Efficient genomic DNA extraction protocol from medicinal rich Passiflora foetida containing high level of polysaccharide and polyphenol

Abstract

In Present work, the main objective is to develop less time consuming protocol for genomic DNA isolation from leaves of Passiflora foetida. Optimized protocol is cost effective, as it avoided use of expensive liquid nitrogen. The important parameters of CTAB buffer composition such as Polyvinylpyrrolidone PVP40000 (without PVP, 1%, 2%, 3.5%, 4.0%, 4.5%, 5.0%), CTAB (w, 1%, 2%, 3%, 4%, 5%), water bath temperature (30°C to 70°C) and duration on water bath for half hr and one and half hr has been optimized. CTAB (2%), PVP (1%), water bath temperature (70%), duration on water bath (1 hr) has efficiently yielded DNA quality of 200-1782 μg/0.5gm from leaf, stem, root, tendril and flower. However, 168 μg - 1782 μg of DNA has been obtained from 0.5 g of leaf of Passiflora foetida. Polyphenol contamination has been overcome using 5M NaCl and PVP. Acetate has been used for obtaining double-stranded DNA in stabilized form. Current DNA extraction protocol takes maximum of four hours for completion, which is many time savings. RAPD-PCR reaction parameters such as DNA concentration (100ng), Primer concentration (2 μM), Dream Taq polymerase (2 U), annealing temperature (29°C) and number of cycles for amplification of DNA has been optimized. Primer fragment Akansha 7 shows high polymorphism of 7 fragments ranges from 200bp – 2500 bp. Current optimized protocol of DNA isolation is specifically for Passiflora foetida, which can be used for downstream molecular techniques.

Comparative analysis of different methods of Hedera helix DNA extraction and molecular evidence of the functionality in PCR

“…Plants also contain substances that have inhibitory effect in PCR, mainly, polysacharides and polyphenolic compounds. Polysacharides are difficult to separate from DNA during the extraction (Porebski et al, 1997;Lade et al, 2014) and in the subsequent PCR they interfere with polymerases. Moreover, phenol, alkaloids, polyphenols Rogers and Bendich (1994) and polysacharides were identified to inhibit enzymes in the PCR or restriction reactions (Barra et al, 2012).…”

Natural polyamines and synthetic analogs modify the growth and the morphology of Pyrus communis pollen tubes affecting ROS levels and causing cell death

“…The longer polymethylene tetramines (Met 6-5-6, Met 6-10-6 and Met 5-10-5) characterized by two 2-methoxybenzyl groups were also effective, but they did not cause a swollen tube tip, pointing out a different mechanism in the inhibition of pollen tube elongation. In a previous study it was found that these polymethylene tetramines act at different cellular levels, as they bind to DNA with a higher affinity than Spm and lead to oxidative stress [40].…”

“…100 mg of germinated pollen was pulverized with liquid nitrogen to a fine powder and DNA was extracted following CTAB-based protocol as described by [40] …”

Leaf conditioning of Brazilian Cerrado species for DNA microextraction

“…This result indicates, mainly for Mangabeira, the necessity of using DNA extraction protocols with optimized anti-oxidant concentrations, to decrease its degradation (Silva, 2010). In this context, it is necessary to test several experimental scenarios considering variations in the composition of the extraction buffer as in the process (Lade et al, 2014). Different from the other conditioning, the product of the extractions resulting from foliar tissues was maintained at -20°C.…”

“…The adequacy of extraction techniques is often carried out for several species of plants (Edge-Garza et al, 2014;Lade et al, 2014;Mogni et al, 2016), but few or no reference has been made to the proper way of conditioning the foliar tissues, obtained in collection expeditions. The way to preserve the tissue for DNA micro extraction is as important as the choice of the DNA extraction protocol so that, optimizations are necessary to facilitate the operation and ensure a better quality of extracted DNA (Tamari and Hinkley, 2016).…”