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Gene Cloning
Transformation
Transformation is the step in the genetic engineering process where a new gene (transgene) is delivered
into the nucleus of a plant cell and inserts into a chromosome where it is passed on to
progeny. Transformation means to genetically change a living thing. A genetically
engineered plant has been transformed and is sometimes referred to as a transgenic
plant.
Transformation is typically done on callus cells (tissue culture) but is occasionally done
to other plant parts, such as germline cells or pollen. Tissue culture is clusters of
undifferentiated plant cells called 'callus', which are grown in culture. This allows them
to be manipulated and then induced to develop into whole plants containing a copy of
the transgene in every cell.
There are many methods used to deliver extra DNA into the nucleus of plant cells.
Gene Gun
This method works by shooting DNA into the plant cells. Microscopic gold or tungsten particles are coated
with hundreds of copies of the gene(s) to be introduced. Tissue culture cells are placed in a vacuum
chamber and the metal particles are propelled with a high-pressure gas that is released in a sudden burst
much like a popped balloon or air gun.
Agrobacteria
Agrobacteria is a soil bacteria that works as a natural genetic engineer. In nature it forces plants to become
good hosts by inserting genes into plant cells that cause the plant to make metabolites they need for
growth. Genetic engineers have taken advantage of Agrobacteria's natural abilities by tricking them to
insert an altered piece of DNA that contains the desired transgene into cells.
Microfibers - 'Whiskers'
The Whiskers method uses microscopic "wiskers" that look like tiny needles with sharp ends. Tissue culture
cells, hundreds of copies of the desired gene(s), and whiskers are suspended in a tube of solution and
shaken vigorously. The tiny whiskers stab the plant cells potentially delivering the desired gene into the
nucleus of the cell without killing it. For expanded information on Whiskers technology click here.
Electroporation
Electroporation uses a quick pulse of electricity to open tiny pores in the walls of plant cells. If DNA is
mixed in a solution with the cells, the DNA molecules are small enough to pass into the cell through these
holes.
The goal of any transformation technique is to transport the new gene into the nucleus of a cell without
destroying it. After that, the genetic engineer has no control as to how or even if the gene will insert into a
chromosome. The successful genetic engineer plays the numbers game. Because they cannot control all of
the steps, they repeat the process hundreds of times in order to produce one genetically engineered plant.
Events
Of the few cells that are actually transformed during the transformation process, very few will have all the
characteristics necessary to become a marketable event. An 'event' is the insertion of a particular
transgene into a specific location on a chromosome. The term "event" is often used to differentiate
genetically engineered crop varieties. Desirable events are rare. Most events are discarded because the
transgene inserted into an existing gene important for plant growth causing the existing gene to be
disrupted and not express, or the transgene inserted into a portion of the chromosome that does not allow
for expression of the transgene or expression that is too low.
Those events that are successful are grown to mature plants. The seed is harvested and passed on to a
plant breeder who performs the last step of the process.
The second step of the genetic engineering process is gene cloning. During DNA
extraction, all of the DNA from the organism is extracted at once. Scientists use
gene cloning to separate the single gene of interest from the rest of the genes
extracted and make thousands of copies of it.
Once a gene has been cloned, genetic engineers begin the third step,
designing the gene to work once inside a different organism. This is done
in a test tube by cutting the gene apart with enzymes and replacing gene
regions that have been seperated./span>
Step 4 : Transformation
The modified gene is now ready for the fourth step in the process,
transformation or gene insertion.
Since plants have millions of cells, it would be impossible to insert a copy
of the transgene into every cell. Therefore, tissue culture is used to
propagate masses of undifferentiated plant cells called callus. These are
the cells to which the new transgene will be added.
The new gene is inserted into some of the cells using various techniques.
Some of the more common methods include the gene gun, agrobacterium,
microfibers, and electroporation. The main goal of each of these methods
is to transport the new gene(s) and deliver them into the nucleus of a cell without killing it. Transformed
plant cells are then regenerated into transgenic plants. The transgenic plants are grown to maturity in
greenhouses and the seed they produce, which has inherited the transgene, is collected. The genetic
engineer's job is now complete. He/she will hand the transgenic seeds over to a plant breeder who is
responsible for the final step.
The fifth and final part of producing a genetically engineered crop is backcross breeding.
Transgenic plants are crossed with elite breeding lines using traditional plant breeding
methods to combine the desired traits of elite parents and the transgene into a single line.
The offspring are repeatedly crossed back to the elite line to obtain a high yielding
transgenic line. The result will be a plant with a yield potential close to current hybrids that
expresses the trait encoded by the new transgene.
