Part
V DNA Technology and Genomics
15
Recombinant DNA Technology
CHAPTER OVERVIEW
This chapter focuses on practical applications of the microbial genetic principles discussed in previous
chapters. Although we have been altering the genetic makeup of organisms for centuries and nature has been
doing it even longer, only recently have we been able to manipulate DNA directly using genetic engineering or
recombinant DNA technology. This chapter discusses the history of DNA technology and major techniques
from restriction digests and electrophoresis to PCR and genomic libraries.
CHAPTER OBJECTIVES
After reading this chapter you should be able to:
•
•
•
•
•
discuss the use of recombinant DNA technology to genetically engineer various organisms
describe PCR, real-time PCR, and RT-PCR and discuss their usefulness to genetic engineering
discuss the key role played by restriction endonucleases and DNA ligase in genetic engineering
discuss how plasmids, phages, cosmids, and artificial chromosomes are used as vectors for insertion and
expression of foreign genes in an organism
discuss the use of both prokaryotes and eukaryotes as target organisms for foreign gene insertion
CHAPTER OUTLINE
I.
II.
Introduction
A. Genetic engineering is the deliberate modification of an organism’s genetic information by directly
changing its nucleic acid
B. Recombinant DNA technology is the collection of methods used to accomplish genetic engineering
C. The generation of a large number of genetically identical DNA molecules is called cloning
D. Biotechnology is defined as those processes in which living organisms are manipulated, particularly
at the molecular genetic level, to form useful products
Key Developments in Recombinant DNA Technology
A. Arber and Smith (late 1960s) discovered restriction endonucleases, which cleave DNA at specific
sequences; Boyer (1969) first isolated the restriction endonuclease EcoRI
B. Baltimore and Temin (1970) independently discovered reverse transcriptase; this enzyme can be
used to construct a DNA copy, called complementary DNA (cDNA), of any RNA molecule
C. Jackson, Symons, and Berg (1972) generated the first recombinant DNA molecules by using DNA
ligase to join DNA fragments together; Cohen and Boyer (1973) produced the first recombinant
plasmid (vector), which was introduced into and replicated within a bacterial host
D. Southern (1975) developed a blotting procedure for detecting (through autoradiography) specific
DNA fragments, using radioactive DNA hybridization probes; this is useful in isolating particular
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genes of interest; nonradioactive, enzyme-linked, or chemiluminescent probes can now replace the
earlier radioactive probes; they are faster and safer
E. By the late 1970s, procedures for rapidly sequencing DNA molecules, synthesizing
oligonucleotides, and expression of eukaryotic genes in bacteria had been developed
III. Polymerase Chain Reaction (PCR)
A. PCR is used to synthesize large quantities of a specific DNA fragment without cloning it
B. Synthetic DNA molecules with sequences identical to those flanking the target sequence are used as
primers for DNA synthesis; replication is carried out in successive cycles using a heat-stable DNA
polymerase
C. Since its initial discovery, PCR has been automated and improved; real-time PCR can be used to
quantitate the amount of target genes in the sample by monitoring the kinetics of amplification using
fluorescent signals; mRNA can be amplified and quantified by creating cDNA prior to PCR using
reverse transcriptase (RT-PCR)
D. PCR has proven valuable in molecular biology, medicine (e.g., PCR-based diagnostic tests), and in
biotechnology (e.g., use of DNA fingerprinting in forensic science; production of insulin)
IV. Gel Electrophoresis
A. Agarose or polyacrylamide gels are used to separate DNA fragments based on size
B. DNA fragments are pulled through the gel by an electric current; small fragments migrate farther
than large fragments, thus separating DNA fragments by size; DNA fragments of similar size form
bands within the gel
V. Cloning Vectors and Creating Recombinant DNA
A. Recombinant DNA technologies require propagation of specific DNA fragments by cloning into
