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gene technology
biology 1 lecture 14
• The importance of plasmids and
restriction endonucleases
• The 4 steps of genetic engineering
• Screening
• PCR and sequencing
• Commercial applications of genetic
engineering
The first artificially created genome used
a bacterium...
• Bacteria have developed a series of
enzymes, restriction endonucleases, to
combat bateriophage viruses
• Restriction enzymes cut DNA into strands at
definitive points deemed by specific
nucleotide sequences
• Such nucleotides sequences are typically
palindromes, e.g.,
G|AATTC
CTTAA|G
• Restriction enzymes cut at the same point: since the
ends created are compatible, ‘sticky ends’, they can
re-associate
• plasmids, circular DNA strands found in prokaryotes,
can be similarly digested by restriction enzymes, and
can be used as vectors
• DNA strands can be spliced into the plasmid, and the
plasmid re-inserted into a bacterium
• As the bacteria divides, the spliced DNA becomes
part of the genome and is also replicated
• Certain viruses can also be used as vectors
The 4 steps of genetic engineering
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DNA cleavage and isolation
Production of recombinant DNA
Cloning
Screening
DNA cleavage and isolation
• Use restriction endonucleases to break
up DNA strands
• Separate strands by electrophoresis
(shorter fragments travel further when
subjected to electric current)
• Excise electrophoresis bands,
redissolve, and use for stage 2
Production of recombinant DNA
• Insert DNA fragments into plasmids or
similar vectors also digested by same
restriction enzymes (that is, same sticky
ends)
• Use vector to ‘infect’ host bacteria (e.g.,
E. coli)
Cloning
• Incubate infected bacterium, which
divides exponentially
• To help identification of correctly
engineered recombinant DNA, choose a
DNA strand that has a characteristic
phenotype e.g.,
– Resistance to penicillin ampr
– Ability to metabolize X-gal (z-gene)
• Arrange recombination to preserve
ampr, but interfere with z-gene
Screening
• Screen for correct phenotype
• Use of probes to identify specific
nucleotide sequences
• Probes can be radioactively labeled:
when the nucleic acid sequence of the
probe links with the DNA sequence of
interest, a radioactive recombinant is
formed that can be detected
Other techniques
• PCR: polymerase chain reaction.
Sequences of DNA are amplified using
repeated sequences of denaturation,
addition of primers, and primer
extension
• Southern blotting is used to identify
DNA strands using a radioactively
labeled probe
• DNA fingerprinting analyzes restriction
fragment length polymorphisms (RFLPs):
point mutations specific to individuals causes
different ‘banding’ in an electrophoresis gel
• DNA can be sequenced using the Sanger
method. Primers (deoxynucleotides and
dioxynucleotides) are added to DNA
fragments. Depending on nucleotide
sequence, primers recombine with DNA in
specific sequence
Applications of genetic technology
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Pharmaceuticals
Gene therapy
Piggyback vaccines
Agricultural applications
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Herbicide resistance
Nitrogen fixation
Insect resistance
Farm animals (growth hormones)
• Cloning