Recombinant DNA Technology Vocabulary

Recombinant DNA Technology

Introduction

• Biotechnology: The utilization of microorganisms.
• Genetic engineering, recombinant DNA technology, and DNA cloning: Methodologies.
• Recombinant DNA technology: Core technique.
• Ethical issues: Considerations surrounding DNA technology.

Key Concepts

• Biotechnology
• Genetic engineering
• Recombinant DNA technology
• Gene cloning
• Cloning vector
• Restriction enzymes
• ‘Sticky ends’
• DNA ligase
• cDNA
• PCR (Polymerase Chain Reaction)

Biotechnology Defined

• Biotechnology: An applied biology field employing living organisms and bioprocesses in engineering, technology, medicine, etc.
• Historical Applications:
  • Microbes in wine and cheese production.
  • Selective livestock breeding.
  • Fruit grafting.
• Practical Applications:
  • Agriculture:
    • Transgenic animals (e.g., enhanced growth via gene transfer).
    • Transgenic plants (e.g., pesticide resistance genes).
  • Medical:
    • Production of antibiotics, insulin, growth hormone, interferon, clotting factor VIII, vaccines, gene therapy.
    • Genetics and regulation research.
  • Industry: Protein production (enzymes, etc.) for manufacturing.
  • Environmental biotechnology: Microbes for remediation (e.g., oil-eating bacteria).

Genetic Engineering

• Gene manipulation and modification: Moving genes between organisms (bacteria, plants, animals).
• Natural Genetic Experiments: Occurring for billions of years.
• Wide application: Spanning agriculture to criminal law.
• Advances: Driven by basic molecular biology and technology.
• Mechanism of Genetic Change: Recombinant DNA technology.
• Capabilities: Isolate, cut, splice genes from different species, produce numerous copies.

Recombinant DNA

• Definition: DNA from two different sources (often species) combined in vitro.
• Application: Understanding gene structure, function, regulation, and protein products.
• Genetic Engineering: Direct manipulation of genes for practical applications.

Definitions

• Genetic engineering: Alteration of the genetic constitution of cells/organisms via direct and selective modification/insertion/deletion of individual genes.
• Recombinant DNA technology: Methodology for inserting a DNA fragment from one organism into the DNA of another for cloning.
• Gene cloning/DNA cloning/molecular cloning: Production of many identical copies of a DNA molecule (or part of it) via replication in a host (e.g., bacteria).
• Gene cloning is NOT animal cloning!

Gene Cloning Process

• Foreign gene (e.g., human) inserted into bacterial plasmid.
• Recombinant DNA plasmid returned to bacterial cell.
• Bacterial cell reproduces, replicating the recombinant plasmid and passing it to offspring.
• Under suitable conditions, bacterial clone produces the protein encoded by foreign gene.

Steps in Gene Cloning

1. Cutting DNA into fragments.
2. Insertion of DNA fragments into a cloning vector (plasmids).
3. Bacterial transformation.
4. Identification of desired clone.

Components for Recombinant DNA Technology

• Plasmid: Vector for introducing gene of interest into host (with antibiotic resistance and other markers for clone identification).
• Restriction enzyme: Cuts plasmid and gene of interest.
• DNA ligase: Joins DNA pieces together to produce recombinant DNA.
• Host cell: (usually bacteria, E. coli) Reproduces rapidly and clones recombinant DNA.

Restriction Enzymes

• Cut DNA at specific locations.
• Cut at specific nucleotide sequences = recognition site.
• > 600 enzymes for > 200 recognition sites.
• Naturally occur in bacteria to cut foreign DNA, protecting from other bacteria or viruses.
• Some enzymes create ‘sticky ends’.
• Can also create ‘sticky ends’ by making a PCR product with specific enzymes in PCR reaction.

Ligation

• DNA fragments cut with the same restriction enzyme will base-pair.
• Restriction enzymes cut the DNA, creating ‘sticky ends’.
• DNA ligase fuses two pieces of DNA together to make recombinant DNA.

Cloning Process

• Restriction enzyme cuts DNA.
• DNA fragments are generated.
• DNA fragments and modification enzymes are mixed together (ligation).
• Recombinant plasmids are produced.
• Host cells (able to divide rapidly) take up recombinant plasmids.

Bacterial Hosts

• Bacteria commonly used as host cells for gene cloning.
• DNA easily isolated and reintroduced into cells.
• Bacterial cultures grow quickly, rapidly replicating foreign genes (e.g., 20 min for E. coli).
• Circular DNA (plasmid) can carry the foreign gene.

Plasmids

• Bacteria often contain plasmids - small circular DNA molecules, in addition to the chromosome.
• Plasmids act as delivery vehicles, or vectors, to introduce foreign DNA into bacteria.
• A plasmid can be modified to carry genes useful to identify recombinant DNA colonies of bacteria on the agar plate.