Gene Libraries
The step following DNA extraction of an organism is the construction of a library to organize the DNA. A
gene library can be defined as a collection of living bacteria colonies that have been transformed with
different pieces of DNA from the organism that is the source of the gene of interest. If a library is to have a
colony of bacteria for every gene, it will consist of tens of thousands of colonies or clones.
Library Construction
Constructing a gene library requires not only the extracted DNA, but also restriction enzymes and a
plasmid.
Step 1: DNA extracted from an organism, with the gene of interest, is cut into gene-size pieces with
restriction enzymes. These enzymes read the nucleotide sequence of the DNA and recognize specific
sequences. The enzymes then cut the DNA sequence by breaking the bonds between nucleotides in a DNA
strand.
Step 2: Bacterial plasmids are cut with the same restriction enzyme. Plasmids are small circles of DNA in
bacterial cells that are naturally present in addition to the bacteria's other DNA.
Step 3: The gene-sized DNA and cut plasmids are combined into one test tube. Some of the enzyme cut
DNA pieces will combine with the cut plasmids and form recombinant DNA (or DNA in a 'new
combination').
Step 4: The recombinant plasmids are then transferred into bacteria using either electroporation or heat
shock. Electroporation uses mild pulses of electricity to disrupt the cell walls of the bacterium and create
small holes. The plasmids are small enough to pass through the holes into the cell. Heat shock works in a
similar fashion. However, rather than using electricity to create holes in the bacterium, it is done by
alternating the temperature between hot and cold.
Step 5: The bacteria is grown on a culture dish and allowed to grow into colonies. All the colonies on all
the plates (cultures) are called a gene library.
Step 6: The gene library is then screened in order to discover which bacterial colony is making copies of
the one gene they are interested in. Library screening identifies colonies, which have that particular gene.
Screening can be based on detecting the DNA sequence of the cloned gene, detecting a protein that the
gene encodes, or the use of linked DNA markers. Therefore before library screening can be done, the
scientist must know either the DNA sequence of the gene, or a very similar gene, the protein that the gene
produces, or a DNA marker that has been mapped very close to the gene. When the bacteria multiply and
replicate the recombinant DNA, the number of gene copies also increases, making gene or protein
detection easier.
When the bacteria colony containing the desired gene is located, the bacteria can be propagated to make
millions of copies of the recombinant plasmid that contains the gene. The plasmids can be extracted for
the next steps of genetic engineering, gene modification, and transformation. Gene cloning is also
important because copies of a gene are needed for these procedures.
Controversies
Bacterial plasmids used in gene cloning naturally contain genes that encode some form of antibiotic
resistance. As a result of the gene cloning process, the cloned genes that are put into plant cells to make a
transgenic plant may also have an antibiotic resistance gene. The antibiotic resistance trait is often used to
help scientists determine which bacteria have been transformed. Those that have antibiotic resistance have
been transformed and are kept. Some critics of genetic engineering claim that the potential risk of
providing an opportunity for organisms in nature to gain antibiotic resistance by taking up the plasmid
outweighs the potential benefits of this technology.
Definitions:
Gene library a collection of all the DNA fragments of a given species that have been taken from one
organism and inserted into a vector for transport (cloning) into a host.
Plasmid- genetic material (DNA) that is located outside the chromosome of a bacterium that usually gives
the organism an evolutionary advantage such as antibiotic resistance.
Restriction enzyme- enzymes that cut DNA molecules at specific code sequences.
http://agbiosafety.unl.edu/education/transformation.htm
Gene Regions
Once a gene has been located and cloned, genetic engineers usually need to modify it so that it expresses
in a specific and desired way when inserted into the plant. The modification involves changing the
sequences in the regions of the gene that control gene expression.
Genes have three regions, the promoter, coding region, and termination sequence. The promoter turns
the gene on. The coding region has the protein building information, and the termination sequence
indicates the end of a gene.
The promoter and the coding region are the gene regions that are normally modified. Using special DNA
modifying enzymes, geneticists can cut apart the gene regions, remove one of them, and replace it with
another that will direct gene expression as desired. The gene is then considered recombinant DNA since it
has a new combination of DNA. This part of the genetic engineering process can be very time consuming
but also gives the genetic engineer a wide array of gene design options to try.
Promoter
The first region of a gene is the promoter. It acts as a 'genetic dimmer switch' in that it turns the gene on
and off and specifies how many copies of the protein will be produced. Two main promoters currently
used in transgenic crops are the 35S promoter and the PEP carboxylase promoter. Each promoter is
turned on differently in plants.
