Recombinant DNA Technology

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1 PowerPoint Lecture Presentations prepared by Mindy Miller-Kittrell, North Carolina State University C H A P T E R 8 Recombinant DNA Technology

2 The Role of Recombinant DNA Technology in Biotechnology Biotechnology the use of microorganisms to make practical products 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

3 The Tools of Recombinant DNA Technology 1) Mutagens Physical and chemical agents that produce mutations 2) The Use of Reverse Transcriptase to Synthesize cdna (RT) Isolated from retroviruses, allows cloning in prokaryotic cells 3) Synthetic Nucleic Acids (NA) 4) Restriction Enzymes REnzyme (a) Cuts with sticky ends (b) or blunt ends 5) Vectors (plasmids, virus, transposons) 6) Gene Libraries

4 Techniques of Recombinant DNA Technology 1) The Polymerase Chain Reaction (PCR) 2) Separating DNA Molecules: Gel Electrophoresis (Gel) 3) Separating DNA Molecules: Southern Blot (Blot) 4) Selecting a Clone of Recombinant Cells 5) DNA Microarrays (Gene Expression) 6) Inserting DNA into Cells

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

6 Figure 8.1 Overview of recombinant DNA technology - Cloning Bacterial cell DNA containing gene of interest Bacterial chromosome Plasmid 1 Isolate plasmid. 2 Gene of interest 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. 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

7 Figure 8.3 An example of the process for producing a recombinant vector. Antibiotic resistance gene Restriction site mrna for human growth hormone (HGH) Reverse transcription Plasmid (vector) cdna for HGH 1 Restriction enzyme Sticky ends Restriction enzyme 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 4 Recombinant plasmid Inoculate bacteria on media containing antibiotic. Bacteria containing the plasmid with HGH gene survive because they also have resistance gene.

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

9 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

10 Techniques of Recombinant DNA Technology 1) The Polymerase Chain Reaction (PCR) 2) Separating DNA Molecules: Gel Electrophoresis 3) Separating DNA Molecules: Southern Blot 4) DNA Microarrays 5) Inserting DNA into Cells

11 Techniques of Recombinant DNA Technology 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

12 Figure 8.5a The use of the polymerase chain reaction (PCR) to replicate DNA. Original DNA 3 3 molecule 5 5 Heat to 94 C 1 Denaturation 2 Priming DNA primer Deoxyribonucleotide triphosphates DNA polymerase 4 Repeat Cool to 65 C DNA polymerase 3 Extension 3 5 DNA primer 5 72 C 5 5 3

13 Figure 8.5b The use of the polymerase chain reaction (PCR) to replicate DNA.

14 Techniques of Recombinant DNA Technology 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

15 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)

16 Techniques of Recombinant DNA Technology 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

17 Figure 8.7 The Southern blot technique. DNA molecules Restriction enzymes Restriction fragments 1 Use gel electrophoresis to separate 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 of interest. 4 Incubate with film; radiation exposes film. Develop film. Developed film 5

18 Techniques of Recombinant DNA Technology 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

19 Figure 8.8 DNA microarray.

20 Techniques of Recombinant DNA Technology Selecting a Clone of Recombinant Cells Must find clone containing DNA of interest Probes are used

21 Techniques of Recombinant DNA Technology Inserting DNA into Cells: Natural methods Transformation Transduction Conjugation Artificial methods Electroporation Protoplast fusion Injection gene gun and microinjection

22 Figure 8.9a-b Artificial methods of inserting DNA into cells. Pores in wall and membrane Chromosome Cell synthesizes new wall Electroporation Electrical field applied Competent cell DNA from another source Recombinant cell Cell walls Cell synthesizes new wall Enzymes remove cell walls Polyethylene glycol Recombinant cell New wall Protoplasts Fused protoplasts Protoplast fusion

23 Figure 8.9c-d Artificial methods of inserting DNA into cells. Micropipette containing DNA Target cell's nucleus Blank.22 caliber shell Nylon 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

24 Applications of Recombinant DNA Technology 1) Genetic Mapping 2) Environmental Studies 3) Pharmaceutical and Therapeutic Applications 4) Agricultural Applications

25 Applications of Recombinant DNA Technology Genetic Mapping Locating genes on a nucleic acid molecule Restriction fragmentation Fluorescent in situ hybridization (FISH)

26 Figure 8.10 Fluorescent in situ hybridization (FISH).

27 Figure 8.11 Automated DNA sequencing.

28 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

29 Figure 8.12 DNA fingerprinting.

30 Applications of Recombinant DNA Technology Pharmaceutical and Therapeutic Applications Protein Synthesis human Insulin gene in bacteria (genetically engineered proteins) Hepatitis B vaccine

31 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

32 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

33 The Ethics of Recombinant DNA Technology Routine screenings? Who should pay? Genetic privacy rights? Profits from genetically altered organisms?

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