PowerPoint Lecture Presentations prepared by Mindy Miller-Kittrell, North Carolina State University C H A P T E R 8 Recombinant DNA Technology The Role of Recombinant DNA Technology in Biotechnology Biotechnology? the use of microorganisms to make practical products Recombinant DNA = DNA from 2 different sources What is Recombinant DNA Technology? modifying genomes of organisms for practical purposes e.g. Eliminate undesirable phenotypic traits, Combine beneficial traits of two or more organisms, Create organisms that synthesize products humans need yeast Fermentation = beer Biotechnology genetically altered yeast Add a bacterial gene for a more efficient enzyme genetically altered yeast Recombinant DNA technology Efficient Fermentation = better beer Recombinant DNA in Biotechnology 1
Microbiology Laboratory Career Vs Nursing Career Figure 8.1 Overview of recombinant DNA technology - Cloning Bacterial cell DNA containing gene of interest Bacterial chromosome Plasmid 1 Isolate plasmid. Gene of interest 2 Enzymatically cleave DNA into fragments. 3 Isolate fragment with the gene of interest. 4 Insert gene into plasmid. 5 Insert plasmid and gene into bacterium. Bacteria for CLONING What is cloning? 6 Culture bacteria. Harvest copies of gene to insert into plants or animals Harvest proteins coded by gene Eliminate undesirable phenotypic traits Create beneficial combination of traits Produce vaccines, antibiotics, hormones, or enzymes The Tools of Recombinant DNA Technology To1) Mutagens To2) Reverse Transcriptase (RT) To3) Synthetic Nucleic Acids (NA) To4) Restriction Enzymes REnzyme To5) Vectors To6) Gene Libraries 2
The Tools of Recombinant DNA Technology To1) Mutagens generate mutants To2) Reverse Transcriptase (RT) RNA to DNA To3) Synthetic Nucleic Acids (NA) generate mutants To4) Restriction Enzymes fragment DNA nonrandomly To5) Vectors carriers for cloning To6) Gene Libraries collection of genome fragments The Tools of Recombinant DNA Technology To1) Mutagens Physical and chemical agents that produce mutations Ultraviolet light Thymine dimer How to create point mutations The Tools of Recombinant DNA Technology To2) The Use of Reverse Transcriptase to Synthesize cdna (Isolated from retroviruses, allows cloning in prokaryotic cells) Reverse Transcriptase cdna Complementary DNA 3
The Tools of Recombinant DNA Technology To3) Synthetic Nucleic Acids To4) Restriction Enzymes (a) Cuts with sticky ends (b) or blunt ends Restriction site (palindrome) G A A T T C C T T A A G C C C G G G G G G C C C G T T A A C C A A T T G Restriction enzyme Restriction enzyme 1 Restriction enzyme 2 G C T T A A Sticky ends Production of sticky ends C C C G G G G G G C C C Blunt ends Production of blunt ends G T T A A C C A A T T G Ligase A A T T C G A T T C G A Restriction fragments from two different organisms cut by the same restriction enzyme C C C A A C G T T G G G G G G T T G C A A C C C Recombinant DNA molecules Recombinants using blunt ends Ligase A A G C T T A A G C T T T T C G A A + T T C G A A Recombinant DNA molecules Recombinants using sticky ends Figure 8.3 An example of the process for producing a recombinant vector. mrna for human growth hormone (HGH) Antibiotic Restriction resistance site gene Reverse transcription To5) Vectors carriers for cloning cdna for HGH Plasmid (vector) 1 Restriction Restriction enzyme enzyme Sticky ends A G C T T HGH A A HGH T T C G A Gene for human growth hormone 2 Ligase Recombinant plasmid 3 Introduce recombinant plasmid into bacteria. Bacterial chromosome Recombinant plasmid 4 Inoculate bacteria on media containing antibiotic. Bacteria containing the plasmid with HGH gene survive because they also have resistance gene. 4
The Tools of Recombinant DNA Technology To6) Gene Libraries 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 To6) Gene Libraries 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 Te1) The Polymerase Chain Reaction (PCR) Te2) Separating DNA Molecules: Gel Electrophoresis (Gel) Te3) Separating DNA Molecules: Southern Blot (Blot) Te4) Selecting a Clone of Recombinant Cells Te5) DNA Microarrays (Gene Expression) Te6) Inserting DNA into Cells 5
Techniques of Recombinant DNA Technology Te1) The Polymerase Chain Reaction (PCR) Multiplying DNA in vitro: amplify DNA in variety of situation Repetitive process consisting of three steps Denaturation Priming Extension Automated Figure 8.5a The use of the polymerase chain reaction (PCR) to replicate DNA. 1 Denaturation Original DNA molecule Heat to 94 C 2 Priming DNA primer Deoxyribonucleotide triphosphates DNA polymerase 4 Repeat Cool to 6C DNA polymerase 3 Extension DNA primer 72 C Figure 8.5b The use of the polymerase chain reaction (PCR) to replicate DNA. 6
Techniques of Recombinant DNA Technology Te2) Separating DNA Molecules: 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 ( ) A B C D E (50) (40) (35) Electrophoresis chamber filled with buffer solution Agarose gel (+) (15) (10) (5) DNA a Movement of DNA b Wire Lane of DNA fragments of known sizes (kilobase pairs) Techniques of Recombinant DNA Technology Te3) Separating DNA Molecules: 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 "fingerprinting" Diagnosis of infectious disease Demonstrate presence of organisms that cannot be cultured 7
