.

To read an old-fashioned Sanger sequencing gel in the 5' to 3' direction, on must read the gel

Explain why DNA polymerase from a thermophilic bacterium is used in PCR instead of DNA polymerase I from E. coli.

Which of the following vectors can accommodate the largest DNA insert?

What is the difference between a restriction endonuclease recognition site and a RFLP?

What aspect of a common recombinant DNA technology tool makes direct use of knowledge acquired from studies of retrovirus replication?

What does it mean when some bands on an autoradiograph are darker than others?

The appropriate hybridization temperature during library screening is calculated according to the G + C content and the percent homology of the probe to target. The more mismatches there are:

You want to make a genomic library with DNA fragments averaging about 85 kb in length. Which vectors discussed would be appropriate to use? Which would be inappropriate? Explain your answer.

Which statement is not true about DNA binding and cleavage by type II restriction endonucleases?

Explain the terms "random walk" and "coupling" in the context of the mode of action of restriction endonucleases.

In a single PCR cycle consisting of 30 seconds at 95C, 1 min at 55C, and 1 min at 72C, what is happening in the step run at 55C?

To prepare a hybridization probe for screening a library, which labeling technique would be most desirable?