E. coli

whooshdisguisingBiotechnology

Dec 14, 2012 (4 years and 6 months ago)

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Copyright
©
The McGraw
-
Hill Companies, Inc. Permission required for reproduction or display.

CHAPTER 17

DNA Manipulation


Restriction endonucleases revolutionized
molecular biology


Enzymes that cleave DNA at specific sites


Used by bacteria against viruses


Restriction enzymes significant


Allow a form of physical mapping that was
previously impossible


Allow the creation of recombinant DNA
molecules (from two different sources)

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3 types of restriction enzymes


Type I and III cleave with less precision
and are not used in manipulating DNA


Type II


Recognize specific DNA sequences


Cleave at specific site within sequence


Can lead to “sticky ends” that can be joined


Blunt ends can also be joined


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DNA ligase


Joins the two fragments forming a stable DNA
molecule


Catalyzes formation of a phosphodiester bond
between adjacent phosphate and hydroxyl
groups of DNA nucleotides


Same enzyme joins Okazaki fragments on
lagging strand in replication

Gel Electrophoresis


Separate DNA fragments by size


Gel made of agarose or polyacrylamide


Submersed in buffer that can carry current


Subjected to an electrical field


Negatively
-
charged DNA migrates towards the
positive pole


Larger fragments move slower, smaller move
faster


DNA is visualized using fluorescent dyes

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Transformation


Introduction of DNA from an outside
source into a cell


Natural process in many species


E. coli

does not


Temperature shifts can induce artificial
transformation in
E. coli



Transgenic organisms are all or part
transformed cells

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Molecular Cloning


Clone


genetically identical copy


Molecular cloning


isolation of a specific
DNA sequence (usually protein
-
encoding)


Sometimes called gene cloning


The most flexible and common host for
cloning is
E. coli



Vector


carries DNA in host and can
replicate in the host


Each host

vector system has particular uses

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Vectors


Plasmids


Small, circular chromosomes


Used for cloning small pieces of DNA


Selectable marker


allows presence of
plasmid to be easily identified

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DNA Libraries


A collection of DNAs in a vector that taken
together represent the complex mixture of
DNA


Genomic library


representation of the
entire genome in a vector


Genome is randomly fragmented


Inserted into a vector


Introduced into host cells


Usually constructed in BACs

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Southern blotting


Sample DNA is digested by restriction
enzymes and separated by gel
electrophoresis


Double
-
stranded DNA denatured into single
-
strands


Gel “blotted” with filter paper to transfer DNA


Filter is incubated with a labeled probe
consisting of purified, single
-
stranded DNA
corresponding to a specific gene


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Northern blotting


mRNA is separated by electrophoresis and
then blotted onto the filter



Western blotting


Proteins are separated by electrophoresis and
then blotted onto the filter


Detection requires an antibody that can bind
to one protein

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RFLP analysis


Restriction fragment length polymorphisms


Generated by point mutations or sequence
duplications


Restriction enzyme fragments are often not
identical in different individuals


Can be detected by Southern blotting

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DNA fingerprinting


Identification technique used to detect
differences in the DNA of individuals


Population is polymorphic for these markers


Using several probes, probability of identity
can be calculated or identity can be ruled out


First used in a U.S. criminal trial in 1987


Tommie Lee Andrews was found guilty of rape


Also used to identify remains

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DNA sequencing


Determination of actual
base sequence of DNA


Basic idea is nested
fragments


Each begin with the same
sequence and end in a
specific base


By starting with the
shortest fragment, one
can then read the
sequence by moving up
the ladder



Automated DNA sequencing


Enzymatic technique is powerful but is labor
-
intensive and time
-
consuming


Automation made sequencing faster and
more practical


Fluorescent dyes are used instead of
radioactive labels


Reaction is done in one tube


Data are assembled by a computer

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Fundamentally new method for DNA sequencing


DNA is cleaved into smaller pieces


Both ends are ligated to adapters that are
complementary to specific primers


DNA fragments are injected into a flow cell


Each of 7 channels contains a solid substrate with
primers that complement the ligated ends of the DNA
fragments


Uses fluorescent tag on dNTPs


Camera records colors after each round of extension

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Copyright © The McGraw
-
Hill Companies, Inc. Permission required for reproduction or display.

NH
2

O

P

O



O

O

CH
2

5
´

4
´

3
´

2
´

1
´

OH

N

N

N

O

A

Reversible terminator

A

A

T

T

G

G

C

A

T

G

C

C

g.

h.

