Chapter 9 Biotechnology and Recombinant DNA Part 2

Restriction Enzymes –DNA cutting enzymes that exist in many bacteria –Cut specific sequences of DNA (recognize 4-, 6-, or 8-base sequences), staggered cuts –Destroy bacteriophage DNA in bacterial cells –Cannot digest (host) DNA with methylated cytosines Tools of Biotechnology

Figure 9.2 Restriction Enzymes

Tools of Biotechnology Vectors –Carry new DNA to desired cell –Plasmids and viruses can be used as vectors –Four properties of vectors Can self-replicate Be a size that allows them to be manipulated outside the cell during recombinant DNA procedures Preservation (circular form of DNA and integrated into host chromosome) Have a marker within the vector for easy selection

Shuttle vectors: a plasmid that can exist in several different species –Very useful in the process of geneticaly modifying multicellular organisms Viral DNA can usually accept much larger pieces of foreign DNA than plasmid –Retroviruses, adenoviruses, & herpesviruses Choice of suitable vector depends on many factors (e.g host & size of the DNA to be cloned) Tools of Biotechnology

Vectors Figure 9.3

Polymerase Chain Reaction (PCR) –To make multiple copies of a piece of DNA enzymatically (limited by the choice of primers used) –Cannot be used to amplify an entire genome –Used to Clone DNA for recombination Amplify DNA to detectable levels Sequence DNA Diagnose genetic disease Detect pathogens Tools of Biotechnology

PCR Figure 9.4.1

PCR Figure 9.4.2

Figure 9.5b Techniques of Genetic Engineering Inserting foreign DNA into cells –Transformation –Electroporation –Protoplast fusion –Gene gun –Microinjection

Techniques of Genetic Engineering –Choice of method is usually determined by the type of vector and host being used Foreign DNA will survive only if it is either present on a self-replicating vector or incorporated into one of the cell’s chromosomes by recombination

Techniques of Genetic Engineering Transformation: used to insert plasmid vector into a cell –many cell types do not naturally transform need to make them competent (able to take up external DNA) Electroporation: uses an electrical current to form microscopic pores in the membranes of cells (DNA enter cells through the pores)

Techniques of Genetic Engineering –Generally applicable to all cells; ones with cell wall must be converted to protoplasts first Protoplast fusion: a method of joining two cells by first removing their cell walls –Protoplasts in solution will fuse at a low but significant rate (can add polyethylene glycol to increase the frequency of fusion) –Valuable in the genetic manipulation of plant and algal cells

Fig. 9.5

Techniques of Genetic Engineering Gene gun: Microscopic particles of tungsten or gold are coated with DNA and propelled by a burst of helium through the plant cell walls –Some of the cells express the introduced DNA as if it were their own if incorporated into host chromosome

Figure 9.6 & 7 Techniques of Genetic Engineering Microinjection: introduce DNA directly into an animal cell using a glass miropipette

Gene library: a collection of cloned DNA fragments created by inserting restriction enzyme fragments in a bacterium, yeast, or phage –Make a collection of clones large enough to ensure that at least one clone exists for every gene in the organism –Pieces of an entire genome stored in plasmids or phage Obtaining DNA

Fig. 9.8

Obtaining DNA Cloning genes from eukaryotic organisms poses a special problems due to introns –Need to use a version of the genes that lacks intron = mRNA cDNA is made from mRNA by reverse transcriptase (mRNA cDNA) cDNA is the most common method of obtaining eukaryotic genes

Fig. 9.9

Obtaining DNA Synthetic DNA is made by a DNA synthesis machine –Chain of over 120 nucleotides can be synthesized –Need to know the sequence of the gene –Rare to clone a gene by synthesizing it directly –Plays a much more useful role selection procedures (add desired restriction sites)

Selecting a clone Use antibiotic resistance genes (marker) on plasmid vectors to screen for cells carrying the desired gene (engineered vector) –e.g. Blue-white screening (2 marker genes on the plasmid vector = amp R and  -galactosidase)

Genetic Engineering Figure Blue-white screening

Genetic Engineering Figure

Selecting a clone Need a second procedure to test if screened bacteria does contain desirable genes –Test clones for desired gene product or ID genes itself in the host bacterium –Colony hybridization: use DNA probe that is complementary to the desired genes DNA probe: short segment of single-stranded DNA that are complementary to the desired gene

Figure Colony hybridization

Figure

Making a gene product Earliest work in genetic engineering used E. coli to synthesize the gene products –E. coli was used because it is easily grown and its genomics are known –Disadvantages of using E. coli: Produce endotoxins (Lipid A, part of LPS layer on the cell wall) Does not secrete protein products need to lyse cells to obtain products Industry prefers Bacillus subtilis because it secretes their products

Use baker’s yeast (Saccharomyces cerevisiae) –Yeast may carry plasmid and has best understood eukaryotic genome –May be more successful in expressing foreign eukaryotic genes than bacteria; likely to secrete products Use mammalian cells in culture –Hosts for growing viruses (vectors) –Often the best suited to making protein products for medical use (e.g. hormones, cytokines, interferon) Making a gene product

Use plant cells in culture –Ti plasmid (from bacterium Agrobacterium tumefaciens), protoplast fusion and gene gun –Use to produce genetically engineered plants May be sources for plant alkaloids (painkiller), isoprenoids (basis for synthetic rubber), and melanin (for sunscreens)

Applications of Genetic Engineering Produce useful substances more efficiently and cheaper Obtain information from the cloned DNA that is useful for either basic research or medical applications Use cloned genes to alter the characteristics of cells or organisms

Subunit vaccines Nonpathogenic viruses carrying genes for pathogen's antigens as vaccines Gene therapy to replace defective or missing genes Human Genome Project –Nucleotides have been sequenced –Human Proteome Project may provide diagnostics and treatments Therapeutic applications

Random Shotgun Sequencing Figure 9.14

Understanding of DNA Sequencing organisms' genomes DNA fingerprinting for identification Scientific Applications Figure 9.16

Southern Blotting Figure

Southern Blotting Figure

Southern Blotting Figure

Agricultural Applications Table 9.2

Genetic Engineering Using Agrobacterium Figure 9.18

Avoid accidental release Genetically modified crops must be safe for consumption and for the environment Who will have access to an individual's genetic information? Safety Issues and Ethics