Biotechnology and DNA Technology

Transcription

1 11/27/2017 PowerPoint Lecture Presentations prepared by Bradley W. Christian, McLennan Community College CHAPTER 9 Biotechnology and DNA Technology Introduction to Biotechnology Learning Objectives Compare and contrast biotechnology, genetic modification, and recombinant DNA technology. Identify the roles of a clone and a vector in making recombinant DNA. 1

2 Introduction to Biotechnology Biotechnology: the use of microorganisms, cells, or cell components to make a product Foods, antibiotics, vitamins, enzymes Recombinant DNA (rdna) technology: the insertion or modification of genes to produce desired proteins An Overview of Recombinant DNA Procedures Vector: self-replicating DNA molecule used to transport foreign DNA into a cell For example plasmid Clone: population of genetically identical cells arising from one cell; each carries the vector 2

3 Figure 9.1 A Typical Genetic Modification Procedure. Tools of Biotechnology Learning Objectives Define restriction enzymes, and outline how they are used to make rdna. List the four properties of vectors. Describe the use of plasmid and viral vectors. Outline the steps in PCR, and provide an example of its use. 3

4 Tools of Biotechnology Selection: selecting for a naturally occurring microbe that produces a desired product Mutation: Mutagens cause mutations that might result in a microbe with a desirable trait Site-directed mutagenesis: a targeted and specific change in a gene Random mutagenesis: a non-targeted and nonspecific change in a gene Restriction Enzymes Cut specific sequences of DNA Origin: destroy bacteriophage DNA in bacterial cells Methylated cytosines in bacteria protect their own DNA from digestion Create blunt ends or staggered cuts known as sticky ends 4

5 Restriction enzymes: from chapter 13 A restriction enzyme or restriction endonuclease is an enzyme that cuts DNA at or near specific recognition nucleotide sequences known as restriction sites. These enzymes are found in bacteria and archaea and provide a defense mechanism against invading viruses. Over 3000 restriction enzymes have been studied in detail, and more than 600 of these are available commercially. Essential tools in recombinant DNA technology! Table 9.1 Selected Restriction Enzymes Used in rdna Technology 5

6 Figure 9.2 A restriction enzyme's role in making rdna. Recognition sites A restriction enzyme cuts (red arrows) double-stranded DNA at its particular recognition sites, shown in blue. DNA Cut Cut Cut Cut These cuts produce a DNA fragment with two sticky ends. DNA from another source, perhaps a plasmid, cut with the same restriction enzyme Sticky end When two such fragments of DNA cut by the same restriction enzyme come together, they can join by base pairing. The joined fragments will usually form either a linear molecule or a circular one, as shown here for a plasmid. Other combinations of fragments can also occur. The enzyme DNA ligase is used to unite the backbones of the two DNA fragments, producing a molecule of rdna. rdna Vectors Carry new DNA to desired cells Must be able to self-replicate Plasmids and viruses can be used as vectors Shuttle vectors exist in several different species and can move cloned sequences among various organisms 6

7 Figure 9.3 A plasmid used for cloning. amp R puc19 lacz HindIII BamHI EcoRI ori Polymerase Chain Reaction Process of increasing small quantities (amplifying) of DNA for analysis Used for diagnostic tests for genetic diseases and detecting pathogens Reverse-transcription PCR (rt-pcr) uses mrna as template Miten paljon kertausta tarvitaan PCR-aiheeseen? Onko tullut selväksi Biotieteen perusteissa? 7

8 Video: PCR Figure 9.4 The polymerase chain reaction. 8

9 Check Your Understanding For what is each of the following used in PCR: primer, DNA polymerase, 94 C? Understand different steps in PCR: - Denaturation - Annealing - Extension Techniques of Genetic Modification Learning Objectives Describe different ways of getting DNA into a cell. Differentiate cdna from synthetic DNA. Explain how each of the following is used to locate a clone: antibiotic-resistance genes, DNA probes, gene products. List one advantage of modifying each of the following: Escherichia coli, Saccharomyces cerevisiae, mammalian cells, plant cells. 9

10 Inserting Foreign DNA into Cells DNA can be inserted into a cell by: Transformation: Cells take up DNA from the surrounding environment (usually requires competent cells = cell walls weakened) Electroporation: Electrical current forms pores in cell membranes Protoplast fusion: Removing cell walls from two bacteria allows them to fuse Figure 9.5 Protoplast fusion. Chromosome Plasma membrane Cell wall Bacterial cells Bacterial cell walls are enzymatically digested, producing protoplasts. Protoplasts In solution, protoplasts are treated with polyethylene glycol. Protoplasts fuse. Segments of the two chromosomes recombine. Recombinant cell Recombinant cell grows new cell wall. 10

