Recombinant DNA Technology

Recombinant DNA

• Recombinant DNA (rDNA) technology's use to produce genetically engineered organisms started in the early 1970s.
• The process began with the pioneering transfer of genes between bacteria of the same Escherichia coli (E. coli) species.
• Cohen and colleagues transferred an insulin synthesis gene into a plasmid of E. coli in 1978, producing the first genetically modified organism (GMO).
• In 1982, the protocol for rDNA received full approval from national drug regulatory authorities, including the US Food and Drug Administration.
• The FDA approval enabled mass production of human insulin, a hormone that regulates blood sugar levels and is naturally made by beta cells in the pancreas.
• rDNA enabled widespread availability of insulin at an affordable price for patients with types 1 and 2 diabetes mellitus.
• These patients either cannot produce or metabolize sufficient insulin.
• rDNA molecules are DNA from two different species inserted into a host organism
• This produces new genetic combinations with value to science and medicine
• rDNA is an artificially-made DNA strand has two or more gene sequences combined
• The new combination may exist naturally or be engineered for a specific application.

Recombinant DNA Applications

• The following are important applications of rDNA through genetic engineering:
• Agricultural (crop improvement)
• Medicinal (medicines)
• Industrial

Agricultural Applications

• Genetic engineering advancements allow gene transfer between distantly related species overcoming gene transfer barriers
• Desirable genes can be transferred from lower to higher organisms using recombinant DNA technology
• Transgenic plants- genetically transformed plants with foreign genes
• These plants are resistant to:
  • Diseases
  • Insects and pests
  • Herbicides
  • Drought
  • Metal toxicity tolerance
  • Induction of male sterility for plant breeding
  • Improved quality can be obtained through recombinant DNA technology
  • BT-cotton resistant to bollworms exemplifies this
• Leguminous plants feature root nodules with nitrogen-fixing Rhizobium bacteria
• Rhizobium converts atmospheric nitrogen into nitrates in the root nodules
• Bacterial genes for nitrogen fixation can be transferred to cereal crops like wheat, rice, maize, and barley.
• This makes them capable of fixing atmospheric nitrogen.
• Recombinant DNA can improve the:
  • Photosynthetic efficiency of crop plants
  • Increased photosynthetic rate when converting C3 plants into C4 plants
  • C4 plants have higher biomass production potential compared to C3 plants
  • C4 plants like sorghum, sugarcane, maize, and some grasses grow in tropical and subtropical zones.

Medicinal Applications

• Penicillium and Streptomyces fungi mass produce antibiotics penicillin and streptomycin
• Genetically efficient fungal strains have been developed to greatly increase antibiotic yields
• Insulin extracted from cow and pig pancreases differs slightly from human insulin and leads to allergic reactions in about 5% of patients
• The human gene for insulin production can be incorporated into bacterial DNA, and genetically engineered bacteria can achieve large-scale insulin production
• Vaccines can be produced with antigen-coding genes transferred to disease-causing bacteria
• In this way antibodies provide protection against infection by same bacteria or virus
• Interferons are virus-induced proteins produced by virus-infected cells that have antiviral action
• They act as a first line of defense against viruses that cause serious infections.
• These infections include breast cancer and lymph node malignancy.
• Natural interferon harvested from human blood cells is costly due to low production volumes
• Recombinant DNA technology makes it possible to produce interferon at a much cheaper rate
• Urokinase used to dissolve blood clots, is an example of a useful enzyme produced by recombinant DNA techniques from genetically engineered microorganisms
• Genetic engineering may enable medical scientists to replace defective genes for hereditary diseases with normal genes in the future.
• The hereditary diseases are hemophilia, phenylketonuria, and alkaptonuria
• The replacement of defective genes for hereditary diseases with normal genes is called gene therapy
• Recombinant technology offers a more accurate method to solve disputed parentage cases than blood tests
• Recombinant DNA technology offers physicians a range of diagnostic tools for diseases.
• These involve constructing probes made of short, single-stranded DNA segments attached to a radioactive or fluorescent marker.
• Probes are used for the identification of infectious agents like food poisoning Salmonella, pus-forming Staphylococcus, hepatitis virus, and HIV
• Testing prospective genetic disorder carrier parents' DNA determines their genotype
• Genetic disorder testing can predict the likelihood of having an afflicted child

Production of Transgenic Animals

• Transgenic animals carry foreign genes
• Examples:
  • Cows, sheep, and goats produce therapeutic human proteins in their milk
  • Common carp, catfish, salmon, and goldfish contain human growth hormone (hGH)

Industrial Applications

• Recombinant DNA techniques can aid in:
  • The production of chemical compounds of commercial importance
  • Improve existing fermentation processes
  • Production of proteins from wastes
• More efficient microorganism strains can achieve this
• Specially developed microorganisms can even be used to clean up pollutants

Genetic Engineering Defined

• Genetic engineering utilizes rDNA technology to alter an organism's genetic makeup
• Genetic engineering is distinct from traditional methods of genetic manipulation such as selective breeding
• Genetic engineering directly changes one or more genes as opposed to indirect breeding manipulation
• Genetic engineering involves the direct manipulation of one or more genes
• Genes from another species are often added to give a desired phenotype

Genetic Engineering

• Genetic engineering artificially modifies an organism's genetic composition
• Genetic engineering transfers genes from one organism into the genome of another to give specific traits
• The organism that results is transgenic (genetically modified organism, GMO)

Genetic Engineering - Basic Stages

• DNA Extraction
• Gene Cloning
• Gene Design
• Transformation
• Backcross Breeding

DNA Extraction

• DNA extraction is the first step in genetic engineering
• Scientists must extract DNA from the desired organism to start with
• A sample from an organism is taken through a series of steps to remove the DNA

Gene Cloning

• Gene cloning is the second step of genetic engineering
• Gene Cloning occurs during DNA extraction, after all the organism's DNA has been extracted
• Scientists use gene cloning to separate a single gene of interest from the extracted genes and make thousands of copies of it

Gene Design

• Gene design is the third phase of genetic engineering
• After a gene is cloned, genetic engineers design the gene to function within a different organism
• Gene design is accomplished in a test tube by using enzymes to cut genes apart and replace removed gene regions

Transformation

• Transformation, or gene insertion is the fourth step in genetic engineering of plants
• Since plants have too many cells to insert a transgene into them, they are propagated via tissue culture
• Tissue culture is used to propagate masses of undifferentiated plant cells called callus.
• Callus will have the transgene added to it
• The new gene gets inserted into some of the cells using techniques like gene gun, agrobacterium, microfibers, and electroporation
• The transformation methods main goal is to transport the new gene and embed them into a cell's nucleus without killing the cell
• Transformed plant cells are regenerated into transgenic plants
• The genetic engineer turns the transgenic seeds over to a plant breeder

Backcross breeding

• Backcross breeding is step give of genetic engineering of crops
• Transgenic plants are crossed with elite breeding lines using traditional plant breeding to combine the desired traits
• The offspring are repeatedly crossed with the elite line.
• An elite line is needed to obtain a high-yielding transgenic line and a plant with the traits encoded by the new transgene.