The 35S promoter originated from a cauliflower mosaic virus gene. This particular gene is continuously
needed in every cell of a plant and its promoter turns on the gene in every cell of a plant that is
metabolically active. Therefore, when genetic engineers use this promoter in a transgene, the protein
encoded by the gene will be produced in every cell all of the time until the cell dies.
The phosphoenolpyruvate (PEP) carboxylase promoter is from a plant gene encoding a photosynthetic
enzyme. As a result, any transgene with this promoter will produce the protein only in cells that are
actively making photosynthetic proteins. Genetic engineers use this promoter to limit gene expression to
cells that make up green tissue. This would not include root, tassel, or ear tissue. Expression also begins
to slow and eventually stop toward the end of the season when the plant is completing its lifecycle and
photosynthesizing less.
A good example of these two promoters in action is in Bt corn. Some Bt hybrids are designed to have
resistance to European corn borer all season long in every part of the plant. Those lines have a transgene
with the 35S promoter. Other Bt hybrids are designed to have resistance only in green tissue but not in
other parts of the plant, such as the seed or roots. These lines contain a transgene with the PEP
carboxylase promoter. These plants also have reduced resistance later in the growing season.
Coding Region
The second region of a gene that is also often modified in transgenes is the coding region. This region
contains the coded information, which designates the amino acid sequence of the protein to be produced.
The amino acid sequence of the protein determines its shape and thus the function of that protein.
During protein production, a complimentary copy of the coding region called mRNA is made which travels
from the nucleus (where the immobile DNA is located) to the cytoplasm (where the amino acids for
building proteins are located).
Many Bt genes have been cloned that have the potential to provide resistance to
ECB and other insect pests. Three coding regions for ECB resistance have been
used in commercial Bt corn; Cry 1A(b), Cry 1A(c), and the Cry 9c. Each region
encodes crystaline proteins in the bacteria that are responsible for insect larvae
toxicity. When eaten by the European corn borer, these crystaline, or Cry
proteins, bind to the insects' midgut causing those cells to burst from a water
imbalance killing the corn borer. Transgenic plants containing a Bt gene produce
these Cry proteins, which the ECB ingest when feeding on the plants. There are
hundreds of naturally occurring Bt proteins each with their own coding regions.
However, not all are toxic to ECB.
The nucleotide sequences of the Cry 1A(b) and Cry 1A(c) coding regions are very similar. The slight
differences are not enough to cause a difference in protein toxicity. The nucleotide sequence of the Cry
9C coding region, on the other hand, is different enough that it produces a protein that is toxic to
European corn borer, but binds to a different site in the midgut killing the corn borer larvae in a slightly
different way. Therefore even if ECB become resistant to the Cry 1A(b) and Cry 1A(c) proteins they may
still be susceptible to the Cry 9(c) protein.
This technique has potential for dealing with the development of insect resistance to the toxin. However,
it is not a silver bullet. It is possible that when ECB develops resistance to one Bt protein, it will also
develop resistance to other Bt proteins. This is called cross-resistance. The alterations of coding regions
to avoid resistance requires an understanding of how the insecticidal proteins interact with the insect on
a biochemical level.
Termination Sequence
3) Plate the colonies on agar plates and let them grow. These are called the master plates.
4) Press a piece of nitrocellulose onto each master plate and lift off. This leaves some of each colony
on the plate and a replica on the filter.
5) Break open (lyse) the bacteria on the filter under conditions that make their plasmid DNA singlestranded, and bind the DNA onto the filter. There are now spots of single-stranded plasmid DNA on
the filter. These spots correspond to the locations of the colonies of bacteria on the master plate.
6-9) Follow steps 5 through 8 of the Southern Blot, using a labeled restriction fragment of the yeast
actin gene from the plasmid as a probe. The result will be a piece of X-ray film with a dark spot
corresponding to a place on the filter where DNA from the human actin gene was present. This spot
on the filter corresponds to a colony from the master plate.
10) Pick up some of the bacteria from the appropriate colony, grow them in broth and extract their
plasmid DNA. It contains a fragment of human DNA containing the actin gene.
It is also possible to clone gene X using an antibody against the protein produced by gene X. In this
case, you make an expression library - a library where the vector contains a strong E. coli promoter
and an ATG codon. Cells containing these plasmids will produce large amounts of protein from
whichever human gene they carry. If you prepare the filter replicas of the library as above, you can
probe them with an antibody to find the colony that contains a plasmid that expresses protein X. This
plasmid will contain gene X.
