Chapter 9
Biotechnology
&
Recombinant DNA
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Recombinant Technologies
Terminology
Recombinant DNA - artificially manipulated DNA
Genetic Engineering – Inserting genes into cells
Biotechnology – Using cells or cell components to create a
useful product
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Typical Genetic Engineering Procedure
Bioremediation
Tools Used in Biotechnology
Restriction enzymes - enzymes which cut DNA (endonucleases)
at specific DNA sequences (fig. 9.2)
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Examples of restriction enzymes and their recognition sequences
Bam H1 – GGATCC
CCTAGG
Hin dIII - AAGCTT
TTCGAA
Eco RI - GAATTC
CTTAAG
Hae III - GGCC
CCGG
Bal I - TGGCCA
ACCGGT
Tools Used in Biotechnology
Vectors - DNA molecules used in the transfer of genes. Must be
self-replicating. (fig. 9.3) Examples: plasmids, some viruses
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pUC19 is a shuttle vector – can exist in several
species of bacteria & carry genes between cells
Methods of Inserting Foreign DNA
Transformation – absorbing naked DNA. Artificially introduce
DNA into bacterial cells through transformation. First necessary
to treat the cells chemically to make them competent (capable of
taking in naked DNA). Ex. E. coli
Electroporation - electrical current makes pores in cell
membrane through which DNA enters (may have to first make
cell into a protoplast)
.
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Methods of Inserting Foreign DNA
Protoplast – a cell which has had its cell wall removed, usually
by enzymatic digestion. This action exposes the plasma
membrane through which DNA can be more easily absorbed
Protoplast fusion - fuse two protoplasts, genetic recombination
can then occur (used in plants)
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Microinjection - directly insert DNA through a micropipette.
Usually inject multiple copies of the gene of interest in the hope
that at least one will incorporate in the chromosome (animal cells)
Sources of DNA
Gene Libraries - collection of clones containing different DNA
fragments (each one a part of the genome)
cDNA (complementary DNA) - eukaryotic genes cannot be
easily cloned in bacteria due to the presence of introns (stretches
of DNA inside a gene which do not code for protein -- the coding
parts are called exons)
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Processing Eukaryotic DNA for
Transferring to Prokaryote
In the eukaryotic cell, the introns are removed by RNA
processing, or splicing, of the mRNA transcript
to clone eukaryotic genes, the processed mRNA is converted to
cDNA by an enzyme called reverse transcriptase
The resulting cDNA can then be inserted in a bacterial cell
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Another Source for DNA
Synthetic DNA - DNA synthesizing machines can produce genes
by creating a desired sequence of nucleotides
DRAWBACKS to synthetic DNA
 can only make short sequences (120 nucleotides) – usually too short for
an entire gene
 must link all the short sequences together to form the whole gene
 must already know the desired sequence to be synthesized (can be
predicted if the amino acid sequence of the desired protein is known)
has been successful with commercial insulin and interferon
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Applications for Genetically
Engineered Products
GENETICALLY ENGINEERED PRODUCTS
FOR MEDICAL THERAPY
Synthetic insulin produced by E. coli
Tissue-plasminogen activator - produced by a geneticallyengineered mammalian cell culture
Streptokinase (natural bacterial product) used in dissolving
blood clots
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Applications for Genetically
Engineered Products
\
Production of subunit vaccines - protein portion of a pathogen
(not entire pathogen itself) used as vaccine free from possibility
of infection (produced in yeast)
Better understanding of the life cycle and method of infection
of pathogens (example, HIV) through cloning of genes
see also Table 9.1 (p263) for product list
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Obtaining DNA Information
DNA sequencing - determining the exact sequence of nucleotide
bases in the DNA – automation makes this a rapid process.
On February 12, 2001 the entire sequence of human genome was
published on Darwin’s birthday
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Obtaining DNA Information
Gel electrophoresis - separation of DNA fragments using a gel
matrix and an electrical current. DNA being negatively charged
moves toward the positive end of the gel. Smaller pieces move
through the dense gel faster than large pieces.
Southern blotting - identifying specific segments of DNA
(genes) by hybridizing complementary DNA probes.
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(-)
(+)
Electrophoresis - Current applied to
gel. DNA moves according to size.
Smaller moves faster/farther.
Southern Blotting –. DNA moves
with solution and is trapped in filter
(read details in figure)
Radioactive (or dye) labeled
probe specific for gene of
interest hybridized to filter.
DNA of interest is labeled
Obtaining DNA Information
RFLPs - restriction fragment length polymorphisms – Inherited
differences in distance between restriction enzyme sites. Can test
for inherited differences in a person’s DNA (can be used to test
for genetic diseases -- genetic screening)
DNA fingerprinting – useful for identification of pathogens, and
for paternity testing
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Polymerase Chain Reaction (PCR)
A method to amplify (make a large quantity of) a specific DNA
sequence in a matter of hours (fig. 9.4, p. 255)
1. uses high temperature denaturizing (melting) of the DNA to
separate the strands
2. anneal known sequence primers to ends of specific DNA sequences
and
3. uses DNA polymerase from Thermus aquaticus to copy the DNA
between the primers. T. acquaticus is thermophilic bacterium, this
polymerase can tolerate high temperature without damage
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4. repeat the process, making more copies from both the original and
the previously made copies
Uses for PCR
Used in diagnostics to identify infectious agents
Used in gene libraries to identify specific genes or clones of
interest
Amplifying small amounts of a sample from a valuable source
such as a crime scene
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Safety and Ethics
Pages 268 - 269
Read over these sections.
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