Deck 17: Recombinant DNA Technology

Question 1

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)consensus sequence
C)multiple cloning site
D)?-galactosidase
E)complementation

Answer 1

C

Question 2

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.

Answer 2

Terminal deoxynucleotidyl transferase extends single-stranded ends by the addition of nucleotide tails.If complementary tails are added,the fragments can hybridize and the recombinant molecules can be ligated.

Question 3

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

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

Answer 3

A

Question 4

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

Question 5

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

Question 6

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

Question 7

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

A)citric acid cycle
B)DNA sequencing
C)restriction digestion
D)electron transport
E)plasmolysis

Question 8

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

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

Question 9

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 10

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

Question 11

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 12

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

Question 13

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

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

Question 14

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

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

Question 15

List two especially useful characteristics of cloning vectors.

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

Question 16

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.

Question 17

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)prototrophic
C)palindromic
D)conjugation
E)insertion

Question 18

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

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

Question 19

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

Question 20

What is a cDNA molecule?

Question 21

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

Question 22

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

Question 23

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

Question 24

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 25

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

Question 26

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

Question 27

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

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

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 30

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

Question 31

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

Question 32

What is meant by the designation EcoRI?

Question 33

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 34

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 35

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?

Question 36

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

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

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

Question 39

Assume that you have cut ? 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 40

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 41

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

Question 42

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

Question 43

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

Question 44

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

Question 45

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

Question 46

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

Question 47

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

Question 48

E.coli is a common YAC.

Question 49

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

Question 50

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