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BCH 401G
Lecture 40/41
Andres
Lecture Summary:
DNA Technology
Human Genome contains approximately 3 x 109 bp of DNA and is
composed of approximately 100,000 different genes.
We will talk about five technologies which have had a major impact
on science this century:
1. Restriction Endonucleases.
2. Plasmid vectors.
3. DNA sequencing.
4. Polymerase Chain Reaction.
5. Complementary DNA (cDNA) Cloning.
1. Restriction Endonucleases
Restriction Endonucleases break the 3',5' phosphodiester
bonds between nucleotides. Different enzymes break this
bond on different sides of the bond.
Most restriction
endonucleases recognize
DNA palindromes.
Restriction enzymes are
used to cleave DNA
molecules at defined points
to yield specific fragments
that are more readily
analyzed and manipulated
than the parental DNA
molecule. DNA ends
generated by digestion with
restriction enzymes are
called "sticky ends" or
overhangs.
These overhangs can
base-pair with ends of DNA
molecules cut with the same
restriction endonuclease.
This is a very useful feature:
This activity can be used to
rejoin fragments.
Restriction Endonucleases
are important because
they allow large pieces
of DNA to be cut into
smaller defined
fragments so we can
isolate and study them.
Most common method for analyzing DNA digestions is by agarose gel
electrophoresis.
Agarose is a material which can be used to separate DNA
molecules on the basis of size.
Mixture of DNA fragments is placed in a trough cut into one end of the
agarose gel. A negative electrode is placed at the end of gel nearest
the DNA and a positive electrode at other end of gel.
Because of negatively charged phosphates in backbone of
DNA, DNA moves toward positive electrode.
II. Plasmid vectors. Restriction enzymes, DNA ligase and plasmid
vectors are the cornerstone of recombinant DNA technologies. They
allow the production of new combinations of unrelated genes to be
constructed in the laboratory. These new genes can be CLONEDamplified many times-by placing them into a suitable host. Theses
new genes are often transcribed and translated. These techniques
are very powerful because once these engineered proteins are
introduced to a host cell, the host cell can be permanently and
profoundly altered.
Cloning a segment of DNA entails five general procedures:
1.
A method for cutting DNA at precise locations (rest. enzymes).
2.
A method for joining two DNA fragments covalently (DNA ligase).
3. Selection of a small molecule of DNA capable of self-replication.
Segments of DNA to be cloned can be joined to plasmids, These
composite molecules are called recombinant DNAs.
4. A method for moving recombinant DNA from the test tube into a
host cell that can provide the enzymatic machinery needed for DNA
replication.
5. Methods to select or identify those host cells that contain
recombinant DNA.
The collection of techniques used to carry out these processes
are collectively referred to as recombinant DNA technology or
genetic engineering.
Plasmid Cloning:
1. Plasmids (vectors) are small (2-6kb) circular DNA molecules found
in bacteria. They are closed circular DNA duplex molecules and act as
accessory chromosomes (see below).
2. They have Origins of Replication -can be replicated
independently within bacteria.
3. Contain a selectable marker- most often provides resistance to
an antibiotic. The drug is used to isolate only those bacteria
which have received the plasmid DNA molecule.
Plasmid vectors are useful - allow scientists to generate large
amounts of specific DNA fragments for study.
3 Steps to using plasmid vectors
1. Cut target DNA (DNA to be propagated) and plasmid DNA with
same restriction enzyme(s). Both DNAs now have same "sticky
ends"
2. Mix cut target DNA and plasmid DNA together and add DNA ligase.
Target DNA is inserted (ligated) into the plasmid.
3. Plasmid DNA with inserted target DNA sequence is then put back
into bacteria. Called Transformation.
Bacteria grow and divide rapidly- also replicate plasmid with inserted
sequence.
Bacteria can divide once every 30 minutes. This means that very
soon you have a lot of bacteria and therefore a lot of plasmid DNA.
You can then extract the plasmid carrying the DNA insert from the
bacteria.
3. DNA Sequencing. How do you determine the sequence of DNA?
The most common method for sequencing DNA is called the "Sanger
Method". Frederick Sanger developed this method in 1976 and
won his second Nobel Prize.
This method depends on dideoxyribonucleotide triphosphates: ddNTPs
There are four: ddATP, ddCTP, ddGTP, and ddTTP.
ddNTPs have a hydrogen at the 3' position of the sugar instead of a
hydroxyl group.
Remember that the 3' hydroxyl group of the nucleotide is critical for the DNA
synthesis reaction.
Since dideoxyNTPs have no 3' hydroxyl group, the growing DNA
chain can not be extended any further upon addition of a ddNTP.
DNA synthesis terminates at that point in the DNA strand.
Basic ingredients required for Sanger Sequencing method:
1. DNA you want to sequence and short single-stranded DNA primer
(usually 17-25 nucleotides) which is complementary to the DNA
to be sequenced.
DNA to be sequenced is often prepared as single-stranded DNA.
2. Four dNTPs (dATP, dCTP, dGTP, and dTTP).
3. Four ddNTPs (ddATP, ddCTP, ddGTP, and ddTTP).
4. One dNTP which has a radioactive Alpha phosphate.
5. A DNA Polymerase -several different ones can be used.
Method:
1. Mix primer-DNA complex with all four dNTPs.
2. Divide into four portions.
To each portion add one (and only one) of the dideoxyNTPs.
One gets ddATP, another gets ddCTP, and so on.
Important: Every reaction contains all four deoxyNTPS and only one
dideoxyNTP.
Terminology: Reaction that contains ddATP is called "A" reaction,
ddCTP is "C" reaction, etc.
