Download PPT File

Mary K. Campbell
Shawn O. Farrell
http://academic.cengage.com/chemistry/campbell
Chapter 13
Nucleic Acid Biotechnology Techniques
Paul D. Adams • University of Arkansas
Purification and Detection of Nucleic Acids
• Gel electrophoresis is
a common technique
used to separate
nucleic acids.
• Based on motion of
charged particles in
an electric field
Purification and Detection (Cont’d)
• Radioactive labeling of sample used to detect
products
• Label or tag allows visualization
• DNA undergo reaction that incorporate radioactive
isotope into the DNA
• Autoradiography used to visualize image that has
been exposed to oligonucleotides that have been
radiolabeled
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, were
being studied when restriction enzymes were
discovered
Methylation of DNA
Restriction Endonucleases (Cont’d)
• Restriction endonuclease (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
• Two As and 2 Ts between breaks in DNA strand which leave
sticky ends
• Sticky ends are joined by by hydrogen bonding between
complementary bases.
• Ligases reseal ends
Restriction Endonucleases and Their
Cleavage Sites
Action of DNA Ligases
Cloning
• Recombinant 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
• 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
Production of Recombinant DNA
The Cloning of a Virus
Plamids
• 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
bacteria colonies contain the plasmid of interest
Plasmid pBR322
• One of the first plasmids used for cloning
Plasmids (Cont’d)
• 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
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
Clone Selection with Blue/White Screening
Cloning Summary
• Cloning refers to creating identical populations
• DNA can be combined by using restriction enzymes
• The target DNA sequence is carried in some type of
vector
• The target DNA sequence 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, it has been
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
• DNA recombination can occur in nature
• The reproductive power of bacteria can be used to
express large quantities of a mammalian protein of
interest, however, process can be complicated
Genetic Engineering (Cont’d)
• Human proteins can be made
by bacteria, but process is not
straight forward. e.g. human
insulin
• An intron is a DNA sequence
that codes for RNA that is
eventually deleted in the
processing of the mRNA that
directs the synthesis of the
protein
• Only the RNA transcribed from
exons appear in the mature
RNA
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 produce protein from the
foreign DNA, vectors are not good
• Instead, expression vectors are used
What is an Expression Vector?
• Have many attributes as
cloning vector:
• The origin of replication
• A multiple cloning site
• At least one selectable
marker
• Must be able to be
transcribed by the
genetic machinery of the
bacteria where it is
transformed
• Must have a transcription
termination sequence
DNA Libraries
• Can we take all the
DNA of an organism
and clone it in chunks
of reasonable size
• The result of this is a
DNA library
• Several steps involved
in construction of the
library
Finding an Individual Clone in a DNA
Library
• After the library has been
constructed, the next
challenge is to find a
single desired clone out
of hundreds of thousands,
or millions
• Technique used to
select depends on
separating and annealing
complementary strands
• Known as Genomic
Library Screening
Finding an Individual Clone in a DNA
Library (Cont’d)
• RNA libraries not
constructed in the same
way
• RNA of interest is used as
template for the synthesis
of complementary DNA
(cDNA)
• Reaction catalyzed by
reverse transcriptase
• cDNA is incorporated into
vector, then process is
identical to the production
of 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, and
transformed into cell lines
• Specific radioactive probes to a sequence of interest
are reacted to filters that have copies of the bacterial
colonies in the library
• 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
The Polymerase Chain Reaction
• It is possible to increase the amount of a given DNA
many times over without cloning the DNA
• This method of amplification is known as the
Polymerase Chain Reaction (PCR)
• Any chosen DNA can be amplified, and it does not
need to be separated from the rest of the DNA in a
sample before the procedure is applied
The Polymerase Chain Reaction (Cont’d)
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, the DNA
fragments can be seen directly under UV light
• 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
• This begins with Southern blotting
The Southern Blot
Restriction-Fragment Length
Polymorphisms
• In organisms with two sets of chromosomes, a given gene on
one chromosome may differ slightly from the corresponding
gene on the paired chromosome
• These are known as alleles
• Organisms are homozygous when they have the same paired
chromosomes
• Organisms are heterozygous when they have different paired
chromosomes
• Restriction fragments of different sizes are obtained by
treatment with endonuclease. They are RestrictionFragment Length Polymorphisms (RFLPs)
The Basis for Restriction-Fragment Length
Polymorphism
Summary
• A DNA fingerprint is created by digesting DNA with
restriction enzymes, separating the pieces on a gel,
and visualizing some of the pieces by using labeled
probes
• Differences in DNA patterns between different
individuals are based on different base sequences of
their DNA
DNA Sequencing
• The nature and order of monomer units determine
the properties of the whole molecule
• The method devised by Sanger and Coulson for
determining the base sequences of nucleic acids
depends on selective interruption of oligonucleotide
synthesis
• A single-stranded DNA fragment whose sequence is
to be determined is used as a template
• The synthesis is interrupted at every possible site in
the population of molecules depending on the
presence of ddNTPs
DNA Sequencing (Cont’d)
• The incorporation of the ddNTP into the growing chain causes
termination at the point of incorporation
• The DNA to be sequenced is mixed with a short
oligonucleotide that serves as a primer for synthesis of the
complementary strand
• Gel electrophoresis is performed on each reaction mixture,
and a band corresponding to each position of the chain
termination appears
• The sequence of the newly formed strand, complementary to
the template DNA, can then be read from the sequencing gel
The Sanger-Coulon Method for Sequencing
DNA
Summary
• 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
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
Microarrays
Summary
• As more DNA sequences become available, it
becomes possible to compare these sequences
• Important medical applications are emerging, and
new methods are making it possible to analyze large
quantities of data
• The proteome is the protein version of the genome.
It refers to all of the proteins being expressed in a
cell
Summary
• DNA or protein microchips is a powerful technique
being used presently, as thousands of samples of
DNA or proteins can be applied and then checked
for binding of biological samples
• The binding can be visualized by using fluorescently
labeled molecules and scanning the chip with a
computer (Figure 13.30). The pattern of fluorescent
labels then indicates which mRNA or proteins are
being expressed in the samples