Download Troubling and Terrific Technology

Troubling and Terrific
Technology
Chapter 20
I. Introductions to DNA
Technology
A.
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Biotechnology - manipulation of organisms or
their components to make useful products.
used to make hundreds of products
can insert genes from one species into another
to make desired proteins
used in solving crimes
used in agriculture
I. Introductions to DNA
Technology
B.
C.
Recombinant DNA - DNA in which genes
from two different sources (can be different
species) are combined in vitro into the same
molecule
Genetic Engineering - direct manipulation of
genes for practical purposes
II. Gene cloning
Preparing well-defined, gene sized pieces of
DNA in multiple copies.
 Can make copies of gene (ex. pest resistance
gene, sequencing)
 Can make protein from the gene (ex. make
growth hormone)
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A. General Method for gene cloning
1.
2.
3.
4.
5.
6.
Isolate the DNA of a plasmid and the DNA
containing the gene you want to clone
Insert the gene into the plasmid (recombinant
DNA)
Put plasmid back into bacteria (recombinant
bacteria)
Allow bacteria to replicate and clone gene
Identify bacteria that have the clone
Many applications: make copies of gene, make
protein from the gene
B. Restriction Enzymes to make
Recombinant DNA
1.
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Characteristics and Terms
enzymes that cut DNA molecules at a limited
number of specific locations
used by bacteria in nature to cut up foreign
DNA
bacterial DNA is not destroyed because it has
methyl groups added where restriction
enzymes work
B. Restriction Enzymes to make
Recombinant DNA
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Restriction site - recognition sequence for restr. enzyme
(the place where it will cut.
Restriction fragments - when DNA is exposed to a
restriction enzyme, the enzyme cuts it in the same place
every time. This leaves the same fragments of DNA
every time it is exposed to the restriction enzyme
Sticky ends - restriction fragments can be staggered,
leaving short stretches of single stranded DNA (sticky
ends). These single stranded areas can hydrogen bond
to different DNA that has been cut with the same
restriction enzyme. (temporary bond)
B. Restriction Enzymes to make
Recombinant DNA
DNA ligase - can be added to mixtures of DNA
to make the temporary bonds permanent
 Cloning vector - DNA molecule (usually a
plasmid) that can carry foreign DNA into a cell
and replicate (usually in a bacterial host)

2. How do you make a Bacteria
express a eukaryotic gene???
Problems in Paradise!
Problem 1: gene expression is different in
prokaryotes and eukaryotes
 Solution: Use an expression vector - a cloning
vector that contains a promoter region upstream
of the restriction site
 When you insert the eukaryotic gene, the
bacteria’s promoter will function and express the
foreign gene

Problem 2: lots of non-coding regions
(introns) in eukaryotic genes (bacteria don’t
have spliceosomes)
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Solution: make artificial eukaryotic genes without
introns
allow transcription of original gene to premRNA
RNA splicing occurs
mRNA is released from nucleus
isolate mRNA from cell
add reverse transcriptase (makes DNA from RNA) the DNA made is called cDNA (complementary) and
contains only the gene of interest (no introns)
cDNA will synthesize complementary strand
Use cDNA to insert into bacteria
DNA Libraries
Genomic libraries are sets of plasmid containing
cell clones
 Bacterial artificial chromosomes (large plasmids)
contain genes for replication and carry larger
inserts
 cDNA libraries contain genes that have already
been transcribed
 To find the correct gene from the library – use
nucleic acid hybridization with a Probe that is
tagged (usually radioactively)
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3. Other ways to avoid
prokaryotic/eukaryotic problems
Use Eukaryotic cell as host - YEAST is a great
eukaryotic host: it grows quickly, is single celled
and even contains plasmids
 To get eukaryotes to take up DNA, scientists
can use electroporation - a brief electrical pulse
that makes a temporary hole in the cell
membrane or they can physically inject DNA
using a microscopic needle

