Genomic DNA extraction method from pearl millet (Pennisetum glaucum) leaves

DNA extraction is difficult in a variety of plants because of the presence of metabolites that interfere with DNA isolation procedures and downstream applications such as DNA restriction, amplification, and cloning. Here we describe a modified procedure based on the hexadecyltrimethylammonium bromide (CTAB) method to isolate DNA from tissues containing high levels of polysaccharides. The procedure is applicable to both dry and fresh leaves of Pennisetum glaucum. This modified CTAB (2%) protocol include the use of 1.4 M NaCl, 1% polyvinylpyrrolidone (PVP), 1% &#946-mercaptoethanol and 100% ethanol in the extraction as well as reducing the centrifugation times during the separation and precipitation of the DNA. This method solved the problems of DNA degradation, contamination, and low yield due to binding and/or coprecipitation with starches and polysaccharides. The isolated DNA proved amenable to PCR amplification and restriction digestion. The technique is fast, reproducible, and can be applied for SSR-PCR markers identification.Key words:Pennisetum glaucum, genomic DNA isolation, leaves. African Journal of Biotechnology Vol. 4 (8), pp. 862-86

Cloning and expression of functional single-chain Fv antibodies directed against NIa and coat proteins of potato virus Y

International audienceThree single-chain variable fragment (scFv) antibodies recognizing the nuclear inclusion a (NIa) and capsid proteins of potato virus Y were obtained from two mouse derived hybridoma clones secreting, respectively, an anti-NIa (22-1) and an anti-coat protein (136-13) monoclonal antibodies. The first monoclonal antibody was able to inhibit in vitro the PVY polyprotein cleavage by blocking the NIa protease activity. The amplified scFv cDNAs were first inserted into the TOPO vector and then sequenced. Several recombinant E. coli clones carrying the accurate scFv sequences were selected and the corresponding cDNAs were subcloned in pHEN phagemid and transferred in E. coli strain. The expressed scFv fragments showed an antibody activity that recognized the viral target proteins in infected tissues. Their activity was comparable to the parental monoclonal antibodies