History and Basics of Genetic Engineering

• Recombinant DNA (rDNA) involves manipulating and isolating DNA segments using enzymes and lab techniques.
• This process can combine DNA from different species or create genes with new functions and resulting copies are often referred to as recombinant DNA.
• Recombinant DNA is propagated in bacterial or yeast cells, whose machinery then copies the engineered DNA.
• Recombinant DNA technology involves joining DNA molecules from different organisms and inserting them into a host organism to produce new combinations.
• It is a biotechnology approach with multidisciplinary applications and the potential to address health issues, food resources, and resistance to environmental effects.
• Recombinant DNA is made from combining DNA from different sources.

History of Genetic Engineering

• The term "genetic engineering," referring to recombinant DNA technology and biotechnology, was coined in 1919 by Karl Ereky, a Hungarian engineer.
• Humans have used artificial selection to manipulate organisms for thousands of years since 8000 and 1000 B.C.
• Yeast was used to make beer as early as 6000 B.C.
• Plants like maize, wheat, and rice were bred from 5000 B.C.
• Socrates speculated on why children do not necessarily resemble their parents, around 420 B.C.
• Hippocrates proposed that males contribute to a child's character through semen around 400 B.C., establishing the idea of heredity.
• Hindu philosophers, from 100-300 A.D., considered reproduction and inheritance laying the foundations of genetics and noting that some diseases run in families.
• In the 19th century, biochemical studies on nucleic acids and amino acids and the speeding up of fermentation led biology in a new direction.
• Mendel presented his work on peas in 1865, but it was largely neglected for some time and the term "gene" was not yet coined.
• The first biotechnology products and the use of agar by the Koch lab occurred in 1882.
• The autoclave was developed in 1884 by a French company called Chamberland's Autoclaves.
• X-rays were discovered by W. Roentgen in 1895.
• X-ray crystallography was developed by physicist Sir William Henry Bragg and his son William Lawrence Bragg and others in 1913.
• William Astbury, a biophysicist, devised the term "molecular biology" in 1945.
• Rosalind Franklin and Maurice Wilkins obtained critical X-ray diffraction data for DNA in the early 1950s.
• Hershey showed that DNA is hereditary material in 1951.
• Werner Arber identified restriction enzymes in bacteria in the late 1960s.
• Temin and Baltimore independently identified reverse transcriptase in 1970 and Paul Berg succeeded in splicing and recombining genetic material in 1971.
• The first recombinant DNA was produced in Boyer Laboratory in 1972.
• The first biotechnology company, Genentech, was born in 1976.
• The polymerase chain reaction (PCR) was invented by Karen Mullis in 1983.
• Genentech's recombinant interferon gamma and Eli Lilly's recombinant human insulin appeared in 1982.
• The Human Genome Project was launched in 1986
• GenPharm International created the first transgenic dairy cow to produce human milk proteins and gene therapy began on a four-year-old with ADA deficiency in the 1990s.
• Dolly the sheep, the first mammalian clone, was born in 1997 via somatic cell nuclear transfer.
• Stanley Cohen, Herbert Boyer, and others built developed recombinant DNA technology at Stanford University and the University of California, San Francisco (1972-1974). This spurred the biotechnology industry.
• Scientists had known since 1959 that bacteria contain extra loops of DNA called "plasmids", able to transfer antibiotic resistance.
• Several plasmids and "restriction endonucleases" were isolated by the early 1970s.
• Herbert Boyer had expertise with restriction endonucleases and Stanley Cohen studied plasmids, and after meeting combine their research efforts to cut open a plasmid loop and insert a gene to create the first GMOs in 1973.
• A year later, the technique inserted a gene from a frog into bacteria. The technology for creating “molecular chimeras” was patented on December 2, 1980.

Plasmids, Restriction Enzymes & DNA Ligase

• Plasmids are physically separate from chromosomal DNA and replicate independently.
• They typically have a small number of genes, notably some associated with antibiotic resistance which can be passed from one cell to another.
• Scientists use methods to splice genes into a plasmid. When the plasmid copies itself, so do the inserted genes.
• Restriction enzymes are molecular scissors in molecular biology for cutting DNA sequences at a specific site to play an important role in gene manipulation.
• Restriction enzymes are useful for rDNA technology recognizing specific short sequences in DNA to produce cohesive (sticky) or blunt-ended fragments.
• DNA Ligase attaches 2 pieces of DNA together.

Process of Recombinant DNA Technology

• The process of recombinant DNA includes multiple sequenced steps.
• Isolate the desired DNA in its pure form free from other macromolecules.
• Restriction enzymes determine where a desired gene is inserted into a vector genome; the reactions are called 'restriction enzyme digestions'.
• Polymerase Chain Reaction (PCR) amplifies a single copy of DNA into copies once the proper gene of interest has been cut using restriction enzymes.
• Ligation joins a cut fragment of DNA and a vector with the help of DNA ligase.
• Recombinant DNA is introduced into a recipient host cell via transformation.
• Once the recombinant DNA is inserted into the cell, it gets multiplied and is expressed as a protein.

Alternate introduction of Recombinant material

• Electroporation is a non-viral delivery system that lets genetic material enter cells, using electrical current to create temporary pores in the cell membrane.
• Protoplast fusion is where somatic plant cells of plants are fused either chemically (polyethylene glycol (PEG) and high calcium at pH 8) or physically by electrofusion to produce hybrid plants.
• Microinjection introduces DNA into animal cells or plant protoplasts via micropipette to produce transgenic mice or other organisms.
• Transduction genetically engineers bacteriophages-viruses that parasitize bacteria-into cells to create the desired recombinant DNA (rDNA).

Applications of Recombinant DNA Technology

• In industry, cyanobacteria have been genetically altered produce plastic (polyethylene) and fuel (butanol).
• E. coli bacteria have been genetically altered to produce diesel fuel.
• Recombinant DNA Tech is used to treat of genetic diseases via gene therapy as well as to remove and replace defective genes with normal ones.
• Examples are Severe Combined Immuno-Deficiency (SCID) and Sickle cell anemia, where SCID is due to a defect in the gene for the enzyme adenosine deaminase (ADA) in 25 per cent of the cases.
• Production of medically useful biologicals is another health application, for example insulin.
• Vaccines and pharmacogenomics, are other applications of recombinant DNA in health; the latter combines pharmacology and genomics to develop effective drugs based on a person's genetic makeup.
• In the environment, uses genetic engineering to develop alternative fuels and uses microorganisms to decompose wastes via bioremediation.
• In agriculture, disease resistant cultivars are used to make crop production more sustainable and reducing agrochemical use and to achieve insect/herbicide/drought resistance, improved yield/nutrition, faster growth and improved shelf life in crops

Explore the origins and fundamental concepts of genetic engineering, including its historical roots in artificial selection and the evolution of related fields like molecular biology. Learn about key experiments, influential figures, and milestones that have shaped our understanding and application of genetic manipulation.