cDNA

igocheddarBiotechnology

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

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Analyzing and Engineering Genes


Chapter 19

Analyzing and

Engineering Genes


genetic engineering:


manipulation of DNA sequences in
organisms


recombinant DNA technology:


techniques used to engineer genes



The goals of genetic engineering:


improve our understanding of how
genes work


advance
biotechnology


the manipulation of organisms to create
products or cure diseases


Reverse transcriptase:


used to make
complementary DNA
(cDNA)


from isolated mRNA



RNA

cDNA


RNA

protein



Genetic cloning:


process of producing many identical
copies of a gene

How Are Plasmids

Used in Cloning?


Plasmids:


small, circular DNA
molecules


replicate independently of
the chromosome


can be used to carry
recombinant genes in
bacteria

Using Restriction Endonucleases
to Cut DNA


Restriction endonucleases


enzymes that cut DNA at specific base
sequences


recognition sites





Most recognition sites are palindromic
sequences



Restriction endonucleases often
make
staggered cuts
in the DNA


sticky ends



Plasmids and cDNAs cut with the
same restriction endonuclease can be
spliced together at their sticky ends

Transformation: Introducing
Recombinant Plasmids into
Bacterial Cells



Plasmids serve as a
vector


vehicle for transferring recombinant
genes to a new host



Plasmids can be introduced into
bacteria by
transformation:


the process of taking up DNA from the
environment and incorporating it into
the genome



The resulting transformed cells make up a
cDNA
library:


a collection of bacterial cells


each containing a vector with one cDNA

Ethical Concerns over Recombinant
Growth Hormone


The increased supply of growth
hormone led to its use to treat
children who were short


but not suffering from pituitary
dwarfism.


Such use for cosmetic purposes
raises ethical questions



The U.S Food and Drug
Administration (FDA) approved use
of the hormone only for children
projected to reach adult heights:



less than 5'3" for males



less than 4'11" for females

The Polymerase Chain Reaction
(PCR)


polymerase chain reaction (PCR)
:


in vitro
(test tube) DNA synthesis
reaction


a section of DNA is amplified millions
of times



A PCR reaction contains just a few
ingredients:


a DNA template


two primers


bracket the region to be amplified


dNTPs


buffer


DNA polymerase



A PCR reaction requires about 30
cycles


each cycle containing three steps
carried out at different temperatures:


Denaturation:


to separate the DNA strands


primer annealing:


to allow the flanking primers to anneal to
the denatured DNA


extension step:



for synthesis of the complementary strand

Improving the Protocol



PCR was improved by the discovery
of a heat
-
stable form of DNA
polymerase called
Taq

polymerase



Use of
Taq

polymerase allowed the
reaction to proceed without having to
add fresh polymerase at each cycle

Dideoxy Sequencing

(DNA Sequencing)


dideoxy sequencing



a method for determining DNA
sequence


The method is based on an
in vitro
DNA synthesis reaction

New Approaches to Therapy


transgenic

organisms


called transgenic because they have
alleles that have been modified by
genetic engineering


engineered to provide an
animal
model

of the disease


makes genetic testing possible


also make novel/unique organisms

-

problems with this…?


GloFish
®

Genetic Tests



In general, genetic tests include
three types:


carrier testing


prenatal testing


adult testing

Ethical Concerns

over Genetic Testing



Genetic testing raises many ethical
issues



whether a pregnancy should be
terminated if a debilitating disease is
found in the fetus



whether health insurance companies
can deny coverage for individuals with
a genetic disease.


Gene therapy:


the introduction of a gene to replace or
augment a mutant gene that is causing
an abnormal phenotype

Ethical Concerns

over Gene Therapy



Gene therapy is highly
experimental, extremely expensive,
and very controversial



Although gene therapy
holds great
promise

for the treatment of a wide
variety of inherited diseases…


fulfilling that promise is almost certain
to require many years of additional
research and testing


as well as the refinement of legal and
ethical guidelines