DNA vectors that will replicate in a host organism; the four major types of vectors are: plasmids,
phages, cosmids, and artificial chromosomes
B. Plasmids
1. Replicate autonomously and are easy to purify; introduced by conjugation or transformation
2. The origin of replication (ori) allows the plasmid to replicate in host cells and determines how
many copies of the plasmid a cell will contain; some plasmids called shuttle vectors have two
origins of replication specific for different hosts
3. Plasmids used for biotechnology typically have a selectable marker such as an antibioticresistance gene so that only cells containing the plasmid can grow under certain conditions
(e.g., presence of the antibiotic)
4. The multicloning site or polylinker is a region of the plasmid that has several unique restriction
sites; this allows the circular plasmid to opened up to insert DNA fragments for cloning
C. Phage vectors are phage genomes engineered to include restriction sites useful for cloning; once
DNA is inserted into the vector the phage can be used to infect host cells
D. Cosmids were created to clone larger fragments of DNA, and contain selectable markers,
polylinkers, and cos sites from  phage that allow for viral packaging; once the phage is introduced
into a host, it replicates as a plasmid
E. Artificial chromosomes were created to clone extraordinarily large pieces of DNA; bacterial
artificial chromosomes (BACs) and yeast artificial chromosomes (YACs) have been engineered to
include the sequences needed to act like natural chromosomes when inserted into host organisms
VI. Construction of Genomic Libraries
A. Genomic libraries are valuable when cloning a gene that has an unknown sequence; all of the DNA
sequences of an entire genome should be represented in the library
B. Making a genomic library:
1. The DNA of an organism is fragmented by endonuclease cleavage and all resulting fragments
are cloned into a vector
2. Plasmid, phage, or cosmid vectors are used to insert the clones into hosts; each host receives
only one vector and hence only one cloned fragment of the genome; the population of host
cells and phages taken together includes every fragment of the genome, with only one per cell
3. The clone containing the desired fragment can be identified by using a nucleic acid
hybridization probe; when no sequence information is available, the desired clone is identified
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by expression in a host, often reversing auxotrophy or another deficiency in a process called
phenotypic rescue
4. Once identified, the vector is extracted, and the desired fragment is purified
C. In eukaryotes, it is best to create a cDNA library (no introns) rather than a genomic library
VII. Introducing Recombinant DNA into Host Cells
A. Transformation and electroporation are popular means to insert recombinant DNA into host
microbes; the hosts typically have been engineered to lack RecA and restriction enzymes
B. Electroporation is a procedure in which target cells are mixed with DNA and are then exposed
briefly to high voltage; this works with bacteria, mammalian cells, and plant cell protoplasts
VIII. Expressing Foreign Genes in Host Cells
A. To express a foreign (heterologous) gene in a host cell, the gene must:
1. Have a promoter that is recognized by the host RNA polymerase
2. Have leader sequences that allow for ribosome binding
B. Expression vectors are designed to provide the above features; in addition they have useful
restriction endonuclease sites and regulatory sequences that can be used to control expression of the
foreign gene
C. Purification and study of recombinant proteins
1. Proteins that can be expressed in E. coli are genetically engineered to have a polyhistidine tag
(series of histidine residues at a terminal); the His-tagged proteins can be purified by
attachment to resin beads that bind histidines
2. Green fluorescent protein (GFP) can be used to detect gene expression by either fusion with
the gene of interest (to make a chimeric protein) or by driving expression of the GFP gene with
the promoter of interest
TERMS AND DEFINITIONS
Place the letter of each term in the space next to the definition or description that best matches it.
