2. The Potential Use of these Techniques
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•
•
•
Diagnosis:
– Test for DNA sequence variation are more sensitive than many
other techniques, such as enzyme assays, Therefore permit
recognition of disease at earlier stage
– Identifying carriers of inherited diseases so they can receive
appropriate counseling
Prevention:
– Vaccination (prevention of hepatitis)
Treatment:
– Diabetes and factor VIII for the treatment of hemophilia
– Gene therapy
Others:
– Determine family relationships
– Help identifying perpetrators of the crime
3. Restriction Endonucleases
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•
•
•
•
Restriction Endonucleases Cleave DNA Molecules at Particular Sites
Most naturally occurring DNA molecules are much larger than can
readily be managed, or analyzed, in the laboratory. If we are to study
individual genes and individual sites on DNA, the large DNA molecules
found in cells must be broken into manageable fragments. This can be
done using restriction endonucleases that cleave DNA at particular
sites by recognizing specific sequences.
Restriction enzymes used in molecular biology typically recognize
short (4 to 8 bp) target sequences, usually palindromic, and cut at a
defined position within those sequences.
Of note, some restriction enzymes are sensitive to methylation. That
is, methylation of a base (or bases) within a recognition sequence
inhibits enzyme activity at that site.
Restriction enzymes differ not only in the specificity and length of
their recognition sequences but also in the nature of the DNA ends
they generate. Thus, some enzymes, such as HpaI, generate flush or
“blunt” ends; others, such as EcoRI, HindIII, and PstI, generate
staggered ends
4. Restriction Endonucleases
- They are of bacterial origin, They protect bacteria from viruses.
- They permit construction of a new type of genetic map (restriction map: map
shows you where each type of RE will cut, its fixed)
-
Some hydrolyze the two strands of DNA in a staggered fashion producing
sticky or cohesive ends, while others cut both strands symmetrically producing
a blunt (Flush) end.
-
Each RS has specific site of cutting (ex. G-A) but it works on more than one
location on the DNA
-
No way to have different enzymes recognizing the same sequence, each RS has
its sequence length and specific cutting location
5.
6.
7.
8.
9. Gel Electrophoresis Separates DNA and RNA
Molecules according to Size
•
•
DNA and RNA molecules are separated by the technique called Gel Electrophoresis.
Linear DNA molecules separate according to size when subjected to an electric field
through jelly-like porous material. Because DNA is negatively charged, when
subjected to an electrical field in this way, it migrates through the gel toward the
positive pole. The gel matrix acts as a sieve through which DNA molecules pass;
large molecules (with a larger effective volume) have more difficulty passing through
the pores of the gel and thus migrate through the gel more slowly than do smaller
DNAs. This means that once the gels have been electrophoresed or “run” for a given
time, molecules of different sizes are separated because they have moved different
distances through the gel. After electrophoresis is complete, the DNA molecules can
be visualized by staining the gel with fluorescent dyes like ethidium bromide, which
binds to DNA and intercalates between the stacked bases. The stained DNA
molecules appear as “bands” that each reveal the presence of a population of DNA
molecules of a specific size.
Two alternative kinds of gel matrices are used: polyacrylamide and agarose.
Polyacrylamide has high resolving capability but can separate DNAs over only a
narrow size range. Thus, electrophoresis through polyacrylamide can resolve DNAs
that differ from each other in size by as little as a single base pair but only with
molecules of up to several hundred ( just under 1000) base pairs. Agarose has less
resolving power than polyacrylamide but can separate DNA molecules of up to tens,
and even hundreds, of kilobases.
14. Restriction maps :
http://www.youtube.com/watch?v=8FqMUF96cPE
- Permit the routine preparation of defined segments
of DNA : which enzyme should we use to cut to get the wanted gene?
- They used to demonstrate sequence diversity:
If we expect an enzyme to cut a specific sequence and it doesn’t cut it, there is
a problem, and that sequence differs (polymorphism)
Ex. 2 people with X gene, the enzyme cuts the X gene in the first person and
doesn’t cut the X gene in the second one, why? X gene differs (polymorphism)
- They can be used to detect deletion mutations.
By Taking 2 different DNA and restrict them to the same enzyme, and observe
the cut parts, if they differ there is a deletion mutation
- They are crucial for cloning and for sequencing genes
and their flanking DNA regions : cut specefic sites we want to
clone
20. DNA Hybridization Can Be Used to Identify
Specific DNA Molecules
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•
The capacity of denatured DNA to reanneal (i.e., to re-form base pairs
between complementary strands) allows for the formation of hybrid
molecules when homologous, denatured DNAs from two different
sources are mixed with each other under the appropriate conditions of
ionic strength and temperature. This process of base pairing between
complementary single-stranded polynucleotides is known as
hybridization.
