Molecular Biotechnology. Bernard R. Glick
22:379−380.
Robbins-Roth C. 2000. From Alchemy to IPO: the Business of Biotechnology. Perseus Publishing, Cambridge, Mass.
OECD Internal Co-ordination Group for Biotechnology. 2015. Biotechnology Update. OECD Publishing, Paris, France.
review questions
1. What is biotechnology?
2. Distinguish between traditional biotechnology and molecular biotechnology.
3. What are the shortcomings of the “mutation and selection” method for developing enhanced organisms for commercial purposes?
4. Why was the work reported by Cohen and Boyer and their coworkers in 1973 considered important?
5. How did recombinant DNA technology enable the production of human insulin?
6. What are some of the problems that molecular biotechnology has the potential to solve?
7. Discuss the statement “Molecular biotechnology is a diverse science.”
8. Discuss some of the social concerns that have been raised about molecular biotechnology.
9. Go to your favorite news website and conduct a search with the word “biotechnology.” Describe and discuss three recent biotechnology news stories.
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Fundamental Technologies
Preparation of DNA for Cloning
Insertion of Target DNA into a Plasmid Vector
Transformation and Selection of Cloned DNA in a Bacterial Host
Genome Engineering Using CRISPR Technology
Assembling Oligonucleotides into Genes
Sequencing Using Reversible Chain Terminators
Sequencing by Single Molecule Synthesis
Preparation of Genomic DNA Sequencing Libraries
High-Throughput Next-Generation Sequencing Strategies
Molecular Cloning
MOLECULAR BIOTECHNOLOGY USES A variety of techniques for isolating genes and transferring them from one organism to another. At the root of these technologies is the ability to join a sequence of deoxyribonucleic acid (DNA) of interest to a vector that can then be introduced into a suitable host. This process is known as recombinant DNA technology or molecular cloning. A vast number of variations on this basic process have been devised to produce products that are important for medicine, food production, environmental remediation, and industrial processes.
Preparation of DNA for Cloning
In theory, DNA from any organism can be cloned. The target DNA may be obtained directly from genomic DNA, derived from messenger ribonucleic acid (mRNA), subcloned from previously cloned DNA, or synthesized in vitro. The target DNA may contain the complete coding sequence for a protein, a part of the protein coding sequence, a random fragment of genomic DNA, or a segment of DNA that contains regulatory elements that control expression of a gene. Prior to cloning, both the source DNA that contains the target sequence and the cloning vector must be cut into discrete fragments, predictably and reproducibly, so that they can be joined (ligated) together to form a stable molecule. Bacterial enzymes known as type II restriction endonucleases, or (more commonly) restriction enzymes, are used for this purpose (see Milestone box on page 12). These enzymes recognize and cut double-stranded DNA molecules at specific base pair sequences and are produced naturally by bacteria to cleave foreign DNA, such as that of infecting bacterial viruses (bacteriophage). A bacterium that produces a specific restriction endonuclease also has a corresponding system to modify the sequence recognized by the restriction endonuclease in its own DNA to protect it from being degraded. Often, methylation of a cytosine within a restriction endonuclease binding site prevents the enzyme from cutting at these sites.
milestone