Download 13.2 – Manipulating DNA

13.2 – Manipulating DNA
Interest Grabber
Section 13-2
The Smallest Scissors in the World
Have you ever used your word processor’s Search function? You can
specify a sequence of letters, whether it is a sentence, a word, or
nonsense, and the program scrolls rapidly through your document,
finding every occurrence of that sequence. How might such a function
be helpful to a molecular biologist who needs to “search” DNA for the
right place to divide it into pieces?
Interest Grabber continued
Section 13-2
1. Copy the following series of DNA nucleotides onto a sheet of paper.
GTACTAGGTTAACTGTACTATCGTTAACGTAAGCTACGTTAACCTA
2. Look carefully at the series, and find this sequence of letters:
GTTAAC. It may appear more than once.
3. When you find it, divide the sequence in half with a mark of your
pencil. You will divide it between the T and the A. This produces short
segments of DNA. How many occurrences of the sequence GTTAAC
can you find?
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Section:
Section Outline
Section 13-2
13–2
Manipulating DNA
A. The Tools of Molecular Biology
1. DNA Extraction
2. Cutting DNA
3. Separating DNA
B. Using the DNA Sequence
1. Reading the Sequence
2. Cutting and Pasting
3. Making Copies
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Section:
The Tools of Molecular Biology
 Genetic engineering – making changes in the DNA code of a
living organism
 DNA extraction – by a simple chemical procedure
 Cutting DNA – most DNA to large to be analyzed
 Can be cut precisely at restriction enzymes
 Separating DNA – gel electrophoresis
Gel Electrophoresis
DNA plus
restriction
enzyme
Power
source
Longer
fragments
Shorter
fragments
Mixture of
DNA
fragments
Gel
The setup
Restriction Enzymes
Section 13-2
Recognition sequences
DNA sequence
Restriction enzyme
EcoRI cuts the DNA
into fragments.
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Section:
Sticky end
Restriction Enzymes
Section 13-2
Recognition sequences
DNA sequence
Restriction enzyme
EcoRI cuts the DNA
into fragments.
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Section:
Sticky end
Using the DNA Sequence
 After the DNA is shortened up it can now be read, studied
and even changed
 The function of different genes can now be discovered
Reading the sequence
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 Small pieces of single-strand DNA
are “tagged” with fluorescent dye
 Using gel-electrophoresis, the
fragments are separated
 The pattern of the colored bands
tells the exact sequence of bases
in the DNA
Cutting and Pasting
 DNA sequences can be changed in many ways
 Short sequences can be “synthesized” and joined with
“natural” DNA and spliced together using enzymes
 The same enzymes can join a gene from one organism and
attach it to the DNA of another
 Recombinant DNA- DNA molecules produced by combining
DNA from different sources
Knockout Genes
Section 13-3
Recombinant DNA
Flanking sequences
match host
Host Cell DNA
Target gene
Recombinant DNA replaces
target gene
Modified Host Cell DNA
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Section:
Making Copies
 What is PCR?
 Sometimes called "molecular photocopying," the polymerase
chain reaction (PCR) is a fast and inexpensive technique used
to "amplify" - copy - small segments of DNA.
 Because significant amounts of a sample of DNA are necessary
for molecular and genetic analyses, studies of isolated pieces of
DNA are nearly impossible without PCR amplification.
 Often heralded as one of the most important scientific advances
in molecular biology, PCR revolutionized the study of DNA to
such an extent that its creator, Kary B. Mullis, was awarded the
Nobel Prize for Chemistry in 1993.
 What is PCR used for?
 Once amplified, the DNA produced by PCR can be used in
many different laboratory procedures. For example, most
mapping techniques in the Human Genome Project (HGP)
rely on PCR.
 PCR is also valuable in a number of newly emerging
laboratory and clinical techniques, including DNA
fingerprinting, detection of bacteria or viruses (particularly
AIDS), and diagnosis of genetic disorders
Figure 13-8 PCR
Section 13-2
DNA polymerase adds
complementary strand
DNA heated to
separate strands
DNA fragment
to be copied
http://www.dnai.org/b/index.html
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Section:
PCR
cycles 1
2
3
4
5 etc.
DNA
copies 1
2
4
8
16 etc.
Interest Grabber
Section 13-3
Sneaking In
You probably have heard of computer viruses. Once inside a
computer, these programs follow their original instructions and
override instructions already in the host computer. Scientists use
small “packages” of DNA to sneak a new gene into a cell, much as a
computer virus sneaks into a computer.