Question #2
A bacterial DNA sequence is transcribed into a complementary copy of RNA, which in turn is
translated into a protein sequence by ribosomes and aminoacyl-tRNA complexes. Starting from each
DNA strand below, indicate the mRNA transcript and predict the protein (amino acid sequence) that
could be synthesized in vitro.
3' ATACGAGTCACAGAGTCGTGTAAC 5'
5' TATGCTCAGTGTCTCAGCACATTG 3'
Question #3
A) Approximately how many molecules of NTPs (nucleoside triphosphates: ATP, GTP, CTP, or UTP)
would be consumed in transcribing and translating a bacterial gene into a protein that is 100 amino
acids long? Explain your reasoning. (You need not give exact numbers but try to include all
categories).
B) Would you expect a protein of 100 amino acids in a typical eukaryotic cell to require the same,
less or more ATP, GTP, CTP, and UTP for transcription and translation compared to a bacterium?
Explain.
To view answers to these questions, jump to Solutions
http://web.mit.edu/esgbio/www/dogma/dogma-problems.html
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5' ATGCC
5' TACGG
5' CTGGA
5' GACCT
5' GGCAT
B.
C.
D.
E.
RFLP profile
F' factor
library
lysogenic phage
Problem 6 Applications
Restriction endonuclease generated DNA fragments separated by gel electrophoresis and blot
transferred onto a membrane filter are probed with a radioactive DNA fragment. This procedure is
called:
A.
B.
C.
D.
E.
Gene cloning
The Southern technique
The polymerase chain reaction
Recombinant DNA
Gene mapping
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13
In the Figure showing data on Huntington's disease, which of the following conclusions is valid:
A. No relationship between age of onset of disease and the migration rate of PCR fragments.
B. A shorter PCR fragment predicts early onset of Huntington's disease.
C. Increased length of the amplified PCR fragment predicts early onset of Huntington's disease.
D. Huntington's disease must be contagious since many of the children have the disease.
E. None of the above.
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Primer design
The gene of interest usually has to be amplified from genomic or vector DNA by PCR (polymerase
chain reaction) before it can be cloned into an expression vector. The first step is the design of the
necessary primers.
Important features are:
Primer sequence. Especially the 3'-end of the primer molecule is critical for the specificity and
sensitivity of PCR. It is recommemded not to have:
3 of more G or C bases at this position. This may stabilize nonspecific annealing of the
primer.
a 3' thymidine, since it is more prone to mispriming than the other nucleotides.
Primer pairs should be checked for complementarity at the 3'-end. This often leads to primer-dimer
formation.
Bases at the 5'-end of the primer are less critical for primer annealing. Therefore, it is possible to add
sequence elements, like restriction sites, to the 5'-end of the primer molecule.
Primer length. Usually a primer length of 18-30 bases is optimal for most PCR applications. Shorter
primers could lead to amplification of nonspecific PCR products.
Melting temperature (Tm). The specificity of PCR depends strongly on the melting temperature (Tm)
of the primers (the temperature at which half of the primer has annealed to the template). Usually
good results are obtained when the Tm's for both primers are similar (within 2-4 C) and above 60C.
The Tm for a primer can be estimated using the following formula:
Tm = 2C * (A + T) + 4C * (C + G)
GC content. The GC content of a primer should be between 40 and 60%.
Restriction site. The restriction site should be the same or provide the same sticky end to the
first of the restriction enzymes in the multiple cloning site of the vector chosen to clone the
gene of interest into. Alternatively, you could pick any restriction enzyme that gives a blunt
end upon cleavage (see cloning). Often Nco I (CCATGG) or Nde I (CATATG) are chosen
because the ATG within these sites can be used directly to create the ATG start codon and/or
the ATG codon for the N-terminal methionine residue (see Utilization of the Nco I cloning
site).
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5'-extension to the restriction site. Restriction enzymes cleave DNA much less efficient
towards the end of a fragment. A 5' extension of the restriction site with 2-10 bases greatly
increases the cleavage efficiency of most enzymes. Data on the effect of the extension length
and sequence on the cleavage efficiencies of the most used restriction enzymes can be found
in the reference appendix of the New England Biolabs catalog.
Start codon. A start codon (usually ATG) should be included when the gene of interest is not
expressed with an N-terminal tag or fusion partner or when an N-terminal methionine residue
is present. It should be checked that the start codon and the gene of interest are in frame with
an eventual N-terminal tag and/or fusion partner.
