5 Molecular Characterization
Molecular characterization techniques are extensively used for the
grouping of organisms globally. A sequence analysis at the molecular level in addition to morphological and morphometric studies is
useful for rapid identification and precise classification of nematode
isolates. Molecular approaches are used to study the systematic
relationships among entomopathogenic nematodes.
The RFLP (restriction fragment length polymorphism) assessment of polymerase chain reaction (PCR), an amplified product
from specific regions of the genome, is an influential taxonomic
tool which can be used for the documentation of single nematodes.
Regions of taxonomic importance comprise the internal transcribed spacer (ITS) of the ribosomal DNA (rDNA) repeat unit
and the region of the mitochondrial genome that divides the cytochrome oxidase subunit II (COII) and the 16S genes. Both of these
regions are extensively used for entomopathogenic nematode identification [172, 173].
Nuclear rDNA has been a beneficial tool for providing markers
associated with delimitation of EPN at diverse taxonomic levels.
The Internal transcribed spacer region (ITS) of rDNA has revealed
adequate genetic variation for differentiating Heterorhabditis and
Steinernema species and has confirmed to be useful for delimitation
and elucidation of evolutionary relationships among species of this
genus. The 28S or large subunit (LSU) of rDNA has been deliberated to evaluate phylogenetic relation among EPN species.
5.1 Isolation of DNA
from
Entomopathogenic
Nematodes
5.1.1 Extraction of DNA
(from Infective Juveniles)
[174]
Materials Required
l
Source: Nematodes (100 IJs).
l
Equipment: UV light, spectrophotometer, water bath, vortex
machine, centrifuge, electrophoresis apparatus, refrigerator.
l
Chemicals: Extraction buffer (0.05 M EDTA, 1% SDS, 400 μl
proteinase K), TE-buffer (0.01 M Tris–Cl pH 7.5, 0.001 M
EDTA), phenol, chloroform, isoamylalcohol, agarose, ethidium
bromide.
l
Miscellaneous: Glass mortar and pestle, microcentrifuge tube,
micropipette.
Procedure
1. Take the infective juveniles (ml) in a microcentrifuge tube.
2. Pour the IJs in a mortar and pestle and grind by adding liquid
nitrogen till a fine white powder is formed.
3. Crush the IJs further using 1 ml of extraction buffer.
4. Keep at 65
C for 1 h in water bath.
5. Centrifuge the sample at 15,000 g for 10–15 min.
6. Collect the supernatant and add similar volume of phenol:
chloroform:isoamylalcohol at the ratio 25:24:1 and mix well.
Molecular Characterization
87
Molecular characterization techniques are extensively used for the
grouping of organisms globally. A sequence analysis at the molecular level in addition to morphological and morphometric studies is
useful for rapid identification and precise classification of nematode
isolates. Molecular approaches are used to study the systematic
relationships among entomopathogenic nematodes.
The RFLP (restriction fragment length polymorphism) assessment of polymerase chain reaction (PCR), an amplified product
from specific regions of the genome, is an influential taxonomic
tool which can be used for the documentation of single nematodes.
Regions of taxonomic importance comprise the internal transcribed spacer (ITS) of the ribosomal DNA (rDNA) repeat unit
and the region of the mitochondrial genome that divides the cytochrome oxidase subunit II (COII) and the 16S genes. Both of these
regions are extensively used for entomopathogenic nematode identification [172, 173].
Nuclear rDNA has been a beneficial tool for providing markers
associated with delimitation of EPN at diverse taxonomic levels.
The Internal transcribed spacer region (ITS) of rDNA has revealed
adequate genetic variation for differentiating Heterorhabditis and
Steinernema species and has confirmed to be useful for delimitation
and elucidation of evolutionary relationships among species of this
genus. The 28S or large subunit (LSU) of rDNA has been deliberated to evaluate phylogenetic relation among EPN species.
5.1 Isolation of DNA
from
Entomopathogenic
Nematodes
5.1.1 Extraction of DNA
(from Infective Juveniles)
[174]
Materials Required
l
Source: Nematodes (100 IJs).
l
Equipment: UV light, spectrophotometer, water bath, vortex
machine, centrifuge, electrophoresis apparatus, refrigerator.
l
Chemicals: Extraction buffer (0.05 M EDTA, 1% SDS, 400 μl
proteinase K), TE-buffer (0.01 M Tris–Cl pH 7.5, 0.001 M
EDTA), phenol, chloroform, isoamylalcohol, agarose, ethidium
bromide.
l
Miscellaneous: Glass mortar and pestle, microcentrifuge tube,
micropipette.
Procedure
1. Take the infective juveniles (ml) in a microcentrifuge tube.
2. Pour the IJs in a mortar and pestle and grind by adding liquid
nitrogen till a fine white powder is formed.
3. Crush the IJs further using 1 ml of extraction buffer.
4. Keep at 65
C for 1 h in water bath.
5. Centrifuge the sample at 15,000 g for 10–15 min.
6. Collect the supernatant and add similar volume of phenol:
chloroform:isoamylalcohol at the ratio 25:24:1 and mix well.
Molecular Characterization
87