1. Recombinant DNA Technology8
Recombinant DNA Technology

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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

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. 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

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

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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

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

19. 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

20. 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

21. 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

22. 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"?