Biotechnology and Recombinant DNA

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23 Οκτ 2013 (πριν από 3 χρόνια και 9 μήνες)

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Biotechnology and Recombinant DNA


Introduction to Biotechnology

(248
-
249)


1.

Biotechnology is the use of microorganisms, cells, or cell components to make a
product.


Recombinant DNA Technology

(249)


1.

Closely related organisms can exchange genes in natural
recombination.

2.

Genes can be transferred among unrelated species via laboratory manipulation,
called genetic engineering.

3.

Recombinant DNA is DNA that has been artificially manipulated to combine
genes from two different sources.


An Overview of Recombinant
DNA technologies
(249)


1.

A desired gene is inserted into a DNA vector, such as a plasmid or a viral
genome.

2.

The vector inserts the DNA into a new cell, which is grown to form a clone.

3.

Large quantities of the gene product can be harvested from the clone.


To
ols of Biotechnology
(249
-
254)


Selection
(249
-
251)


1.

Microbes with desirable traits are selected for culturing by artificial selection.


Mutation

(251
-
252)


1.

Mutagens are used to cause mutations that might result in a microbe with
desirable traits.

2.

Site
-
dir
ected mutagenesis is used to change a specific codon in a gene.



Restriction Enzymes

(252)


1.

Prepackaged kits are available for many genetic
-
engineering techniques.

2.

A restriction enzyme recognizes and cuts only one particular nucleotide
sequence in DNA.

3.

So
me restriction enzymes produce sticky ends, short stretches of single
-
stranded
DNA at the ends of the DNA fragments.

4.

Fragments of DNA produces by the same restriction enzyme will spontaneously
join by hydrogen bonding. DNA ligase can covalently link the DN
A backbone.


Vectors

(252
-
254)




1.

Deliver the gene of interest (the gene to be cloned) to the host.

2.

Properties:

a.

Self
-
replicating

b.

Small enough to be manipulated outside the cell without breaking

c.

Preservation


must be protected from degradation by host enzyme
s

i.

Circular

ii.

Viral, which can insert into the host genome quickly

d.

Selectable markers


must have some gene that allows selection of host
cells hat have taken up the vector.

3.

Shuttle vectors are plasmids that can exist in several different species.

4.

A plasmid c
ontaining a new gene can be inserted into a cell by transformation.

5.

A virus containing a new gene can insert the gene into a cell.


Polymerase Chain Reaction
(254)


1.

The polymerase chain reaction (PCR) is used to make multiple copies of a
desired piece of D
NA enzymatically.

2.

Requires primers, deoxynucleotide triphosphates, and DNA polymerase (Taq).

3.

Steps:

4.

Denature target DNA with heat (94


C for 1 minute) to separate strands

5.

Cool to appropriate temperature for primers to anneal to separated strands (55


-

65


C for 1 minute)

6.

Increase temperature to optimal for polymerase to synthesize (72


C for 1
minute)

7.

Repeat


each cycle produces 2 new copies from each template strand, the
number of copies increases exponentially in each round of synthesis (cycle)


can ca
lculate the number of copies, assuming faithful replication and abundance
of dNTPs and primers, as 2
n
, where n = the number of cycles.

8.

PCR can be used to increase the amount of DNA in samples to detectable levels.
This may allow sequencing of genes, the d
iagnosis of genes, or the detection of
viruses.


Techniques of Genetic Engineering

(254
-
262)


Inserting Foreign DNA into Cells
(254
-
257)


1.

Cells can take up naked DNA by
transformation
. Chemical treatments are used
to make cells that are not naturally trans
formed competent to take up DNA.

2.

Pores made in protoplasts and animal cells by electric current in the process of
electroporation

can provide entrance for new pieces of DNA

3.

Protoplast fusion is the joining of cells whose cell walls have been removed.

4.

Forei
gn DNA can be introduced into plant cells by shooting DNA
-
coated particles
into the cells with a “
gene gun
”.

5.

Foreign DNA can be
injected

into animal cells by using a fine glass micropipette.

6.

Infection

with genetically engineered bacteriophage or virus.

a.

Co
smids


combinations of plasmids and phage DNA, packaged into


phage coats
in vitro
; allows insertion of larger pieces of DNA.


Obtaining DNA
(257
-
259)


1.

Gene libraries can be made by cutting up an entire genome with restriction
enzymes and inserting the fra
gments into bacterial plasmids or phages.

2.

Vectors containing the genomic DNA are transferred into bacteria and the
colonies screened for genes of interest

3.

cDNA made from mRNA by reverse transcription can be cloned in gene libraries.

4.

The mRNA has no introns
, the resulting cDNA can be expressed in bacteria.

5.

Reverse transcription in vitro often fails to complete, giving incomplete DNA
fragments.

6.

Synthetic DNA can be made
in vitro

by a DNA synthesis machine.

a.

Can only make strands about 120 bases long due to fra
gility.

b.

