Life Science I Dr. Ekaterina (Kate) Vorotnikova Office: Olsen 413b Lecture 26 Genetically modified organisms. DNA profiling. (Page 231; )

GENETICALLY MODIFIED ORGANISMS: Copyright © 2009 Pearson Education, Inc. Genetically Modified Bacteria Genetically Modified Plants Genetically Modified Animals

Recombinant cells and organisms can mass-produce gene products –Cells and organisms containing cloned genes are used to manufacture large quantities of gene products –Capabilities of the host cell are matched to the characteristics of the desired product –Prokaryotic host: E. coli –Can produce eukaryotic proteins that do not require post- translational modification –Has many advantages in gene transfer, cell growth, and quantity of protein production –Can be engineered to secrete proteins Copyright © 2009 Pearson Education, Inc.

–Eukaryotic hosts –Yeast: Saccharomyces cerevisiae –Can produce and secrete complex eukaryotic proteins –Mammalian cells in culture –Can attach sugars to form glycoproteins –“Pharm” animals –Will secrete gene product in milk Copyright © 2009 Pearson Education, Inc.

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“Pharm” animals that produce a human protein. Examples of products from “pharm” animals include: goats producing tissue plasminogen activator (TPA) for heart attack victims, sheep producing alpha-1-antitrypsin to treat emphysema, and pigs producing hemoglobin as a blood substitute.

DNA technology has changed the pharmaceutical industry and medicine –Products of DNA technology –Therapeutic hormones –Insulin to treat diabetes –Human growth hormone to treat dwarfism –Diagnosis and treatment of disease –Testing for inherited diseases –Detecting infectious agents such as HIV Copyright © 2009 Pearson Education, Inc.

–Products of DNA technology –Vaccines –Stimulate an immune response by injecting –Protein from the surface of an infectious agent –A harmless version of the infectious agent –A harmless version of the smallpox virus containing genes from other infectious agents Copyright © 2009 Pearson Education, Inc.

Equipment used in the production of a vaccine against hepatitis B.

–Advantages of recombinant DNA products –Identity to human protein –Purity –Quantity Copyright © 2009 Pearson Education, Inc.

Genetically modified organisms are transforming agriculture –Genetically modified (GM) organisms contain one or more genes introduced by artificial means –Transgenic organisms contain at least one gene from another species –GM plants –Resistance to herbicides –Resistance to pests –Improved nutritional profile –GM animals –Improved qualities –Production of proteins or therapeutics Copyright © 2009 Pearson Education, Inc.

Agrobacterium tumefaciens DNA containing gene for desired trait Ti plasmid Insertion of gene into plasmid Recombinant Ti plasmid 1 Restriction site A modified form of the Ti (tumor inducing) plasmid is used in plant genetic engineering. The tumor inducing genes have been removed from the plasmid but genes required for insertion into plant chromosomes have been retained. The gene of interest has been inserted into the plasmid under the control of a bacterial promoter. Using the Ti plasmid as a vector for genetically engineering plants.

Agrobacterium tumefaciens DNA containing gene for desired trait Ti plasmid Insertion of gene into plasmid Recombinant Ti plasmid 1 Restriction site Plant cell Introduction into plant cells 2 DNA carrying new gene

Agrobacterium tumefaciens DNA containing gene for desired trait Ti plasmid Insertion of gene into plasmid Recombinant Ti plasmid 1 Restriction site Plant cell Introduction into plant cells 2 DNA carrying new gene Regeneration of plant 3 Plant with new trait

A mix of “golden rice” and standard rice.

Gene therapy aims to treat a disease by supplying a functional allele One possible procedure –Clone the functional allele and insert it in a retroviral vector –Use the virus to deliver the gene to an affected cell type from the patient, such as a bone marrow cell –Viral DNA will insert the functional allele into the patient ’ s chromosome –Return the cells to the patient for growth and division Gene therapy may someday help treat a variety of diseases

–SCID (severe combined immune deficiency) was the first disease treated by gene therapy –First trial in 1990 was inconclusive –Second trial in 2000 led to the development of leukemia in some patients due to the site of gene insertion –Challenges –Safe delivery to the area of the body affected by the disease –Achieving a long-lasting therapeutic effect –Addressing ethical questions Copyright © 2009 Pearson Education, Inc.

DNA PROFILING Copyright © 2009 Pearson Education, Inc.

–DNA evidence was used to solve a double murder in England –Showed that two murders could have been committed by the same person –Showed the innocence of someone who confessed to one of the murders –Showed the absence of a match in 5,000 men tested when the murderer persuaded another man to donate blood in his name –Showed a match with the murderer and DNA found with both victims Media_26\12_15ADNAForensics_DV.mpg Media_26\12_15ADNAForensics_DV.mpg DNA and Crime Scene Investigations Copyright © 2009 Pearson Education, Inc.

DNA profiling is the analysis of DNA fragments to determine whether they come from a particular individual –Compares genetic markers from noncoding regions that show variation between individuals –Involves amplification (copying) of markers for analysis –Sizes of amplified fragments are compared

Polymerase chain reaction (PCR) is a method of amplifying a specific segment of a DNA molecule Relies upon a pair of primers –Short DNA molecules that bind to sequences at each end of the sequence to be copied –Used as a starting point for DNA replication Repeated cycle of steps for PCR –Sample is heated to separate DNA strands –Sample is cooled and primer binds to specific target sequence –Target sequence is copied with heat-stable DNA polymerase The PCR method is used to amplify DNA sequences

–Advantages of PCR –Can amplify DNA from a small sample –Results are obtained rapidly –Reaction is highly sensitive, copying only the target sequence Copyright © 2009 Pearson Education, Inc.

Compare STR (short tandem repeats) from individuals.

Gel electrophoresis separates DNA molecules based on size –DNA sample is placed at one end of a porous gel –Current is applied and DNA molecules move from the negative electrode toward the positive electrode –Shorter DNA fragments move through the gel pores more quickly and travel farther through the gel –DNA fragments appear as bands, visualized through staining or detecting radioactivity or fluorescence –Each band is a collection of DNA molecules of the same length

DNA profiling has provided evidence in many forensic investigations –Forensics –Evidence to show guilt or innocence –Establishing family relationships –Paternity analysis –Identification of human remains –After tragedies such as the September 11, 2001, attack on the World Trade Center –Species identification –Evidence for sale of products from endangered species Copyright © 2009 Pearson Education, Inc.