Overlap with the gene of interest. The overlap between the primer and the gene of interest
should be long enough to give a Tm of 60C or more (calculated as shown above).
Restriction site. The restriction site should be the same or provide the same sticky end to the
second of the restriction enzymes in the multiple cloning site of the vector chosen to clone the
gene of interest into. Alternatively, you could pick any restriction enzyme that gives a blunt
end upon cleavage (see cloning).
5'-extension to the restriction site. Restriction enzymes cleave DNA much less efficient
towards the end of a fragment. A 5' extension of the restriction site with 2-10 bases greatly
increases the cleavage efficiency of most enzymes. Data on the effect of the extension length
and sequence on the cleavage efficiencies of the most used restriction enzymes can be found
on pp. 210-211 of the 2000/01 catalog of New England Biolabs.
Stop codon(s). A stop codon (TAA is preferred because it is less prone to read-through than
TAG and TGA) should be included when no C-terminal tag is used. To increase the
efficiency of termination it is possible to use 2 or 3 stop codons in series.
Overlap with the stand complement to the 3'-end of the gene of interest. The overlap
between the primer and the strand complement to the 3'-end of the gene of interest should be
long enough to give a Tm of 60C or more (calculated as shown above). It should be checked
that the gene of interest is in frame with an eventual C-terminal tag.
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Basic Definitions
Blots are named for the target molecule.
Southern Blot
DNA cut with restriction enzymes - probed with radioactive DNA.
Northern Blot
RNA - probed with radioactive DNA or RNA.
Western Blot
Protein - probed with radioactive or enzymatically-tagged antibodies.
Overview
The formation of hybrids in solution is of little experimental value - if you mix a solution of DNA
with a solution of radioactive probe, you end up with just a radioactive solution. You cannot tell the
hybrids from the non-hybridized molecules. For this reason, you must first physically separate the
mixture of molecules to be probed on the basis of some convenient parameter.
These molecules must then be immobilized on a solid support, so that they will remain in position
during probing and washing. The probe is then added, the non-specifically bound probe is removed,
and the probe is detected. The place where the probe is detected corresponds to the location of the
immobilized target molecule. This process is diagrammed below:
In the case of Southern, Northern, and Western blots, the initial separation of molecules is done on
the basis of molecular weight. (Cloning uses a different technique.)
In general, the process has the following steps, detailed below:
- Gel electrophoresis
- Transfer to Solid Support
- Blocking
- Preparing the Probe
- Hybridization
- Washing
- Detection of Probe-Target Hybrids
Gel Electrophoresis
This is a technique that separates molecules on the basis of their size.
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First, a slab of gel material is cast. Gels are usually cast from agarose or poly-acrylamide. These gels
are solid and consist of a matrix of long thin molecules forming sub-microscopic pores. The size of
the pores can be controlled by varying the chemical composition of the gel. The gel is cast soaked
with buffer.
The gel is then set up for electrophoresis in a tank holding buffer and having electrodes to apply an
electric field:
The pH and other buffer conditions are arranged so that the molecules being separated carry a net (-)
charge so that they will me moved by the electric field from left to right. As they move through the
gel, the larger molecules will be held up as they try to pass through the pores of the gel, while the
smaller molecules will be impeded less and move faster. This results in a separation by size, with the
larger molecules nearer the well and the smaller molecules farther away.
Note that this separates on the basis of size, not necessarily molecular weight. For example, two 1000
nucleotide RNA molecules, one of which is fully extended as a long chain (A); the other of which can
base-pair with itself to form a hairpin structure (B):
As they migrate through the gel, both molecules behave as though they were solid spheres whose
diameter is the same as the length of the rod-like molecule. Both have the same molecular weight, but
because B has secondary (2') structure that makes it smaller than A, B will migrate faster than A in a
gel. To prevent differences in shape (2' structure) from confusing measurements of molecular weight,
the molecules to be separated must be in a long extend rod conformation - no 2' structure. In order to
remove any such secondary or tertiary structure, different techniques are employed for preparing
DNA, RNA and protein samples for electrophoresis.
Preparing DNA for Southern Blots
DNA is first cut with restriction enzymes and the resulting double-stranded DNA fragments
have an extended rod conformation without pre-treatment.
Preparing RNA for Northern Blots
Although RNA is single-stranded, RNA molecules often have small regions that can form
base-paired secondary structures. To prevent this, the RNA is pre-treated with formaldehyde.