Many techniques rely on the specificity of hybridization between two
DNA molecules of complementary sequence. For example, this
property is the basis for detecting specific sequences within
complicated mixtures of nucleic acids. In this case, one of the
molecules is a probe of defined sequence—either a purified fragment
or a chemically synthesized DNA molecule. The probe is used to
search mixtures of nucleic acids for molecules containing a
complementary sequence. The probe DNA must be labeled so that it
can be readily located once it has found its target sequence.
Labeling by Radioactive( P32,S35) or chemically (Fluorescent)
Radioactive cause less damage than chemical labeling
21. DNA hybridization
• DNA hybridization can be used to identify specific DNA
molecules
• Probes are needed: either a purified fragment or a
chemically synthesized DNA molecule. The probe is used to
search mixtures of nucleic acids. probes should be labeled.
• Southern blot
• Northern blot
22. DNA labeling
There are three basic methods for labeling DNA. The first involves adding a
label to the end of an intact DNA molecule. Thus, for example, the enzyme
polynucleotide kinase adds the gamma-phosphate from ATP to the 5’-OH
group of DNA. If that phosphate is radioactive, this process labels the DNA
molecule to which it is transferred. Labeling by incorporation (the other
mechanism) involves synthesizing new DNA in the presence of a labeled
precursor. This approach is often performed by using PCR with a labeled
precursor, or even by hybridizing short random hexameric
oligonucleotides to DNA and allowing a DNA polymerase to extend them.
The labeled precursors are most commonly nucleotides modified with
either a fluorescent moiety or radioactive atoms. In addition, DNA can be
labeled by so called nick translation.nicking (cutting) DNA with specific enzymes
.and introducing labeled (florescent) nucleotide
•
DNA labeled with fluorescent precursors can be detected by illuminating
the DNA sample with appropriate wavelength UV light and monitoring the
longer-wavelength light that is emitted in response. Radioactively labeled
precursors typically have radioactive P32 or S35 incorporated into the alphaphosphate of one of the four nucleotides. This phosphate is retained in the
product DNA. Radioactive DNA can be detected by exposing the sample of
interest to X-ray film or by photomultipliers that emit light in response to
excitation by the beta particles emitted from P32and S35.
26. Hybridization Probes Can Identify
Electrophoretically Separated DNAs and RNAs
•
•
It is often desirable to monitor the abundance or size of a particular
DNA or RNA molecule in a population of many other similar molecules.
For example, this can be useful when determining the amount of a
specific mRNA that is expressed in two different cell types or the
length of a restriction fragment that contains the gene being studied.
This type of information can be obtained using blotting methods that
localize specific nucleic acids after they have been separated by
electrophoresis.
Suppose that the yeast genome has been cleaved with the restriction
enzyme EcoRI and the investigator wants to identify or know the size
of the fragment that contains the gene of interest. When stained with
ethidium bromide, the thousands of DNA fragments generated by
cutting the yeast genome are too numerous to resolve into discretely
visible bands, and they look like a smear centered around 4 kb. The
technique of Southern blot hybridization (named after its inventor
Edward Southern) will identify within the smear the size of the
particular fragment containing the gene of interest.
27. Southern Blot Hybridization http://www.youtube.com/watch?v=RsMsAiNVIGw
•
•
In this procedure, the cut DNA is separated by gel electrophoresis, and the
gel is soaked in alkali to denature the double-stranded DNA fragments.
These fragments are then transferred from the gel to a positively charged
membrane to which they adhere, creating an imprint, or “blot,” of the gel.
During the transfer process, the DNA fragments are bound to the
membrane in positions that mirror their corresponding positions in the gel
after electrophoresis. After DNAs of interest are bound to the membrane,
the charged membrane is incubated with a mixture of nonspecific DNA
fragments to saturate all of the remaining binding sites on the membrane.
Because the DNA in this mixture is randomly distributed on the membrane
and, if chosen properly, will not contain the sequence of interest (e.g.,
from a different organism than the probe DNA), it will not interfere with
subsequent detection of a specific gene.
The DNA bound to the membrane is then incubated with probe DNA
containing a sequence complementary to a sequence within the gene of
interest. Because all of the nonspecific binding sites on the membrane are
occupied with unrelated DNA, the only way that the probe DNA can
associate with the membrane is by hybridizing to any complementary DNA
present on the membrane. This probing is performed under conditions of
salt concentration and temperature close to those at which nucleic acids
denature and renature.
28. Southern Blot Hybridization…cont
•
•
Under these conditions, the probe DNA will hybridize tightly to only its
exact complement. Often the probe DNA is in high molar excess
compared with its immobilized target on the filter, thereby favoring
probe hybridization rather than reannealing of the denatured DNA on
the blot. In addition, the immobilization of the denatured DNA on the
filter tends to interfere with renaturation.
