AP Biology
Ch. 20
Biotechnology
DNA Cloning
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DNA cloning and other techniques can be
used to manipulate and analyze DNA and to
produce useful new products and organisms.
DNA cloning permits production of multiple
copies of a specific gene or other DNA
segment.
Restriction Enzymes to Make
Recombinant DNA
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Bacterial restriction enzymes cut DNA
molecules within short, specific nucleotide
sequences to yield a set of double-stranded
DNA fragments with single-stranded sticky
ends.
The sticky ends on fragments from one DNA
source can base-pair with complementary
sticky ends on fragments from other DNA
molecules.
Using restriction
enzymes and DNA
ligase to make
recombinant DNA
Cloning a Eukaryotic Gene in a
Bacterial Plasmid
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A recombinant plasmid is made by inserting
restriction fragments from DNA containing a
gene of interest into a plasmid vector that has
been cut open by the same enzyme.
Gene cloning results when the recombinant
plasmid is introduced into a host bacterial cell
and the foreign genes are replicated along
with the bacterial chromosome as the host
cell reproduces.
Overview of gene cloning with a bacterial plasmid
Cloned genes, cont.
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A clone of cells carrying the desired gene can
be identified with a radioactively labeled
nucleic acid probe.
Eukaryotic cells can also serve as host cells
for gene cloning.
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Nucleic Acid Probe
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Cloning a human gene
in a bacterial plasmid
Cloned Genes
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Cloned genes are stored in genomic libraries, which
are collections of recombinant vector clones
produced by cloning DNA fragments derived from an
entire genome.
A cDNA library is made by cloning DNA made in
vitro by reverse transcription of all the mRNA
produced by a particular kind of cell.
Libraries of cDNA are especially useful for working
with eukaryotic genes and for studying gene
expression.
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Genomic Libraries
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Expressing Eukaryotic genes
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Several technical difficulties hinder the
expression of cloned eukaryotic genes in
bacterial host cells.
The use of cultured eukaryotic cells as host
cells and yeast artificial chromosomes
(YACs) as vectors helps avoid these
problems.
Polymerase Chain Reaction
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The PCR can be used to produce many
copies of a specific target segment of DNA,
using primers that bracket the desired
sequence and a heat-resistant DNA
polymerase.
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Polymerase Chain Reaction
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Restriction fragment analysis
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Restriction fragment analysis detects DNA
differences that affect restriction sites.
Gel electrophoresis can separate DNA
restriction fragments of different lengths.
Specific fragments can be identified using
Southern blotting, which uses labeled probes
that hybridize to the DNA immobilized on a
“blot” of the gel.
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Gel Electrophoresis
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Southern Blotting
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Restriction Fragment Length
Polymorphisms (RFLP)
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RFLPs are differences in DNA sequence on
homologous chromosomes that result in restriction
fragments of different lengths, which can be
determined using Southern blotting.
The thousands of RFLPs present throughout
eukaryotic noncoding DNA can serve as genetic
markers.
RFLP analysis has many applications, including
genetic mapping and diagnosis of genetic disorders.
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RFLP Allele Analysis
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RFLP Markers
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Mapping Genomes
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Entire genomes can be mapped at the DNA level.
The Human Genome Project is an international
research effort that involves linkage mapping,
physical mapping, and DNA sequencing of the
human genome and the genomes of other
organisms.
An alternative approach starts with sequencing of
random DNA fragments, relying especially heavily
on computers to assemble the sequences.
The human genome is thought to have 30,000 to
40,000 genes, fewer than what was once estimated.
Genome Size of Different Organisms
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Sanger Method of Sequencing DNA
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Alternative Sequencing Strategies
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Gene Sequences
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Genome sequences provide clues to
important biological questions; helping
researchers find new genes, probe details of
gene organization and control, and answer
questions about evolution.
DNA microarrays allow researchers to
compare patterns of expression in different
tissues and under different conditions.
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DNA Microarray
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Gene sequences, cont.
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Genomics is the study of entire genomes;
proteomics is the study of proteins coded for
by a genome.
Single nucleotide polymorphisms (SNPs)
provide useful markers for studying human
genetic variation.
Practical Applications of DNA
technology
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DNA technology is reshaping medicine and the
pharmaceutical industry.
Medical applications include diagnostic tests for
genetic and other diseases, safer, more effective
vaccines, and the prospect of treating or even curing
certain genetic disorders.
Pharmaceutical applications include the large-scale
production of many new, and some previously
scarce, drugs and other medicines.
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Gene Therapy
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DNA tech applications, cont.
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DNA technology offers forensic,
environmental, and agricultural applications.
DNA “fingerprints” obtained from RFLP or
STR analysis of tissue found at the scenes of
violent crimes provide evidence in trials.
Such fingerprints are also helpful in resolving
parenthood disputes.
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DNA Fingerprints
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DNA tech applications, cont.
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Genetic engineering can modify the
metabolism of microorganisms so that they
cab be used to extract minerals from the
environment or degrade waste materials.
In agriculture, transgenic plants and animals
are being designed to improve food
productivity and quality.
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Using plasmid vectors in agriculture
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DNA Technology: Ethical and
safety concerns
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Several U.S. government agencies are
responsible for setting policies about and
regulating recombinant DNA technology.
The potential benefits of genetic engineering
must be carefully weighed against the
potential hazards of creating products or
developing procedures that are harmful to
humans or the environment.