- (A method for multiplying DNA)
PCR is a wonderful
technology for amplifying DNA. It allows you to take a specific region of DNA on the
chromosome and through the use of primers, copy back and forth, only a particular desired
segment, making two, then four, then eight, then sixteen, and so on, up to millions of
copies. It is possible to start from the DNA segment of a single cell and produce enough
of it for use in DNA typing or fingerprinting.
PCR relies on the ability
of DNA-copying enzymes to remain stable at high temperatures. The process was sparked by a
high temperature bacterium (Thermus aquaticus) inhabiting the hot springs of
Yellowstone National Park. In nature, most organisms copy their DNA in the same way. PCR
mimics this process, only it does it in a test tube. A PCR vial contains all the necessary
components for DNA duplication: a DNA segment, a large quantity of the four nucleotides
(A,T,G & C), large quantities of the primer sequence (DNA duplication cannot take
place without first, a short sequence of nucleotides to "prime" the process or
get it started), and DNA polymerase (enzymes that make a copy of all the DNA in each
chromosome). The polymerase (enzyme) is the "Taq" polymerase, named for the
bacterium Thermus aquaticus, from which it was isolated.
The three parts of the
polymerase chain reaction are carried out in the same vial, but at different temperatures.
The first part of the process separates the two DNA chains in the double helix by heating
the vial to 90-95 degrees centigrade for 30 seconds. But the primers cannot bind to the
DNA strands at such high temperature, so the vial is cooled to 55 degrees C. At this
temperature, the primers bind or "anneal" to the ends of the DNA strands, taking
about 20 seconds. The final step of the reaction is to make a complete copy of the
templates. Taq polymerase works best at around 75 degrees C (the temperature of the hot
springs where the bacterium was found), hence the temperature of the vial is raised. The
Taq polymerase begins adding nucleotides to the primer and eventually makes a
complementary copy of the template. (If the template contains an A nucleotide, the enzyme
adds on a T nucleotide to the primer. If the template contains a G, it adds a C to the new
chain, and so on to the end of the DNA strand). This completes one PCR cycle.
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The three steps in
polymerase chain reaction (separation of the strands, annealing the primer to the
template, and the synthesis of new strands) will take less than two minutes and the
process can be carried out in the same vial. At the end of a cycle, each piece of DNA in
the vial has been duplicated. This cycle can be repeated 30 or more times. Each newly
synthesized DNA piece can act as a new template. So, after 30 cycles, about a million
copies of a single piece of DNA can be produced. Taking into account the time it takes to
change the temperature of the reaction vial, 1 million copies can be made in about three
hours.
Amplified DNA fragments can
then be isolated or separated on the basis of size by a process of electrophoresis, in
which the fragments are drawn through a thin, flat gel by an electric potential that spans
the length of the gel. The gel matrix impedes the larger DNA fragments to a greater degree
than it does the smaller ones, and the fragments become distributed on the basis of size.
At this point, DNA can be made visible by bathing the gel in chemicals, making it (the
location of DNA on the gel) intensely fluorescent when irradiated with ultraviolet light.
By performing this process over and over for different DNA fragments and digitally
scanning the information for storage, a species library of DNA sequence locations is
generated. Variation or uniqueness of sequence locations serve as markers of economic
importance once statistically associated with frequency of observed (desirable) traits.
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Approaches for DNA Typing
There are a number of
approaches for determining and deciphering DNA sequences. The degree of success with
any given method tends to be related to the level of technology by which results are
produced and this tends to influence the philosophies of reliance on any given
approach by the researcher.
The approaches (allozyme,
RAPD, RFLP, AFLP and microsatellite technology), are all based upon identification of
polymorphic loci. Polymorphic loci are regions or points in the genetic structure that may
vary from individual to individual. This variation is basically differences in the number
of repeats (of nucleotides) in the same location. However, polymorphism may be relative in
that the loci or gene may be variable in one strain but not another, or in one individual
(heterozygous) but not in another (homozygous). The major approaches are:
Allozymes: proteins
produced from DNA that may vary due to polymorphism in the underlying DNA. This is the
oldest form of molecular markers (early 1970’s). This approach suffers from the
difficulty in requiring the use of enzymes that can only be identified on the basis of the
chemical product from their activity and in finding enough polymorphic allozymes to
provide the needed resolution. proteins
produced from DNA that may vary due to polymorphism in the underlying DNA. This is the
oldest form of molecular markers (early 1970’s). This approach suffers from the
difficulty in requiring the use of enzymes that can only be identified on the basis of the
chemical product from their activity and in finding enough polymorphic allozymes to
provide the needed resolution.
