Genetic Engineering Lecture 13

spikydoeBiotechnology

Dec 11, 2012 (4 years and 11 months ago)

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INTRODUCTION TO
GENETIC ENGINEERING
Introduction
Recombinant DNA technology or genetic engineering
comprises of a mixture of techniques such as:
•a) Fragmentation, separation and sequencing of DNA
molecules.
•b) Nucleic acid hybridisation, which makes it possible to
find a specific sequence of DNA or RNA with great
accuracy and sensitivity on the basis of its ability to find
complementary nucleic acid sequence.
•c) DNA cloning, where a single DNA molecule can be
copied to generate many billions of identical molecules.
•d) DNA engineering, by which DNA sequences are
altered to make modified versions of genes, which are
inserted back into cells or organisms.
Fragmentation, Separation and Sequencing of
DNA Molecules
One of the major obstacles to molecular analysis of
genomic DNA is the immense size of the molecules
involved.
The discovery of restriction nucleases that cleave dsDNA
into smaller more manageable fragments, opened the
way for DNA analysis.
Many bacteria make restriction nucleases (enzymes that
hydrolyseDNA molecules at specific nucleotide
sequences), which protect the bacterial cell from viruses
by degrading the viral DNA.
Each such nuclease recognisesa specific sequence of 4-8
nucleotides in DNA
These sequences, where they occur in the genome of the
bacterium itself, are protected from cleavage by
methylationat an A or a C residue but when the
sequence appears in a foreign DNA, they are generally
not methylatedand so are cleaved by the restriction
nucleases.
Many restriction nucleases have been purified from
bacteria which recognisesdifferent nucleotide
sequences and are available commercially.
Some restriction nucleases like Tag I produce staggered
cuts, which leave short sstails at the 2 ends of each
fragment.
Fig 7-1
•End of this type are known as cohesive or sticky ends,
as each tail can form complementary basepairswith tail
at any other.
•Fig 33.4 and 7.2
•Other restriction nucleases such as HaeIII (it is the 3rd
restriction nuclease isolated from the bacterium
Haemophilusaegyptics), cleave in the middle of their
recognition sequence (that is, the axis of symmetry) and
produce fragments that have blunt ends that do not form
hydrogen bonds with each other.
•Fig. 33-3 and Fig 33-4
•Using the enzyme DNA ligase, sticky ends of a DNA
fragment of interest can be covalently joined with other
DNA fragments that have sticky ends produced by
cleavage with the same restriction nucleases.
•Fig 33-5
•Another ligase, encoded by bacteriophageT4, can
covalently join blunt ended fragments. The hydrid
combination of 2 fragments is called recombinant DNA
molecule.
•A DNA sequence that is recognisedby a restriction
nuclease is called restriction site.
•Because these sites occur at random, restriction
nucleases cleave DNA into fragments of different sizes
called restriction fragments.
•By comparing the sizes of the DNA fragments produced
after treatment with a combination of different restriction
nucleases, a restriction map of that genetic region can
be constructed showing the location of each cut.
•Fig 7-3
•The different short DNA sequences recognisedby
different restriction nucleases serve as convenient
markers, and the restriction map reflects their
arrangement in the region.
•This allows one to compare the same region of DNA in
different individuals (by comparing their restriction maps)
without having to determine the nucleotide sequences in
detail.
•Fig 7.4
•Fig 7-4 shows the restriction maps of the chromosomal
region that codes for Hbchains in humans and various
other primates and it indicates that the chromosomal
regions have remained unchanged for the past 5-10
million years and that the chinpanzeeis the most closely
related to us.
Gel Electrophoresis to Separate DNA molecules of
different Sizes
•For DNA fragments less than 500 nucleotides long
specially designed polyacrylamidegels allow molecules
that differ in length by as little as a single nucleotide to
be separated from each other.
•Fig 7-5A
•The pores in polyacrylamidegels, however are too small
to permit very large DNA molecules to pass so to
separate these by size, the much more porous agarose
gels are used.
•Fig 7-5B
•The DNA bands on agaroseof polyacrylamidegels are
invisible unless the DNA is labelledor stained.
•One sensitive method of staining is to expose the DNA
to the dye ethidiumbromide, which fluoresces under UV
light when bound to DNA.
•Fig 7-5B
•An even more sensitive detection method involves
incorporating a radioisotope into DNA molecules befire
electrophoresis. 32p is often used and is incorporated into
DNA phosphates and emits a an energetic βarticle that
is detected by autoradiography.