First round of

synthesis with

labeled dNTPs

Image capture for each

round of synthesis

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Copyright © The McGraw
-
Hill Companies, Inc. Permission required for reproduction or display.

b: © 2007, Illumina Inc. All rights reserved

Adapter

Adapter

Attached

Clusters

Adapters

a.

b.

c.

d.

e.

f.

Flow cell

1 cm

DNA

DNA fragment

Dense primer lawn

in flow cell

35 cycles

of bridge

amplification

Bridge

amplification

with unlabeled

dNTPs

Free end

binds to

primer

Fragments

become

double
-

stranded

Free

terminus

Denature

double
-

stranded

molecules

Attached


Polymerase chain reaction (PCR)


Developed by Kary Mullis


Awarded Nobel Prize


Allows the amplification of a small DNA
fragment using primers that flank the region


Each PCR cycle involves three steps:

1.
Denaturation (high temperature)

2.
Annealing of primers (low temperature)

3.
DNA synthesis (intermediate temperature)


Taq polymerase

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After 20 cycles, a
single fragment
produces over one
million (2
20
) copies!

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Applications of PCR


Allows the investigation of minute samples of
DNA


Forensics


drop of blood, cells at base of a
hair


Detection of genetic defects in embryos by
analyzing a single cell


Analysis of mitochondrial DNA from early
human species

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Medical Applications


Medically important proteins can be
produced in bacteria


Human insulin


Interferon


Atrial peptides


Tissue plasminogen activator


Human growth hormone


Problem has been purification of desired
proteins from other bacterial proteins

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Genetically engineered mouse

with human growth hormone


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Vaccines


Subunit vaccines


Genes encoding a part of the protein coat are
spliced into a fragment of the vaccinia (cowpox)
genome


Injection of harmless recombinant virus leads to
immunity


DNA vaccines


Depend on the cellular immune response (not
antibodies)

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Gene therapy


Adding a functional copy of a gene to correct
a hereditary disorder


Severe combined immunodeficiency disease
(SCID) illustrates both the potential and the
problems


On the positive side, 15 children treated
successfully are still alive


On the negative side, three other children treated
have developed leukemia (due to therapy)

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Agricultural Applications


Ti (tumor
-
inducing) plasmid


Most used vector for plant genetic
engineering


Obtained from
Agrobacterium tumefaciens,
which normally infects broadleaf plants


Part of the Ti plasmid integrates into the plant
DNA and other genes can be attached to it


However, bacterium does not infect cereals
such as corn, rice, and wheat

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Other methods of gene insertion


Gene guns


Uses bombardment with tiny gold particles coated
with DNA


Possible for any species


Copy number of inserted genes cannot be
controlled


Modification of
Agrobacterium
system


Use of other bacteria like
Agrobacterium

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Herbicide resistance


Broadleaf plants have been engineered to be
resistant to the herbicide glyphosate


Benefits


Crop resistant to glyphosate would not have to be
weeded


Single herbicide instead of many types


Glyphosate breaks down in environment


In the United States, 90% of soy currently
grown is GM soy

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Bt crops


Insecticidal proteins have been transferred
into crop plants to make them pest
-
resistant


Bt toxin from
Bacillus thuringiensis



Use of Bt maize is the second most common
GM crop globally


Stacked crops


Both glyphosate
-
resistant and Bt
-
producing

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Golden rice


Rice that has been genetically modified to
produce
b
-
carotene (provitamin A)


Converted in the body to vitamin A


Interesting for 2 reasons


Introduces a new biochemical pathway in tissue of
the transgenic plants


Could not have been done by conventional
breeding as no rice cultivar known produces these
enzymes in endosperm


Available free with no commercial
entanglements

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Adoption of genetically modified (GM)
crops has been resisted in some areas
because of questions


Crop safety for human consumption


Movement of genes into wild relatives


No evidence so far but it is not impossible

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Biopharming


Transgenic plants are used to produce
pharmaceuticals


1990


Human serum albumin produced in
genetically engineered tobacco and potato
plants


In development


Recombinant subunit vaccines against Norwalk
and rabies viruses


Recombinant monoclonal antibodies against tooth
decay
-
causing bacteria

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Transgenic animal technology has not
been as successful as that in plants


Molecular techniques combined with the
ability to clone domestic animals could
produce improved animals for
economically desirable traits


Main use thus far has been engineering
animals to produce pharmaceuticals in
milk (also biopharming)

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