Genomics, Proteomics and Genetic Engineering

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14 Δεκ 2012 (πριν από 4 χρόνια και 8 μήνες)

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Genomics, Proteomics and
Genetic Engineering

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Genomics and Proteomics




The field of

genomics

deals with the DNA sequence,
organization, function, and evolution of genomes





Proteomics

aims to identify all the proteins in a cell or
organism including any posttranslationally modified
forms, as well as their cellular localization, functions,
and interactions






Genomics was made possible by the invention of
techniques of
recombinant DNA,

also known as
gene
cloning

or
genetic engineering

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Genetic Engineering


In genetic engineering, the immediate goal of an
experiment is to insert a particular fragment of
chromosomal DNA into a plasmid or a viral DNA
molecule



This is accomplished by breaking DNA molecules at
specific sites and isolating particular DNA fragments



DNA fragments are usually obtained by the treatment of
DNA samples with
restriction enzymes



Cloning from mRNA molecules depends on an unusual
polymerase,
reverse transcriptase
, which can use a
single
-
stranded RNA molecule as a template and
synthesize a
complementary DNA

(
cDNA
)

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cDNA Cloning



The resulting full
-
length cDNA contains an
uninterrupted by introns coding sequence for the
protein of interest


If DNA sequence is known at both ends of the
cDNA for design of appropriate primers,
amplification of the cDNA produced by reverse
transcriptase is possible by
reverse transcriptase
PCR
(
RT
-
PCR)

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Bioinformatics


Rapid automated DNA sequencing was instrumental in the
success of the
Human Genome Project
, an international
effort begun in 1990 to sequence the human genome and
that of a number of organisms


However, a genomic sequence is like a book using an
alphabet of only four letters, without spaces or punctuation.
Identifying genes and their functions is a major challenge


The annotation of genomic sequences at this level is one
aspect of
bioinformatics
, defined broadly as the use of
computers in the interpretation and management of
biological data

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Functional Genomics


Genomic sequencing has made possible a new approach to genetics
called
functional genomics
, which focuses on genome
-
wide patterns of
gene expression and the mechanisms by which gene expression is
coordinated



DNA microarray

(or
chip
)
-

a flat surface about the size of a postage
stamp with up to 100,000 distinct spots, each containing a different
immobilized DNA sequence suitable for hybridization with DNA or RNA
isolated from cells growing under different conditions



DNA microarrays

are used to estimate the relative level of gene
expression of each gene in the genome

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Fig. 10.13

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Reverse Genetics


Mutation has traditionally provided the raw material
needed for genetic analysis. The customary procedure
has been to use a mutant phenotype to recognize a
mutant gene and then to identify the wildtype allele
and its normal function



Recombinant DNA technology has made possible
another approach, often called
reverse genetics
, in
which wildtype genes are cloned, intentionally mutated
in specific ways, and introduced back into the
organism to study the phenotypic effects of the
mutations

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People that are unable
to remove UV
-
induced
DNA lesions


are sensitive to
sunlight

WT

uvrA uvrB uvrC

UV dose

J/m
2

0

5

10

20

40

80

120

E. coli

that are unable
to remove UV
-
induced
DNA lesions


are also sensitive to
sunlight

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Gene Targeting


The procedure for introducing mutations into specific
genes is called
gene targeting



Gene targeting in embryonic stem cells involves
homologous recombination between target gene in vector
and target gene in genome



Target gene in vector contains unrelated DNA so that
recombination disrupts function of targeted gene




Cells with targeted gene mutations can be selected by
including an selectable marker in the sequences that are
incorporated into the genome

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Transgenic Animals


Germ
-
line transformation
involves the insertion of
genes into the
reproductive cells of an
organism, which
permanently alters the
genetic content of the
individual and all offspring
=
transgenic

animals



Transgenic animals are
used to study the functions
of specific genes in
development or disease
processes

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Applied Genetic Engineering



Crop plants with
improved nutritional
qualities can be
created


Animal growth rate can
be genetically
engineered


Engineered microbes
can help degrade toxic
waste


The production of
useful proteins is a
primary impetus for
recombinant DNA

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Biomedical Applications


Recombinant DNA technology is used to produce large
amounts of medically important proteins



Animal viruses such as retroviruses may prove useful
vectors for gene therapy to treat single gene disorders



Recombinant DNA probes detect mutant genes in
hereditary disease



A major breakthrough in disease prevention would
come through the development of synthetic vaccines
produced by recombinant DNA