Plasmid Constructs

• ampⓇ: Confers resistance to ampicillin antibiotic.
• lacZ: Beta-galactosidase enzyme, catalyzes sugar hydrolysis.
• Steps:
  1. Plasmid and foreign DNA cut with same restriction enzyme. Plasmid has genes for lactose hydrolysis (lacZ gene encodes the enzyme B-galactosidase) and ampicillin resistance.
  2. Foreign DNA inserts into lacZ gene. Bacterium receiving plasmid vector will not produce B-galactosidase if foreign DNA has been inserted into the plasmid.
  3. Recombinant plasmid introduced into bacterium, which becomes ampicillin resistant.
  4. All treated bacteria are spread on nutrient agar plate containing ampicillin and a B-galactosidase substrate (X-gal) and incubated.
  5. Only bacteria that picked up the plasmid will grow in presence of ampicillin. Bacteria that hydrolyze X-gal produce galactose and an indigo compound (turns colonies blue). Bacteria that cannot hydrolyze X-gal produce white colonies.

Transformation

• Transferring exogenous DNA into cells.
• Bacteria carrying the recombinant plasmid reproduce, and plasmid DNA is also reproduced.
• Bacteria cultures grow quickly, rapidly replicating the foreign genes.
• During bacteria reproduction – recombinant plasmid is replicated => CLONED.
• DNA can be easily isolated and reintroduced into other cells.
• Two general methods for transforming bacteria:
  • Chemical method utilizing CaCl_2 and heat shock (42°C) to promote DNA entry into cells.
  • Electroporation based on a short pulse of electric charge to facilitate DNA uptake.

Chemical Transformation

• Steps:
  • Aliquot competent cells
  • Centrifuge
  • Resuspend bacterial pellet in CaCl_2 solution
  • Chill on ice
  • Store at -80°C
  • Add amp plasmid DNA
  • Heat shock in 42°C H_2O bath
  • Chill on ice
  • Plate on LB + ampicillin
  • Result: 10^6-10^8 amp colonies/μg DNA

Electroporation

• Current pulses create micropores in cell membrane.
• Exogenous DNA enters the cell, and the membrane reseals.

Screening Colonies

• Molecular cloning requires screening colonies for the presence of an insert.
• Traditionally done with restriction enzyme digest, but colony PCR can accomplish the same thing faster and cheaper.

Screening Methods

• DNA sequencing: Correct DNA inserted
• Gene (RNA) expression
• Protein production screening: Cell Lysate test by Western Blot, ELISA

Mammalian Cells

• Recombinant DNA can be transfected into mammalian cells (tissue culture) by Transfection
• Transient or stable
• Methods of transfection:
  • Biological (viral vectors)
  • Chemical (e.g., lipofection)
  • Physical (e.g., electroporation, ‘gene gun’)

Multiple Host Systems

• Multiple host systems commonly used to produce recombinant human protein
  • E.coli cells
  • Yeast cells
  • Insect cells
  • Mammalian cells

Applications and Implications

• DNA technology reshaping medicine and pharmaceutical industry (insulin production, growth hormone, etc.).
• Forensic, environmental (oil degraders), and agricultural applications (pesticide control).
• Raises important safety and ethical questions.

Modern Biotechnology Contributions

• Enormous contributions to both the diagnosis of disease and in development of pharmaceutical products.
• Identification of genes whose mutations are responsible for genetic disease could lead to ways to diagnose, treat, or even prevent these conditions.
• Diseases of all sorts involve changes in gene expression.
• DNA technology can identify these changes, and lead to the development of targets for prevention or therapy.

Tracking Pathogens

• PCR and labelled probes can track down the pathogens responsible for infectious diseases.
• For example, PCR can amplify and thus detect viral DNA in blood and tissue samples, detecting an otherwise elusive infection.
• Medical scientists can use DNA technology to identify individuals with genetic diseases before the onset of symptoms, even before birth.
• It is also possible to identify symptomless carriers.
• Genes have been cloned for many human diseases, including haemophilia, cystic fibrosis, and Duchenne muscular dystrophy.

Practical Applications

• Scientific Research.
• Medicine and pharmaceutical industry.
• Gene for pest resistance inserted into plants.
• Gene inserted into plasmid.
• Recombinant bacterium.
• Cell multiplies with gene of interest.
• Clone of cells.
• Copies of gene.
• Copies of protein.
• Gene used to alter bacteria for cleaning up toxic waste.
• Protein used to dissolve blood clots in heart attack therapy.
• Protein used to make snow form at higher temperature.

