1. Recombinant DNA Technology
Chapter 8
Recombinant DNA Technology

2. The Role of Recombinant DNA technology in Biotechnology
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. Overview of recombinant DNA technology
Figure 8.1

4. 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

5. 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
mRNA of eukaryotes has introns removed
Allows cloning in prokaryotic cells

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

7. 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

8. Actions of restriction enzymes
Figure 8.2

9. The Tools of Recombinant DNA Technology
Animation: Recombinant DNA Technology

10. 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

11. Producing a recombinant vector
Figure 8.3

12. 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

13. Production of a gene library
Figure 8.4

14. Techniques of Recombinant DNA Technology
Multiplying DNA in vitro: The Polymerase Chain Reaction (PCR)
Large number of identical molecules of DNA produced in vitro
Critical to amplify DNA in variety of situations
Epidemiologists use to amplify genome of unknown pathogen
Amplified DNA from Bacillus anthracis spores in 2001 to identify source of spores

15. Techniques of Recombinant DNA Technology
Multiplying DNA in vitro: The Polymerase Chain Reaction (PCR)
Repetitive process consisting of three steps
Denaturation
Priming
Extension
Can be automated using a thermocycler

16. Techniques of Recombinant DNA Technology
Animation: Polymerase Chain Reaction: Overview

17. Techniques of Recombinant DNA Technology
Animation: Polymerase Chain Reaction: Components

18. Polymerase chain reaction (PCR)
Figure 8.5a

19. Polymerase chain reaction (PCR)
Figure 8.5b

20. Techniques of Recombinant DNA Technology
Animation: PCR: The Process

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

22. 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

23. Gel electrophoresis
Figure 8.6

24. Techniques of Recombinant DNA Technology
Separating 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 – used to detect RNA
Uses of Southern blots
Genetic “fingerprinting”
Diagnosis of infectious disease
Demonstrate incidence and prevalence of organisms that cannot be cultured

25. The Southern blot technique
Figure 8.7

26. 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

27. DNA microarray
Figure 8.8

28. 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

29. Artificial methods of inserting DNA into cells
Figure 8.9a/b

30. Artificial methods of inserting DNA into cells
Figure 8.9c/d

31. 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
Locating Genes
Until 1970, genes identified by labor-intensive methods
Simpler and universal methods now available
Restriction fragmentation
Fluorescent in situ hybridization (FISH)

32. Fluorescent in situ hybridization
Figure 8.10

33. Automated DNA sequencing
Figure 8.11

34. Sequencing Valuable for diagnostic identification as well
Provides definitive pathogen identification (usually)
depending on the accuracy of library, size of target- resolution is better

35. The steps for 16S rRNA (Sanger Sequencing)
Template preparation (extraction)
Amplification of region of interest (PCR)
Cycle Sequencing (dye terminator)

36. The steps for 16S rRNA (Sanger Sequencing)
Template preparation (extraction)
Amplification of region of interest (PCR)
Cycle Sequencing (dye terminator)
Extension Product Purification
Separation of the products (electrophoresis)

37. Common Sequencing Targets
16S Ribosomal RNA (rRNA)
Heat shock proteins (hsp)
Internal transcribed spacer (ITS)
RNA polymerase subunit B (rpoB)

38. 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

39. Applications of Recombinant DNA Technology
Pharmaceutical and Therapeutic Applications
Protein synthesis
Creation of synthetic peptides for cloning
Vaccines
Production of safer vaccines
Subunit vaccines
Introduce genes of pathogens into common fruits and vegetables
Injecting humans with plasmid carrying gene from pathogen
Humans synthesize pathogen’s proteins

40. Applications of Recombinant DNA Technology
Pharmaceutical and Therapeutic Applications
Genetic screening
DNA microarrays used to screen individuals for inherited disease caused by mutations
Can also identify pathogen’s DNA in blood or tissues
DNA fingerprinting
Identifying individuals or organisms by their unique DNA sequence

41. DNA fingerprinting
Figure 8.12

42. Applications of Recombinant DNA Technology
Pharmaceutical and Therapeutic Applications
Gene therapy
Missing or defective genes replaced with normal copies
Some patients’ immune systems react negatively
Medical diagnosis
Patient specimens can be examined for presence of gene sequences unique to certain pathogens
Xenotransplants
Animal cells, tissues, or organs introduced into human body

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

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

45. 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 used to reduce insect damage to crops
Gene for Bt toxin inserted into various crop plants
Genes for Phytophthora resistance inserted into potato crops

46. Applications of Recombinant DNA Technology
Agricultural Applications
Improvements in nutritional value and yield
Tomatoes allowed to ripen on vine and shelf life increased
Gene for enzyme that breaks down pectin suppressed
BGH allows cattle to gain weight more rapidly,
Have meat with lower fat content and produce 10% more milk
Gene for β-carotene (vitamin A precursor) inserted into rice
Scientists considering transplanting genes coding for entire metabolic pathways

47. The Ethics and Safety of Recombinant DNA Technology
Supremacist view – humans are of greater value than animals
Long-term effects of transgenic manipulations are unknown
Unforeseen problems arise from every new technology and procedure
Natural genetic transfer could deliver genes from transgenic plants and animals into other organisms
Transgenic organisms could trigger allergies or cause harmless organisms to become pathogenic

48. 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

49. Molecular Diagnostics

50. Pathogen Identification
Accurate identification important
Patient to patient:
Isolate causing infection?
colonizer/ contaminant
choice and duration of antibiotic treatment
appropriate infection control procedure
Large Scale:
Epidemiology
antibiotic resistance patterns
treatment plans and outcomes associated with a particular bacterium

51. Conventional Identification

52. Advantages Disadvantages Inexpensive Can be time consuming
Allow id for most organisms
Can be time consuming
Ambiguous profile

53. Limitations require additional equipment and expertise?
What to do when organism:
does not fit biochemical pattern?
slow growing?
Unable to culture?
require additional equipment and expertise?
(ex. anaerobes and mycobacterium)
Phenotypic characteristics are not static and can change due to stress or environment

54. Alternative to Conventional Method Identification..
...molecular
Alternative to Conventional Method Identification..

55. Examples of Molecular Tests
DNA hybridization
Nucleic acid amplification
Sequencing
Melt curve analysis
Strand Displacement Amplification
Single Stranded Conformation Polymorphism
Ligase Chain Amplification