7.1 – Techniques for Producing and Analyzing DNA
(Taken from Biology 12, MHR, 2011)
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Molecular biologists use tools to complete a project like investigating genetic
disorders, altering the genetic makeup of organisms so that they can produce
useful products (such as, insulin) or analyzing DNA evidence in a criminal
investigation.
Recombinant DNA Technology
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Recombinant DNA is DNA that has been prepared in the laboratory by combining
fragments of DNA from more than one source.
Restriction Endonucleases
(a.k.a. – Restriction Enzymes (R.E.))
To protect themselves from infection by foreign viruses, bacteria manufacture one
or more types of enzymes known as restriction enzymes.
Restriction endonucleases are enzymes that have been isolated from bacteria.
Restriction enzymes are molecular scissors that cut double-stranded DNA at a
specific base-pair.
Each restriction enzyme notices a particular base sequence as its recognition site
where it does the ‘cutting’.
Most recognition sites are about 4 to 8 base pairs long and are characterized by
palindromic sequences.
R.E.’s bind to their recognition sites and disrupt the phosphodiester bond between
nucleotides, via hydrolysis, which in turn disrupts the H-bonds between nitrogenous
bases.
See Figure 7.1, pg 286.
EcoRI is a restriction endonuclease that recognizes and cleaves the sequence
GAATTC. The cut DNA fragment that is produced now has single-stranded sticky
ends at each end of the molecule.
Some restriction endonucleases produce blunt ends. This allows two fragments of
DNA that have blunt ends to be combined.
Sticky-end fragments are generally more useful because they can be easily joined
with other sticky end fragments.
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Restriction enzymes are named according to the bacteria from which they
originate. The first letter is the initial of the genus name of the organism from
which the enzyme is isolated. The second and third letters are usually the initial
letters of the species name. The fourth letter indicates the strain, and the
numerals indicate the order of discovery of that particular enzyme from the strain
of bacteria.
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For example, the restriction enzyme BamHI is named as follows:
B
represents the genus Bacillus
am
represents the species amyloliquefaciens
H
represents the strain
I
means that it was the first endonucleases isolated from this strain
Methylases
Restriction enzymes must be able to recognize the difference between foreign DNA
and their own.
In prokaryotes, methylases (specific enzymes) modify the recognition site of a
respective R.E. by placing a methyl group on one of the bases, preventing the R.E.
from cutting the DNA into fragments.
Methylases allow molecular biologists to protect a gene fragment from being
cleaved in an undesired location.
DNA Ligase
Genes that are cut out from DNA sources must be rejoined back into a sequence of
existing DNA.
DNA ligase is the enzyme that is used as the tool to join sticky-ended fragments of
DNA together – using a condensation reaction, DNA ligase drives out a molecule of
water and reforms the phosphodiester bond of the backbone of the DNA.
Steps for Producing a Recombinant DNA molecule
1) A restriction endonuclease is selected that can cut both DNA fragments to be
combined. Enzymes that produce sticky ends are the best.
2) Each piece of DNA is reacted with the restriction endonuclease enzyme to produce cut
DNA fragments.
3) The two cut DNA fragments are joined together with the help of DNA ligase.
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See Figure 7.3, pg 287.
To make a recombinant DNA molecule, two fragments from different sources are
often cut with the same enzyme to produce complementary single-stranded sticky
ends.
Base pairing between the complementary sticky ends brings the molecules
together.
DNA ligase then covalently joins the strands to produce double-stranded
recombinant DNA.
Gene Cloning in Bacteria
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Gene cloning is an indirect method of making many identical copies of a gene.
Scientists often use bacteria as host systems when cloning a gene.
Plasmids
Bacteria are able to express foreign genes inserted into plasmids.
Plasmids are small, circular, double-stranded DNA molecules lacking a protein
coat that naturally exist in the cytoplasm of many strains of bacteria.
Using the enzymes and ribosomes that the bacterial cell houses, DNA contained in
plasmids can be replicated and expressed.
The relationship between bacteria and plasmids is endosymbiotic; both the bacteria
and the plasmid benefit from the mutual arrangement.
Plasmids often carry genes for antibiotic resistance.
Restriction enzymes are used to splice a foreign gene into a plasmid.
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Artificial plasmids have been engineered to contain a unique region that can be cut
by many restriction enzymes. This region is called the multiple-cloning site.
Restriction enzymes make one cut in the circular plasmid making it linear.
