Download Nucleic Acid Biotechnology Techniques

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 sitetranslation
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.
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