Deck 20: Recombinant DNA Technology

Question 1

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 1

A

Question 2

What is meant by the designation EcoRI?

Answer 2

one of the first restriction enzymes isolated from E.coli

Question 3

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.

Answer 3

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 4

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 above is correct.

Question 5

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

Question 6

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 7

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 8

Some vectors such as pUC18 and others of the pUC series contain a large number of restriction enzyme sites clustered in one region.Which term is given to this advantageous arrangement of restriction sites?

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

Question 9

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 10

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

Question 11

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 12

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 95ᵒC.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 13

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

Question 14

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 15

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

Question 16

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 17

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 18

Words such as level,civic,and kayak have something in common compared to 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 19

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.

Question 20

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 21

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

Question 22

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

Question 23

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.

Question 24

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 25

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 26

What is a cDNA molecule?

Question 27

What is the fundamental purpose of creating a knockout organism?

Question 28

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 29

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

Question 30

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

Question 31

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

Question 32

What method might be used to study a knockout mouse that,by virtue of the lost gene or genes,generates a lethal condition?

Question 33

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

Question 34

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 35

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

Question 36

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?

Question 37

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

Question 38

What term is used to refer to the process in which DNA can be introduced into host bacterial cells?

Question 39

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.

Question 40

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

Question 41

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

Question 42

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

Question 43

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

Question 44

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

Question 45

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

Question 46

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

Question 47

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

Question 48

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

Question 49

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

Question 50

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 51

E.coli is a common YAC.

Question 52

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

Question 53

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

Question 54

A knockout animal,in the context of recombinant DNA technology,is one that has been completely anesthetized.