Deck 17: Recombinant Dna Technology

Question 1

A ddNTP, used often in DNA sequencing, lacks a(n) carbons.
A) OH; 2'; 3'

A) carboxyl; 5'; 3'
B) methyl; 2'; 3'
D) OH; 2'; 5'
E) None of the listed answers is correct.

Answer 1

A

Question 2

Molecular biologists rely on many, often sophisticated, techniques to pursue their
discipline. Ultracentrifugation, electron microscopy, X -ray diffraction, electrophoresis, and computer interfacing are 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.

Answer 2

Bacteriophage: useful vector, relatively simple genome, short generation time. Bacteria: relatively simple, short generation time, simple growth requirements, well understood genetics, transformable. Viruses: capable of carrying
recombinant DNA and infecting eukaryotic cells. Yeast: relatively simple for a eukaryote, short generation time, simple growth requirements, transformable.

Question 3

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.

A) 300, 700, 2200
B) 400, 800, 1000 (2 of these)
C) 400, 1200, 1600
D) 300, 700, 1000, 1200
E) 700, 400, 1400, 2600

Answer 3

D

Question 4

Speculate on the origin of the restriction enzyme called EcoRI.

Question 5

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.

Question 6

What is recombinant DNA technology? What are the safety issues related to recombinant DNA technology?

Question 7

What might be a reasonable function of restriction endonucleases in a bacterium, distinct from their use by molecular biologists?

Question 8

List two especially useful characteristics of cloning vectors.

A) reverse transcriptase and ligase activities
B) nonautonomous replication and transposition
C) high copy number and antibiotic resistance gene(s)
D) virulence and lysogenicity
E) ability to integrate into the host chromosome and then causing a lytic cycle

Question 9

One of the primary reasons for generating a large number of clones in a eukaryotic genomic library is that _.

A) each vector can take up only a relatively small fraction of the eukaryotic DNA
B) the host range of the vector is limited
C) each ligation product is sequence specific
D) lysogenic phages continue to integrate their DNA into the host chromosome, thus reducing the number of desired recombinant clones
E) each cosmid replicates in coordination with the host chromosome

Question 10

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?

Question 11

The PCR (polymerase chain reaction) protocol that is currently used in laboratories was facilitated by the discovery of a bacterium called Thermus aquaticus in a hot spring inside Yellowstone National Park, in Wyoming. This organism contains a heat -stable form of DNA polymerase known as Taq polymerase, which continues to function even after it has been
Heated to 95°C. Why would such a heat -stable polymerase be beneficial in PCR?

A) Each cycle includes a "hot" denaturation phase (95°C), which separates the hydrogen bonds that hold the strands of the template DNA together.
B) Each cycle includes a "hot" denaturation phase (95°C), which serves to sterilize the culture.
C) Each cycle includes a "hot" saturation phase (95°C), which allows the primers to anneal to the target DNA.
D) Each cycle includes a "hot" denaturation phase (95°C), which activates the Taq polymerase.
E) More than one of the above are correct.

Question 12

Nucleic acid blotting is widely used in recombinant DNA technology. In a Southern blot, one generally _.

A) examines amino acid substitutions with radioactive probes
B) ligates DNA with DNA ligase
C) hybridizes filter -bound RNA with a DNA probe
D) hybridizes filter -bound DNA with a DNA probe
E) cleaves RNA with restriction endonucleases

Question 13

List, in order, the steps usually followed in producing recombinant DNA molecules in a plasmid vector.

Question 14

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

A) transfection
B) immunoprecipitation
C) DNA sequencing
D) plasmolysis
E) restriction digestion

Question 15

In the context of molecular genetics, reverse transcription PCR (RT -PCR) refers to _.

A) translating in the 3' to 5' direction
B) assembling a DNA sequence from an mRNA
C) making an amino acid sequence from a DNA sequence
D) assembling an RNA sequence from a DNA sequence
E) transcribing first, then translating

Question 16

Restriction endonucleases are especially useful if they generate "sticky" ends. What makes an end sticky?

A) 5' cap
B) poly -A sequences
C) blunt ends
D) single -stranded complementary tails
E) 3' cap

Question 17

Vectors such as pUC18 and others of the pUC series contain a large number of restriction enzyme sites clustered in one region. Which term describes this advantageous arrangement of restriction sites?

A) palindrome
B) multiple cloning site
C) consensus sequence
D) fi -galactosidase
E) complementation

Question 18

In the context of recombinant DNA technology, what is meant by the term vector?

Question 19

Words such as did, mom, and pop have something in common with the fundamental tool of recombinant DNA technology. In the context of recombinant DNA technology, which term would be used to describe such words?

A) lysogenic
B) conjugation
C) palindromic
D) prototrophic
E) insertion

Question 20

Some restriction enzymes cleave DNA in such a manner as to produce blunt ends.
Ligation of blunt end fragments is most often 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.

Question 21

Assume that a researcher conducted a 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 more likely contain the recombinant plasmid? Why?

Question 22

What is a cDNA molecule?

Question 23

Assume that you have cut h 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.

Question 24

In general, the main goal of cloning is to include as many different genes as possible in a single cloning vector.

Question 25

Restriction endonucleases typically recognize palindromic DNA sequences and often generate "sticky ends" or single -stranded DNA overhangs at cut sites.

Question 26

Nucleic acid blotting is commonly used in molecular biology. Two types, Southern blots and Northern blots, involve gel electrophoresis and a filter, which holds the nucleic acid. Briefly describe the procedure of "blotting" in this context and
differentiate between Southern and Northern blots.

Question 27

Of what advantage is it to have a multiple cloning site (multiple unique restriction sites) embedded in the lacZ component of a plasmid?

Question 28

In the polymerase chain reaction, what is the purpose of the initial high temperature? What is the purpose of cooling in the second step?

Question 29

A common term for a plasmid or other DNA element that serves as a cloning vehicle is vector.

Question 30

Name at least two typical characteristics of a DNA cloning plasmid.

Question 31

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

Question 32

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

Question 33

How might cDNA libraries be used to assign particular genes to the diseased state of a cell?

Question 34

Which term refers to the process in which DNA can be introduced into host bacterial cells?

Question 35

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

Question 36

Some restriction endonucleases are capable of producing blunt ends; others can generate "sticky" ends.

Question 37

To isolate a bacterium with a plasmid that carries a desired DNA fragment cloned within the ampicillin resistance gene, we should grow bacteria in a medium that contains ampicillin.

Question 38

If a researcher wishes to clone a gene using typical restriction endonucleases, how does the restriction endonuclease recognize genes in the genome?

Question 39

When propagating a clone in the lambda phage, would you have more immediate success if the phage entered the lysogenic cycle or the lytic cycle?

Question 40

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?

Question 41

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

Question 42

In recombinant DNA technology, YAC, RFLP, and h have identical uses.

Question 43

The main purpose of a probe is its insertion in plasmid DNA.

Question 44

Reverse transcriptase is often used as the heat -stable enzyme in PCR.

Question 45

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

Question 46

During a PCR, heat is provided to inactivate the polymerase enzyme.

Question 47

The function of a ddNTP in DNA sequencing is to methylate guanine.

Question 48

E. coli is a common YAC.

Question 49

In recombinant DNA technology, a YAC is an enzyme isolated from a large South American, four -legged mammal.

Question 50

A restriction map provides the location of sites cleaved by restriction enzymes.