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Chapter 17: Recombinant DNA Technology
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- University Of South Africa (Unisa)
Chapter 17: Recombinant DNA Technology focuses on practical applications of microbial genetic principles
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CHAPTER 17: Recombinant DNA TechnologyCHAPTER 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
● 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
● 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
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
● 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
● 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)
● PCR is used to synthesize large quantities of a specific DNA fragment without cloning it
● 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
● 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)
● 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
● 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
● Plasmids
1. Replicate autonomously and are easy to purify
2. introduced by conjugation or transformation
● 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 antibiotic-
resistance gene so that only cells containing the plasmid can grow under certain conditions
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