Recombinant DNA technology

Essential vocabulary

Biotechnology/ GEBiotechnology/ GE RDNA/ RDNA techRDNA/ RDNA tech Restriction enzymes/endonucleasesRestriction enzymes/endonucleases CloningCloning DNA ligaseDNA ligase VectorsVectors PlasmidPlasmid BacteriophagesBacteriophages DNA libraryDNA library

Biotechnology

The manipulation of biological processes and/or The manipulation of biological processes and/or organisms for the benefit of mankindorganisms for the benefit of mankind

.

RDNA

DNA that has been created DNA that has been created artificially (not natural). DNA from two or more sources is DNA from two or more sources is

incorporated into a single recombinant into a single recombinant moleculemolecule

RDNA tech

Procedures by which DNA from different species can Procedures by which DNA from different species can be isolated, cut and spliced together be isolated, cut and spliced together

New "recombinant " molecules are then multiplied in New "recombinant " molecules are then multiplied in quantity in populations of rapidly dividing cells (e.g. quantity in populations of rapidly dividing cells (e.g. bacteria, yeast). bacteria, yeast).

Uses methods derived from biochemistry of nucleic Uses methods derived from biochemistry of nucleic acids coupled with genetic techniques originally used acids coupled with genetic techniques originally used for the study of bacteria and virusesfor the study of bacteria and viruses

RDNA tech process

1.1. A gene is located on a chromosome map (RFLP)

2. A DNA library is constructed – plasmid are obtained, cleavage occurs and RDNA prod.

3. The gene of interest is isolated (cloned) from the library via plasmid DNA isolation, restriction digestion and electrophoresis.

4. Multiple copies of gene interest are produced for study.

steps

Restriction enzymes “cuts” DNA “cuts” DNA

at specific sites (restriction fragments)at specific sites (restriction fragments)

DNA ligase “pastes” the DNA “pastes” the DNA

fragments togetherfragments together

The cut segments are inserted into other DNA The cut segments are inserted into other DNA molecules that serves as molecules that serves as vectors

Vectors

Carrier DNA mol = transfers the RDNA into Carrier DNA mol = transfers the RDNA into the host cell. the host cell.

Within host cells, vectors can replicate Within host cells, vectors can replicate producing many DNA segments = identical producing many DNA segments = identical copies (CLONES)copies (CLONES)

Host cells pass the recombinant DNA mol on Host cells pass the recombinant DNA mol on their progeny = population of cells.their progeny = population of cells.

Cloned DNA segments recovered from host Cloned DNA segments recovered from host cells for purification and analysiscells for purification and analysis

Plasmids

Molecules of DNA that are found in bacteria Molecules of DNA that are found in bacteria Act as a system to Act as a system to transfer genetic material to other to other

bacteria, allowing those to express the transmitted bacteria, allowing those to express the transmitted genes.genes.

small (a few thousand base pairs) small (a few thousand base pairs) usually carry only one or a few genes usually carry only one or a few genes circular circular have a single origin of replicationhave a single origin of replication

Resctriction enzymes DNA cutting enzymes. Restriction endonucleases cuts DNA at a cuts DNA at a specific site defined by a sequence of defined by a sequence of

bases in the DNA (recog.site) forming “sticky ends” bases in the DNA (recog.site) forming “sticky ends” (ss)(ss)

Palindromic sites Palindromic sites Eg. BamHI cuts 5' GGATCC 3'

3' CCTAGG5’ Eg.2 HaeIII cuts 5'GGCC3'3'CCGG5'

several hundred endonucleases have been extracted several hundred endonucleases have been extracted from bacteria and many are used in recombinant from bacteria and many are used in recombinant DNA research. eg DNA research. eg EcoR1,Hind III, HaeIII, TaqA1, Sau3A

Specific palindromic sites

Library construction

Because each cloned DNA segemnt is Because each cloned DNA segemnt is relatively small, many separate clones must be relatively small, many separate clones must be constructed .constructed .

A set of cloned DNA segments derived from a A set of cloned DNA segments derived from a single individual represents a single individual represents a library

Cloned libraries could be an entire genome, a Cloned libraries could be an entire genome, a singe chromosome, or a set og genes compiled singe chromosome, or a set og genes compiled together in a single cell type.together in a single cell type.

summary

Applications of recombinant DNA Applications of recombinant DNA technologytechnology

Used widely in research and hospital laboratories.Used widely in research and hospital laboratories.Broad applications - medicine, agriculture, Broad applications - medicine, agriculture,

??..

1.1. The processes of inheritance and gene The processes of inheritance and gene expression.expression.

2.2. Process and tx of various diseasesProcess and tx of various diseases 3.3. Generation of economic benefits eg. Generation of economic benefits eg.

improved agricultural products.improved agricultural products.

