Abstract
The advent of recombinant DNA technology has offered new opportunities for innovations to produce a wide range of bioproducts in food and agriculture, health and disease, and environment. Biotechnology is recognized universally as one of the key enabling technologies for the 21st century forming the basis of genetic engineering where genes are isolated, modified, and inserted into organisms. The new CRISPR-Cas9 technology has made it easier to make direct changes to a DNA strand called gene editing. In applied sciences such as clinical medicine, biotechnology, forensics, molecular, and evolutionary biology, sequencing DNA has become an important tool. Gene therapy is a technique used to correct single gene disorders where a cloned normal gene is separated and inserted into a cloning vector. Biotechnology has called for oversight and regulation in ways that makes its application and products safe for human use and operating within human ethical and social guidelines. This chapter explores recombinant DNA technology.
TopChapter Outline
10.1 Science of Recombinant DNA
10.2 Recombinant DNA Tool-Kit
10.2.1 Restriction Enzymes
10.2.2 Cloning Vectors
10.2.3 CRISPR Cas9 System
10.3 DNA Libraries
10.3.1 Genomic
10.3.2 Complimentary DNA
10.3.3 Randomized Mutant
10.4 Genetic Sequencing
10.4.1 DNA Microarrays
10.4.2 Next Generation sequencing
10.5 DNA Amplification and Fingerprinting
10.6 Genomics and Proteomics
10.7 Gene Therapy
10.8 Biotechnology and its Applications
10.9 Biotechnology Safety and Ethical Considerations
Chapter Summary
TopLearning Outcomes
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With examples, explain what recombinant DNA technology means
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Be able to illustrate what the DNA tool kits are comprise of
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Explain the meaning of DNA libraries and how they are made
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Outline the process of DNA amplification, fingerprinting, and gene therapy
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With examples demonstrate the process of gene sequencing
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Present information of how humans use biotechnology and its ethical and safety implications
Top10.1 Science Of Recombinant Dna
In the last two decades or so, the advent of recombinant DNA technology has offered new opportunities for innovations to produce a wide range of bio-products. These products have wide applications in food and agriculture, health and disease (gene therapy, antibody production, drug metabolism research, vaccine development, medical nutrition), and environment (phytoremediation and energy applications) (Khan et al., 2016). DNA recombination is done through the modification of microorganisms, animals, and plants to yield biomedically useful products and medical therapies in a field of study called transgenics. Some of these products include biopharmaceuticals (like vaccines, growth hormones, antibodies, recombinant protein and enzymes), vitamins, organic acids, amino acids, glucosamines, solvents, anti-tumour agents, cholesterol-lowering agents, and a host of other compounds (Adrio & Gemain, 2010).
Recombinant DNA technology involves altering genetic material outside an organism to obtain enhanced and desired characteristics in living organisms or their products. DNA fragments with the desirable gene sequence from a variety of sources are inserted using several types of vectors like bacteria, plasmids or retroviruses (Figures 1, 2, 3).
Figure 1. Cloning a gene of interest into a bacterial plasmid
The organism’s genome is then manipulated by either the introduction of one or several new genes and regulatory elements (Figure 1); or by decreasing or blocking the expression of original genes through recombining genes and elements (Bazan-Peregrino et al., 2013). When different genes are incorporated in different plasmids as shown, it creates a plasmid library (Figure 2).
Key Terms in this Chapter
Sanger Sequencing: The original sequencing technology used to sequence short stretches of DNA.
Next Generation Sequencing (NGS): A sequencing method used to measure large numbers of individual DNA sequences in a short period of time.
Restriction Endonucleases (REases): DNA-cleaving enzymes that biologically function to protect the host genome against foreign DNA.
Genomics: A study of how bases on DNA determine whole sets of genes and how they function together.
DNA Library: An assortment of DNA fragments that have carefully been cloned into vectors.
Variable Number of Tandem Repeats (VNTRs or Microsatellite DNA): Specific regions of DNA that demonstration variations in the number of tandem repeats.
Guide RNA: An RNA sequence used in CRISPR Cas9 technology to direct enzymes to specific DNA sequences.
Bioremediators: Biological organisms such as bacteria and fungi—microorganisms that are used to reduce the amount of environmental pollutants.
Whole Genome Sequencing: A sequencing method used to determine whole genomes of an organism.
Industrial Biotechnology: The implementation of biotechnology-based tools into traditional industrial processes as a means to improve and sustain global sustainability.
Protein-Based Therapeutics: A type of biotechnology-based product that aims to prevent and treat a variety of medical issues that have yet to be addressed.
Transgenics: The process of isolating genetic material from one organism and introducing it into another organism's genome.
Genetic Engineering: The process of altering the characteristics of an organism by insertion of modified genes.
Real-Time Polymerase Chain Reaction (RT-PCR): A newer, faster technique of PCR amplification that does not require post-PCR analysis.
Pharmacogenomics: A sector within biomedicine and biotechnology that aims to revolutionize modern medicine by advancing pharmaceutical development in vaccines, therapies, and biological molecules.
Forensic Evidence: Evidence that is obtained by scientific methods and is often used in criminal and legal proceedings.
Gene Therapy: The replacement of defective genes with functional genes or the introduction of new genes to prevent or treat diseases.
Whole Exome Sequencing: A sequencing method used to determine all the exons in a genome of an organism.
Polymerase Chain Reaction (PCR): A technique used to make copies of a target piece of DNA.
DNA Fingerprint: A unique pattern of repeated sequences, or RFLPs, obtained via gel electrophoresis, that can be used to identify an individual’s DNA.
Complementary DNA (cDNA): DNA synthesized from mature mRNA transcripts that can be used to analyze the structure, organization, and expression of eukaryotic genes.
DNA Microarrays (Nucleic Acid Arrays): Specific DNA sequences that are deposited and bound to a solid surface such as a glass microscope slide.
Bioprocessing: The process and application of biotechnology-based tools as a means for commercial use.
CRISPR Arrays: Short segments of DNA that allow bacteria to remember and recognize when an infectious agent invades again.
Bioeconomy: An economy in which biological processes and biological based products are used to reduce environmental impacts and improve overall global sustainability.
Biotechnology: The utilization and application of knowledge, techniques, and procedures from a variety of biology-related disciplines.
Proteomics: A study and characterization of proteins expressed by a genome at one given time in a cell.
Reverse Transcriptase: An enzyme that produces a copy of DNA from mRNA.
Recombinant DNA Technology: Biomedical technology that focuses on altering genetic material outside an organism as a means to enhancing and improving characteristics inside living organisms or their products.
Retroviruses: A group of RNA viruses that replicate by producing a DNA copy of their genome and inserts it into a host cell.
Plasmids: Small circular pieces of double stranded DNA that can replicate independently of the host’s chromosomal DNA, usually found in bacteria.
Gel Electrophoresis: A method used in the lab to separate DNA fragments based on their size.
Restriction Length Fragment Polymorphisms (RFLPs): A set of DNA sequence fragments of varying length.