11 Inserting Foreign DNA into Cells Eukaryotes: DNA can be inserted into a cell by: Gene gun Microinjection Transformation (e.g. yeast, requires competent cells) Prokaryotes (bacteria): Transformation (requires competent cells) Figure 9.6 A gene gun, which can be used to insert DNA-coated "bullets" into a cell. 11

12 Figure 9.7 The microinjection of foreign DNA into an egg. Figure 9.9 Making complementary DNA (cdna) for a eukaryotic gene. Exon Intron Exon Intron Exon DNA Nucleus RNA transcript A gene composed of exons and introns is transcribed to RNA by RNA polymerase. Processing enzymes in the nucleus remove the intron-derived RNA and splice together the exon-derived RNA into mrna. mrna mrna is isolated from the cell, and reverse transcriptase is added. Cytoplasm DNA strand being synthesized First strand of DNA is synthesized. The mrna is digested by reverse transcriptase. cdna of gene without introns DNA polymerase is added to synthesize second strand of DNA. Test tube 12

13 Synthetic DNA Builds genes using a DNA synthesis machine Several manufacturers on the market (GeneArt, IDT, GenScript etc.) Figure 9.10 A DNA synthesis machine. 13

14 Making a Gene Product E. coli Advantages: easily grown and its genomics are known Disadvantages: produces endotoxins and does not secrete its protein products Figure 9.13 E. coli genetically modified to produce gamma interferon, a human protein that promotes an immune response. 14

15 Making a Gene Product Saccharomyces cerevisiae Easily grown and has a larger genome than bacteria Expresses eukaryotic genes easily Plant cells and whole plants Express eukaryotic genes easily Plants are easily grown, large-scale, and low-cost Mammalian cells Express eukaryotic genes easily Can make products for medical use Harder to grow Applications of DNA Technology Learning Objectives List at least five applications of DNA technology. Discuss the value of genome projects. Define the following terms: random shotgun sequencing, bioinformatics, proteomics. 15

16 Applications of DNA Technology Learning Objectives Diagram DNA fingerprinting, and provide an example of its use. Outline genetic engineering with Agrobacterium. Therapeutic Applications Human enzymes and other proteins such as insulin Subunit vaccines: made from pathogen proteins in genetically modified yeasts Nonpathogenic viruses carrying genes for pathogen's antigens as DNA vaccines Gene therapy to replace defective or missing genes 16

17 Table 9.2 Some Pharmaceutical Products of rdna (1 of 2) Table 9.2 Some Pharmaceutical Products of rdna (2 of 2) 17

18 Therapeutic Applications Gene silencing Small interfering RNAs (sirnas) bind to mrna, which is then destroyed by RNA-induced silencing complex (RISC) RNA interference (RNAi) inserts DNA encoding sirna into a plasmid and transferred into a cell Figure 9.14 Gene silencing could provide treatments for a wide range of diseases. 18

19 Scientific Applications Bioinformatics: understanding gene function via computer-assisted analysis Proteomics: determining proteins expressed in a cell Reverse genetics: discovering gene function from a genetic sequence Forensic Microbiology DNA fingerprinting is used to identify pathogens PCR microarrays and DNA chips can screen samples for multiple pathogens Differs from medicine because it requires: Proper collection of evidence Establishing a chain of custody 19

20 DNA fingerprinting e.g. in forensics: video Nanotechnology Bacteria can make molecule-sized particles Nanospheres used in drug targeting and delivery 20

21 Figure 9.18 Bacillus cells growing on selenium form chains of elemental selenium. Figure 9.20 Using the Ti plasmid as a vector for genetic modification in plants. Agrobacterium tumefaciens bacterium Inserted T-DNA carrying foreign gene Restriction cleavage site T-DNA Ti plasmid The plasmid is removed from the bacterium, and the T-DNA is cut by a restriction enzyme. Foreign DNA is cut by the same enzyme. The plasmid is reinserted into a bacterium. Recombinant Ti plasmid The foreign DNA is inserted into the T-DNA of the plasmid. The bacterium is used to insert the T-DNA carrying the foreign gene into the chromosome of a plant cell. A plant is generated from a cell clone. All of its cells carry the foreign gene and may express it as a new trait. The plant cells are grown in culture. 21

22 Agricultural Applications Bt toxin Herbicide resistance Suppression of genes Antisense DNA Nutrition Human proteins Table 9.3 Some Agriculturally Important Products of rdna Technology 22

23 Safety Issues and Ethics of Using DNA Technology Learning Objective List the advantages of, and problems associated with the use of genetic modification techniques. Safety Issues and Ethics of Using DNA Technology Need to avoid accidental release into the environment Genetically modified crops must be safe for consumption and for the environment Who will have access to an individual's genetic information? 23