•Fig 7-5A
Purified DNA Molecules Can be Specifically Labelled
with Radioisotopes or Chemical Markers in vitro
2 procedures are widely used to add distinct lablesto
isolate DNA molecules.
In the first, the DNA is copied by an E.colienzyme, DNA
polymerase I, in the presence of nucleotides that are
either radioactive (32p) or chemically tagged.
Fig 7-6A
•In this way, DNA probes containing many labeled
nucleotides can be produced for nucleic acid
hybridisationreactions.
•The second procedure uses the bacteriophageenzyme
polynucleotide kinaseto transfer a single 32p-labelled
phosphate from ATP to the 5’end of each DNA chain.
•Fig 7-6B
•Because only one 32p atom is incorporated by the
kinaseinto each DNA strand, the DNA molecules
labelledthis way are often not radioactive enough to
be used as DNA probes.
Isolated DNA Fragments can be Rapidly Sequenced
2 powerful DNA sequencing methods are used:
a)Chemically method (Fig 7-7)
b)Enzymatic method (Fig 7-8)
•The enzymatic method, carried out in the presence of
chain terminator nucleoside triphosphateshas become
the standard procedure for sequencing DNA today.
Nucleic Acid Hybridisation
When an aqueous solution of DNA is heated at 100C or
exposed at very high pH (pH >13), the complementary
basepairswill breakdown and the double helix will
dissociate into 2 ss. This process is called denaturation.
In 1961, it was discovered that complementary ssof DNA
will readily re-form double helices if they are kept for a
prolonged period at 65C. This process is called DNA
renaturationor hydridization.
Similar hydridizationwill occur between any 2 single
stranded nucleic acid chains (DNA/DNA, RNA/RNA, or
RNA/DNA), provided that they have complementary
nucleotide sequence.
DNA Probes
Cleavage of large DNA molecules by restriction nucleases
produces a large array of fragments.
How can a specific gene or DNA sequence of interest be
picked out of all the fragments?
The answer lies in the use of a probe –a sspiece of DNA,
usually labelledwith a radioisotopsuch as 32p.
The nucleotide sequence of a probe is complementary to
the DNA of interest, called the target DNA.
Probes are used to identify which clone of a library or which
band on a gel contains the target DNA
Northern Blotting and Southern Blotting Facilitate
Hybridization with ElectrophoreticallySeparated
Nucleic Acid Molecules.
•Southern and Northern blotting are techniques that can
be used to detect mutations in DNA.
•It combines the use of restriction nucleases and DNA
probes.
•Suppose you wish to determine the nature of the defect
in a mutant mouse that produces very low amounts of
albumin, a protein that liver cells normally secrete into
the blood in large amounts.
•First, you collect identical liver tissue samples from the
mutant and normal (control) mice.
•Disrupt the cells in a strong detergent to inactivate
cellular nucleases that might degrade the nucleic acids.
•Then, separate the RNA and DNA from all the other cell
components. The proteins are completely denatured and
removed by repeated extractions with phenol –a potent
organic solvent that is partly miscible in water.
•The nucleic acids which remain in the aqueous phase
are then precipitated with alcohol to separate them from
the small molecules of the cell.
•Then, you can separate the DNA from RNA by the
different solubilitiesin alcohols and degrades any
contaminating nucleic acid of the unwanted type with a
highly specific enzyme –the DNaseor RNase
•Fig 7-13
a)Northern Blotting
To analysethe albumin-encoding RNAswith a DNA probe,
a technique called Northern Blotting is used.
First the intact RNA molecules purified from the mutant and
control liver cells are fragmented by restriction
enzymes and fractionated according to their size into a
series of bands by gel electrophoresis.
Then, to make RNA molecules accessible to DNA probes,
a replica of the pattern of RNA bands on the gel is
made by transferring (blotting) the fractionated RNA
molecules onto a sheet of nitrocellulose paper (nylon).
The RNA molecules then hybridisedto the labelledDNA
probe (because they are complementary to prtof the
normal albumin gene sequence) are then located by
incubating the paper with a solution containing the
probe and detecting the hybridisedprobe by
autoradiography.
•The size of the RNA molecules in each band that bind to
the probe can be determined by reference to bands of
RNA molecules of known size(RNAstandards) that are
electrophoresedside by side with the experimental
sample.
•In this way, you can discover that the presence of a
mutation affecting a restriction site causes the patterns
of bands to differ between the mutant and normal gene.
b) Southern Blotting
Similar to Northern blotting except that it analyses DNA
rather than RNA.