Figure 8.7 The Southern blot technique. DNA molecules Restriction enzymes Restriction fragments Use gel electrophoresis to separate 1 fragments by size; denature DNA into single strands with NaOH. DNA The DNA fragments are invisible to the investigators at this stage. DNA bands Gel Nitrocellulose membrane Absorbent material 2 Side view Electrophoresis gel Nitrocellulose membrane Absorbent material Nitrocellulose membrane with DNA fragments at same locations as in gel (still invisible) is baked to permanently affix DNA. 3 Add radioactive probes complementary to DNA nucleotide sequence of interest. Probes bind to DNA 4 of interest. Incubate with film; radiation exposes film. Develop film. Developed film 5 Techniques of Recombinant DNA Technology Te4) DNA Microarrays Consist of molecules of immobilized single-stranded DNA Fluorescently labeled DNA washed over array will adhere only at locations where there are complementary DNA sequences Variety of scientific uses of DNA microarrays Monitoring gene expression Diagnosis of infection Identification of organisms in an environmental sample Figure 8.8 DNA microarray. 8
Techniques of Recombinant DNA Technology Te5) Selecting a Clone of Recombinant Cells Must find clone containing DNA of interest Probes are used The Tools of Recombinant DNA Technology To3) Synthetic Nucleic Acids Figure 8.8 DNA microarray. To3) Synthetic Nucleic Acids 9
Techniques of Recombinant DNA Technology Te6) Inserting DNA into Cells: Natural methods Transformation Transduction Conjugation Artificial methods Electroporation Protoplast fusion Injection gene gun and microinjection Figure 8.9a-b Artificial methods of inserting DNA into cells. Pores in wall and membrane Chromosome Cell synthesizes new wall Electrical field applied Competent cell Recombinant cell Electroporation DNA from another source Cell walls Cell synthesizes new wall Enzymes remove cell walls Polyethylene glycol Recombinant cell New wall Protoplasts Fused protoplasts Protoplast fusion Figure 8.9c-d Artificial methods of inserting DNA into cells. Micropipette containing DNA Target cell's nucleus Blank.22 Nylon caliber shell projectile Vent Plate to stop nylon projectile Target cell DNA-coated beads Target cell Suction tube to hold target cell in place Gene gun Nylon projectile Microinjection 10
Figure 8.1 Overview of recombinant DNA technology - Cloning Bacterial cell DNA containing gene of interest Bacterial chromosome Plasmid 1 Isolate plasmid. Gene of interest To5 2 Enzymatically cleave DNA into fragments. 3 Isolate fragment with the gene of interest. 4 Insert gene into plasmid. 5 Te6 Insert plasmid and gene into bacterium. To4 6 Culture bacteria. Harvest copies of gene to insert into plants or animals Harvest proteins coded by gene Eliminate undesirable phenotypic traits Create beneficial combination of traits Produce vaccines, antibiotics, hormones, or enzymes Figure 8.3 An example of the process for producing a recombinant vector. mrna for human growth hormone (HGH) Antibiotic Restriction resistance site gene Reverse transcription To5) Vectors carriers for cloning cdna for HGH Plasmid (vector) 1 Restriction Restriction enzyme enzyme Sticky ends A G C T T HGH A A HGH T T C G A Gene for human growth hormone 2 Ligase Recombinant plasmid 3 Introduce recombinant plasmid into bacteria. Bacterial chromosome Recombinant plasmid 4 Inoculate bacteria on media containing antibiotic. Bacteria containing the plasmid with HGH gene survive because they also have resistance gene. The problem Make lots of human Insulin Insert human Insulin gene in bacteria to make genetically engineered insulin protein But first need to find human Insulin gene 11
How to find human Insulin gene Isolate human pancreatic RNA Te5 Convert RNA to DNA Make DNA microarray To2 screen To6 Screen Gene library Te2 To3 Synthetic Nucleic Acid got human Insulin gene Te1 Clone human Insulin gene Make lots of it got human Insulin gene Clone human Insulin gene Make lots of it To4 To5 Te6 Applications of Recombinant DNA Technology 1) Genetic Mapping 2) Environmental Studies 3) Pharmaceutical and Therapeutic Applications 4) Agricultural Applications 12
Applications of Recombinant DNA Technology Genetic Mapping Locating genes on a nucleic acid molecule Restriction fragmentation Fluorescent in situ hybridization (FISH) Figure 8.10 Fluorescent in situ hybridization (FISH). 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 13
Applications of Recombinant DNA Technology Pharmaceutical and Therapeutic Applications Protein Synthesis human Insulin gene in bacteria (genetically engineered proteins) Hepatitis B vaccine Applications of Recombinant DNA Technology Agricultural Applications Production of transgenic organisms Recombinant plants and animals altered by addition of genes from other organisms Herbicide tolerance Gene from Agrobacterium conveys resistance to herbicide glyphosate (Roundup) Farmers can kill weeds without killing crops Transgenic Fish 14
Transgenic what? PROS and CON of GMO 15
The Safety of Recombinant DNA Technology Long-term effects of transgenic manipulations are unknown Transgenic organisms could trigger allergies or cause harmless organisms to become pathogenic Can create biological weapons using same technology The Ethics of Recombinant DNA Technology Routine screenings? Who should pay? Genetic privacy rights? Profits from genetically altered organisms? 16