____ 1.
Enzymes that recognize and cleave DNA at specific base
pair sequences
____ 2. A DNA copy of an mRNA that is produced by reverse
transcriptase
____ 3. A carrier of foreign DNA into the cloning host
____ 4. A piece of detectably labeled nucleic acid that hybridizes
with complementary DNA fragments and is used to locate
them
____ 5. The phenomenon of movement of charged molecules in an
electrical field; it is used to separate nucleic acid fragments
(and/or proteins)
____ 6. A vector that has sequences necessary for packaging into
bacteriophage lambda capsids
____ 7. A vector that has all of the necessary transcription and
translation start and stop signals, and that has nearby useful
restriction endonuclease sites to enable the insertion of
foreign DNA fragments in proper orientation
____ 8. The process in which a high-voltage electric current induces
target cells to take up DNA
____ 9. The process in which recombinant phage DNA is taken up
directly by the target cell without using complete phage
particles
____ 10. A vector with all of the features necessary for chromosomal
replication in yeast; it can carry very large pieces of foreign
DNA into a host organism
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____ 11. A short piece of
DNA, often
synthesized for use
as a probe or
primer
____ 12. A technique that
can be used to
amplify and
quantify mRNA
____ 13. Target genes in a
sample can be
quantified by
monitoring the
kinetics of the
reaction
____ 14. A type of vector
that can be used to
clone large
fragments of DNA
a.
b.
c.
d.
bacterial artificial
chromosome
complementary
DNA (cDNA)
cosmid
electrophoresis
e.
f.
g.
h.
i.
j.
k.
l.
electroporation
expression vector
oligonucleotide
probe
real-time PCR
restriction enzymes (endonucleases)
RT-PCR
transfection
m.
n.
vector
yeast artificial
chromosomes
FILL IN THE BLANK
1.
2.
3.
4.
5.
6.
7.
8.
9.
Enzymes called
cleave DNA at specific sequences.
Successful isolation of recombinant clones is dependent on the availability of suitable ____________,
which can be obtained in a variety of ways. Frequently they are constructed from____________ clones,
which are produced from isolated mRNA molecules by the action of the enzyme reverse transcriptase.
Once a
has been created, it is labeled, usually with a radioactive label such as 32P. Radioactive
labels are easily detected by
, in which energy released by the radioactive isotope causes
formation of dark-silver grains on a sheet of photographic film.
A common technique called _______________ can be used separate DNA fragments based on ______ by
screening the fragments through an _________ ________. The DNA fragments will form _______ that
can be used to determine the ________ of the fragments.
A collection of techniques called
can be used to deliberately modify
an organism by directly changing its genome (a process called
.)
The development of these techniques resulted from the discovery of several enzymes, including
enzymes, DNA ligase, and reverse transcriptase.
The problem of recombinant gene expression in host cells is overcome with the help of special cloning
vectors called __________ vectors that contain the necessary ____________ and ____________ start
signals.
When making a genomic library, DNA is fragmented using __________ __________ and then the
fragments are _____________ into a ___________. The library can be screened by using a hybridization
__________ or by __________ in a host.
A procedure in which target cells are mixed with DNA and briefly exposed to high voltage in order to
introduce the DNA into the target cell is called
.
In many instances, a gene is cloned by finding it in a set of cloned fragments representing the entire
genome of an organism. Such a set of DNA fragments is called a
.
Short pieces of either DNA or RNA are called
. They are useful as
for
identifying genes of interest, in creating mutants with known defects by
,
and as primers for amplifying DNA by the
.
MULTIPLE CHOICE
For each of the questions below select the one best answer.
1.
2.
Which of the following enzymes is used to
produce complementary DNA (cDNA)?
a. restriction endonucleases
b. DNA polymerase
c. reverse transcriptase
d. DNA ligase
Which of the following is NOT normally
used as a cloning vector?
a. transposon
b. plasmid
3.
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c. cosmid
d. bacteriophage
Which of the following is NOT effective for
inserting foreign DNA into a plasmid?
a. Cut plasmid and foreign DNA with the
same enzyme to produce the same
sticky ends, which can then be used to
hold the fragment of foreign DNA in
place for insertion.
b.
4.
5.
Cut plasmid and foreign DNA with an
enzyme to produce blunt ends, and then
add complementary tails by using
terminal transferase to produce sticky
ends.
c. Cut plasmid and foreign DNA with an
enzyme that produces blunt ends, and
then use T4 DNA ligase to do a bluntend ligation.
d. All of the above are effective for
inserting foreign DNA into a plasmid.