C. PCR - Polymerase Chain
Reaction
method to copy any piece of DNA (even small
quantities) without using cells
 incubate DNA in test tubes with DNA
polymerase (special type able to withstand high
heat), short pieces of ssDNA for primers, and a
supply of nucleotides (to make new DNA)
 useful for making a limited quantity of DNA
(for testing crime scenes, diagnosing prenatal
diseases, mammoth cloning etc)
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6. Gel Electrophoresis
separates macromolecules (nucleic acids and
proteins) based on size, electrial charge and
other physical properties
 DNA (separated by size) is sorted into a mixture
of bands of DNA that are the same length
(negatively charged DNA moves toward positive
end of gel)
 Can be used to compare genes in individuals or
between species
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A. Procedure
1.
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2.
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Prepare restriction fragments
Mix DNA samples to be tested with the same restriction
enzyme
Electrophoresis
Put the samples into different wells of a gel (see picture)
Attach electrodes at both ends. DNA has a negative charge
and will be attracted to the positive end of the gel
Longer molecules move more slowly through the gel and
shorter molecules move quickly
Each sample will produce a different banding pattern
A. Procedure
3.
Southern Blotting
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4.
Transfer DNA from gel to special nitrocellulose paper
This denatures the DNA and makes it single strands
Hybridization
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Expose the paper to radioactively labeled probe that is
complementary to the sequence you are trying to find
Even if you start with an entire genome (i.e. all 46
chromosomes), only the sequence you are looking for will
be hybridized
A. Procedure
5.
Autoradiography
 put photographic film over the
paper.
 radioactivity exposes the film and
forms an image
 banding patterns are produced
RFLP - restriction fragment length
polymorphisms
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Differences found in non-coding regions of DNA
Serves as a genetic marker for a particular location
These markers can have many variants within a
species (like different phenotypes)
These can serve as genetic markers used to make
linkage maps (like the fruit fly maps we made
before)
The more often a RFLP and a gene are inherited
together, the closer they are on a chromosome
Human Genome Project
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An effort to map the entire human genome,
determining the complete nucleotide sequence
of the DNA of each human chromosome.
Done using three methods
1. Genetic (linkage) mapping
make a linkage map of several thousand genetic
markers spread evenly throughout the
chromosomes
 markers can be genes, satellite DNA or RFLP’s
 markers used as a reference point for mapping
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2. Physical mapping
ordering DNA fragments
 distance between markers expressed in a
physical measure
 done by cutting DNA with restriction enzymes
and determining the order of the fragments
produced
 must make fragments that overlap and use
probes to find overlap
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3. DNA sequencing

Use the technology of cloning fragments of
DNA
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use a sequencing machine that combines DNA
labeling, DNA synthesis and electrophoresis
A. Sanger method= prepare a single stranded
DNA with unknown sequence, divide it into 4
portions
B. Incubate with all necessary components to
make new DNA strand
3. DNA sequencing
C. Put the mixtures into four different tubes with special
nitrogen bases called didoxyribonucleotides (ddATP,
ddCTP, ddTTP, ddGTP)
D. When a dd____ nucleotide is added, the strand stops
growing. This happens at random points along the
new DNA strand
E. Eventually, a set of labeled strands of various lengths
is generated and can be run and separated on a gel
F. The bands on the gel are in direct correspondence
with the sequence of DNA
IV. How is all this info used???
A.
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Analyzing DNA sequences- scientists can use
computers to exam stretches of DNA and find
the parts that actually code for genes (look for
start/stop codons etc)
Belief now is that there are only 30-40000
genes - most of our genome is non coding
Most vertebrate genes can code for 2 or 3
polypeptides by changing the splicing of
mRNA
How is all this info used???
B. Comparing genes - scientists are using DNA to
compare genes in different organisms and within the
same species
 Many genes are seen in multiple organisms even those very
distantly related
C. Studying gene expression - DNA can be used to see
which genes are active in which cells, and when in
development these genes might be turned on and off
D. In vitro mutagenesis - if scientists don’t know the
function of a gene they have found, they can mutate
the gene and look for changes in the cell or organism
Cloning
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Producing one or more organisms that is
genetically identical to the parent
Plants – can be cloned by growing from cuttings
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Used to reproduce plants with favorable
characteristics like pest resistance
Cloning - Animals
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Done by nuclear transplantation
Remove nucleus of unfertilized egg
 Replace it with nucleus from cell you are cloning
 Let it divide in culture
 Put egg in surrogate mother
 May lead to cells that are not as healthy as normal
cells
 Can lead to other problems like obesity, pneumonia,
liver failure, premature death (all these found in
cloned mice)
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Stem Cells
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Relatively unspecialized cell that can reproduce
itself and can differentiate into specialized cells
Embryonic Stem cells – come from early
embryo
Reproduce indefinitely
 Can become variety of specialized cells, even eggs
and sperm
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Adult Stem Cells – used to replace
nonreproducing specialized cells as needed
Practical Applications
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A. Diagnosis of Diseases - using PCR and probes,
certain pathogens can be detected
*hundreds of diseases are diagnosed using this
technology
i.e. HIV, hemophilia, cystic fibrosis
B. Human Gene Therapy - alteration of problem genes
- may be possible by replacing or repairing the problem
gene
i.e. defective bone marrow cells repaired and returned
to patient. As the cells divide, regular bone marrow
cells are formed
Practical Applications
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C. Pharmaceutical Products - many protein products
have been created by transferring genes into bacterium
or yeast, growing it in culture and producing large
quantities
D. Forensics - blood or tissue left at scene can be
tested using DNA sequencing. RFLP’s are used to
compare DNA samples from suspects and victims
yielding a DNA fingerprint. The probability that two
people have the same fingerprint is very small
Practical Applications
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E. Environmental - microorganisms can be genetically
engineered to extact heavy metals, clean oil spills
F. Agriculture - DNA technology is used to make
vaccines and growth hormones and even transgenic
organisms (organisms whose genomes carry genes from
another species
*genes for human proteins can be inserted into animals
to make them express and produce these proteins
Ethical concerns
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*What happens if we “create” deadly pathogens
by accident?
*What if genetically modified food is hazardous
to people?
*What if genetically modified crops hybridize
with wild plants (like weeds) and create
“superweeds” that are hard to control?