Molecular Characterization in Using Dot-Blot Analysis and Cross Hybridization Through Species-Specificity of the Non-Radioactive Digoxigenin (DIG) Labeled Specific DNA Probe

摘要

The recombinant plasmid (pTK302) containing an insert of 302 bp DNA fragment of was isolated from recombinant clone and the size was determined. The species-specificity of the non-radioactive Digoxigenin (DIG) labeled specific DNA probe was also analyzed by dot-blot analysis and cross hybridization along with DNA from and three other spp. such as , Only showed a positive signal. The sensitivity of the assay with positive control was up to 10 pg DNA. This first study in fish deal to demonstrate the utility of non-radioactive DIG labeled technique in the development of species-specific DNA marker for identification and stock characterization of among the spp. Thus, technique can also be applied in other genera having many species: for molecular taxonomy, stock characterization and conservation management of fish germplasm. The non-radioactive Digoxigenin (DIG) labeled specific DNA probe was cross hybridization along with DNA from and three other spp.; , and This first study in fish demonstrate the use of DIG in the development of species-specific DNA marker for identification of among the spp. The technique can also be applied in other genera having many species for molecular taxonomical identification and thereby implifications in stock characterization and conservation management. Dot-blot hybridization specificity of 302 bp DNA probe with different spp. (1) Control DNA (442 bp) hybridized with DIG labeled 442 bp; (2) r-plasmid (302 bp) hybridized with DIG labeled 302 bp; (3) Plain vector pTZ57R-2.886 kb hybridized with 1&2 DIG labeled probes; (4) Genomic DNA of hybridized with DIG labeled 302 bp probe; (5) Plain vector pTZ57R-2.886 kb hybridized with 1&2 DIG labeled probes; (6) Genomic DNA of hybridized with 1&2 DIG labeled probes; (7) Genomic DNA of hybridized with 1&2 DIG labeled probes; (8) Genomic DNA of hybridized with 1&2 DIG labeled probes.