Chapter 8

Recombinant DNA Technology

Recombinant DNA Technology

Recombinant DNA technology

Intentionally modifying genomes of organisms, by natural and artificial processes, for practical purposes

Three Goals of Recombinant DNA Technology

Eliminate undesirable phenotypic traits in humans, animals, plants, and microbes

Combine beneficial traits of two or more organisms to create valuable new organisms

Create organisms that synthesize products humans need

Tools of Recombinant DNA Technology

Mutagens

Reverse transcriptase

Synthetic nucleic acids

Restriction enzymes

Vectors

Creation of gene libraries

Restriction Enzymes

Bacterial enzymes that cut DNA molecules only at specific locations (restriction sites)

Categorized in two groups based on type of cut

Cuts with sticky ends

Cuts with blunt ends

Gene Libraries

A collection of bacterial or phage clones; each contains a portion of the genetic material of interest

Typically, each clone in library contains one gene of an organism’s genome

Library may contain all genes of a single chromosome

Library may contain set of cDNA complementary to mRNA

Techniques of Recombinant DNA Technology

Polymerase Chain Reaction

Clone Selection

Separation of DNA Molecules

Inserting DNA into Cells

Multiplying DNA in vitro: The Polymerase Chain Reaction (PCR)

Large number of identical molecules of DNA produced in vitro

Critical to amplify DNA in variety of situations

Epidemiologists use to amplify genome of unknown pathogen (West Nile virus)

Amplify DNA from Bacillus anthracis spores in 2001 to identify source of spores

Multiplying DNA in vitro: The Polymerase Chain Reaction (PCR)

Repetitive process consisting of three steps

Denaturation

Priming

Extension

Can be automated using a thermocycler

Gel Electrophoresis

DNA has negative charge; drawn by electric current toward positive electrode

Agarose makes up gel; acts as molecular sieve

Smaller fragments migrate faster and further than larger ones

Determine size by comparing distance migrated to that of standards and constructing standard curve

Applications of Recombinant DNA Technology

Pharmaceutical and Therapeutic Applications

Protein synthesis

Vaccines

Genetic screening

DNA fingerprinting

Gene therapy

Xenotransplants

Protein Synthesis

Creation of synthetic peptides for cloning

Vaccines

Production of safer vaccines

Subunit vaccines

Introducing genes of pathogens into common fruits and vegetables

Injecting humans with plasmid carrying gene from pathogen; humans synthesize pathogen’s proteins

Genetic Screening

Southern blots used to screen patients, prospective parents, and fetuses for inherited disease caused by mutations

Can also identify pathogen’s DNA in blood or tissues

Gene Therapy

Missing or defective genes replaced with normal copies

Some patients’ immune systems reacted negatively

Successfully treated patients with severe combined immunodeficiency disease

Others that may respond well – cystic fibrosis, sickle cell anemia, some types of hemophilia, some types of diabetes

Xenotransplants

Animal cells, tissues, or organs introduced into human body

Theoretically feasible to insert human genes into animals to direct them to produce organs and tissues for transplantation into humans

Applications of Recombinant DNA Technology

Agricultural Applications

Herbicide resistance

Salt tolerance

Freeze resistance

Pest resistance

Improvements in nutritional value and yield

Transgenic organisms – recombinant plants and animals altered by addition of genes from other organisms

Herbicide Resistance

Glyphosate (Roundup) kills all plants

Gene from Salmonella that conveys resistance to glyphosate

Now farmers can kill weeds without killing crops

Also reduces tilling and soil erosion

Salt Tolerance

Irrigation has led to salt buildup in some farmland; useless for farming

Gene for salt tolerance removed and inserted into tomato and canola plants

Transgenic plants survive, produce fruit, and remove salt from soil (restore soil)

Scientists want to create salt tolerant wheat and corn

Freeze Resistance

Crops sprayed with genetically modified bacteria can tolerate mild freezes

Pest Resistance

Bt-toxin naturally occurring, harmful to insects only, and biodegradable

Organic farmers have used to reduce insect damage to crops

Gene for Bt-toxin inserted into potatoes, cotton, and corn

Improvements in Nutritional Value and Yield

Tomatoes allowed to ripen on vine and shelf life increased

Gene for enzyme that breaks down pectin suppressed

BGH allows cattle to gain weight more rapidly, have meat with lower fat content, and produce 10% more milk

Gene for β-carotene (precursor to vitamin A) inserted into rice

Scientists considering transplanting genes coding for entire metabolic pathways

Ethics and Safety of Recombinant DNA Technology

Supremacist view – humans are of greater value than animals

Long-term effects of transgenic manipulations are unknown

Unforeseen problems arise from every new technology and procedure

Natural genetic transfer could deliver genes from transgenic plants and animals into other organisms

Transgenic organisms could trigger allergies or cause harmless organisms to become pathogenic

Ethics and Safety of Recombinant DNA Technology

Studies have not shown any risks to human health or environment

Standards imposed on labs involved in recombinant DNA technology

Can create biological weapons using same technology

Ethics and Safety of Recombinant DNA Technology

Ethical issues

Routine screenings?

Who should pay?

Genetic privacy rights?

Profits from genetically altered organisms?

Required genetic screening?

Forced correction of “genetic abnormalities?”