Recombinant DNA Technology 8 Recombinant DNA Technology

The Role of Recombinant DNA Technology in Biotechnology Biotechnology – the use of microorganisms to make practical products Recombinant DNA technology Intentionally modifying genomes of organisms for practical purposes Three goals Eliminate undesirable phenotypic traits Combine beneficial traits of two or more organisms Create organisms that synthesize products humans need

The Tools of Recombinant DNA Technology Mutagens Physical and chemical agents that produce mutations Scientists utilize mutagens to Create changes in microbes' genomes to change phenotypes Select for and culture cells with beneficial characteristics Mutated genes alone can be isolated

The Tools of Recombinant DNA Technology The Use of Reverse Transcriptase to Synthesize cDNA Isolated from retroviruses Uses RNA template to transcribe molecule of cDNA Easier to isolate mRNA molecule for desired protein first cDNA generated from mRNA of eukaryotes has introns removed Allows cloning in prokaryotic cells

The Tools of Recombinant DNA Technology Synthetic Nucleic Acids Molecules of DNA and RNA produced in cell-free solutions Uses of synthetic nucleic acids Use genetic code Creating genes for specific proteins Synthesizing DNA and RNA probes to locate specific sequences of nucleotides

The Tools of Recombinant DNA Technology Restriction Enzymes Bacterial enzymes that cut DNA molecules only at restriction sites Categorized into two groups based on type of cut Cuts with sticky ends Cuts with blunt ends

Figure 8.2 Actions of restriction enzymes. Restriction site (palindrome) 5 G A A T T C 3 5 C C C G G G 3 5 G T T A A C 3 C T T A A G G G G C C C C A A T T G Restriction enzyme Restriction enzyme 1 Restriction enzyme 2 A A T T 5 C C C G G G 3 5 G T T A A C 3 G C G G G C C C C A A T T G C T T A A G Blunt ends Sticky ends Production of blunt ends Production of sticky ends Ligase A A G G C C T T A T A T 5 C C C A A C 3 5 G T T G G G 3 A A T T C G A T T C G A G G G T T G C A A C C C Recombinant DNA molecules Restriction fragments from two different organisms cut by the same restriction enzyme Recombinants using blunt ends Ligase 5 A A G C T T 3 5 A A G C T T 3 + T T C G A A T T C G A A Recombinant DNA molecules Recombinants using sticky ends

The Tools of Recombinant DNA Technology Vectors Nucleic acid molecules that deliver a gene into a cell Useful properties Small enough to manipulate in a lab Survive inside cells Contain recognizable genetic marker Ensure genetic expression of gene Include viral genomes, transposons, and plasmids

The Tools of Recombinant DNA Technology Gene Libraries A collection of bacterial or phage clones Each clone in library often contains one gene of an organism's genome Library may contain all genes of a single chromosome Library may contain set of cDNA complementary to mRNA

Figure 8.4 Production of a gene library. Genome 1 Isolate genome of organism. 2 Generate fragments using restriction enzymes. 1 2 3 4 5 6 7 8 9 10 11 3 Insert each fragment into a vector. 1 2 3 4 5 6 7 8 9 10 11 4 Introduce vectors into cells. 1 2 3 4 5 6 7 8 9 10 11 5 Culture recombinant cells; descendants are clones. 1 2 3 4 5 6 7 8 9 10 11

Techniques of Recombinant DNA Technology Separating DNA Molecules: Gel Electrophoresis and the Southern Blot Gel electrophoresis Separates molecules based on electrical charge, size, and shape Allows scientists to isolate DNA of interest Negatively charged DNA drawn toward positive electrode Agarose makes up gel; acts as molecular sieve Smaller fragments migrate faster and farther than larger ones Determine size by comparing distance migrated to standards

Figure 8.6 Gel electrophoresis. Wells (–) Electrophoresis chamber filled with buffer solution E D C (50) (40) Agarose gel (+) B A (35) (15) (10) a (5) Movement of DNA DNA b Lane of DNA fragments of known sizes (kilobase pairs) Wire

Techniques of Recombinant DNA Technology parating DNA Molecules: Gel Electrophoresis and the Southern Blot Southern blot DNA transferred from gel to nitrocellulose membrane Probes used to localize DNA sequence of interest Northern blot – similar technique used to detect RNA Uses of Southern blots Genetic "fingerprSeinting" Diagnosis of infectious disease Demonstrate presence of organisms that cannot be cultured

Techniques of Recombinant DNA Technology Inserting DNA into Cells Goal of DNA technology is insertion of DNA into cell Natural methods Transformation Transduction Conjugation Artificial methods Electroporation Protoplast fusion Injection – gene gun and microinjection

Applications of Recombinant DNA Technology Genetic Mapping Locating genes on a nucleic acid molecule Provides useful facts concerning metabolism, growth characteristics, and relatedness to others

Applications of Recombinant DNA Technology Environmental Studies Most microorganisms have never been grown in a laboratory Scientists know them only by their DNA fingerprints Allowed identification of over 500 species of bacteria from human mouths Determined that methane-producing archaea are a problem in rice agriculture

Applications of Recombinant DNA Technology Pharmaceutical and Therapeutic Applications Protein synthesis Creation of synthetic proteins by bacteria and yeast cells Vaccines Production of safer vaccines Introduce genes of pathogens into common fruits and vegetables Injecting humans with plasmid carrying gene from pathogen Humans synthesize pathogen's proteins

Applications of Recombinant DNA Technology Agricultural Applications Production of transgenic organisms Recombinant plants and animals altered by addition of genes from other organisms

Applications of Recombinant DNA Technology Agricultural Applications Herbicide tolerance Gene from Salmonella conveys resistance to glyphosate (Roundup) Farmers can kill weeds without killing crops Salt tolerance Scientists have inserted a gene for salt tolerance into tomato and canola plants Transgenic plants survive, produce fruit, and remove salt from soil

Applications of Recombinant DNA Technology Agricultural Applications Freeze resistance Crops sprayed with genetically modified bacteria can tolerate mild freezes Pest resistance Bt toxin Naturally occurring toxin only harmful to insects Organic farmers use to reduce insect damage to crops Gene for Bt toxin inserted into various crop plants Genes for Phytophthora resistance inserted into potato crops

The Ethics and Safety of Recombinant DNA Technology Studies have not shown any risks to human health or environment Standards imposed on labs involved in recombinant DNA technology Can create biological weapons using same technology

The Ethics and Safety of Recombinant DNA Technology Ethical issues Routine screenings? Who should pay? Genetic privacy rights? Profits from genetically altered organisms? Required genetic screening? Forced correction of "genetic abnormalities"?