Download learning outcomes - McGraw Hill Higher Education

Prescott’s Microbiology, 9th Edition
17
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.
LEARNING OUTCOMES
After reading this chapter you should be able to:
•
•
•
•
•
•
explain how restriction enzymes recognize and digest DNA to create either blunt or sticky ends
explain the general principles by which molecules are electrophoretically separated
draw a sample agarose gel in which molecular weight markers and digested DNA can be visualized
discuss the use of single-stranded oligonucleotide probes to identify specific fragments of DNA (for RNA)
diagram the reaction catalyzed by reverse transcriptase and describe its application to biotechnology
differentiate between a PCR cycle and step, and define the function of each of the three steps used in a PCR
cycle
explain why PCR results in the amplification of a specific DNA sequence despite many competing sequences
explain why PCR generates billions of products that are all the same size
contrast real-time, quantitative PCR to end-point collection PCR and identify an application for each
summarize the importance of PCR in biology
list the three features of a cloning vector and why these elements are needed
differentiate between plasmids, phage-based cloning vectors, cosmids, and artificial chromosomes in terms of
structure and application
explain how a piece of foreign DNA is recombined in vitro into a cloning vector
explain why genomic libraries are useful
outline the construction of a genomic library and how the gene of interest might be selected
identify commonly used host cells
compare two common techniques by which recombinant DNA constructed in vitro is introduced into host cells
explain the utility of expression vectors
outline the procedure by which a His-tagged protein is generated in vivo and purified in vitro
summarize the role of GFP in in protein analysis, and differentiate between a transcriptional and a translational
GFP fusion
•
•
•
•
•
•
•
•
•
•
•
•
•
•
CHAPTER OUTLINE
I.
Key Developments in Recombinant DNA Technology
A. Restriction enzymes
1. Arber and Smith (late 1960s) discovered restriction endonucleases, which cleave DNA at specific
sequences
2.
Boyer (1969) first isolated the restriction endonuclease EcoRI
B. Genetic cloning and cDNA synthesis
1. 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
1
© 2014 by McGraw-Hill Education. This is proprietary material solely for authorized instructor use. Not authorized for sale or distribution in any
manner. This document may not be copied, scanned, duplicated, forwarded, distributed, or posted on a website, in whole or part.
Prescott’s Microbiology, 9th Edition
2.
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. Gel Electrophoresis
1. Agarose or polyacrylamide gels are used to separate DNA fragments based on size
2. 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
D. Southern Blotting
1. Southern (1975) developed a blotting procedure for detecting (through autoradiography) specific
DNA fragments, using radioactive DNA hybridization probe
2.
this is useful in isolating particular genes of interest; nonradioactive, enzyme-linked, or
chemiluminescent probes can now replace the earlier radioactive probes; they are faster and safer
II. 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)
III. 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
2.
introduced by conjugation or transformation
C. The origin of replication (ori)
1. allows the plasmid to replicate in host cells
2. determines how many copies of the plasmid a cell will con
3. some plasmids called shuttle vectors have two origins of replication, each recognized by
different host organisms
4. 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)
5.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
D
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
E
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
F
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
IV. 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
© 2014 by McGraw-Hill Education. This is proprietary material solely for authorized instructor use. Not authorized for sale or distribution in any
manner. This document may not be copied, scanned, duplicated, forwarded, distributed, or posted on a website, in whole or part.
Prescott’s Microbiology, 9th Edition
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 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
V. 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
VI. 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
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?
3.
The PCR process has made the technique of Southern blotting obsolete for many applications. Explain
how the polymerase chain reaction can be used to determine if a particular DNA sequence is present in a
sample of DNA and how this could be extended to determine if a particular bacterium is present in a soil
sample.
3
© 2014 by McGraw-Hill Education. This is proprietary material solely for authorized instructor use. Not authorized for sale or distribution in any
manner. This document may not be copied, scanned, duplicated, forwarded, distributed, or posted on a website, in whole or part.
Prescott’s Microbiology, 9th Edition
CONCEPT MAPPING CHALLENGE
Prepare a concept map that includes the following terms: DNA Replication
Genomic Library Expression vector
Translational fusion
PCR
Cloning
Plasmid
4
© 2014 by McGraw-Hill Education. This is proprietary material solely for authorized instructor use. Not authorized for sale or distribution in any
manner. This document may not be copied, scanned, duplicated, forwarded, distributed, or posted on a website, in whole or part.