ISOLATION AND CHARACTERIZATION OF MICROSATELLITES FROM THE WILD SILKMOTH Antheraea mylitta
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Date
2004
Authors
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ACHARYA N. G. RANGA AGRICULTURAL UNIVERSITY RAJENDRANAGAR, HYDERABAD
Abstract
The study is aimed at establishing a standard method for the isolation of
Microsatellites from the wild silkmoth Antheraea mylitta, ecorace Daba trivoltine.
Microsatellites were isolated by the selective hybridization method in which DNA
fragments containing repeat motifs specific to repeat probe used in hybridization
were captured.
In the present study six biotinylated oligos of the Di-, Tri- and
Tetranucleotide repeat types were used as probes in individual hybridization
reactions. Genomic DNA was digested with Sau3A, dephosphorylated, size selected
from 100-1000 bp to which Sau3A ds linkers were ligated. The linker ligated
genomic digest was hybridized against biotinylated repeat oligo and eluted
microsatellite enriched DNA fragments were subjected to linker digestion with
Sau3A, purified and cloned into BamH I digested pBluescript (KS+) plasmid and
transformed into INVF competent cells.
Totally 110 white colonies were screened and the clones having insert size
above 100 bp were selected and sequenced. Totally 81 clones were sequenced of
which 18 were found to be positive for microsatellites. Out of 18 microsatellites 15
are dinucleotide repeats and 3 are trinucleotide repeats and no tetranucleotide repeats
were captured.
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Keywords
ISOLATION, CHARACTERIZATION, MICROSATELLITES, WILD SILKMOTH