A RAPID, SIMPLE AND LOW COST MODIFIED CTAB METHOD FOR HIGH QUALITY GENOMIC DNA EXTRACTION FROM SHIP BARNACLE Balanus sp.

Ship Barnacle (Balanus sp.) (Superorder- Thoracica) is an intertidal marine crustacean with very diverse ecological distribution found throughout the world. Barnacles take integral part in the coastal food web- thus maintains a steady balance in the ecosystem. Like many other commercially irrelevant marine organisms, barnacles also have been studied poorly from genetics prospective. The primary reason of this lack of study can be the unavailability of a simple, efficient and cheap method of DNA extraction process for marine organisms. Commercial kit-based methods have been successfully used with a disadvantage of cost for a species like barnacle.

High molecular weight good quality DNA often fails to isolate from marine crustaceans like barnacle due to the presence of high polysaccharide and polyphenolic compounds which co-purifies with the DNA. In this study, we represent a modified CTAB method which is simple, efficient and quick enough to be carried out within two hours. The traditional phenol-chloroform and salting out method failed to even isolate the DNA from barnacle sample. The modified procedure uses common laboratory instruments like water bath, centrifuge and reagents like sodium phosphate buffer, proteinase K, CTAB, SDS, phenol-chloroform etc. The DNAzol ^® reagent was used as a reference to compare the usefulness of current protocols. From 10 mg of tissue, ~45 µg of DNA with 260/280 value of 1.8-1.9 was extracted by modified CTAB method whereas in case of DNAzol ^®, the amount and ratio was 27 µg and 1.9-2.0 respectively. Restriction digestion and polymerase chain reaction analysis was performed to prove that the DNA was free from any enzyme inhibitory substances. Restriction digestion with two frequent 6 base pair cutters (BamHI and HindIII) was carried out separately and the presence of uniform smear in agarose gel electrophoresis represented that both the DNA (modified CTAB and DNAzol ^®) were properly digested. The CrustDF1/R1 degenerate primers for mitochondrial COI region was used for amplification whereas the universal primers LCO1490 and HC03198 failed to amplify the DNA. It was suspected that the degeneracy of CrustD primers helped in proper annealing. An amplicon of around 730 bp were successfully amplified from the DNA extracted from both modified CTAB method and DNAzol ^® method by PCR. Furthermore,the molecular weight of the DNA was precisely detected by myImageAnalysis software (Thermo Fisher Scientific), taking 1 kb ladder as a reference.The molecular weight of the modified CTAB extracted DNA was higher than that of DNAzol ^® extracted DNA which makes the first one better for analyses like whole genome sequencing.