Plant Modification

• Foreign genes can be inserted into the Ti plasmid (a version that does not cause disease) using recombinant DNA techniques.
• The recombinant plasmid can be put back into Agrobacterium, which then infects plant cells, or introduced directly into plant cells.
  • Delayed ripening and resistance to spoilage and disease.
  • Cotton plant having a gene for herbicide resistance.
  • Gene transfer to improve the nutritional value of crop plants; e.g: a transgenic golden rice plant has been developed that produces yellow grains containing beta-carotene (vitamin A precursor).

Protein Production

• Production of human proteins; e.g. recombinant vaccines for hepatitis B and antibodies that block the bacteria that cause tooth decay.
• Insulin:-
  • Insulin is a hormone made up of protein. It is secreted in the pancreas by some cells called as islet cells.
  • If a person has decreased amount of insulin in his body, he will suffer from a disease called diabetes.
  • Recombinant DNA technology has allowed the scientists to develop human insulin by using the bacteria as a host cell and it is also available in the market.
  • It is believed that the drugs produced through microbes are safer.

Synthesis of Human Insulin

• Why synthesize human insulin?
  • Immune system of diabetic patients will not produce antibodies against human insulin as they do with bovine or porcine insulin.
  • Need for a reliable and sustainable method of obtaining the product.
• How?
  • Restriction enzymes used to cut out human insulin gene.
  • Same enzyme used to cut E.coli plasmid.
  • Enzyme produces ‘sticky ends’.
  • Insulin gene is inserted into the vector/plasmid.
  • Recombinant plasmid carrying human insulin gene is replicated in E.coli to make many copies of this gene.
  • The gene produces human insulin protein within bacteria cells.
  • This protein can be purified from E.coli cells.
  • Final product is Humulin – chemically identical to human insulin

Vaccines

• Recombinant DNA technology enables the scientists to develop vaccines by cloning the gene used for protective antigen protein.
• Viral vaccines are most commonly developed through this technology for example, Human Papilloma Virus (HPV), Influenza, Hepatitis and Foot and Mouth Diseases Virus like particles (VLPs)—empty viral capsids with no viral DNA inside L1 gene.

Stem Cells

• Gene delivery for generating induced Pluripotent Stem Cells (iPSC).
• Gene manipulation to confer specific trade to the Stem Cells.

Gene Therapy

• Bone marrow cells, which include the stem cells that give rise to blood and immune system cells, are prime candidates for gene therapy.
• A normal allele could be inserted by a viral vector into some bone marrow cells removed from the patient.
• If the procedure succeeds, the returned modified cells will multiply throughout the patient’s life and express the normal gene, providing missing proteins.

Gene Therapy Limitations

• Little scientifically strong evidence of effective gene therapy to date.
• Even when genes are successfully and safely transferred and expressed in their new host, their activity typically diminishes after a short period.
• Potential risks.
• Still a work in progress…

Exondys 51

• Skips the dystrophin gene.

Duchenne Muscular Dystrophy

• Exon 51 cannot join up with exon 53, which prevents the rest of the exons being assembled.
• For the dystrophin protein to work, it must have both ends of the protein.
• Therefore, this mutation results in a completely non-functional dystrophin protein and the severe symptoms of Duchenne muscular dystrophy.

Molecular Plaster

• Exon 51 can now join up to exon 54 and continue to make the rest of the protein, with exons 52 and 53 missing in the middle.

CRISPR-Cas9

• New Technology
• Components:
  • DNA target sequence
  • Guide RNA
  • Cas9 enzyme
• Mechanism:
  • Guide RNA binds to target sequence
  • Cas9 enzyme binds to guide RNA
  • Cas9 enzyme cuts both strands of DNA
  • The cut is repaired, introducing mutation

Safety and Ethical Concerns

• The power of DNA technology has led to worries about potential dangers.
• For example, recombinant DNA technology may create hazardous new pathogens that are antibiotic resistant.
• Today, most public concern centres on genetically modified (GM) organisms used in agriculture.
• Advocates of a cautious approach fear that GM crops might somehow be hazardous to human health or cause ecological harm.
• In Europe, safety concerns have led to pending new legislation regarding GM crops and bans on the import of all GM foodstuffs.
• To date there is little good data against any special health or environmental risks posed by genetically modified crops.
• Gene testing/gene therapy eligibility for clinical trials

Summary

• Genetic engineering involves the isolation, modification, and insertion of genes.
• Recombinant DNA technology allows the isolation, cutting, and splicing of gene regions.
• Restriction enzymes and DNA ligase allow new DNA to be inserted into plasmids.
• Amplification (cloning) of selected gene/ DNA fragment can be done using bacteria or yeast or by PCR.
• Recombinant DNA technology and genetic engineering greatly benefit research and have applications in medicine, agriculture, and industry.

Review Questions

1. Explain what recombinant DNA technology is.
2. Detail steps of the experiment involving genetic manipulation using recombinant DNA technology and gene cloning.
3. List the necessary components required for these experiments and describe their function.
4. Give examples of applications of recombinant DNA technology.