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Exam 20: Recombinant Dna Technology

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Question 1

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In what way are specific DNA sequences of the template amplified in the polymerase chain reaction? In other words, how does one target the target?

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Oligonucleotide primers associate by hydrogen bonding to specific sections; primers are then extended.

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In a typical PCR, primers are used to cleave specific regions of the DNA template.

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Molecular biologists rely on many, often sophisticated, techniques to pursue their discipline. One may list ultracentrifugation, electron microscopy, X-ray diffraction, electrophoresis, and computer interfacing as fundamental tools. Model organisms provide the raw materials for study. List three "organisms" (or organismic groups) often used by recombinant DNA technologists and describe a major advantage of each group.

What is meant by the designation EcoRI?

Assume that you have cut λ DNA with the restriction enzyme HindIII. You separate the fragments on an agarose gel and stain the DNA with ethidium bromide. You notice that the intensity of the stain is less in the bands that have migrated closer to the "+" pole. Give an explanation for this finding.

What is recombinant DNA technology? What would you list as safety issues associated with recombinant DNA technology?

In a PCR, primers are complementary to stretches of DNA with which they anneal.

In which of the following biochemical reactions is it common to use ddNTPs (dideoxyribonucleoside triphosphates) ?

Some restriction enzymes cleave DNA in such a manner as to produce blunt ends. Most often ligation of blunt end fragments is enhanced by the use of the enzyme terminal deoxynucleotidyl transferase. Speculate on the function of deoxynucleotidyl transferase in terms of using blunt end fragments in cloning.

Over the years, sophisticated plasmid vectors have been developed for use in recombinant DNA technology. List at least two features that have been introduced in particularly useful vectors.

List two especially useful characteristics of cloning vectors.

A ddNTP, used often in DNA sequencing, lacks a(n) ________ at the ________ and ________ carbons.

Following are four processes common to most cloning experiments: a) transforming bacteria b) plating bacteria on selective medium c) cutting DNA with restriction endonucleases d) ligating DNA fragments Place components of this list in the order in which they would most likely occur during a cloning experiment.

Assume that a given plasmid vector to be used in a cloning experiment contains 4000 base pairs of DNA. Assume also that the restriction endonuclease Cuj cuts this plasmid at the following sites (starting from an arbitrary zero point): 1000, 1500, and 3000. Given complete digestion of the plasmid with the endonuclease so that only linear fragments are produced, what sizes of DNA are expected?

Assume that one conducted a typical cloning experiment using a typical plasmid, transformed an appropriate host bacterial strain, and plated the bacteria on an appropriate X-gal medium. Blue and white colonies appeared. Which of the two types of colonies, blue or white, would most likely contain the recombinant plasmid?

Under ideal conditions, how many copies of all the sequences of the host genome should be represented in a genomic library?

What is the name of the process by which bacterial colonies (cells) are transferred from one agar plate to another, maintaining the same spatial pattern?

Assume that a plasmid (circular) is 3200 base pairs in length and has restriction sites at the following locations: 400, 700, 1400, 2600. Give the expected sizes of the restriction fragments following complete digestion.

What is the specific application of reverse transcriptase in the preparation of cDNA?

What is a popular approach that is often used to introduce the targeting vector into cells?