in vivo

igocheddarBiotechnology

Dec 14, 2012 (4 years and 8 months ago)

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Foundations in Microbiology

Chapter

10

PowerPoint to accompany

Fifth Edition

Talaro

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

2

Genetic Engineering:
A
Revolution in Molecular Biology

Chapter 10

3

Genetic engineering


direct, deliberate modification of an
organism’s genome


bioengineering



Biotechnology



use of an organism’s
biochemical and metabolic pathways for
industrial production

4

I. Tools & Techniques of genetic
engineering


enzymes for dicing, splicing, & reversing
nucleic acids


analysis of DNA

5

Enzymes for dicing, splicing, &
reversing nucleic acids

1.
restriction endonucleases



recognize
specific sequences of DNA & break
phosphodiester bonds

2.
ligase



rejoins phosphate
-
sugar bonds cut
by endonucleases

3.
reverse transcriptase



makes a DNA
copy of RNA
-

cDNA

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7

Analysis of DNA


gel electrophoresis
-

separates DNA fragments
based on size


nucleic acid hybridization & probes


probes base
pair with complementary sequences; used to detect
specific sequences


DNA Sequencing


reading the sequence of
nucleotides in a stretch of DNA


Polymerase Chain Reaction


way to amplify
DNA


8

Gel electrophoresis

9

Southern blot hydridization

10

In situ hybridization

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Sanger DNA sequence technique

12

Polymerase chain reaction (PCR)

13

II. Methods in Recombinant
DNA Technology


concerned with transferring DNA from
one organism to another


1.
Cloning vectors & hosts

2.
Construction of a recombinant plasmid

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Characteristics of cloning vectors


must be capable of carrying a significant piece of
donor DNA


must be readily accepted by the cloning host



plasmids



small, well characterized, easy to
manipulate & can be transferred into appropriate
host cells through transformation


bacteriophages



have the natural ability to inject
their DNA into bacterial hosts through
transduction

16

Vector considerations


origin of replication


size of donated DNA vector will accept


gene which confers drug resistance to their
cloning host



17

pBR322

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Characteristics of cloning hosts

1.
rapid overturn, fast growth rate

2.
can be grown in large quantities using ordinary culture
methods

3.
nonpathogenic

4.
genome that is well delineated

5.
capable of accepting plasmid or bacteriophage vectors

6.
maintains foreign genes through multiple generations

7.
will secrete a high yield of proteins from expressed
foreign genes


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III. Biochemical Products of
Recombinant DNA Technology


enables large scale manufacturing of life
-
saving hormones, enzymes, vaccines


insulin for diabetes


human growth hormone for dwarfism


erythropoietin for anemia


Factor VIII for hemophilia


HBV vaccine


22

IV. Genetically Modified Organisms
(GMO)


Recombinant microbes


Pseudomonas syringae



prevents ice crystals


Bacillus thuringienisis


encodes an insecticide


Transgenic plants


Rice that makes beta
-
carotene


Tobacco resistant to herbicides


Peas resistant to weevils


Transgenic animals


Mouse models for CF, Alzheimer’s, sickle cell anemia


Sheep or goats that make medicine in their milk semen

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Bioengineering of plants

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Transgenic mice

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V. Genetic Treatments


Gene therapy


Antisense DNA


Triplex DNA

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Gene therapy


correct faulty gene in human suffering from
disease


ex vivo



normal gene is is added to tissues taken from
the body, then transfected cells are reintroduced into the
body


in vivo



naked DNA or viral vector is directly
introduced into patient’s tissue


Most trials target cancer, single gene defects &
infections


Most gene deliveries are carried out by viral
vectors


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Gene therapy

28

Antisense DNA: targeting mRNA


Antisense


a nucleic acid strand with a base
sequence that is complementary to the translatable
strand


Antisense DNA gets into the nucleus and binds to
mRNA, blocking the expression of an unwanted
protein


cancers


Alzheimer’s disease


autoimmune diseases

29

Triplex DNA


A triple helix formed when a third strand of
DNA inserts into the major groove, making
it inaccessible to normal transcription


oligonucleotides have been synthesized to
form triplex DNA


oncogenes


viruses


receptor for IL
-
2



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Antisense DNA & triplex DNA

31

VI. Genome Analysis


Gene Mapping


DNA Fingerprinting


Microarray analysis

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Gene Mapping


determining the location of specific genes
on the chromosomes


Human Genome Project



to determine
the nucleotide sequence of the >30,000
genes in the genome & the importance of
these sequences & how they relate to human
disease

33

Map of chromosome 16

34

DNA Fingerprinting


Every individual has a unique sequence of
DNA


Used to:


identify hereditary relationships


study inheritance of patterns of diseases


study human evolution


identify criminals or victims of disaster

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DNA fingerprints

36

Pedigree analysis

37

Microarray analysis


Method of determining which genes are
actively transcribed in a cell under various
conditions


health vs disease


growth vs differentiation


could improve accuracy of diagnosis and
specificity of treatment

38

Microarray