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Chapter 20 DNA Technology
DNA Cloning
 Gene cloning allows scientists to work
with small sections of DNA (single
genes) in isolation.
– Exactly what does the gene code for?
 Much of a DNA molecule is noncoding,
and scientists are mostly interested in
the genes.
 Cloning makes identical copies of the
same gene (or genes)
Figure 20.1 An overview of how bacterial plasmids are used to clone genes
Bacterial Plasmids
 Plasmids are small, circular DNA molecules
in bacteria.
 By inserting genes into plasmids, scientists
can combine eukaryotic and prokaryotic
DNA. (Recombinant DNA)
 Bacterial cells continually replicate the
foreign gene along with their DNA.
 Cloning using plasmids can be used to:
– Identify a particular protein a gene makes
(ie: for study)
– Produce large amounts of a particular
protein/gene (ie: for use in medicine)
Restriction Enzymes
 Also used
to make
recombinan
t DNA.
 Specifically
cut DNA
molecules
at precise
base
locations.
(restriction)
Making Recombinant DNA (Fig 20.3)
Making Recombinant DNA (Fig 20.3)
…Still Making Recombinant DNA
…Almost Recombinant
DNA Technology Files
Restriction Enzyme and Cloning Movies
Why Use Bacteria as vectors?
1. Plasmids are easy to use to
manipulate which genes are
expressed in clones.
2. Bacteria replicate very quickly and
allow you to produce a large number
of a desired gene.
Identifying Clones
 Not all of the reproduced bacteria are
clones carrying the desired gene.
 Two ways to identify which are clones:
–Look for the gene
–Look for the protein the gene codes
for
Nucleic Acid Hybridization (find gene)
 If you know the sequence of the cloned gene
you are looking for, you can make a nucleic
acid probe with a complementary sequence.
 The probe is radioactively labeled and
allowed to base pair with the denatured
(separated strands) DNA.
 The probes H-bond with their complement
(cloned gene), thus identifying the cloned
cells.
 Identified cells are cultured to produce more.
Figure 20.4 Using a nucleic acid probe to identify a cloned gene
Expressing Euk. Proteins in Bacteria
 It is more difficult to get the bacteria to
translate the proteins because of
differences in promotor sequences b/t
prokaryotes and eukaryotes.
 Expression vectors are plasmids that
contain the promotor sequence just before
the restriction site.
 This allows the insertion of a eukaryotic
gene right next to the prokaryotic
promotor.
Expressing Euk. Proteins in Bacteria
 Bacteria also lack the enzymes needed to
remove introns from DNA.
 Therefore, cDNA (no introns) is inserted
into plasmids to allow expression of the
eukaryotic gene.
 Reverse transcriptase is the enzyme used
to make cDNA from a fully processed
mRNA strand.
Figure 20.5 Making complementary DNA (cDNA) for a eukaryotic gene
Another Solution: Use Yeast (eukaryotic)
 Why?
–They grow quickly like bacteria
–They are eukaryotes (similar enzymes,
metabolic mechanisms, protein mods)
–They have plasmids (rare for
eukaryotes)
–Can replicate artificial chromosomes as
well as DNA in plasmids
Genomic Libraries
 Plasmids and phages used to store
copies of specific genes.
Polymerase Chain Reaction (PCR)
PCR
 Faster and more specific method for
amplifying short DNA sequences
 After DNA is denatured (split), primers start
new complementary strands with each
strand producing more molecules of the
sequence.
 In vitro = doesn’t require living cells
– In test tube: denatured DNA, free
nucleotides, DNA primers (specific to gene
desired), “special” DNA polymerase (can
withstand high heat w/o denaturing)
PCR
Analyzing DNA
 Gel electrophoresis separates molecules
based on size, charge, density, etc.
 Linear DNA – mainly separated by
fragment length (size)
 Molecules of DNA are separated into
bands of molecules of the same length.
Gel Electrophoresis
Restriction Fragment Analysis
Southern Blotting
Southern Blotting
1. Produce restriction fragments of DNA
(rest. enzyme used)
2. Separate fragments (gel electrophoresis)
3. Blotting
 Transfer DNA to nitrocellulose paper
via cap. action
4. Hybridize with radioactive probes (know
seq.)
5. Autoradiography to identify which have
probes.
RFLPs (rif-lips)
 Polymorphisms that result from differences in
noncoding regions of DNA.
 Restriction enzymes cut DNA into different
fragments in each variant.
 RFLP markers allowed scientists to more
accurately map the human genome.
 Genetic studies do not have to rely on
phenotypic (appearance/proteins) differences to
guide them anymore.
In Situ (on a slide) Hybridization
 Radioactively (or fluorescently) labeled
probes base pair with complementary
denatured DNA on a microscope slide.
 Autoradiography and staining identify the
location of the bound probe.
Human Genome Project
 Attempt to map the genes on every
human chromosome as well as noncoding
information.
 Three stages
–Genetic Mapping (linkage)
–Physical Mapping
–Gene (DNA) Sequencing
 Genomes of species that give insight to
human codes are also being done (fruit
fly, E coli, yeast)
Genetic Mapping (Stage 1)
 Linkage maps based on recombination
frequencies created.
 Linkage maps portray gene sequences as
you physically move along a chromosome.
 Genetic markers along the chromosome
allow researchers to use them as
reference points while studying other
genes.
Physical Mapping (Stage 2)
 Determines the actual distance between
the markers along a chromosome (# of
bases)
 Utilizes chromosome walking to identify
the distance between.
–Use a series of probes to identify the
DNA sequence of various restriction
fragments, and ultimately the entire
length of DNA sample.
Chromosome Walking
DNA Sequencing (Stage 3)
 As of 1998, 3% of the human genome had been
sequenced using automation. (Sanger Method)
 Once the sequences of all the genes are
known, scientists can begin to study all of their
functions, and manipulate their products in
many ways.
Applied Genetics
 Diagnosis of Genetic Disorders
–Sequence individuals before birth to
know if their DNA contains abnormalities
 Human Gene Therapy
–Replace missing or fix damaged genes
in affected individuals
Gene Therapy
Pharmaceuticals
 Hormone production (ie: Human Growth)
 Protein supplements
– HIV treatment: “decoy” receptor protein
used to inhibit HIV virus’ ability to enter cell
 Vaccines
– Proteins that stimulate immune response
can be used instead of traditional vaccines
 Antisense Nucleic Acids
– Block translation of certain proteins
Other Uses of DNA Tech
 DNA Fingerprinting for forensic cases
 Environmental cleanup
 Agriculture
–Animal Husbandry
–Genetic Engineering of Plants
The Future of Genetics
 The future of science lies in genetics.
 The question is not whether or not we
can do the things discussed in this
chapter, but whether or not we should.
This is a question you will ultimately
have to help answer.