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Section:
Interest Grabber continued
Section 13-3
1. Computer viruses enter a computer attached to some other file.
What are some ways that a file can be added to a computer’s
memory?
2. Why would a person download a virus program?
3. If scientists want to get some DNA into a cell, such as a bacterial
cell, to what sort of molecule might they attach the DNA?
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Section:
Section Outline
Section 13-3
13–3
Cell Transformation
A. Transforming Bacteria
B. Transforming Plant Cells
C. Transforming Animal Cells
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Cell Transformation
 During transformation, a cell takes in DNA from outside the
cell
 This external DNA becomes part of the cell’s DNA
Transforming Bacteria
 Use recombinant DNA
Figure 13-9 Making Recombinant DNA
Section 13-3
Recombinant
DNA
Gene for human
growth hormone
Gene for human
growth hormone
Human Cell
Sticky
ends
DNA
recombination
DNA
insertion
Bacterial Cell
Bacterial
chromosome
Plasmid
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Bacterial cell for
containing gene for
human growth hormone
Plant Transformation
 Agrobacterium tumefaciens – Causes plant tumors.
Figure 13-10 Plant Cell
Transformation
Section 13-3
Tumor causing bacteria
Agrobacterium
tumefaciens
Gene to be
transferred
Recombinant
plasmid
Cellular
DNA
Inside plant cell,
Agrobacterium inserts part of
its DNA into host cell
chromosome
Plant cell colonies
Transformed bacteria introduce
plasmids into plant cells
Complete plant is
generated from
transformed cell
http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/bio40.swf::The%20Ti%20Plasmid
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13.4 – Applications of
Genetic Engineering
Interest Grabber
Section 13-4
The Good With the Bad
The manipulation of DNA allows scientists to do some interesting
things. Scientists have developed many transgenic organisms, which
are organisms that contain genes from other organisms. Recently,
scientists have removed a gene for green fluorescent protein from a
jellyfish and tried to insert it into a monkey.
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Section:
Interest Grabber continued
Section 13-4
1. Transgenic animals are often used in research. What might be the
benefit to medical research of a mouse whose immune system is
genetically altered to mimic some aspect of the human immune
system?
2. Transgenic plants and animals may have increased value as food
sources. What might happen to native species if transgenic animals
or plants were released into the wild?
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Section:
Flowchart
Section 13-4
Cloning
A body cell is taken from a donor animal.
An egg cell is taken from a donor animal.
The nucleus is removed from the egg.
The body cell and egg are fused by electric shock.
The fused cell begins dividing, becoming an embryo.
The embryo is implanted into the uterus of a foster mother.
The embryo develops into a cloned animal.
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Section:
Figure 13-13 Cloning of the First
Mammal
Section 13-4
A donor cell is
taken from a
sheep’s udder.
Donor
Nucleus
Egg Cell
These two cells
are fused using
an electric shock.
Fused Cell
Cloned Lamb
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Section:
The nucleus of
the egg cell is
An egg cell
The fused
removed.
is taken from
cell begins
an adult
dividing
female
normally.
sheep.
Embryo
The embryo
The embryo is
develops normally
Foster
into a lamb—Dolly
Mother placed in the
uterus of a
foster mother.
Transgenic Organisms
 Transgenic Organisms – contain genes from other organisms
 These are produced by genetic engineering
 Have helped bacteria produce things like insulin, growth
hormone, and clotting factors that were once very expensive
 Allowed animals immune system to act more like humans which
allows them to study effects of disease
 Can grow animals that are more lean
Cloning
 Clone – member of a population of genetically identical cells
produced from a single cell
 Bacteria can be cloned quite easily
 In 1997, a Scottish scientist (Ian Wilmut) announced he had
cloned a sheep - Dolly
Cloning of the First Mammal
A donor cell is taken from
a sheep’s udder.
Donor
Nucleus
These two cells are fused using an
.
electric shock
Fused Cell
Egg Cell
An egg cell is
taken from an
adult female
sheep.
The nucleus of the
egg cell is removed.
The fused cell
begins dividing
normally.
Embryo
Cloned Lamb
The embryo
develops
normally into a
lamb—Dolly
Foster
Mother
The embryo is placed
in the uterus of a
foster mother.
Injaz the Camel
April 2009 in Dubai
Carbon Copy Cat
Cc surprised many when she was born and did not look or
act exactly like the genetic mother
Daisy the Cow
Daisy in July of 1999
Dolly
Snuppy
Human Cloning
 In 2005 the UN approved a non-binding global ban on human
cloning
 In US it is legal for non-embryonic stem cell research under
certain regulations