ABSTRACT
DNA is the prerequisite for life's inception that transfers hereditary information, past several years; various types of commercial kits are made available which vary depending on the type of the biological sample being used. The present study is focused on developing an improvised methodology for the isolation of genomic DNA from stored bovine blood samples. DNA was isolated by using the conventional Phenol: Chloroform: Isoamyl alcohol (PCI) method and Detergent method. The aim of the study was to make a comparative analysis and evaluation of these two methods to identify the one that gives a superior quality and quantity of genomic DNA. Total (n=48) each duplicate blood samples from three different buffalo(Bubalus bubalis) breeds Banni, Surti, Murrah, three zebu cattle (Bos indicus) breeds Kankerj, Gir, Sahiwal were collected from the jugular vein. The quantity, purity of the genomic DNA was assessed based on the total DNA yield, purity ratios, spectral profile, agarose gel electrophoresis analysis and polymerase chain reaction amplification of MC1R gene product without any inhibitors. The results of our study suggest that detergent method is also suitable for extraction of genomic DNA from the bovine blood and results were significant (*P>0.05). The total mean yield was found to be 329.05±11 µg/5ml for all six breeds while the PCI method was employed. The total mean yield of the gDNA for all six breeds was 406.6±43 µg/5ml of blood when the detergent method was used. One way ANOVA test showed that the total DNA yield varied depending on the isolation method used. The DNA yield obtained from the DG method was (***P< 0.001) significant as compared to the PCI method (**P<0.01).
ABSTRACT
Cathelicidins are an ancient class of antimicrobial peptides (AMPs) with broad spectrum bactericidal activities. In this study, we investigated the diversity and biological activity of cathelicidins of buffalo, a species known for its disease resistance. A series of new homologs of cathelicidin4 (CATHL4), which were structurally diverse in their antimicrobial domain, was identified in buffalo. AMPs of newly identified buffalo CATHL4s (buCATHL4s) displayed potent antimicrobial activity against selected Gram positive (G+) and Gram negative (G-) bacteria. These peptides were prompt to disrupt the membrane integrity of bacteria and induced specific changes such as blebing, budding, and pore like structure formation on bacterial membrane. The peptides assumed different secondary structure conformations in aqueous and membrane-mimicking environments. Simulation studies suggested that the amphipathic design of buCATHL4 was crucial for water permeation following membrane disruption. A great diversity, broad-spectrum antimicrobial action, and ability to induce an inflammatory response indicated the pleiotropic role of cathelicidins in innate immunity of buffalo. This study suggests short buffalo cathelicidin peptides with potent bactericidal properties and low cytotoxicity have potential translational applications for the development of novel antibiotics and antimicrobial peptidomimetics.
