Nucleic Acid Biotechnology Techniques - Workforce3one.org

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23 Οκτ 2013 (πριν από 3 χρόνια και 11 μήνες)

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Nucleic Acid Biotechnology
Techniques

Chapter 13

Separation techniques of Nucleic Acids



Gel
electrophoresis
-
used
to separate
nucleic
acids based
on charge and size.



Proteins


SDS PAGE



Done in an
electric
field


Detection of Nucleic Acids


Radioactive labeling
of sample used to detect
products



Label or tag allows visualization



DNA
undergoes
reaction that
incorporates
radioactive isotope into the DNA



Autoradiography

used to visualize image that has
been exposed to
radiolabeled

oligonucleotides

Detection of Nucleic Acids


Fluorescence



Ethidium

bromide intercalates

between
bases



Under UV light glows orange

Restriction Endonucleases


Nucleases
-

catalyze the hydrolysis of the
phosphodiester

backbone of nucleic acids




-

Endonuclease
: cleavage in the middle of the chain


-

Exonuclease
: cleavage from the ends of the molecule



Restriction
Endonucleases

-

Have a crucial role in
development of recombinant DNA technology



Bacteriophages

-

viruses that infect
bacteria

-

Led to discovery of restriction enzymes


Methylation of DNA

Restriction
Endonucleases


Restriction
endonucleases

(RE) hydrolyzes only a
specific bond of a specific
sequence in DNA



Sequences recognized by RE
read the same from left to
right as from right to left,
known as
palindrome



Sticky and Blunt ends


Resealing by DNA
ligase



Sticky ends are joined
by hydrogen bonding
between
complementary bases.



Ligases

reseal ends


Recombinant DNA Technology


Recombinant/
Chimeric

DNA
-

DNA molecules that
contain covalently linked
segments derived from 2 or
more DNA sources



Sticky Ends

can be used to
construct Recombinant DNA



DNA
Ligase
-

seals nicks in
the covalent structure


What is Cloning?


Plasmid
-

small circular DNA that is not part of
the main circular DNA chromosome of the
bacterium.



Cloning
-

The process of making identical
copies of DNA


Transformation


Bacteria take up
recombinant DNA



Heat shock method



Electroporation



Transformed bacteria


scaled up

pBR322


One of the first plasmids
used for cloning


E.coli



Foreign DNA must be
inserted at unique
restriction sites



Confers resistance to two
antibiotics


Tetracycline
and
ampicillin

Plasmids


As the technology to design plasmids improved,
regions were created that had many different
restriction sites in a small place



This region is known as a
multiple cloning site
(MCS
)

or
polylinker



Selection


How do we know which bacteria takes up the
desired plasmid?



Selection
-

Each plasmid chosen for cloning has
a selectable marker that indicates that the
growing bacterial colonies contain the plasmid
of interest


Clone Selection with Blue/White
Screening


Basis for selection



pUC

plasmids contain
lacZ

gene



lacZ

gene codes for the

-
subunit of

-
galactosidase
, which
cleaves disaccharides



This procedure helps with
selection


Cloning Summary


Cloning

refers to creating identical populations


DNA can be combined by using
restriction
enzymes +
Ligases


The
target DNA sequence
is carried in some type
of
vector/plasmid


The
target plasmid
is inserted into host organism


Organisms that carry the target DNA are
identified through a process called
selection


Genetic Engineering


When an organism is intentionally altered at
the molecular level to exhibit different
traits
-

genetically
engineered


One focus of genetic engineering has been
gene
therapy
-

where cells of specific tissues
in a living person are altered in a way that
alleviates the affects of a
disease

Protein Expression Vectors


Plasmid vectors pBR322 and
pUC

are
cloning
vectors


Vectors are used to
insert foreign DNA and
amplify it



If we want to produce protein from the
foreign DNA
-

Expression vectors


What is an Expression Vector?


Have many attributes as
cloning vector:


-

The origin of
replication


-
A multiple cloning site


-
At least one selectable
marker





What is an Expression Vector?


Must be able to be
transcribed by the
genetic machinery of
the

bacteria where it is
transformed


Must have a
transcription initiation
and termination
sequence


Ribosomal binding site
-
translation


Producing Large Numbers of Transformed
Cells





DNA libraries


All the DNA of an
organism
-

clone it in
chunks of reasonable
size


The result of this is a
DNA library


Several steps involved in
construction of the
library


How do we find the piece of DNA we
want in a library?


Genomic Library
Screening


A
nitrocellulose disc
is
put on the dish and
removed


Disc treated with
denaturing agent to
unwind DNA


DNA is permanently fixed
to disc by treatment with
heat or UV light

How do we find the piece of DNA we
want in a library?


Expose DNA on disc to a
solution that contains
single stranded
complementary DNA or
RNA
(radioactive
probing)



Wash

the disc


Identify the colonies


Making
cDNA

library


RNA of interest is used as
template for the synthesis
of complementary DNA
(
cDNA
)


Reaction catalyzed by
reverse transcriptase


cDNA

is incorporated into
vector



cDNA

library
construction is identical
to genomic DNA library


Summary


A DNA library is a collection of clones of an
entire genome


The genome is digested with restriction
enzymes and the pieces are cloned into
vectors


A
cDNA

library is constructed by using reverse
transcriptase to make DNA from the mRNA in
a cell. This
cDNA

is then used to construct a
library similar to a genomic DNA library

Polymerase Chain Reaction



It is possible to increase
the amount of a given
DNA many times over
without cloning the DNA




Any chosen DNA can be
amplified, and it does not
need to be separated
from the rest of the DNA
in a sample

DNA fingerprinting



DNA samples can be studied and compared by
DNA fingerprinting


DNA is digested with restriction enzymes and
then run on an
agarose

gel


When soaked in
ethidium

bromide


can be
seen directly under UV light


Southern blotting



If greater sensitivity
needed or if number of
fragments would be too
great to distinguish the
bands, technique can
be modified to show
only selected DNA
sequences


Sequencing


DNA can be sequenced by using several
techniques, the most common being the
chain
termination method


Dideoxy

nucleotides
are used to terminate DNA
synthesis. Multiple reactions are run with
different
dideoxy

nucleotide in each reaction mix


The reactions produce a series of DNA fragments
of different length that can be run on a gel and
the sequence determined by tracking the
different length fragments in the lanes with the
four different
dideoxy

nucleotides


Fig. 17.11

Genomics and Proteomics


Knowing the full DNA sequence of the human
genome allows for the investigation for the
causes of disease in a way that has not been
possible until now



The
proteome

is a protein version of a genome



Proteomics

is the study of interactions among all
the proteins in a cell

Open book take home quiz for 30
points



1. Name the two kinds of gels used in electrophoresis and what
molecules do those separate. Explain the original charges of those
molecules and in which direction do they move in an electric field.
In other words explain the effect of charge and size on the
biomolecules
.


2. What is
methylation

of DNA in Bacteria?


3. Write names of any two enzymes and the name of the bacteria
from which it has been extracted. Explain how these enzymes have
been named or their naming procedures.


4. Draw a recombinant DNA plasmid showing the sites of
BamHI

and
HindIII

along with same RE sites on your DNA of interest.


5. Explain the whole procedure of PCR along with factors required
to run a PCR reaction.


6. Explain the procedure of RNA interference.


7. Explain the
Agrobacterium

transformation done in plants.



This project is funded by a grant awarded under the President’s Community Based Job Training Grant as implemented by the
U.S. Department of Labor’s Employment and Training Administration (CB
-
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-
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-
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political affiliation or belief; and



against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998
(WIA), on the basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in the United

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