A variety of films or other media sensitive to the light or electrons
emitted by the labeled DNA can detect where on the blot the probe
hybridizes. For example, when a radioactively labeled probe DNA is
used and X-ray film is exposed to the filter and then developed, an
autoradiogram is produced in which the pattern of exposure on the
film corresponds to the position of the hybrids on the blot.
If we end up with one band, its called HIGH stingency, more than one,
we call it low stringency
29. Northern Blot Hybridization
•
•
Used to identify a particular mRNA in a population of RNAs. Because
mRNAs are relatively short (typically ,5 kb), there is no need for them to be
digested with any enzymes (there are only a limited number of specific
RNA-cleaving enzymes). Otherwise, the protocol is similar to that
described for Southern blotting. The separated mRNAs are transferred to a
positively charged membrane and probed with a probe DNA of choice. (In
this case, hybrids are formed by base pairing between complementary
strands of RNA and DNA).
An investigator might perform northern blot hybridization to ascertain the
amount of a particular mRNA present in a sample rather than its size. This
measure is a reflection of the level of expression of the gene that encodes
that mRNA. Thus, for example, one might use northern blot hybridization
to ask how much more mRNA of a specific type is present in a cell treated
with an inducer of the gene in question compared with an uninduced cell.
As another example, northern blot hybridization might be performed to
compare the relative levels of a particular mRNA (and hence the
expression level of the gene in question) among different tissues of an
organism. Because an excess of DNA probe is used in these assays, the
amount of hybridization is related to the amount of mRNA present in the
original samples, allowing the relative amounts of mRNA to be determined.
35. Isolation of Specific Segments of DNA
•
Much of the molecular analysis of genes and their function requires
the separation of specific segments of DNA from much larger DNA
molecules and their selective amplification. Isolating a large amount of
a single pure DNA molecule facilitates the analysis of the information
encoded in that particular DNA molecule. Thus, the DNA can be
sequenced and analyzed, or it can be cloned and expressed to allow
the study of its protein product.
•
Recombinant DNA molecules can be created and used to alter the
expression of a particular gene (e.g., by fusing its coding sequence to
a heterologous promoter). Alternatively, purified DNA sequences can
be recombined to generate DNAs that encode so-called fusion protein
that is, hybrid proteins made up of parts derived from different
proteins. The techniques of DNA cloning and amplification by PCR
have become essential tools in asking questions regarding the control
of gene expression, maintenance of the genome, and protein function.
Note : this part will be explained in slide number 47
36. DNA cloning
http://www.youtube.com/watch?v=MIfDx417SDs
•
The ability to construct recombinant DNA(2 DNA molecules from
different origins) molecules and maintain them in cells is called DNA
cloning. This process typically involves a vector that provides the
information necessary to propagate the cloned DNA in the replicating
host cell. Key to creating recombinant DNA molecules are the
restriction enzymes that cut DNA at specific sequences and other
enzymes that join the cut DNAs to one another. By creating
recombinant DNA molecules that can be propagated in a host
organism, a particular DNA fragment can be both purified from other
DNAs and amplified to produce large quantities.
•
Cloning DNA in plasmid vector: Once DNA is cleaved into fragments, it
typically needs to be inserted into a vector for propagation. That is,
the DNA fragment must be inserted into a second DNA molecule (the
vector) to be replicated in a host organism. The most common host
used to propagate DNA is the bacterium E. coli. Many common vectors
are small (3 kb) circular DNA molecules called plasmids. These
molecules were originally derived from extrachromosomal circular
DNA molecules that are found naturally in many bacteria and singlecell eukaryotes.
37. DNA cloning…cont
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•
A fragment of DNA, generated by cleavage with EcoRI, is inserted into
the plasmid vector linearized by the same enzyme. Once ligated, the
recombinant plasmid is introduced into bacteria by transformation.
Cells containing the plasmid can be selected by growth on the agar
plates that contain growth media including antibiotic to which the
plasmid confers resistance.
Some vectors not only allow the isolation and purification of a
particular DNA but also drive the expression of genes within the insert
DNA. These plasmids are called expression vectors and have
transcriptional promoters, derived from the host cell, immediately
adjacent to the site of insertion. If the coding region of a gene (without
its promoter) is placed at the site of insertion in the proper orientation,
then the inserted gene will be transcribed into mRNA and translated
into protein by the host cell. Expression vectors are frequently used to
express heterologous or mutant genes to assess their function. They
can also be used to produce large amounts of a protein for
purification. In addition, the promoter in the expression vector can be
chosen such that expression of the insert is regulated by the addition
of a simple compound to the growth media (e.g., a sugar or an amino
acid)
38. DNA Cloning
In standard cloning protocols, the cloning of any
DNA fragment essentially involves seven steps:
1- Choice of host organism and cloning vector,
2- Preparation of vector DNA,
3- Preparation of DNA to be cloned,
4- Creation of recombinant DNA,
5- Introduction of recombinant DNA into the host
organism,
6- Selection of organisms containing recombinant
DNA,
7- Screening for clones with desired DNA inserts and
biological properties.