First DNA is extracted from cells, egpatients’sleucocytes.
Second, the DNA is cut with restriction nucleases into
many fragments.
The resulting fragments are separated on the basis of size
by electrophoresis with the larger fragments moving
more slowly than the smaller fragments.
•Therefore, the length of the fragments, usually
expressed in the no of basepairscan be calculated from
comparison of the position of the band relative to
standards.
•Then, probes are used to identify the DNA fragments of
interest.
•Fig 33-12
DNA Cloning
In DNA cloning, a DNA fragment that contains a
gene of interest is inserted into the purified DNA
genome of a self-replicating genetic element-
generally a virus or a plasmid.
A DNA fragment of a human gene can be joined in
a test tube to the chromosome of a bacterial
virus, and the new recombinant DNA molecule
can then be introduced into a bacterial cell.
•Starting from one such recombinant DNA molecule that
infects a single cell, the normal replication mechanisms
of the virus can produce many identical virus DNA
molecules in less than a day, thereby amplifying the
amount inserted human DNA fragments.
•A virus or plasmid used in this way is known as the
cloning vector and the DNA propagated by insertion into
it is said to have been cloned.
A DNA library can be made using either viral or
plasmid vector.
In order to clone a specific gene, one begins by
constructing a DNA library –a comprehensive collection
of cloned DNA fragments, including at least one
fragment that contains the gene of interest.
•The library can be constructed using a viral or plasmid
vector and the principles for preparing them are the
same.
•Plasmids are circular pieces of dsDNA which are
replicated within the host cell (usually bacteria).
•They generally account for only a minor fraction of the
total host bacterial cell DNA, and they can be easily
separated on the basis of their small size from
chromosomal DNA molecules, which are large and
precipitate as a pellet upon centrifugation.
•For use as cloning vectors, the purified plasmid DNA
circles are first cut with a restriction nuclease to create
linear DNA molecules.
•Fig 7-21
•The cellular DNA to be used in constructing the library is
cut with the same restriction nuclease and the resulting
restriction fragments (including the gene to be cloned)
are then added to the cut plasmids and annealed via
their cohesive ends to form recombinant DNA circles.
•These recombinant molecules containing foreign DNA
inserts are then covalently sealed with the enzyme DNA
ligase.
•Later, the recombinant DNA circles are introduced into
bacterial cells that have been made transiently
permeable to DNA. Such cells are said to be transfected
with the plasmids.
•As these cells grow and divide, doubling in number every
30 minutes, the recombinant plasmids also replicate to
produce enormous no of copies of DNA circles
containing the foreign DNA.
•Many bacterial plasmids carry genes for antibiotic
resistance, a property that is exploited to select those
cells that have been successfully transfected. If the
bacteria are grown in the presence of the antibiotic, only
cells containing plasmids will survive.
•The mixture of different surviving bacteria contains the
DNA library, composed of a large no of different DNA
inserts. The problem is only a few of these bacteria will
contain the particular recombinant plasmids with the
desired gene.
•One needs to be able to identify these rare cells in order
to recover the DNA of interest in pure form and in useful
quantities.
•This will be described later
Two types of DNA libraries can be Constructed
•Cleaving the entire genome of a cell with a specific
restriction nuclease as just described is sometimes
called the “shotgun”approach to gene cloning.
•It produced a very large of DNA fragments which will
generate millions of different colonies of transfected
bacterial cells.
•Such a plasmid is said to contain a genomic DNA clone,
and the entire collection of plasmids is said to constitute
a genomic DNA library.
•But because the genomic DNA is cut into fragments at
random, only some fragments will contain genes. Many
will contain only portion of a gene, while most of the
genomic DNA clone will contain noncoding(introns)
DNA, which make up most of the DNA in such genomes.
•An alternative strategy is to begin the cloning process by
selecting only those DNA sequences that are transcribed
into RNA and thus presumed to correspond to genes.
•This is done by extracting the mRNA from cells and then
making complementary DNA (cDNA) copy of each
mRNA molecule present.
•The above reaction is catalysedby reverse transcriptase
enzyme of retrovirus, which synthesisesa DNA chain on
an RNA template.
•The ssDNA molecules synthesisedby the reverse
transcriptase are converted to dsDNA molecules by
DNA polymerase and these molecules are inserted into
plasmid or virus vector and cloned.