If the desired foreign DNA fragment is excised out of its source DNA with EcoRI
and the circular plasmid is cut open at the multiple cloning site by digesting with
the same restriction enzyme (EcoRI), then they will both possess the same
complementary sticky ends.
The desired fragment and plasmid fragments are brought together, where they
anneal because of the complementary sticky ends produced by digestion with the
same enzyme.
DNA ligase is then added to re-form the phosphodiester bond between fragments.
This results in another circular piece of DNA that now carries the foreign gene
fragment.
The Major Steps in the Cloning of DNA
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Many diseases are caused by gene alterations. Our understanding of genetic
diseases was greatly increased by information gained from DNA cloning.
In DNA cloning, a DNA fragment that contains a gene of interest is inserted into a
cloning vector or plasmid.
The plasmid-carrying genes for antibiotic resistance, and a DNA strand, which
contains the gene of interest, are both cut with the same restriction enzyme
endonuclease.
The plasmid is opened up and the gene is freed from its parent DNA. They have
complementary ‘sticky ends’. The opened plasmid and the freed gene are mixed
with DNA ligase, which reforms the two pieces as recombinant DNA.
This recombinant DNA is allowed to transform (transformation) a bacterial culture,
which is then exposed to antibiotics (genetic markers).
DNA cloning allows a copy of any specific part of a DNA (or RNA) sequence to be
selected among many others and produced in an unlimited amount.
This technique is the first stage of most of the genetic engineering experiments:
production of DNA libraries, PCR (Polymerase Chain Reaction), DNA sequencing, et
al.
See Figure 7.4, pg 289.
Polymerase Chain Reaction (PCR)
PCR is direct method of making multiple copies of a desired gene or section of
DNA.
The process of making large amounts of DNA for use or analysis is called DNA
amplification.
This process was discovered by Kary Mullis in 1983 and in 1993 he shared the
Nobel Prize in Chemistry for his invention of the polymerase chain reaction method.
PCR is useful in forensic criminal investigations, medical diagnosis, and genetic
research and only requires a small amount of DNA to work.
STEPS
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DNA sample is heated to 95oC (94oC to 96oC)  H-bonds break and strands
separate.
Temperature is cooled to approximately 55oC (50oC – 65oC range) for primers to
anneal with template DNA.
Sample is heated to 72oC which is optimal for Taq polymerase.
Taq polymerase, a DNA polymerase that is isolated from a bacterium that lives in
hot springs therefore, can withstand high heat, can build complementary strands
using free nucleotides that were added to solution, once primers anneal.
Synthesis of a DNA strand takes place at 72oC.
With complementary strand made, process repeats itself, doubling the number of
strands each time.
After 30 cycles, more than 1 billion copies of targeted area will exist.
Amplified DNA is run on a polyacrylamide gel using gel electrophoresis.
Gel is stained using ethidium bromide and viewed under UV light to visualize the
bands of DNA.
See Figure 7.6, pg 290.
PCR is carried out in special thermocycler machines, which allow the process to be
automated. These machines are programmed to change temperature quickly and
accurately at set times and to do so for a set numbers of cycles. Typically, about 30
cycles are used when performing PCR in the laboratory.
Analyzing DNA Fragment Size
Gel Electrophoresis
Once the desired gene has been excised from its source DNA, it must be separated
from the remaining unwanted fragments.
DNA fragments can be separated using the process of gel electrophoresis.
Gel electrophoresis takes advantage of DNA’s negative charge.
The gel is submerged in a buffer solution to maintain pH of the solution.
A solution containing different-size fragments to be separated is placed in a well. A
well is a depression at one end of the gel. (Figure 7.7, pg 291)
Using direct current, a negative charge is placed at one end of the gel where the
wells are, and a positive charge is placed at the opposite end of the gel.
The negatively charged DNA will migrate toward the positively charged electrode,
with the shorter fragments migrating faster than the longer fragments, achieving
separation.
Once gel electrophoresis is complete, the DNA fragments are made visible by
staining the gel with ethidium bromide (which fluoresces under UV light and is able
to insert itself among the rungs of the ladder of DNA).
The size of the fragments is then determined using a molecular marker as a
standard.
See Figure 7.8, pg 292.
Using a DNA Fingerprint for Identification
DNA fingerprinting or DNA profiling is a technique used to identify individuals
by analyzing the DNA sequence of certain regions of their genome.
In DNA fingerprinting, a sample of DNA from an individual is fragmented
(restriction endonucleases), amplified (PCR), and then separated by gel
electrophoresis to produce a unique “fingerprint’ that can be compared to reference
samples for identification or matching.