40. Applications of recombinant DNA technology
:Medicine
- Recombinant human insulin: before they used to isolate the insulin from
pancreatic tissue, now they use DNA recombination technology
- Recombinant human growth hormone (HGH,
somatotropin)
- Recombinant blood clotting factor VIII
- Recombinant hepatitis B vaccine
- Diagnosis of infection with HIV: each of the three widely
used methods for diagnosing HIV infection has been developed
using recombinant DNA. The antibody test (ELISA or
western blot) uses a recombinant HIV protein to test for the
presence of antibodies that the body has produced in response to
an HIV infection. The DNA test looks for the presence of HIV
genetic material using reverse transcriptase polymerase chain
reaction (RT-PCR). Development of the RT-PCR test was made
possible by the molecular cloning and sequence analysis of HIV
genomes.
41. Recombinant DNA Technology
- It is a combination of recombinant DNA, replication, separation and
identification that permits the production of large quantities of purified DNA
fragments.
- Cloning vectors: Plasmids, Bacteriophage-λ, Cosmid, and yeast cloning vectors.
we choose the vector depending on the SIZE of DNA fragment you want to clone
Yeast > Cosmid > Bacteriophage-λ > plasmid
- Plasmids: have the ability to confer antibiotic resistance to the bacterium, used
to select the bacteria that has the recombinant DNA
Ex. Bacteria selection : adding antibiotic solution to all Bacteria .
Bacteria with recombinant DNA with plasmid vector tend to have
antibiotic –resistant feature used to help it in surviving the antibiotic solution
thus it will be selected
- Most vectors contain an inserted sequence of DNA termed polylinker,
restriction site bank or polycloning site.
The site where the RE make the specific cuts
42. Desirable features of a plasmid vector
- Relatively low molecular weight (3-5kb) to accommodate
larger fragments of DNA of interest
- Several different restriction endonuclease sites.
- Multiple selectable markers (antibiotic-resistance) , to aid in
selecting bacteria with recombinant DNA molecule.
- High rate of replication.
47. For amplification purposes, you can use or better to use only one type of
restriction enzyme. Why ? – because we don’t care how we introduce the DNA into the
plasmid vector (either 3'-5' or 5'-3') ; the way the DNA introduce in the vector will not
affect the process of replication because we have origin of replication and when the
plasmid starts to replicate (because its highly replicatable ) it well replicate all DNA that
contained in that plasmid including the DNA of interest.
When we need to express the gene of interest to get protein, this way we cannot
introduce the piece of cDNA anywhere in the plasmid ,,we have to have an orientation
for this insertion. Why orientation ? because we cannot express a given protein unless
we have the gene of interest located downstream the promoter of a given DNA that
included in the plasmid or the plasmid promoter the directional cloning, we have the plasmid
which should have a promoter
in this situation we have for the plasmid two restriction sites (site A , site B ) this means that
you have to use two different restriction enzymes in order to make sure that the piece
of DNA that inserted is inserted in a given orientation not in the other.
In this situation we use the Lac Z with its promoter ,, what is the Lac Z ? its Lac operon
and it presents in E.coli (remember Lac Z ,, Lac A,, Lac Y) Lac Z with its promoter will be
introduced in this plasmid why? Because the bacteria recognize the Lac Z promoter and
it is very active promoter in bacteria and this will get benefit out of this in term of
amplification more and more, production of the gene of interest that located
downstream of the promoter and the Lac Z gene,, By this you will form the protein from
the Lac Z gene and you form the protein for the DNA of interest ,,these together we call
fusion protein – see slide 35, now you will understand it insha allah
48. Example of the usage of recombinant DNA
technology in medicine
49. Insulin-amplification
First you get a DNA from tiny pancreatic tissue
Bacteria only can produce proteins out of DNA without introns, so
if you want to amplify the insulin gene without concern about expression
you can introduce the whole gene and amplify it ..
for expression purposes you have to be exact in direction and have
cDNA(DNA without introns) instead of whole DNA ,,we will talk about how
we make cDNA
from RNA 'from DNA that will result in mature RNA' we have the human
insulin
complementary DNA (cDNA) is DNA synthesized from a messenger RNA (mRNA) template
DNA polymerase in a reaction catalysed by the enzymes reverse transcriptase and
50.
51. DNA sequencing by Sanger
:Watch this amazing video for this method
http://www.youtube.com/watch?v=bEFLBf5WEtc
56. DNA sequencing
The outcome sequence you get is the sequence of the polymerized one, if you
want the sequence of the DNA of interest you take the complementary one
