Extraction of Genomic DNA from Dried mature leaves of Eclipta alba (L.) suitable for ISSR-PCR and other DNA fingerprinting and Barcoding techniques

Abstract

Eclipta alba (L.) belongs to the Asteraceae family and is commonly known as false daisy in English and bhringoraj or bhringraj in Bangladesh and India. It is regarded as a weed of ethno medicinal significance. The plant contains polysaccharides, polyphenols, and other secondary metabolites in high concentration. The presence of these compounds inhibits enzymatic activities, extraction and downstream PCR amplification of DNA. Since, DNA is stable and is the basic component of all living cells, DNA based marker analysis has been proven to be an important tool in herbal drug standardization. The characteristics, traits and morphological features of various plants are determined by the specific arrangement of DNA base pair sequences in their cell and helps in various genetic studies such as DNA based fingerprinting techniques which plays a crucial role in the authentication of botanicals which are medicinally important. Therefore, DNA of high quality, free from PCR-inhibitors is of prime importance. In present study, a modified CTAB protocol was developed for isolating the DNA from dried mature leaves of E. alba by varying concentrations of CTAB, PVP and NaCl constituting the Extraction buffer which resulted in high quality of DNA and yield suitable for diverse molecular studies. Dried mature leaves were used in the study as availability of fresh leaves for the molecular and phylogenetic studies is often quite challenging. The PCR inhibitor-free quality of the isolated DNA was also cross-examined by PCR amplification using ISSR (Inter Simple Sequence Repeats) primers.

Keywords: Eclipta alba, CTAB, PVP, ISSR, NaCl