Which of the following sequences of steps
cannot be used to clone a desired DNA
fragment?
a. Cleave the DNA; isolate the fragment;
clone the isolated fragment.
b. Cleave the DNA; clone all the resulting
fragments; isolate a clone containing the
desired fragment.
c. Synthesize the desired fragment; clone
the synthesized fragment.
d. All of the above can be used to clone a
desired DNA fragment.
Which of the following processes can be
used to create mutants with known sequence
alterations?
a. polymerase chain reaction
b. site-directed mutagenesis
c. genomic library mutagenesis
d. none of the above
6.
7.
8.
9.
Which of the following cloning vectors
carries the least amount of foreign
DNA?
a. artificial chromosome (e.g.,
YAC or BAC)
b. bacteriophage
c. cosmid
d. plasmid
Which of the following cloning vectors
can carry the most foreign DNA?
a. artificial chromosome (e.g.,
YAC or BAC)
b. bacteriophage
c. cosmid
d. plasmid
Which of the following is a way to
detect DNA fragments of interest on a
Southern blot?
a. enzyme-linked probes
b. chemiluminescent probes
c. radioactive probes
d. any of the above
Which of the following is not a
characteristic of plasmids used for
biotechnology?
a. replicate with host
chromosome
b. small circular DNA
c. contain a multiclonal site
d. have a selectable marker
TRUE/FALSE
____ 1.
____ 2.
____ 3.
____ 4.
____ 5.
____ 6.
____ 7.
____ 8.
The Southern blotting technique was named after the person who developed the procedure, E. M.
Southern.
In electrophoresis, DNA fragments separate according to size, with the smallest fragments migrating
the farthest.
Regardless of the specific technique used in recombinant DNA technology, one of the keys to
successful cloning is choosing the right vector.
Cosmids are so named because they can be used to express foreign genes in a variety of different
cloning hosts.
It is not necessary to remove introns from eukaryotic genes before cloning them in a prokaryotic
organism, because when the eukaryotic RNA transcript is produced in the prokaryote, the introns
are removed by the same posttranscriptional processing inherent in the eukaryotic cell of origin.
Although the term “biotechnology” can be used in many ways, this text uses the term to refer to the
manipulation of organisms, particularly at the molecular genetic level, to form useful products.
The polymerase chain reaction is used to detect small DNA fragments in a Southern blot.
Typical PCR reactions are useful in quantifying genes.
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CRITICAL THINKING
1.
Expression vectors have been extensively modified to allow for efficient expression of recombinant
genes. List as many of these modifications as possible and explain the advantages of each.
2.
You have been asked to clone the gene for the enzyme aspartate transcarbamylase from a newly
discovered organism. What experimental schemes could you use to clone this gene given that this gene
already has been cloned from other organisms? What are the advantages and disadvantages of these
schemes? How would purify the resultant protein and study control of the expression of this gene?
ANSWER KEY
Terms and Definitions
1. j, 2. b, 3. m, 4. h, 5. d, 6. c, 7. f, 8. e, 9. l, 10. n, 11. g, 12. k, 13. i, 14. a
Fill in the Blank
1. restriction endonucleases 2. probes; cDNA; probe; autoradiography 3. electrophoresis; size; agarose gel;
bands; sizes 4. recombinant DNA technology; genetic engineering; restriction 5. expression; transcription;
translation 6. restriction endonucleases; cloned; vector; probe; expression 7. electroporation 8. genomic library
9. oligonucleotides; probes; site-directed mutagenesis; polymerase chain reaction
Multiple Choice
1. c, 2. a, 3. d, 4. d, 5. b, 6. d, 7. a, 8. d, 9. a
True/False
1. T, 2. T, 3. T, 4. F, 5. F, 6. T, 7. F, 8. F
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