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RAPD: (Random
Amplified Polymorphic DNA), a random amplification of anonymous loci by PCR (polymerase
chain reaction – amplification of DNA fragments that may be unique to a loci or
gene). In the process, many bands (e.g. 30 or more) might appear simultaneously on the
electrophoretic gel, some of which are not constant from individual to individual. RAPD
markers allow creation of genomic markers from species of which little is known about
target sequences to be amplified. This methodology has some disadvantages which include
difficulty in reproducing results, subjective determination of whether a given band is
present or not, and difficulty in analysis due to the large number of products. (Random
Amplified Polymorphic DNA), a random amplification of anonymous loci by PCR (polymerase
chain reaction – amplification of DNA fragments that may be unique to a loci or
gene). In the process, many bands (e.g. 30 or more) might appear simultaneously on the
electrophoretic gel, some of which are not constant from individual to individual. RAPD
markers allow creation of genomic markers from species of which little is known about
target sequences to be amplified. This methodology has some disadvantages which include
difficulty in reproducing results, subjective determination of whether a given band is
present or not, and difficulty in analysis due to the large number of products.
RFLP: RFLP: (Restriction
Fragment Length Polymorphism), polymorphism represented by the presence or absence of
"restriction" sites, which are short sequences along the DNA that can be cut by
commercially available "restriction enzymes." The length of the cut fragment
depends on whether particular restriction sites are present or not (polymorphic). The
presence and absence of fragments resulting from changes in recognition sites are used to
identify species or populations. This is the oldest DNA-based method for finding
polymorphic loci, (which are difficult to find using this methodology), and the analysis
may be awkward. The technique requires large amounts of DNA material which may be invasive
and lethal to small aquatic organisms.
AFLP: (Amplified
Fragment Length Polymorphism), this is kind of a combination of RAPD and RFLP. DNA is cut
with restriction enzymes and short fragments that support PCR which are added to the cut
ends. PCR is then performed to produce many fragments, some of which vary in length from
individual to individual (polymorphic) and is based upon tightly linked markers flanking
the desired gene locus (positional cloning). This methodology is difficult to analyze due
to the large number of unrelated fragments that are visible (on the gel) along with the
polymorphic fragments (as with RAPD’s). (Amplified
Fragment Length Polymorphism), this is kind of a combination of RAPD and RFLP. DNA is cut
with restriction enzymes and short fragments that support PCR which are added to the cut
ends. PCR is then performed to produce many fragments, some of which vary in length from
individual to individual (polymorphic) and is based upon tightly linked markers flanking
the desired gene locus (positional cloning). This methodology is difficult to analyze due
to the large number of unrelated fragments that are visible (on the gel) along with the
polymorphic fragments (as with RAPD’s).
Microsatellites or
STR’s (Short Tandem Repeats): A microsatellite is a simple DNA sequence that is
repeated several times at various points in the organism’s DNA. Such repeats are
highly variable enabling that location (polymorphic locus or loci) to be tagged or used as
a marker. This has quantitative value when the location is associated with gene traits of
value or importance. Microsatellites have much more information than allozymes, yet offer
the same advantages of analysis. Ambiguity (RAPD’s and AFLP’s), or scarcity
(RFLP’s) are not a problem with microsatellites, given appropriate enrichment
technologies. Technical expertise required for detection and scoring/analysis once the
polymorphic loci are identified is similar for all the methods. ASICo’s microsatellite library forms the basis for marker
identification, characterization and association to traits of economic importance.
Selection of polymorphic loci (markers) from this library and comparison with those from
captive or wild populations produces the molecular tools used for assisting selective
breeding programs. |
