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Chapter 9
Biotechnology and
Recombinant DNA
Part 2
Tools of Biotechnology
• Restriction Enzymes
– DNA cutting enzymes that exist in many
bacteria
– Cut specific sequences of DNA (recognize 4-,
6-, or 8-base sequences), staggered cuts
– Destroy bacteriophage DNA in bacterial cells
– Cannot digest (host) DNA with methylated
cytosines
Restriction Enzymes
Figure 9.2
Tools of Biotechnology
• Vectors
– Carry new DNA to desired cell
– Plasmids and viruses can be used as vectors
– Four properties of vectors
• Can self-replicate
• Be a size that allows them to be manipulated outside
the cell during recombinant DNA procedures
• Preservation (circular form of DNA and integrated
into host chromosome)
• Have a marker within the vector for easy selection
Tools of Biotechnology
• Shuttle vectors: a plasmid that can exist
in several different species
– Very useful in the process of geneticaly
modifying multicellular organisms
• Viral DNA can usually accept much larger
pieces of foreign DNA than plasmid
– Retroviruses, adenoviruses, & herpesviruses
• Choice of suitable vector depends on many
factors (e.g host & size of the DNA to be
cloned)
Vectors
Figure 9.3
Tools of Biotechnology
• Polymerase Chain Reaction (PCR)
– To make multiple copies of a piece of DNA
enzymatically (limited by the choice of primers used)
– Cannot be used to amplify an entire genome
– Used to
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•
•
•
•
Clone DNA for recombination
Amplify DNA to detectable levels
Sequence DNA
Diagnose genetic disease
Detect pathogens
PCR
Figure 9.4.1
PCR
Figure 9.4.2
Techniques of Genetic Engineering
• Inserting foreign
DNA into cells
–
–
–
–
–
Transformation
Electroporation
Protoplast fusion
Gene gun
Microinjection
Figure 9.5b
Techniques of Genetic Engineering
– Choice of method is usually determined by the
type of vector and host being used
• Foreign DNA will survive only if it is either
present on a self-replicating vector or
incorporated into one of the cell’s
chromosomes by recombination
Techniques of Genetic Engineering
• Transformation: used to insert plasmid
vector into a cell
– many cell types do not naturally transform
need to make them competent (able to take up
external DNA)
• Electroporation: uses an electrical current
to form microscopic pores in the
membranes of cells (DNA enter cells
through the pores)
Techniques of Genetic Engineering
– Generally applicable to all cells; ones with cell
wall must be converted to protoplasts first
• Protoplast fusion: a method of joining two
cells by first removing their cell walls
– Protoplasts in solution will fuse at a low but
significant rate (can add polyethylene glycol to
increase the frequency of fusion)
– Valuable in the genetic manipulation of plant
and algal cells
Fig. 9.5
Techniques of Genetic Engineering
• Gene gun: Microscopic particles of
tungsten or gold are coated with DNA and
propelled by a burst of helium through the
plant cell walls
– Some of the cells express the introduced DNA
as if it were their own if incorporated into host
chromosome
Techniques of Genetic Engineering
• Microinjection: introduce DNA directly into
an animal cell using a glass miropipette
Figure 9.6 & 7
Obtaining DNA
• Gene library: a collection of cloned DNA
fragments created by inserting restriction
enzyme fragments in a bacterium, yeast, or
phage
– Make a collection of clones large enough to
ensure that at least one clone exists for every
gene in the organism
– Pieces of an entire genome stored in plasmids or
phage
Fig. 9.8
Obtaining DNA
• Cloning genes from eukaryotic organisms
poses a special problems due to introns
– Need to use a version of the genes that lacks
intron = mRNA
• cDNA is made from mRNA by reverse
transcriptase (mRNA
cDNA)
• cDNA is the most common method of
obtaining eukaryotic genes
Fig. 9.9
Obtaining DNA
• Synthetic DNA is made by a DNA synthesis
machine
– Chain of over 120 nucleotides can be
synthesized
– Need to know the sequence of the gene
– Rare to clone a gene by synthesizing it directly
– Plays a much more useful role selection
procedures (add desired restriction sites)
Selecting a clone
• Use antibiotic resistance genes (marker) on
plasmid vectors to screen for cells carrying
the desired gene (engineered vector)
– e.g. Blue-white screening (2 marker genes on
the plasmid vector = ampR and -galactosidase)
Genetic Engineering
Blue-white screening
Figure 9.11.1
Genetic Engineering
Figure 9.11.2
Selecting a clone
• Need a second procedure to test if screened
bacteria does contain desirable genes
– Test clones for desired gene product or ID
genes itself in the host bacterium
– Colony hybridization: use DNA probe that is
complementary to the desired genes
• DNA probe: short segment of single-stranded DNA
that are complementary to the desired gene
Colony hybridization
Figure 9.12.1
Colony hybridization
Figure 9.12.2
Making a gene product
• Earliest work in genetic engineering used
E. coli to synthesize the gene products
– E. coli was used because it is easily grown and
its genomics are known
– Disadvantages of using E. coli:
• Produce endotoxins (Lipid A, part of LPS layer on
the cell wall)
• Does not secrete protein products
need to lyse
cells to obtain products
• Industry prefers Bacillus subtilis because it secretes
their products
Making a gene product
• Use baker’s yeast (Saccharomyces cerevisiae)
– Yeast may carry plasmid and has best understood
eukaryotic genome
– May be more successful in expressing foreign
eukaryotic genes than bacteria; likely to secrete
products
• Use mammalian cells in culture
– Hosts for growing viruses (vectors)
– Often the best suited to making protein products for
medical use (e.g. hormones, cytokines, interferon)
Making a gene product
• Use plant cells in culture
– Ti plasmid (from bacterium Agrobacterium
tumefaciens), protoplast fusion and gene gun
– Use to produce genetically engineered plants
• May be sources for plant alkaloids (painkiller),
isoprenoids (basis for synthetic rubber), and melanin
(for sunscreens)
Applications of Genetic Engineering
• Produce useful substances more efficiently
and cheaper
• Obtain information from the cloned DNA
that is useful for either basic research or
medical applications
• Use cloned genes to alter the characteristics
of cells or organisms
Therapeutic applications
• Subunit vaccines
• Nonpathogenic viruses carrying genes for
pathogen's antigens as vaccines
• Gene therapy to replace defective or missing
genes
• Human Genome Project
– Nucleotides have been sequenced
– Human Proteome Project may provide diagnostics
and treatments
Random Shotgun Sequencing
Figure 9.14
Scientific Applications
• Understanding
of DNA
• Sequencing
organisms'
genomes
• DNA
fingerprinting
for identification
Figure 9.16
Southern Blotting
Figure 9.15.1
Southern Blotting
Figure 9.15.2
Southern Blotting
Figure 9.15.3
Agricultural Applications
Table 9.2
Genetic Engineering Using
Agrobacterium
Figure 9.18
Safety Issues and Ethics
• Avoid accidental release
• Genetically modified crops must be safe for
consumption and for the environment
• Who will have access to an individual's
genetic information?