ApplicationsApplications

Molecular Biology/ResearchMolecular Biology/Research DiagnosticsDiagnostics Genetic CounselingGenetic Counseling Criminology/ForensicsCriminology/Forensics Paternity testingPaternity testing ArcheologyArcheology Food testingFood testing Evolutionary studiesEvolutionary studies

Medicine Medicine –production of industrial and commercial production of industrial and commercial

compoundscompounds Insulin – DiabetesInsulin – Diabetes drugs (angiostation and endostatin) ,drugs (angiostation and endostatin) , Factor VIII – Haemophilia AFactor VIII – Haemophilia A Factor IX – Haemophilia BFactor IX – Haemophilia B EPO – AnaemiaEPO – Anaemia Interleukins and interferonsInterleukins and interferons Tissue plasminogen activator – dissolve blood clotsTissue plasminogen activator – dissolve blood clots Hormones = GH, parathyroid Hormones = GH, parathyroid OxytocinOxytocin Adenosine deaminase –sev. Com. Imm (SCID)Adenosine deaminase –sev. Com. Imm (SCID)

,

Diagnostic kitsDiagnostic kits – Hep, AIDS – Hep, AIDS AntibioticsAntibiotics genetic testing,–Mapping the chromosomal location –Mapping the chromosomal location

of genetic disorders. RFLP, DNA fingerprinting – the of genetic disorders. RFLP, DNA fingerprinting – the HG projectHG project

Gene therapy – manipulation of DNA to tx diseases by altering individuals genes. (CF, PKU, DMD) ETHICAL ISSUES

Forensic applications - All individuals are genetically All individuals are genetically unique = a distinct "genetic fingerprint“, all types of unique = a distinct "genetic fingerprint“, all types of specs, old and newspecs, old and new

Animals = models of genetic diseases Eg GE mice models of genetic diseases Eg GE mice

Vaccines Vaccines :vVaccines :vare created by transferring the genes that are created by transferring the genes that

determine a pathogen's surface configuration to a determine a pathogen's surface configuration to a microorganism.microorganism.

When the GM microorganism is used in a vaccine, its When the GM microorganism is used in a vaccine, its surface stimulates the production of antibodies.surface stimulates the production of antibodies.

These antibodies protects an individual against the These antibodies protects an individual against the pathogen.pathogen.

Successful vaccines have been produced for Successful vaccines have been produced for influenza, cold sores, and hepatitis B(HBsAg). influenza, cold sores, and hepatitis B(HBsAg).

Animals

Models of GDModels of GD : GA to be vulnerable to a : GA to be vulnerable to a disease so that the mechanism of the disease disease so that the mechanism of the disease can be investigated and potential therapeutic can be investigated and potential therapeutic agents tested. agents tested.

Eg. mice GA with a cancer-predisposing Eg. mice GA with a cancer-predisposing gene. Research to test the consequences of gene. Research to test the consequences of exposure to various potential carcinogens and exposure to various potential carcinogens and to test the efficacy of preventive drugs.to test the efficacy of preventive drugs.

Agriculture

Animals – improved quality/quantity of – improved quality/quantity of meat productsmeat products

Plants – Gene altered plants : improved – Gene altered plants : improved crops – quantity, quality and pest crops – quantity, quality and pest resistance. Eg golden rice – GM to resistance. Eg golden rice – GM to contain beta carotene – vit A deficiency.contain beta carotene – vit A deficiency.

Spoilage of delicate products. Eg tomatoes if ripened are too Eg tomatoes if ripened are too soft for shipping. In the past, tomatoes were harvested while soft for shipping. In the past, tomatoes were harvested while green and hard, refrigerated while shipping, and artificially green and hard, refrigerated while shipping, and artificially ripened in ethylene gas.ripened in ethylene gas.

The softening of tomatoes is caused by the enzyme The softening of tomatoes is caused by the enzyme polygalacturonase. polygalacturonase.

Now = The polygalacturonase gene has been inserted, in Now = The polygalacturonase gene has been inserted, in reverse order, into tomato plants. This inactivates the original reverse order, into tomato plants. This inactivates the original enzyme-producing gene and produces a reduction in softening enzyme-producing gene and produces a reduction in softening of the tomatoes. of the tomatoes.

GA tomatoes can be shipped without expensive refrigeration, GA tomatoes can be shipped without expensive refrigeration, which allows them to be produced more cheaply and sold at which allows them to be produced more cheaply and sold at lower price.lower price.

Agriculture

Environment. Many waste products of agriculture/industry do not break down naturally/break down slowly. PROBLEM.

Many bacteria have been GE capable of breaking down oil and other organic wastes

Cheese making industry : GE Saccharomyces cerevisiae able to dispose of whey by converting lactose to alcohol.

Agricultural waste products, eg. corn husks, contain cellulose that normally decomposes slowly. Can be converted into sugar by cellulase. Cellulase has been inserted in E.coli making it useful in waste management/disposal programs..