Microbiology

1. PowerPoint® Lecture
Presentations prepared by
Mindy Miller-Kittrell,
North Carolina State University
C H A P T E R
© 2015 Pearson Education, Inc.
Recombinant
DNA
Technology
8

2. © 2015 Pearson Education, Inc.
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. © 2015 Pearson Education, Inc.
Figure 8.1 Overview of recombinant DNA technology.
Bacterial cell
Bacterial
chromosome
Plasmid
Isolate plasmid.
DNA containing
gene of interest
Gene of interest
Enzymatically cleave
DNA into fragments.
Isolate fragment
with the gene of
interest.
Insert gene into plasmid.
Insert plasmid and gene into
bacterium.
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
1
2
3
4
5
6

4. © 2015 Pearson Education, Inc.
The Role of Recombinant DNA Technology in
Biotechnology
• Tell Me Why
• Why aren't the terms genetic engineering and
biotechnology synonymous?

5. © 2015 Pearson Education, Inc.
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

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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 prokaryotic cells to produce eukaryotic proteins

7. © 2015 Pearson Education, Inc.
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

8. © 2015 Pearson Education, Inc.
The Tools of Recombinant DNA Technology
• Restriction Enzymes
• Bacterial enzymes that cut DNA molecules only at
restriction sites
• Restriction site sequences are usually palindromes
• Categorized into two groups based on type of cut
• Cuts with sticky ends
• Cuts with blunt ends

9. © 2015 Pearson Education, Inc.
Figure 8.2 Actions of restriction enzymes.
Restriction site
(palindrome)
5′
Restriction enzyme
Sticky ends
Production of sticky ends
Restriction
enzyme 1
Restriction
enzyme 2
Blunt ends
Production
of blunt ends
Restriction fragments from two different organisms
cut by the same restriction enzyme
Ligase
Recombinant DNA molecules
Recombinants using blunt ends
Ligase
Recombinants using sticky ends
Recombinant DNA molecules
3′G A A T T C
C T T A A G
5′ 3′C C C G G G
G G G C C C
5′ 3′C C C G G G
G G G C C C
5′ 3′G T T A A C
C A A T T G
5′ 3′G T T A A C
C A A T T G
5′ 3′C C C A A C
G G G T T G
5′ 3′G T T G G G
C A A C C C
5′ 3′A A G C T T
T T C G A A
5′ 3′A A G C T T
T T C G A A
A
T T C G A
A
T T C G A
G
C T T A A
+

10. © 2015 Pearson Education, Inc.

11. © 2015 Pearson Education, Inc.
Recombinant DNA Technology

12. © 2015 Pearson Education, Inc.
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

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Figure 8.3 An example of the process for producing a recombinant vector.

14. © 2015 Pearson Education, Inc.
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

15. © 2015 Pearson Education, Inc.
Figure 8.4 Production of a gene library.
Genome
Isolate genome
of organism.
Generate fragments using
restriction enzymes.
Insert each fragment
into a vector.
Introduce vectors
into cells.
Culture recombinant cells;
descendants are clones.
1
2
3
4
5
1 2 3 4 5 6 7 8 9 10 11
1 2 3
4 5 6
7 8 9
10 11
1 2 3 4 5 6
7 8 9 10 11
1 2 3 4 5 6
7 8 9 10 11

16. © 2015 Pearson Education, Inc.
The Tools of Recombinant DNA Technology
• Tell Me Why
• Why did the discovery of restriction enzymes speed up
the study of recombinant DNA technology?

17. © 2015 Pearson Education, Inc.
Techniques of Recombinant DNA Technology
• Multiplying DNA in vitro: The Polymerase
Chain Reaction (PCR)
• Large number of identical molecules of DNA are
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

18. © 2015 Pearson Education, Inc.
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

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Polymerase Chain Reaction (PCR): Overview

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PCR: Components

21. © 2015 Pearson Education, Inc.
Figure 8.5a The use of the polymerase chain reaction (PCR) to replicate DNA.
Denaturation
Priming
Extension
Original DNA
molecule
DNA primer
Deoxyribonucleotide
triphosphates
DNA polymerase
Heat to 94°C
Cool to 65°C
DNA polymerase
DNA primer
72°C
1
2
3
3′
5′
3′
5′
3′
5′
3′
5′
5′
5′
Repeat4

22. © 2015 Pearson Education, Inc.
Figure 8.5b The use of the polymerase chain reaction (PCR) to replicate DNA.

23. © 2015 Pearson Education, Inc.
PCR: The Process

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

25. © 2015 Pearson Education, Inc.
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

26. © 2015 Pearson Education, Inc.
Figure 8.6 Gel electrophoresis.
Electrophoresis
chamber filled with
buffer solution
Lane of DNA
fragments of
known sizes
(kilobase pairs)
Agarose gel
DNA
Wire
Wells
Movement
of DNA
A
B
C
D
E
a
b
(50)
(40)
(35)
(15)
(10)
(5)
(+)
(–)

27. © 2015 Pearson Education, Inc.
Techniques of Recombinant DNA Technology
• Separating DNA Molecules: Gel Electrophoresis and
the Southern Blot
• Southern blot
• DNA is transferred from gel to nitrocellulose membrane
• Probes are used to localize DNA sequence of interest
• Northern blot – similar technique used to detect RNA
• Uses of Southern blots
• Genetic "fingerprinting"
• Diagnosing infectious disease
• Demonstrating presence of organisms that cannot be
cultured

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Figure 8.7 The Southern blot technique.