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Traditionally, DNA fingerprinting has been done by subjecting the sample of DNA to
restriction endonucleases and then separating the fragments by gel
electrophoresis.
Restriction length polymorphism (RFLP) analysis is then used to compare
the pattern of bands produced when the fragments are separated by gel
electrophoresis.
A more recent method of DNA fingerprinting involves short tandem repeat
(STR) profiling.
STRs are repeating short sequences of DNA in the genome that vary in length
between individuals depending on how many copies of a particular STR are
present.
With STR profiling, STRs of an individual are amplified and then separated by gel
electrophoresis.
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The more repeats at an STR locus, the longer the DNA fragment is and the shorter
the distance it travels through the gel.
The fewer the repeats, the shorter the DNA fragment is and the farther it travels
through the gel.
See Figure 7.10, pg 293.
DNA fingerprinting using STR profiling produces a series of peaks that represent
STRs of differing molecular mass and differing lengths.
Each individual has a unique series of peaks in their STR profile.
Applications
DNA fingerprinting is used in forensic sciences.
A small sample of blood, hair, or skin tissue may be found at a crime scene.
The DNA from this sample is amplified using PCR and the DNA fragments separated
using gel electrophoresis. A DNA fingerprint is then created.
The DNA fingerprint can be compared with the DNA fingerprint of a suspect in the
crime.
A match in DNA fingerprints tells investigators that there is strong evidence that
the suspect was present at the crime scene. However, it does not prove guilt.
DNA fingerprints can also be used to solve disputes over parentage.
A child’s DNA fingerprint will show some matches with the DNA fingerprints of both
parents.
Analyzing DNA Sequences
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DNA sequencing is a method for determining the nucleotide sequence, base by
base, of a fragment of DNA.
Manual DNA Sequencing
Dideoxy sequencing is a method for determining the sequence of a DNA
fragment using dideoxynucleotides, which cause termination of DNA synthesis
during the procedure.
Dideoxy sequencing relies on DNA replication.
DNA polymerase is used to synthesize a series of DNA fragments of differing
lengths, using the DNA to be sequenced as the template.
The different sized fragments occur because replication is stopped due to the
incorporation of one of four possible dideoxynucleotides (ddA, ddG, ddC, or ddT).
Steps in Dideoxy Sequencing
See Figure 7.12, pg 296.
The DNA being sequenced by dideoxy sequencing is shown in the top panel, with
the primer annealed. Each of the dideoxynucleotides is placed in a separate
reaction tube containing the denatured DNA and primer, DNA polymerase, and
deoxynucleotides used in the synthesis of DNA. The fragments generated from
each reaction show up as bands on the gel. Because the final base of each
fragment and the length of each fragment are known, the nucleotide sequence of
the original DNA can be determined. Note that the sequence read from the gel is
the complement of the DNA fragment being sequenced.
Early Automated DNA Sequencing
Manual sequencing was an important technological advancement however, it was
laborious and time-consuming.
Early automated sequencing technologies made the Human Genome Project
possible.
The Human Genome Project is a project that sequenced the human genome
and identified all the genes within it.
See Figure 7.13, pg 297.
Using early automated sequencing technologies, the dideoxynucleotides are
fluorescently dyed a particular colour. A detector reads each coloured band that is
run through the gel. Since each colour represents a particular base, the printout of
coloured peaks from the detector represents the sequence of the fragment that is
complementary to the DNA being sequenced.
Next-generation Automated DNA Sequencing
Next-generation sequencing improves the data output per year.
The ability to sequence DNA has numerous applications, particularly in medicine.
Doctors and molecular biologists can use a person’s genome sequence to diagnose
and treat various diseases.
Next-generation sequencing is being applied to cancer diagnosis and treatment.
It is now possible to determine the DNA sequence of a tumour. This is known as
tumour profiling.
Tumour profiling allows physicians to know exactly what type of genetic change
has caused a particular cancer.
This can help physicians decide on the best course of treatment for the patient.
Making Sequence-Specific Mutations
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Site-directed mutagenesis is a technology that enables researchers to create
specific mutations in the DNA sequence of a gene.
As a result, researchers can study the structure and function of DNA, genes, and
the proteins coded by these genes.
HOMEWORK:
pg 300 #1-12
7.2 – Production and Regulation of Genetically Engineered Organisms
(Taken from Biology 12, MHR, 2011)
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Gene therapy is a method for treating genetic disorders by introducing a correct form
of the disease-related gene into an individual’s genome.