Preparing Proteins for Western Blots
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Proteins have extensive 2' and 3' structures and are not always negatively charged. Proteins
are treated with the detergent SDS (sodium dodecyl sulfate) which removes 2' and 3' structure
and coats the protein with negative charges.
If these conditions are satisfied, the molecules will be separated by molecular weight, with the high
molecular weight molecules near the wells and the low molecular weight molecules far from the
wells. The distance migrated is roughly proportional to the log of the inverse of the molecular weight
(the log of 1/MW). Gels are normally depicted as running vertically, with the wells at the top and the
direction of migration downwards. This leaves the large molecules at the top and the smaller
molecules at the bottom. Molecular weights are measured with different units for DNA, RNA, and
protein:
DNA: Molecular weight is measured in base-pairs, or bp, and commonly in kilobase-pairs
(1000bp), or kbp.
RNA: Molecular weight is measured in nucleotides, or nt, and commonly in kilonucleotides
(1000nt), or knt. [Sometimes, bases, or b and kb are used.]
Protein: Molecular weight is measured in Daltons (grams per mole), or Da, and commonly in
kiloDaltons (1000Da), or kDa.
On most gels, one well is loaded with a mixture of DNA, RNA, or protein molecules of known
molecular weight. These 'molecular weight standards' are used to calibrate the gel run and the
molecular weight of any sample molecule can be determined by interpolating between the standards.
Below is a gel stained with a dye: a colored molecule which binds to a specific class of
macromolecules in a sequence-independent manner (probes bind in a sequence-dependent manner).
Sample 1 contains only one size class of macromolecule - it could be a plasmid, a pure mRNA
transcript, or a purified protein. In this case, you would not have to use a probe to detect the molecule
of interest since there is only one type of molecule present. Blotting is usually necessary for samples
that are not complex mixtures. By interpolation, its molecular weight is roughly 3.
Sample 2 is what a sample of total DNA cut with a restriction enzyme, total cellular RNA, or total
cellular protein would look like in a gel stained with a sequence-independent stain. There are so many
bands that it is impossible to find the one we are interested in. Without a probe (which acts like a
sequence-dependent stain) we cannot get very much information from a sample like this.
Different stains and staining procedures are used for different classes of macromolecules:
Staining DNA
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DNA is stained with ethidium bromide (EtBr), which binds to nucleic aids. The DNA-EtBr
complex fluoresces under UV light.
Staining RNA
RNA is stained with ethidium bromide (EtBr), which binds to nucleic aids. The RNA-EtBr
complex fluoresces under UV light.
Staining Protein
Protein is stained with Coomassie Blue (CB). The protein-CB complex is deep blue and can
be seen with visible light.
2) Capillary blotting, where the molecules are transferred in a flow of buffer from wet filter paper to
dry filter paper:
Note: In a Southern Blot, the DNA molecules in the gel are double-stranded, so they must be made
single stranded in order for the probe to hybridize to them. To do this, the DNA is transferred using a
strongly alkaline buffer, which causes the DNA strands to separate - this process is called
denaturation - and bind to the filter as single-stranded molecules. RNA an protein are run in the gels
in a state that allows the probe to bind without this pre-treatment.
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Blocking
At this point, the surface of the filter has the separated molecules on it, as well as many spaces
between the lanes, etc., where no molecules have yet bound. If we added the probe directly to the
filter now, the probe would stick to these blank parts of the filter, like the molecules transferred from
the gel did. This would result in a filter completely covered with probe which would make it
impossible to locate the probe-target hybrids. For this reason, the filters are soaked in a blocking
solution which contains a high concentration of DNA, RNA, or protein. This coats the filter and
prevents the probe from sticking to the filter itself. During hybridization, we want the probe to bind
only to the target molecule.
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This produces a radioactive single-stranded DNA copy of both strands of the template for use as a
probe.
Radioactive Antibodies for Westerns
Antibodies are raised by injecting a purified protein into an animal, usually a rabbit or a mouse. This
produces an immune response to that protein. Antibodies isolated from the serum (blood) of that
rabbit will bind to the protein used for immunization. These antibodies are protein molecules and are
not themselves radioactive.
They are labeled by chemically modifying the side chains of tyrosines in the antibody with iodine125 (125I), which is radioactive. A set of enzymes catalyzes the following reaction:
antibody-tyrosine + 125I- + H2O2 ---------> H2O + 125iodo-tyrosine-antibody
Enzyme-conjugated Antibodies for Westerns
Antibodies against a particular protein are raised as above and labeled by chemically cross-linking
the antibody molecules to molecules of an enzyme. The resulting antibody-enzyme conjugate is still
able to bind to the target protein.
