Recombinant DNA BiotechnologyEthical Concerns over Gene TherapyThe Effort to Cure Pituitary DwarfismWhy Did Early Efforts to Treat the Disease Fail?Genetic engineering: Engineering a Safe Supply of Growth HormoneRECOMBINANT DNA & CLONINGFrom 7. Prokaryotic Cells – Genetic InformationUsing Plasmids in CloningUsing Reverse Transcriptase to Produce cDNAsCutting and Pasting DNARestriction Endonucleases (RE): “molecular scissors”Slide 12Transformation=Introducing chimeric plasmid into bacteriaSlide 14Slide 15BCMB 419: pGLO and pGLO-transformed E.ColiGenetic Engineering – synthetic genesExample of a molecular cloning experiment: Synthetic Insulin - Use of Bacteria as “Protein Factories”:Some recombinant DNA products in medicine:Slide 20Slide 21Rice as a Target Crop-Synthesizing b-Carotene in RiceThe Agrobacterium Transformation SystemSlide 24Using the Ti Plasmid to Produce Golden RiceSlide 26Slide 27Recombinant DNA technology yields new products and choices© 2011 Pearson Education, Inc.Recombinant DNA Biotechnology i) The use of gene-replacement therapy to treat individuals with diseases caused by dysfunctional genes  gene therapyii) Identification of biological specimens at crime scenes  PCRiii) Using bacterial cells to produce large quantities of proteins for medical or industrial use. example: INSULIN, Growth hormoneManipulation of DNA sequences in organisms is known as genetic engineering, and techniques used to engineer genes are called recombinant DNA technology.© 2011 Pearson Education, Inc.Ethical Concerns over Gene Therapy•Researchers are attempting to insert genes into humans to cure genetic diseases. The use of gene-replacement therapy to treat individuals with diseases caused by dysfunctional genes  Gene therapy•Gene therapy is highly experimental, extremely expensive, and intensely controversial.•Although gene therapy holds great promise for the treatment of a wide variety of devastating inherited diseases, fulfilling that promise is almost certain to require many years of additional research and testing, as well as the refinement of legal and ethical guidelines.© 2011 Pearson Education, Inc.The Effort to Cure Pituitary Dwarfism•Pituitary dwarfism results from the lack of production of growth hormone, encoded by the GH1 gene.Humans affected by pituitary dwarfism grow slowly, reaching a maximum adult height of about 4 feet.© 2011 Pearson Education, Inc.Why Did Early Efforts to Treat the Disease Fail?•Early trials showed that people with pituitary dwarfism could be treated successfully with growth hormone therapy, but only if the protein came from humans. •Growth hormone purified from the pituitary glands of human cadavers is scarce and expensive. •Human treatment with growth hormone from cadavers has been banned due to possible contamination with prions—protein particles that have been implicated as the cause of various neurodegenerative disorders.© 2011 Pearson Education, Inc.Genetic engineering: Engineering a Safe Supply of Growth Hormone•The recombinant DNA strategy for producing human growth hormone involved cloning the human gene, introducing the gene into bacteria, and having the recombinant cells (selection) produce the hormone.Recombinant Growth Hormone- Use of Bacteria as “Protein Factories”:© 2011 Pearson Education, Inc.RECOMBINANT DNA & CLONING3 Components in a cloning experiment: 1. “GENE” of interest – example: GH1 gene2. VECTOR - example: Plasmid3. HOST – E. Coli, Yeast cells, Plant cells, Mammalian cells Ligation and Transformation:-Using “sticky” ends to construct recombinant DNA (chimeric DNA)© 2011 Pearson Education, Inc.From 7. Prokaryotic Cells – Genetic Information •Most prokaryotic species have one supercoiled circular chromosome (DNA) found in the nucleoid region of the cell. •In addition to the large chromosome, many bacteria contain plasmids.–Small, supercoiled, circular DNA molecules–Plasmids usually contain genes that help the cell adapt to unusual environmental conditions.© 2011 Pearson Education, Inc.Using Plasmids in Cloning•Plasmids are small, circular double stranded DNA molecules often found in bacteria. They replicate independently of the chromosome.•Plasmids can serve as a vector—a vehicle for transferring recombinant genes to a new host.•If a recombinant plasmid can be inserted into a bacterial or yeast cell, the foreign DNA will be copied and transmitted to new cells as the host cell grows and divides. In this way plasmids can be used to produce millions of identical copies of specific genes.© 2011 Pearson Education, Inc.Using Reverse Transcriptase to Produce cDNAs•DNA of Interest: mRNA   cDNA•The enzyme reverse transcriptase can synthesize DNA from an RNA template. •Researchers used reverse transcriptase to make complementary DNA (cDNA) from mRNA isolated from pituitary cells. (cDNA is any DNA made from an RNA template.)•They then used DNA cloning—the process of producing many identical copies of a gene—to copy the cDNAs for analysis to determine which encoded the growth hormone protein.© 2011 Pearson Education, Inc.Cutting and Pasting DNA•Restriction endonucleases are bacterial enzymes that cut DNA at specific base sequences called recognition sites. •The first step in cloning genes into plasmids is to cut the plasmid and the cDNA with the same restriction endonuclease.–Restriction endonucleases often make staggered cuts in the DNA, resulting in sticky ends, complementary single-stranded ends.•The sticky ends of the plasmids and cDNAs will bind by complementary base pairing.•DNA ligase then seals the recombinant pieces of DNA together.© 2011 Pearson Education, Inc.Restriction Endonucleases (RE): “molecular scissors”•Recognize Palindromic sequences on ds DNA—it reads the same in both directions from the 5′ end.•4-8 base pair sequence•Cuts both strands•“Sticky” and “blunt” ends.•DNA Ligase can seal these ends.•Important tool in DNA cloning© 2011 Pearson Education, Inc.© 2011 Pearson Education, Inc.Transformation=Introducing chimeric plasmid into bacteriaIf a recombinant plasmid can be inserted into a bacterial or yeast cell, the foreign DNA will be copied and transmitted to new cells as the host cell grows and divides. In this way, researchers can obtain millions or billions of copies of specific genes. •Plasmid vectors can be introduced into bacteria by transformation, the process of