2 Chapter 9 Biotechnology & Recombinant DNA

3 Recombinant Technologies Terminology Recombinant DNA - artificially manipulated DNA Genetic Engineering – Inserting genes into cells Biotechnology – Using cells or cell components to create a useful product

Typical Genetic Engineering Procedure

Bioremediation

6 Restriction enzymes - enzymes which cut DNA (endonucleases) at specific DNA sequences (fig. 9.2) Tools Used in Biotechnology

Hae III - GGCC CCGG Examples of restriction enzymes and their recognition sequences Bam H1 – GGATCC CCTAGG Hin dIII - AAGCTT TTCGAA Eco RI - GAATTC CTTAAG Bal I - TGGCCA ACCGGT

10 Tools Used in Biotechnology Vectors - DNA molecules used in the transfer of genes. Must be self-replicating. (fig. 9.3) Examples: plasmids, some viruses

pUC19 is a shuttle vector – can exist in several species of bacteria & carry genes between cells

12 Methods of Inserting Foreign DNA Transformation – absorbing naked DNA. Artificially introduce DNA into bacterial cells through transformation. First necessary to treat the cells chemically to make them competent (capable of taking in naked DNA). Ex. E. coli Electroporation - electrical current makes pores in cell membrane through which DNA enters (may have to first make cell into a protoplast).

13 Methods of Inserting Foreign DNA Protoplast – a cell which has had its cell wall removed, usually by enzymatic digestion. This action exposes the plasma membrane through which DNA can be more easily absorbed Protoplast fusion - fuse two protoplasts, genetic recombination can then occur (used in plants)

Microinjection - directly insert DNA through a micropipette. Usually inject multiple copies of the gene of interest in the hope that at least one will incorporate in the chromosome (animal cells)

15 Sources of DNA Gene Libraries - collection of clones containing different DNA fragments (each one a part of the genome) cDNA (complementary DNA) - eukaryotic genes cannot be easily cloned in bacteria due to the presence of introns (stretches of DNA inside a gene which do not code for protein -- the coding parts are called exons)

16 Processing Eukaryotic DNA for Transferring to Prokaryote In the eukaryotic cell, the introns are removed by RNA processing, or splicing, of the mRNA transcript to clone eukaryotic genes, the processed mRNA is converted to cDNA by an enzyme called reverse transcriptase The resulting cDNA can then be inserted in a bacterial cell

18 Another Source for DNA Synthetic DNA - DNA synthesizing machines can produce genes by creating a desired sequence of nucleotides DRAWBACKS to synthetic DNA  can only make short sequences (120 nucleotides) – usually too short for an entire gene  must link all the short sequences together to form the whole gene  must already know the desired sequence to be synthesized (can be predicted if the amino acid sequence of the desired protein is known) has been successful with commercial insulin and interferon

19 Applications for Genetically Engineered Products GENETICALLY ENGINEERED PRODUCTS FOR MEDICAL THERAPY Synthetic insulin produced by E. coli Tissue-plasminogen activator - produced by a genetically- engineered mammalian cell culture Streptokinase (natural bacterial product) used in dissolving blood clots

20 Applications for Genetically Engineered Products \ Production of subunit vaccines - protein portion of a pathogen (not entire pathogen itself) used as vaccine free from possibility of infection (produced in yeast) Better understanding of the life cycle and method of infection of pathogens (example, HIV) through cloning of genes see also Table 9.1 (p264) for product list

21 Obtaining DNA Information DNA sequencing - determining the exact sequence of nucleotide bases in the DNA – automation makes this a rapid process. On February 12, 2001 the entire sequence of human genome was published on Darwin’s birthday

22 Obtaining DNA Information Gel electrophoresis - separation of DNA fragments using a gel matrix and an electrical current. DNA being negatively charged moves toward the positive end of the gel. Smaller pieces move through the dense gel faster than large pieces. Southern blotting - identifying specific segments of DNA (genes) by hybridizing complementary DNA probes.

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Electrophoresis - Current applied to gel. DNA moves according to size. Smaller moves faster/farther. (+) (-) Southern Blotting –. DNA moves with solution and is trapped in filter (read details in figure) Radioactive (or dye) labeled probe specific for gene of interest hybridized to filter. DNA of interest is labeled

25 Obtaining DNA Information RFLPs - restriction fragment length polymorphisms – Inherited differences in distance between restriction enzyme sites. Can test for inherited differences in a person’s DNA (can be used to test for genetic diseases -- genetic screening) DNA fingerprinting – useful for identification of pathogens, and for paternity testing

26 Polymerase Chain Reaction (PCR) A method to amplify (make a large quantity of) a specific DNA sequence in a matter of hours (fig. 9.4, p. 255) 1.uses high temperature denaturizing (melting) of the DNA to separate the strands 2.anneal known sequence primers to ends of specific DNA sequences and 3.uses DNA polymerase from Thermus aquaticus to copy the DNA between the primers. T. acquaticus is thermophilic bacterium, this polymerase can tolerate high temperature without damage 4.repeat the process, making more copies from both the original and the previously made copies

29 Uses for PCR Used in diagnostics to identify infectious agents Used in gene libraries to identify specific genes or clones of interest Amplifying small amounts of a sample from a valuable source such as a crime scene

30 Safety and Ethics Pages Read over these sections.

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