The ability to alter the human genome is very controversial.
The development of recombinant DNA technology has given rise to various disciplines
that focus on bioethics and societal impact associated with these advances in genetics.
Applications of Genetically Engineered Organisms
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Genetic engineering is the alteration of the genetic material of an organism in a
specific manner.
Transgenic organisms are organisms that are produced from the introduction of
foreign DNA into its genome, providing it with a new phenotype.
Genetically modified organisms (GMOs) are organisms whose genetic material
has been modified, often through the insertion of a foreign gene into its genome.
Biotechnology is the use of an organism, or a product from an organism, for the
benefit of humans.
Private companies that use recombinant techniques to produce GMOs want to claim
ownership for the organism and its genome. They are applying for patents for these
techniques and organisms.
A patent is a government ruling giving an individual or organization the sole title or
right to make, use, or sell a particular invention.
Applications of Transgenic Bacteria in Pharmaceuticals
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Transgenic, or genetically modified, bacteria can be used to produce pharmaceutical
products such as insulin.
To produce transgenic bacteria, an expression vector is used.
An expression vector is a plasmid vector that is transformed into a host cell for the
purpose of producing a foreign protein.
See Figure 7.17, pg 303.
Other examples of medicinal proteins produced in bacteria include: human growth
hormone, tissue plasminogen activator (used to treat blood clots), erythropoietin (used
to stimulate red blood cell production), and a hepatitis B vaccine.
Transgenic Bacteria and Bioremediation
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GM bacteria are also used in bioremediation of environmental pollutants.
Some bacteria break down pesticides and herbicides that have been released into
water systems.
Others were developed to remove sulfur from coal to produce cleaner emissions when
the coal is burned.
Transgenic Plants
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Transgenic crops have increased tolerance to herbicides and increased resistance to
disease and pests, and many have been approved for human consumption (soybeans,
corn, canola, tomatoes, and potatoes).
Techniques for Introducing Foreign DNA into Plant Cells
Biolistic method:
Ti plasmid method:
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a method used for producing transgenic plants that involves
bombarding plant cells with particles coated in DNA that become
integrated into the plant genome
a method of producing transgenic plants that uses the tumourinducing plasmid from Agrobacterium tumefaciens as the vector for
the insertion of a foreign gene into a plant genome
Part of the Ti plasmid, called the T-DNA integrates into the plant genome and causes
the uncontrolled cell growth that results in a tumour.
This T-DNA region has been altered so that it no longer causes tumours but it can still
integrate into the plant genome.
Steps in the Ti plasmid Method
1) Recombinant DNA is produced by inserting gene of interest into the altered T-DNA
region of the Ti plasmid. The recombinant DNA has a selectable marker that provides
cells that have taken up the plasmid with resistance to the antibiotic kanamycin.
2) The recombinant Ti plasmid is taken up by the bacterium Agrobacterium tumefaciens.
3) Plant cells are infected with the bacterium. The recombinant DNA carrying the gene of
interest integrates into the plant cells.
4) The selectable marker is used to determine which cells have taken up the recombinant
DNA. Those that survive when exposed to the antibiotic kanamycin have taken up the
DNA. An antibiotic is also used to kill cells of the bacteria so only plant cells remain. A
transgenic plant is grown from these cells.
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See Figure 7.19, pg 306.
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Plants are also engineered to produce medicinal products such as human growth
hormone, clotting factors, and antibodies.
Transgenic Animals and Related Controveries
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Transgenic animals can be used to produce pharmaceutical products such as human
proteins, and some, such as pigs and salmon, are being considered for human
consumption.
To produce transgenic animals, a foreign gene is inserted into the genome of an
animal oocyte (egg) that is then fertilized.
The fertilized egg is implanted in a host female and allowed to develop.
The use of transgenic animals to produce human therapeutic proteins
(pharmaceuticals) is called gene pharming.
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See Figure 7.20, pg 308
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The production and use of transgenic plants and animals are controversial, in terms of
the safety of human consumption and the possibility of negative environmental effects.
Health Canada requires that all GMOs intended for human consumption undergo seven
to 10 years of health-and-safety research. (See pg 306,307)
Mammals Cloning from Somatic Cells
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Mammals can be cloned by fusing a somatic cell, including its genetic material, with an
oocyte from which the nucleus has been removed, and then fertilizing the resulting
embryo and implanting it in a host. Although sheep, cows, and other mammals have
been cloned, human cloning remains highly controversial.
HOMEWORK:
pg 311 #1-12