Biotechnology notes

Introduction to Biotechnology

Biotechnology uses biological systems for the benefit of humans. Traditional biotechnology includes the use of microorganisms in bread, yoghurt and fermentation. Modern biotechnology uses cell culture, recombinant DNA technology and genetic engineering.

Genetic engineering involves changing the genetic material of an organism. A useful gene may be isolated, copied, joined to another DNA molecule and introduced into a host cell. The host then expresses the gene and produces the required product.

• Biotechnology uses organisms, cells, enzymes or biological processes to make useful products.
• Genetic engineering is the artificial modification and transfer of genetic material.
• An organism containing a gene from another organism is called a transgenic organism.
• Recombinant DNA contains DNA molecules joined together from different sources.
• Organic chemicals are synthesised when catalysts act on precursor molecules.
• Applications include medicine, agriculture, industry, food production and environmental management.

Enzymes Used in Genetic Engineering

Restriction enzymes are bacterial enzymes that cut DNA at specific nucleotide sequences. These sequences are usually called recognition sites. A restriction enzyme is used to isolate a required gene from DNA.

DNA ligase joins DNA fragments by forming bonds in the sugar-phosphate backbone. It is used to join a desired gene with a vector. Other enzymes help copy or modify DNA during genetic engineering.

Restriction enzymes may produce sticky ends or blunt ends. Sticky ends have short, unpaired DNA sequences that can pair with complementary ends, making joining easier.

• Restriction enzyme: an enzyme that cuts DNA at a specific recognition sequence.
• DNA ligase: an enzyme that joins DNA fragments together.
• Restriction enzymes are used to isolate a gene from DNA.
• A gene and a vector with matching sticky ends can join by complementary base pairing.
• DNA polymerase adds nucleotides to form a new DNA strand.
• The inserted gene, vector and host cell together form the basic system of recombinant DNA technology.

Vectors and Genomic Libraries

A vector carries a foreign gene into a host cell. Plasmids and bacteriophages are common vectors. A vector should be able to replicate in the host and should contain suitable sites for insertion of foreign DNA.

Plasmids are small, circular, double-stranded DNA molecules found mainly in bacteria. They replicate independently of the bacterial chromosome. Their circular DNA structure allows them to be joined with foreign DNA and used in genetic engineering.

A genomic library is a collection of cloned DNA fragments that represents the complete genome of an organism. A cDNA library is made from complementary DNA produced from mature mRNA, so it contains expressed genes and generally lacks introns.

• Plasmids are important vectors in genetic engineering.
• A plasmid is small, circular DNA separate from the bacterial chromosome.
• Plasmids can carry foreign genes into bacterial cells.
• Lambda phage is a commonly used bacteriophage vector.
• A genomic library contains DNA fragments representing the whole genome.
• A cDNA library is prepared from mRNA and represents genes expressed in particular cells.
• Plasmids became recognised during investigations of genetic exchange and the sex-life of Escherichia coli.
• A vector normally has an origin of replication, a selectable marker and a site for insertion of foreign DNA.

Polymerase Chain Reaction and DNA Fingerprinting

Polymerase chain reaction, or PCR, is an in vitro method for making many copies of a selected DNA sequence. It requires template DNA, primers, free nucleotides, a suitable buffer and a heat-stable DNA polymerase.

Each PCR cycle includes denaturation, annealing and extension. During denaturation, the two DNA strands separate. During annealing, primers attach to complementary sequences. During extension, the polymerase makes new DNA strands.

Taq polymerase is obtained from the thermophilic bacterium Thermus aquaticus. It remains active during the high temperatures used in PCR. PCR can detect a very small amount of target DNA, even less than one part in a million of the total DNA sample.

• PCR is used to amplify DNA.
• Taq polymerase is the enzyme used in PCR to synthesise DNA.
• Denaturation separates the two strands of DNA by heating.
• Annealing allows primers to attach to the target sequences.
• Extension is the stage in which Taq polymerase adds nucleotides.
• PCR is specific because primers select the DNA sequence to be copied.
• DNA fingerprinting identifies individuals through differences in selected repetitive DNA regions.
• DNA fingerprints can be prepared from blood, hair roots, semen, saliva and other cells containing DNA.
• Applications include forensic identification, paternity testing and identification of remains.

Biotechnology in Health Care

Genetic engineering is used to produce medicines and to diagnose and treat diseases. Human genes can be inserted into microorganisms so that the microorganisms produce useful proteins. Examples include human insulin, growth hormone and some vaccines.

Gene therapy attempts to treat a genetic disorder by supplying a normal copy of a defective gene or by modifying the activity of a gene. In cystic fibrosis, an attempt has been made to deliver a normal gene into respiratory cells using gene-coated liposomes.

The in vivo treatment of cystic fibrosis using gene-coated liposomes has not yet been successful because gene transfer is limited. The delivered gene may not reach enough target cells or remain active for a sufficient time.

• Adenosine deaminase deficiency can cause severe immunodeficiency.
• Gene therapy may be used to replace or supplement a defective gene.
• Liposomes are small lipid vesicles that can carry genetic material into cells.
• A major problem in cystic fibrosis gene therapy is limited gene transfer.
• Recombinant DNA technology can produce therapeutic proteins.
• Transgenic organisms may be used to study human diseases and test medicines.
• Transgenic mice have been produced for testing the safety of polio vaccine before use in humans.
• Technical problems concern delivery, expression and safety, while ethical problems concern the effects and acceptability of the procedure.