Hybridization
In all three blots, the labeled probe is added to the blocked filter in buffer and incubated for several
hours to allow the probe molecules to find their targets.
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Washing
After hybrids have formed between the probe and target, it is necessary to remove any probe that is
on the filter that is not stuck to the target molecules. Because the nitrocellulose is absorbent, some of
the probe soaks into the filter and must be removed. If it is not removed, the whole filter will be
radioactive and the specific hybrids will be undetectable.
To do this, the filter is rinsed repeatedly in several changes of buffer to wash off any un-hybridized
probe.
Note: In Southerns and Northerns, hybrids can form between molecules with similar but not
necessarily identical sequences (For example, the same gene from two different species.). This
property can be used to study genes from different organisms or genes that are mutated. The washing
conditions can be varied so that hybrids with differing mismatch frequencies are maintained. This is
called 'controlling the 'stringency' - the higher the wash temperature, the more stringent the wash, the
fewer mismatches per hybrid are allowed.
Summary:
The procedure for these three blots is summarized below:
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Solving Problems
A typical Southern Blot interpretation problem:
You are studying a gene responsible for pigment biosynthesis in plants. RR individuals are red; Rr are
pink; and rr are white. The restriction map of the R allele is:
You extract genomic DNA from a red, a pink, and a white plant; digest with EcoRI, HindIII and
EcoRI+HindIII; run on a gel, and transfer to nitrocellulose. You have a cDNA for the R gene which
you use as a probe. The results of the autoradiogram are as follows:
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therefore bind probe and appear on But the probe hybridizes to only part of fragments e2 and e4, why
do they appear in their full lengths in the Southern?
- The probe only needs to find a short region of the target (20 - 30 nt) to hybridize to, so the length of
the overlap is usually not important. Also, since hybridization occurs after the DNA fragments have
been separated by molecular weight, the length of hybridization overlap is irrelevant to the size that
the fragments migrate. (This is a subtle, but very important point!)
HindIII: Digestion of this region of DNA should produce 5 fragments in addition to the thousands of
others produced from the rest of the genome. These are, from left to right:
h1) A band of unknown size which begins off the left end of the map, includes the 0.2kbp EcoRIHindIII fragment, and ends with the leftmost HindIII site on the map.
h2) A 1.75kbp band including exon 1, the intron, and part of exon 2.
h3) A 0.3kbp band from exon 2.
h4) A 0.95 kbp band including part of exon 2 and the 0.4kbp EcoRI-HindIII fragment.
h5) A band of unknown size which begins with the rightmost HindIII site and extends off the right
end of the map.
Since only fragments h2, h3, and h4 contain sequences found in the probe, only they will appear on
the autoradiogram.
EcoRI+HindIII: Digestion with both enzymes will result in cleavage of the DNA at all EcoRI sites
and at all HindIII sites. This should produce 9 fragments in addition to the thousands of others
produced from the rest of the genome. These others produced from the rest of the genome. These are,
from left to right:
eh1) A fragment of unknown size which begins off the left end of the map.
eh2) A 0.2kbp EcoRI-HindIII fragment.
eh3) A 1.0kbp HindIII-EcoRI fragment.
eh4) A 0.1kbp EcoRI fragment.
eh5) A 0.65kbp EcoRI-HindIII fragment.
eh6) A 0.3kbp HindIII fragment.
eh7) A 0.55 kbp HindIII-EcoRI fragment.
eh8) A 0.4kbp EcoRI-HindIII fragment.
eh9) A fragment of unknown size extending off the right end of the map.
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Fragments eh3, eh5, eh6, and eh7 will bind the probe and show up on the autoradiogram.
1) To find the difference between r and R, find the bands that are altered between the two DNA's.
The differences are:
EcoRI: The 1.5kbp band (e4) has disappeared and a 1.25kbp band has appeared. The simplest
explanation for this is that 0.25kbp has been deleted from this region in the R allele to produce the r
allele.
HindIII: The 0.3kbp band (h3) has disappeared and a 0.05kbp band has appeared. The simplest
explanation for this is that 0.25kbp has been deleted from this region of the R allele to give the r
allele.
EcoRI+HindIII: The 0.3kbp band (eh6) has disappeared and a 0.05kbp band has appeared. The
simplest explanation for this is that 0.25kbp has been deleted from this region of the R allele to give
the r allele.