Have to know sequence of gene before you start.

c.

Can work backward from protein sequence but genetic code is degenerate
and you won’t choose the exact base sequence.

d.

Good for making probes and primers.


Selecting a Clone
(259
-
260)


1.

Antibiotic
-
resis
tance markers on plasmid vectors are used to identify cells
containing the engineered vector by direct selection.

2.

In blue
-
white screening, the vector contains the genes for
amp
R

and
β
-
galactosidase (
lacZ
).

3.

The desired gene is inserted into the
β

-
galactosidase gene site, destroying the
gene.

4.

Cells are cultured on media containing ampicillin and X
-
gal (a chromogenic
analog of galactose, 5
-
bromo
-
4chloro
-
3
-
indoyl
-
β


galactopyranoside, w
hich turns
blue when broken down by
β


galactosidase)

5.

Clones containing the recombinant vector will be resistant to ampicillin and
unable to hydrolyze X
-
gal (white colonies). Clones containing the vector without
the new gene will be blue. Clones lacking th
e vector will not grow.

6.

Clones containing foreign DNA can be tested for the desired gene product.

7.

A short piece of labeled DNA called a DNA probe can be used to identify clones
carrying the desired gene.


Making a Gene Product
(260
-
262)


1.

E. coli
is used t
o produce proteins by genetic engineering because it is easily
grown and its genomic are well understood.

2.

Efforts must be made to ensure the
E. coli
’s endotoxin does not contaminate a
product intended for human use.

3.

To recover the product,
E. coli
must be
lysed or the gene must be linked to a
gene that produces a naturally secreted protein.

4.

Yeasts can be genetically engineered and are likely to continuously secrete a


gene product.

5.

Mammalian cells can be engineered to produce proteins such as hormones for
me
dical use.

6.

Plant cells can be engineered and used to produce plants with new properties.


Applications of Genetic Engineering

(262
-
268)


1.

Cloned DNA is used to produce products, study the cloned DNA, and alter the
phenotype of an organism.


Therapeutic Appl
ications
(262
-
264)


1.

Synthetic genes linked to the
β

-
galactosidase gene (
lacZ
) in a plasmid vector
were inserted into
E. coli
, allowing
E. coli
to produce and secrete the two
polypeptides used to make human insulin.

2.

Cells can be engineered to produce a pat
hogen’s surface protein, which can be
used as a subunit vaccine.

3.

Animal viruses can be engineered to carry a gene for a pathogen’s surface
protein. When the virus is used as a vaccine, the host develops immunity to the
pathogen.

4.

Gene therapy can be used to

cure genetic diseases by replacing the defective or
missing gene.


Scientific Applications
(264
-
266)


1.

Recombinant DNA techniques can be used to increase understanding of DNA,
for genetic fingerprinting, and for gene therapy.

2.

DNA sequencing machines are us
ed to determine the nucleotide
-
based
sequence in a gene.

3.

Southern blotting can be used to locate a gene in a cell.

4.

Genetic screening uses Southern blotting to look for mutations responsible for
inherited diseases in human.

5.

Southern blotting is used in DNA
fingerprinting to identify bacterial or viral
pathogens.

6.

DNA probes can be used to quickly identify a pathogen in body tissue or food.


Agricultural Applications
(266
-
268)


1.

Cells from plants with desirable characteristics can be cloned to produce many
iden
tical cells. These cells can then be used to produce whole plants from which
seeds can be harvested.

2.

Plant cells can be engineered by using the Ti plasmid vector. The tumor
-
producing T genes are replaced with desired genes and the recombinant DNA is
insert
ed into the
Agrobacterium
. The bacterium naturally transforms its plant
hosts.

3.

Genes for glyphosate resistance, Bt (
Bacillus thuringiensis
)

toxin, pectinase


suppression have been engineered into crop plants.

4.

Rhizobium
has been engineered for enhanced nitr
ogen fixation.

5.

Pseudomonas

has been engineered to produce
Bacillus thuringiensis
toxin
against insects.

6.

Bovine growth hormone is being produced by
E. coli
.


Safety Issues and the Ethics of Genetic Engineering
(268
-
269)


1.

Strict safety standards are used to
avoid the accidental release of genetically
engineered microorganisms.

2.

Some microbes used in genetic engineering have been altered so that they
cannot survive outside the laboratory.

3.

Microorganisms intended for use in the environment may be engineered to
contain suicide genes so that the organisms do not persist in the environment.

4.

Genetic technology raise ethical questions such as: Should employers and
insurance companies have access to a person’s genetic records? Will some
people be targeted for either
breeding or sterilization? Will genetic counseling be
available to everyone?

5.

Genetically
-
engineered crops must be safe for consumption and release in the
environment.

6.

Genetic engineering techniques
are being

were used to map the human genome
through the H
uman Genome Project.

7.

This will provide tools for diagnosis and possibly the repair of genetic diseases.