Vaccines, ELISA and Disease Diagnosis

Vaccination stimulates the immune system to develop protection against a specific disease. Vaccines may contain weakened organisms, killed organisms, purified antigens, toxoids or genetic material that produces an antigen.

Vaccination can be carried out against both viral and bacterial diseases. After vaccination, B lymphocytes produce antibodies and memory cells. On later exposure to the pathogen, the immune response is faster and stronger.

ELISA, or enzyme-linked immunosorbent assay, detects a specific antigen or antibody. In virus detection, antibodies may bind to viral antigens. An enzyme label produces a visible colour reaction when its substrate is added. Alkaline phosphatase is an important enzyme used as the label in ELISA.

• Vaccines provide active acquired immunity.
• Vaccination is possible against viral and bacterial diseases.
• ELISA stands for enzyme-linked immunosorbent assay.
• ELISA depends on specific antigen-antibody binding.
• Alkaline phosphatase may be used as the key enzyme reagent in ELISA.
• A positive ELISA result is shown by a detectable colour change after substrate addition.
• ELISA is used in diagnosis, screening and detection of specific proteins or pathogens.
• Antibodies are produced by B lymphocytes and are specific to particular antigens.

Plant Biotechnology and Crop Improvement

Plant biotechnology produces crops with useful characteristics. Genes may be transferred to plants to provide resistance against herbicides, insects, viruses or environmental stresses. Tissue culture allows the production of many genetically similar plants from a small piece of plant tissue.

Somatic embryogenesis is the formation of embryos from somatic cells. These embryos can develop into complete plants. Somatic embryos do not develop from microspores, because microspores are reproductive cells and are not somatic cells.

Hybridisation combines desirable characteristics from two different plants. Triticale is a hybrid obtained by crossing wheat with rye. It combines selected features of both parent plants.

• Sugarbeets have been made resistant to herbicides through genetic modification.
• Somatic embryogenesis produces embryos from somatic, or non-reproductive, cells.
• Somatic embryos can develop into complete plants under suitable culture conditions.
• Somatic embryos do not develop from microspores.
• Micropropagation uses tissue culture to produce many plants from a small explant.
• Triticale is obtained by crossing wheat with rye.
• Norman Borlaug is associated with the Green Revolution.
• The Green Revolution involved improved crop varieties, better cultivation methods and increased agricultural production.

Transgenic Organisms, Cloning and Ethics

A transgenic organism carries and expresses a gene introduced from another organism. Transgenic animals can be used to study diseases, produce useful proteins and test drugs or vaccines. Transgenic plants may have resistance to herbicides, insects or diseases.

Cloning produces genetically identical copies of a cell or organism. In reproductive cloning, the aim is to produce a complete organism. In therapeutic cloning, cells or tissues may be produced for medical purposes.

Human cloning raises ethical questions in addition to technical problems. An ethical objection is that a cloned child would be created as a copy of another person rather than as an individual. This is different from a technical objection, such as difficulty in transferring genes or developing a healthy embryo.

• Transgenic means containing a gene from another organism.
• Transgenic mice have been used in testing the safety of polio vaccine before human use.
• Cloning aims to produce genetically identical cells or organisms.
• An ethical objection concerns the identity, rights and individuality of a cloned child.
• A technical objection concerns the biological difficulty or risk of the procedure.
• Genetic engineering and cloning are different: genetic engineering changes genetic material, while cloning produces a genetic copy.
• The use of genetically modified organisms requires attention to safety, environmental effects and ethical responsibility.

Industrial Biotechnology and Downstream Processing

Industrial biotechnology uses microorganisms or cultured cells to produce antibiotics, enzymes, alcohols, organic acids, vaccines and other chemicals. The desired product is made by a biological system under controlled conditions in a fermenter or bioreactor.

Some industrial products are formed from precursor molecules. Enzymes act as biological catalysts and convert the precursors into the required organic chemicals. Conditions such as temperature, pH, oxygen supply and nutrient concentration must be controlled.

Downstream processing includes the recovery and purification of a product after it has been produced. Expression is the production of a protein from a gene and is not itself a component of downstream processing.

• A fermenter provides controlled conditions for the growth of microorganisms or cells.
• Important conditions include temperature, pH, oxygen and nutrient supply.
• Downstream processing includes separation, recovery, purification and formulation of the product.
• Expression means production of a protein from the information in a gene.
• Expression is not a component of downstream processing.
• Enzymes act as biological catalysts in industrial synthesis.
• Industrial biotechnology can produce medicines, food additives, enzymes and organic chemicals.

Other Applications and Terms

Biotechnology has applications beyond medicine and agriculture. Environmental and geographical information may also support biological production and crop planning. An isohel is a line on a map joining places that receive equal solar radiation.

Solar radiation affects photosynthesis, crop growth and the productivity of agricultural systems. Knowledge of environmental conditions helps in selecting suitable crops and improving their production.

The uses of biotechnology must be balanced with safety. Products should be tested for effectiveness, unwanted effects and possible effects on organisms and the environment.

• An isohel joins places receiving equal solar radiation.
• Solar radiation provides the energy required for photosynthesis.
• Crop improvement may involve genetic engineering, hybridisation or tissue culture.
• Biotechnology can be used in medical, agricultural, industrial and environmental fields.
• Safety assessment is required before a genetically modified product or biological treatment is widely used.