3. Then add DNA polymerase enzyme.
Polymerase extends primer and synthesizes DNA sequence by
template DNA and incorporates one or more radioactive dNTPs. This
allows you to detect the DNA that is being newly synthesized.
ddNTPs are occasionally incorporated and act to terminate DNA
synthesis.
4. Separate DNA fragments in each reaction by gel electrophoresis.
DNA fragments separated by size.
Length of the terminated fragments indicates the positions
where the dideoxyNTP was incorporated in place of the
deoxyNTP.
Have "A" Lane (ddATP), "G"Lane (ddGTP), "C" Lane (ddCTP), and "T"
Lane (ddTTP).
Can then read DNA sequence from the gel.
Important to understand:
1. There are very many template DNA molecules in each of
these reactions.
2. Concentration of ddNTPs is such that they will only
occasionally incorporate and terminate the synthesis.
3. Therefore, when each sequencing reaction is completed,
there will be a variety of DNA products of different lengths.
Can vary the amounts of dideoxyNTPs in the sequencing reaction.
If you increase the amounts of dideoxy NTPs in the reaction the
dideoxyNTPs will be more likely to be incorporated. Synthesis
will terminate more often. You will be able to read the sequence
close to the primer (near the start) on the sequencing gel.
If you decrease the amounts of the dideoxyNTPs in the
reaction, synthesis will proceed further because it will not
terminate as often. This will allow you to read sequence
further from the primer.
4. Another very powerful DNA Technology is the Polymerase Chain
Reaction or PCR.
PCR allows you to reproduce specific DNA sequences. The process is
termed "Amplification".
PCR generates large amounts of a particular DNA sequence from very
small amounts of starting material (can achieve a billion fold amplification in a
very short period of time).
For PCR you need:
1. DNA which contains the sequence you want to amplify.
2. DNA Primers flanking the sequence you want to amplify.
3. dNTPs.
4. Heat-stable DNA polymerase.
Three steps in PCR:
1. Denaturation. Heat to 95°C. Double stranded template DNA denatures
(the double stranded DNA helix becomes two separate single stranded
templates for PCR).
2. Annealing. Reaction is cooled to temperature below the Annealing
temperature of the primer. Say 60°C. Primers now can base-pair with
single-stranded DNA template.
3. Extension. Polymerase extends both primers and replicates DNA
sequence (just a DNA polymerase reaction, need template, primer with a
free 3'-OH, and dNTPs).
Three steps called one cycle of PCR.
Each cycle doubles the number of copies of the DNA sequence that lies
between the primers.
PCR can analyze small amounts of DNA. 40 cycles of PCR can allow you to
detect 50 molecules of DNA (thirty cycles will produce about 1 million replica
sequences from a single starting DNA sample).
Since each cell has two copies of DNA, this means you can easily
detect any DNA sequence starting with only 25 cells.
Diagnostic and Forensic Medicine. Application of Restriction
Mapping and PCR.
Most of the DNA sequence in all humans is identical. However,
there are differences between all of us that make us unique.
Some of these differences create or remove Restriction
Enzyme cleavage sites. This creates differences in sizes of
fragments resulting from digestion of chromosomal DNA with
restriction enzymes between individuals in any population.
Called Restriction Fragment Length Polymorphism, or
RFLP.
This technique can be used to map the position of genes responsible
for inherited human diseases. Many mutations either create or
destroy a previously existing restriction enzyme site(s). If such a
mutation occurs within or near a particular gene, the restriction
enzyme pattern will change, and can be detected using radioactively
labeled DNA probes.
RFLP Analysis can be combined with PCR for Forensic
Medicine.
Examples:
1.
Crime scene analysis: Can use PCR to produce a large
amount of DNA from a small amount of initial material. RFLP analysis
could then be used to compare digestion's of this DNA with a large
number of known human markers to determine whether the DNA from
a suspect is related to the crime scene sample. Must worry about
contamination and analysis of a person from a small and isolated
gene pool (an isolated group of individuals will tend to become inbred,
this will tend to distort the representation of individual RFLP markers
within this group relative to a larger population).
2.
Test for disease, virus or microorganism. Specific DNA
primers could be designed that would amplify the DNA from a foreign
pathogen but not the human hosts DNA.
3.
Medical Tests: If a specific gene mutation is correlated
with a disease, PCR primers can be designed to detect the mutation.
Infants or embryos (very early term) can then be tested for the
potential of carrying a defective gene leading to illness.
5. Complementary DNA (cDNA) prepared from mRNA can be
used to isolate active gene products:
Remember that most Eukaryotic genes are mosaics of
exons and introns. These interrupted genes can not be expressed in
bacteria, however, this difficulty can be overcome by providing
bacterial cells with recombinant DNA which is complementary to the
mRNA.
The key to forming complementary DNA (cDNA) is the enzyme
reverse transcriptase.
cDNA molecules can be inserted into plasmids that contain the
necessary DNA regions for efficient expression in hosts such as
bacteria, yeast, or mammalian cells grown in culture. In bacterial
expression vectors, the cDNA is inserted into the vector in the correct
reading frame near a strong bacterial promoter. In addition, to assure
efficient translation a shine-dalgarno sequence is positioned near the
AUG initiation codon.
If cDNAs are made from the entire collection of mRNAs being
expressed within a tissue or organism the collective cDNA clones
when placed in a suitable plasmid are called a "cDNA library." This
cDNA library should contain the entire repertoire of proteins being
used by the host cells just before mRNA was collected. These
"libraries" can then be used to isolate gene products of interest.
This allows a researched to work in the opposite direction of the
information flow we have studied. If a protein sequence is known, you
could guess at the DNA sequence and using this "guess" look for the
gene that encoded the protein you wished to study (think about how
this could be done - it is often accomplished using PCR).