3.3 Cloning of a Target Gene
The term “clone” refers to groups of organisms arising from asexual reproduction
and organisms with the same genetic composition. “Cloning” refers to the creation
of such groups of individual with identical genetic composition. Gene cloning is
thus a phenomenon of separating out a particular gene and increasing its number by
insertion in a prokaryote or eukaryote cell. It is therefore possible to use cells with
the right gene for some purpose to create large quantities of the proteins that it
encodes or to produce large quantities of the gene itself.
Several methods of gene cloning exist. First, there is the method of using
restriction endonuclease to truncate the genome DNA in some organism to the
length of approximately one gene. The recombinant DNA procedures through
joining of that DNA fragment to a vector is introduced to E. coli or a similar host
cell to function as a genome library, or mRNA is extracted from a donor and reverse
transcriptase is used to synthesize complementary DNA (cDNA) to create a cDNA
library. After that, a specific DNA probe is used to select and separate cells with the
target gene through the genome library or cDNA by means of hybridization. Target
cDNA may also be screened with antibodies.
Recently, target DNA has been cloned from organisms besides fish. Base
sequences from the best preserved of these regions serve as a reference for creating
primer, with the polymerase chain reaction (PCR) technique used to proliferate
some or all of the cDNA or gene DNA region from the cDNA or genome library.
When the amino acid sequence for the protein produced by target DNA is at least
partially determined, that sequence can serve as a reference for creating
multi-primer, allowing for multiplication of the gene through PCR. Discovered by
Kary Mullis in 1986, PCR is a technique by which DNA fragment becomes
amplified. The principle behind PCR is simple. The process requires a particular
form of DNA polymerase obtained from prokaryotes living in hot spring water.
Unlike other proteins, the enzyme is capable of withstanding the high-temperature
treatment needed to separate DNA strands in the PCR process. The DNA sample to
be amplified is mixed with this characteristic polymerase, nucleotide monomers,
and small pieces of DNA to serve as primers in DNA replication. Heat treatment of
the mixture results in the DNA’s replication, and as two new strands of DNA form,
that new DNA undergoes its own separation and replication due to the heat in a
repeating process. The result has been to make gene cloning extremely simple, as
large numbers of DNA molecules can be obtained in a short time from a DNA
fragment with the same base sequence (Fig. 3.5) (Rı us et al. 1998).
PCR is in wide use today, with applications not only in gene cloning but also in
fish diagnosis, species and genus identification, and diagnosis of microbial
infections.
Figure 3.6 shows the various gene cloning methods described above. Progress in
gene manipulation has been accelerating, and staggering advancements continued
to be made in the field.
3.3 Cloning of a Target Gene
61
The term “clone” refers to groups of organisms arising from asexual reproduction
and organisms with the same genetic composition. “Cloning” refers to the creation
of such groups of individual with identical genetic composition. Gene cloning is
thus a phenomenon of separating out a particular gene and increasing its number by
insertion in a prokaryote or eukaryote cell. It is therefore possible to use cells with
the right gene for some purpose to create large quantities of the proteins that it
encodes or to produce large quantities of the gene itself.
Several methods of gene cloning exist. First, there is the method of using
restriction endonuclease to truncate the genome DNA in some organism to the
length of approximately one gene. The recombinant DNA procedures through
joining of that DNA fragment to a vector is introduced to E. coli or a similar host
cell to function as a genome library, or mRNA is extracted from a donor and reverse
transcriptase is used to synthesize complementary DNA (cDNA) to create a cDNA
library. After that, a specific DNA probe is used to select and separate cells with the
target gene through the genome library or cDNA by means of hybridization. Target
cDNA may also be screened with antibodies.
Recently, target DNA has been cloned from organisms besides fish. Base
sequences from the best preserved of these regions serve as a reference for creating
primer, with the polymerase chain reaction (PCR) technique used to proliferate
some or all of the cDNA or gene DNA region from the cDNA or genome library.
When the amino acid sequence for the protein produced by target DNA is at least
partially determined, that sequence can serve as a reference for creating
multi-primer, allowing for multiplication of the gene through PCR. Discovered by
Kary Mullis in 1986, PCR is a technique by which DNA fragment becomes
amplified. The principle behind PCR is simple. The process requires a particular
form of DNA polymerase obtained from prokaryotes living in hot spring water.
Unlike other proteins, the enzyme is capable of withstanding the high-temperature
treatment needed to separate DNA strands in the PCR process. The DNA sample to
be amplified is mixed with this characteristic polymerase, nucleotide monomers,
and small pieces of DNA to serve as primers in DNA replication. Heat treatment of
the mixture results in the DNA’s replication, and as two new strands of DNA form,
that new DNA undergoes its own separation and replication due to the heat in a
repeating process. The result has been to make gene cloning extremely simple, as
large numbers of DNA molecules can be obtained in a short time from a DNA
fragment with the same base sequence (Fig. 3.5) (Rı us et al. 1998).
PCR is in wide use today, with applications not only in gene cloning but also in
fish diagnosis, species and genus identification, and diagnosis of microbial
infections.
Figure 3.6 shows the various gene cloning methods described above. Progress in
gene manipulation has been accelerating, and staggering advancements continued
to be made in the field.
3.3 Cloning of a Target Gene
61