Thus, all three digests agree with the model that a 250bp deletion has occurred in the 0.3kb HindIIIHindIII region of exon 2 in the R alleles given rise to the r allele. A deletion of this type would be
likely to cause an inactive protein to be produced by the r allele, leading to reduced red pigment
production.
Note: You cannot be certain that alterations have not occurred in the intron regions as well - without
an intron sequence probe, you cannot tell for sure. Also, it is possible that other models may be able
to explain this data. However, this is the simplest reasonable explanation - you could be more certain
if you did a Southern using: other restriction enzymes; a probe to exon 2 only; or even by sequencing
the DNA of the R and r alleles to find the exact differences.
2) What about the pink (Rr) strain?
There are two types of bands in this part of the blot. This is because this strain is a heterozygote - it
contains one copy of the R allele and one copy of the r allele.
i) Heavy bands at positions which are not altered between the R and r alleles. These represent
fragments that are present in two copies per cell. They are the same intensity as the bands from the
RR and rr strains because in the RR and rr, the alleles are present in two identical copies per cell.
ii) Light bands at positions representing both R and r alleles. These represent fragments that are
present in only one copy per cell. They are lighter and thinner than the other bands because there is
half as much DNA in each band.
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You extract RNA from male and female flies and run duplicate samples of both on several northern
blots. You have the entire 3.85kbp EcoRI-HindIII fragment containing the gene on a plasmid, from
which you generate probes by restriction enzyme digestion. Assuming that the DNA is the same in
both sexes (How could you tell this?), explain the difference between the mRNA for this gene in
males and females using the data below.
Probe
Restriction Fragment
1.0kbp EcoRI-HindIII
0.6kbp HindIII-EcoRI
0.1kbp EcoRI-EcoRI
0.5kbp EcoRI-HindIII
- You could tell if males and females have identical copies of this gene by doing a Southern blot of
DNA from males and from females, probed with cloned fragments from this region, and make sure
that there are no differences in the restriction maps of the regions in both sexes. Note that this cannot
guarantee that the sequences are identical throughout - that would require sequencing the DNA from
both - but it can show that the sequences are very similar.
- There are at least two ways to analyze this data:
i) First, what do the blots show? Since the target molecule is mRNA, only exon regions of the probes
will form hybrids. Each probe will then detect the presence of a different exon or exons in the final
mRNA.
Probe
Exon(s) Detected
1 and 2
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From this, we can determine which exons are present in the mature mRNA's.
Blot A shows that exon 1 is present in both male and female mRNA for this gene. Blot B shows that
exon 1 and/or exon 2 are present in both mRNA species.
Blot C shows that exon 2 is present in the female mRNA but not in the male mRNA - note that this
does not conflict with Blot B's result.
Blot D shows that exon 3 is present in both male and female mRNA for this gene.
Summarizing: female mRNA for this gene contains exon 1, 2, and 3 - this is expected pattern of
splicing; male mRNA for this gene contains only exon 1 and 3 - exon 2 is skipped. These models are
confirmed by the sizes of the mRNA's in both sexes. We would predict that in females, mature
mRNA = 0.5 + 0.2 + 0.6 = 1.3knt; and in males, mature mRNA = 0.5 + 0.6 = 1.1knt.
Note that the size of the mRNA observed in each sex does not change even with different probes.
ii) You could also look at this another way:
You would predict that the mature mRNA for this gene would be 0.5 + 0.2 + 0.6 = 1.3knt. This size is
observed in females and represents apparently normal splicing of the pre- mRNA. Therefore, in
females, the gene is expressed as we would expect.
But what about the males?
In males, the mature mRNA is 0.2knt shorter. There are three possible models that can explain this:
i) Transcription in males starts 0.2kbp to the right of where it normally starts in females (2/5 of the
way through exon 1).
ii) Transcription in males ends 0.2kbp to the left of where it normally ends in females (2/3 of the way
through exon 3).
iii) Exon 2 is skipped in males - the spliceosome starts with the start of intron 1 and uses the end of
intron 2.
In the case of i) and ii), complete or partial sequences from all three exons would be present in the
mature mRNA. This is not observed - blot C shows that males lack exon 2. This confirms iii) and we
must conclude that exon 2 is missing in the mature mRNA from this gene in males.
In either method of analysis, you are forced to a surprising conclusion: that splicing can skip an
intron under certain circumstances. You are not required to know that this process occurs, nor would
you be expected to come up with it on your own. However, when given this data, you are forced to
conclude - even with out knowing a mechanism - that all of exon 2, and only exon 2 is somehow
missing from the mature mRNA in males.
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Protein
Isolated From:
1
wild-type
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