29. © 2015 Pearson Education, Inc.
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
• Diagnosing infection
• Identifying organisms in an environmental sample

30. © 2015 Pearson Education, Inc.
Figure 8.8 DNA microarray.

31. © 2015 Pearson Education, Inc.
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

32. © 2015 Pearson Education, Inc.
Figure 8.9a-b Artificial methods of inserting DNA into cells.
Chromosome
Pores in wall and membrane
Electrical
field applied
Electroporation
Competent cell
DNA from
another source
Cell synthesizes
new wall
Recombinant cell
Cell walls
Enzymes remove
cell walls
Protoplasts
Protoplast fusion
Polyethylene
glycol
Fused protoplasts
Recombinant cell
Cell synthesizes
new wall
New wall

33. © 2015 Pearson Education, Inc.
Figure 8.9c-d Artificial methods of inserting DNA into cells.
Blank .22-
caliber shell
Nylon
projectile
Vent Plate to stop
nylon projectile
DNA-coated beads Target cell
Gene gun
Nylon
projectile
Micropipette
containing DNA
Target cell’s
nucleus
Target cell
Suction tube
to hold target
cell in place
Microinjection

34. © 2015 Pearson Education, Inc.
Techniques of Recombinant DNA Technology
• Tell Me Why
• Why wasn't polymerase chain reaction (PCR) practical
before the discovery of hyperthermophilic bacteria?

35. © 2015 Pearson Education, Inc.
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

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Applications of Recombinant DNA Technology
• Genetic Mapping
• Locating genes
• Until 1970, genes were identified by labor-intensive
methods
• Simpler and universal methods are now available
• Restriction fragmentation
• Determine relative location of DNA fragments
produced by cleavage with restriction enzymes
• Fluorescent in situ hybridization (FISH)
• Fluorescent probe used to visualize location of a gene

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Figure 8.10 Fluorescent in situ hybridization (FISH).

38. © 2015 Pearson Education, Inc.
Applications of Recombinant DNA Technology
• Genetic Mapping
• Nucleotide sequencing
• Genomics – sequencing and analysis of the nucleotide
bases of genomes
• Elucidation of the genomes of pathogens is a priority
• Used to relate DNA sequence to protein function

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Figure 8.11 Automated DNA sequencing.

40. © 2015 Pearson Education, Inc.

41. © 2015 Pearson Education, Inc.
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

42. © 2015 Pearson Education, Inc.
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
• Subunit vaccines
• New approaches to stimulate immunological memory
• Introducing genes of pathogens into fruits and vegetables
• Injecting humans with plasmid carrying gene from pathogen
• Humans synthesize pathogen's proteins

43. © 2015 Pearson Education, Inc.
Applications of Recombinant DNA Technology
• Pharmaceutical and Therapeutic Applications
• Genetic screening
• DNA microarrays are 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

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Figure 8.12 DNA fingerprinting.

45. © 2015 Pearson Education, Inc.
Applications of Recombinant DNA Technology
• Pharmaceutical and Therapeutic Applications
• Gene therapy
• Missing or defective genes are 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

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

47. © 2015 Pearson Education, Inc.
Applications of Recombinant DNA Technology
• Agricultural Applications
• Herbicide tolerance
• Gene from Agrobacterium tumefaciens 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

48. © 2015 Pearson Education, Inc.
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 harmful only to insects
• Used by organic farmers to reduce insect damage to crops
• Gene for Bt toxin is inserted into various crop plants
• Genes for Phytophthora resistance are inserted into potato
crops

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Figure 8.13 Genetically modified papaya plants.

50. © 2015 Pearson Education, Inc.
Applications of Recombinant DNA Technology
• Agricultural Applications
• Improvements in nutritional value and yield
• Enzyme that breaks down pectin is suppressed in some
tomatoes
• Allows tomatoes to ripen on vine and increases shelf life
• 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) are inserted into rice
• Scientists considering transplanting genes coding for entire
metabolic pathways

51. © 2015 Pearson Education, Inc.
Applications of Recombinant DNA Technology
• Tell Me Why
• Why don't doctors routinely insert genes into their
patients to cure the common cold, flu, or tuberculosis?

52. © 2015 Pearson Education, Inc.
The Ethics and Safety of Recombinant DNA
Technology
• 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

53. © 2015 Pearson Education, Inc.
The Ethics and Safety of Recombinant DNA
Technology
• Studies have not shown any risks to human health
or environment
• Standards are imposed on labs involved in
recombinant DNA technology
• Can create biological weapons using same
technology

54. © 2015 Pearson Education, Inc.
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"?

55. © 2015 Pearson Education, Inc.
The Ethics and Safety of Recombinant DNA
Technology
• Tell Me Why
• Why don't scientists who work with recombinant DNA
know all the long-term effects of their work?