•Each clone obtained this way is called a cDNAclone,
and the entire collection of clones derived from one
mRNA preparation constitutes a cDNAlibrary.
Detection of the Cloned Gene of Interest
The technique most frequently used to identify the rare
colony that contains the DNA fragment of interest is
called in situ hybridisationthat takes advantage of the
basepairinginteractions between 2 complementary
between nucleic acid molecules.
Culture dishes containing the growing bacteria colonies are
blotted with a piece of filter paper, to which some
members of each bacterial colony adhere.
The adhering colonies, known as replicas, are treated with
alkali to disrupt the cells and to separate the strands of
their DNA molecules.
•The paper is then incubated with a radioactive labeled
DNA probe containing part of the sequence of gene
being sought.
•The colonies on the master culture dish can then be
identified.
Polymerase Chain Reaction (PCR)
PCR is a test tube method for amplifying a selected DNA
sequence and does not rely on the biologic cloning
(living cell) method describe before.
PCR allows the synthesis of millions of copies of a specific
nucleotide sequence in a few hours.
It can amplify the sequence even when the targeted
sequence makes up less than one part in a million of the
total initial sample.
•This method can be used to amplify DNA from any
source –bacterial, viral, animal or plant.
Steps of a PCR
PCR uses DNA polymerase to repetitively amplify targeted
portions of DNA.
Each cycle of amplification doubles the amount of DNA in
the sample, leading to an exponential increase in DNA
with repeated cycles of amplification.
It is not necessary to know the nucleotide sequence of the
target DNA in the PCR method.
However, it is necessary to know the nucleotide sequence
of short segments on each side of the target DNA called
the flanking sequences.
The flanking sequences bracket the DNA sequence of
interest.
•The nucleotide sequences of the flanking regions are
used to construct 2 sssynthetic oligonucleotides(not
discussed here), usually 20-35 nucleotides long that are
complementary to the respective flanking sequences
where the 3’-hydroxyl end of each primer points toward
the target sequences.
•Fig 33-9
•These synthetic oligonucleotidesfunction as primers in
the PCR reactions.
•The DNA to be amplified is heated to separate the ds
target DNA into ss.
•The separated strands are cooled and allowed to anneal
to the 2 primers (one for each strand).
•DNA polymerase and excess of deoxyribonucleoside
triphosphatesare added to the mixture to initiate the
synthesis of 2 new chains complementary to the original
DNA chains.
•Fig 33-9.
•DNA polymerase adds nucleotides to the 3’-OH end of
the primer, and strand growth extends across the target
DNA, making complementary copies of the target.
•At the completion of one cycle of replication, the reaction
mixture is heated again to denature the DNA strands (of
which there are now four).
•Each DNA strand binds a complementary primer and the
cycle of chain extension is repeated.
•By using a hat stable DNA polymerase from a
thermophilicbacterium, the polymerase is not denatured
and therefore, does not have to be added at each
successive cycle.
•Typically, 20-30 cycles are run during DNA amplification.
•Fig 33-10.
Application of PCR
1.Comparison of a normal cloned gene with an uncloned
mutant form of the gene
PCR allows the synthesis of the mutant DNA in sufficient
quantities for a sequencing protocol without laboriously
cloning the altered DNA.
2. Detection of low-abundance nucleic acid sequences
Viruses that have a long latency period such as HIV are
difficult to detect at the early stages of infection using
conventional methods. PCR offers a rapid and
sensitive method for detecting viral DNA sequences
even when only a small proportions of cells is
harboring the virus.
3. Forensic analysis of DNA samples
DNA isolated from a single human hair is sufficient to
determine whether the sample is from a specific
individual. Humans contain DNA known as mini satellite
DNA. This DNA consists of multiple repeats of certain so
called “core”nucleotide sequence which is different
between one individual to another. The distribution of
these core sequence patterns is like a finger print that is
related to that of his/her parents and close relatives.
DNA Engineering
•All the methods described so far have revolutionised
all other aspects of cell biology. Some examples are:
1.New DNA molecules of any sequence can be formed
by joining together DNA fragments.
2.The use of mutant organisms to reveal the function of
a gene.
3.Cells containing mutated genes can be made to order.
4.Genes can be redesigned to produce proteins of any
desired sequence.
5.Engineered genes can be used to create specific
dominant mutations in diploid organisms.
6. Engineered genes can be permanently inserted into the
germ line of mice or fruit flies to produce transgenic
animals.
7. The production of transgenic plants important to
agriculture.