fragments produced when they are expressed to restriction enzymes, a phenomenon
known as restriction fragment length polymorphisms (RFLP). RFLP is exhibited
randomly throughout chromosomal DNA and transmitted along a Mendelian
model. Accordingly, several different RFLP expression patterns may be combined
to distinguish organisms, lines, populations, and species.
Animal DNA includes series-type repetition sequences that are prone to changes
in frequency through repetition. Restriction enzyme fragments including these
sequences exhibit RFLP according to differences in the number of sequence repetitions. These forms are known as variable tandem pair repeats (VTPR).
With RFLP, each organism exhibits different numbers of repeats as pairs of 10–
20 bases showing the same sequence at a given gene site are repeatedly established,
producing differing forms of mutations.
Polymorphism refers to diversity in the forms and traits exhibited by organisms
within the same species (Taniguchi 2000).
5.4 DNA Polymorph Detection Methods
The following is a brief explanation of the chief DNA polymorph detection
methods (Table 5.1).
(1) DNA Extraction: VNTR (as in DNA fingerprinting), which is a DNA polymorph detection method that does not use PCR, requires large volumes of
highly pure DNA.
(2) Blood is often used as a sample in this case. It is subjected to manipulation
including extraction with phenol, purification, and precipitation with ethanol
to extract DNA from the blood cell nuclei.
With other methods of DNA polymorph detection, PCR may be used to amplify
DNA during the manipulation process, which means that a small amount of DNA
will suffice. This results in a simpler DNA detection method that allows for
extraction and processing from multiple organisms. (Taniguchi and Takag 1997)
(3) Variable number of tandem repeat (VNTR) method
Minisatellite region polymorphs (Fig. 5.2): These may be divided into multiple
gene locus and single gene locus methods, depending on the probe used. Probes used
in minisatellite region polymorph detection include M13 phage DNA (GAGGGTG
GNGGNTCT) and the human-derived minisatellite 33.15 (AGAGGTGGGCAGG
TGG). (Vassart 1987; Jeffreys et al. 1985)
These methods require a sample with large quantities of highly pure DNA, with
around 60 h of electrophoresis following restriction enzyme truncation. After
electrophoresis comes additional manipulation, including Southern blotting,
hybridization with a probe marked with a radioactive isotope, and band detection
through autoradiography. A drawback of this approach is that the manipulation
sequence is somewhat complex and the analytical method demanding, with a total
process lasting seven days (Fig. 5.2). Time reductions and simplification of the
5.3 What Are Genetic Markers?
115
known as restriction fragment length polymorphisms (RFLP). RFLP is exhibited
randomly throughout chromosomal DNA and transmitted along a Mendelian
model. Accordingly, several different RFLP expression patterns may be combined
to distinguish organisms, lines, populations, and species.
Animal DNA includes series-type repetition sequences that are prone to changes
in frequency through repetition. Restriction enzyme fragments including these
sequences exhibit RFLP according to differences in the number of sequence repetitions. These forms are known as variable tandem pair repeats (VTPR).
With RFLP, each organism exhibits different numbers of repeats as pairs of 10–
20 bases showing the same sequence at a given gene site are repeatedly established,
producing differing forms of mutations.
Polymorphism refers to diversity in the forms and traits exhibited by organisms
within the same species (Taniguchi 2000).
5.4 DNA Polymorph Detection Methods
The following is a brief explanation of the chief DNA polymorph detection
methods (Table 5.1).
(1) DNA Extraction: VNTR (as in DNA fingerprinting), which is a DNA polymorph detection method that does not use PCR, requires large volumes of
highly pure DNA.
(2) Blood is often used as a sample in this case. It is subjected to manipulation
including extraction with phenol, purification, and precipitation with ethanol
to extract DNA from the blood cell nuclei.
With other methods of DNA polymorph detection, PCR may be used to amplify
DNA during the manipulation process, which means that a small amount of DNA
will suffice. This results in a simpler DNA detection method that allows for
extraction and processing from multiple organisms. (Taniguchi and Takag 1997)
(3) Variable number of tandem repeat (VNTR) method
Minisatellite region polymorphs (Fig. 5.2): These may be divided into multiple
gene locus and single gene locus methods, depending on the probe used. Probes used
in minisatellite region polymorph detection include M13 phage DNA (GAGGGTG
GNGGNTCT) and the human-derived minisatellite 33.15 (AGAGGTGGGCAGG
TGG). (Vassart 1987; Jeffreys et al. 1985)
These methods require a sample with large quantities of highly pure DNA, with
around 60 h of electrophoresis following restriction enzyme truncation. After
electrophoresis comes additional manipulation, including Southern blotting,
hybridization with a probe marked with a radioactive isotope, and band detection
through autoradiography. A drawback of this approach is that the manipulation
sequence is somewhat complex and the analytical method demanding, with a total
process lasting seven days (Fig. 5.2). Time reductions and simplification of the
5.3 What Are Genetic Markers?
115