Terminalia arjuna is commonly called as Roxburgh and grows to a height of 20 to 30 meters. It belongs to the plant family of Combretaceae. The bark of T.arjuna is useful in treating heart failure, coronary artery diseases and hypercholesterolemia. It also seems to possess antioxidant activity through the chloroform extract in diabetic rats. T.arjuna bark is also found to be essential in the treatment of ulcers, cancer, heart diseases, fractures, urinary discharges, tumors, asthma, biliousness, anemia and Leucoderma. This plant is utilized in Unani medicine as well as shows antiviral activity against HSV-2. Terminalia bark functions as antifungal and antibacterial against the bacteria like Klebseila aerogenes, E.coli, Pseudomonas aeruginosa, Proteus vulgaris, Salmonella typhi.
Terminalia arjuna is known to constitute compounds in it like tannins, triterpenoid saponins, ardenolide, ellagic acid, gallic acid, phytosterols, oligomeric proanthocyanidins, calcium, zinc, magnesium and copper. It was investigated earlier and was also reported that the polyphenolic components in T.arjuna work as blockers of DNA isolation. They get precipitated along with DNA and spoil the purity of DNA that is isolated.
The previous literature has reported that HEPES based DNA isolation is the best in isolating high quality pure DNA from the dried leaves of T.arjuna and other species of this genus from the same family. Polymerase chain reaction is considered as a versatile method for generating large amount of DNA for the molecular analysis purpose. The DNA is quantified in this study using UV-Vis spectrophotometer at 260nm and 280nm. There are several methods used in this study for DNA isolation like CTAB based procedure, modified CTAB based procedure, Method based on HEPES, and FTA plant saver card method.
Terminalia arjuna accessions were gathered from a few places in India and they were stored at Human Herbal Health Care Garden and MPCST Bhopal at Madhya Pradesh in India. The methods that are used for isolating DNA from young leaves of this plant were modified CTAB based methods and HEPES based methods. The DNA isolated was quantified with the help of Nano Drop UV-Spectrophotometer at the wavelength of 260 nm and by using the water free of DNase-RNase, as the blank. DNA is isolated also using FTA method which is an easy way to isolate and store the DNA for a longer period.
In the first FTA method, Terminalia leaves are pressed on the FTA card. In the FTA card second and third methods, 10mg of Terminalia leaves are ground properly using mortar and pestle along with PBS and water without DNase and RNase. This homogenate is applied on the FTA card using micropipette. The card is dried later at room temperature. In the 4th and 5th methods, the isolated DNA from modified CTAB method was made to dissolve in the water free of DNase RNase and then is dropped on the FTA card. The card is dried for some time. The 6th FTA card procedure involves collecting DNA from the first phenol: chloroform: isoamyl alcohol precipitation of DNA by modified CTAB method. The collected DNA is mixed thoroughly with DNase RNase free water and placed on FTA card. The FTA sample was also purified effectively. Primers for RAPD PCR amplification procedure were collected and certain amplification enhancement agents were used for generating quality DNA in large quantities.
Results of the study
Both the modified procedures based on CTAB were not able to isolate pure DNA or quality DNA from the young leaves of Terminalia arjuna. The DNA that was isolated consisted of brownish compounds precipitated along with DNA. The DNA that was isolated by HEPES based method consisted of protein precipitated along with DNA identified by spectrophotometric analysis.
Analysis of DNA
The isolated DNA amount (467.9+/-82.87 55 ng/ul) by HEPES based procedure was comparatively good in quality. When it is dissolved in 50 micro liters of water free of DNase-RNase, the recovery of DNA from one sample was 997.15 ng/ul while in another sample the DNA was 900.55 ng/ul. The lowest recovery of DNA was measured as 20.49 ng/ul from another sample which is different from the above two. The ratio of Absorbance at 260 nm/Absorbance at 280 nm was found to be 1.27+/-0.090 through spectrophotometric analysis. This value indicates that the samples have protein in them along with DNA.
RAPD of FTA
The Terminalia leaves homogenized with PBS and water and the method in which the leaves are placed on the FTA card were not amplified. The direct application of the DNA that is isolated from the modified CTAB methods on the FTA card was amplified properly by RAPD PCR. The application of DNA on the FTA card that was dissolved in DNase-RNase free water after being removed from the precipitation from phenol: chloroform: isoamyl alcohol method of modified CTAB procedure was also amplified properly.
PCR enhancers in RAPD amplification
The use of RPI primer-8 in the polymerase chain reaction, addition of BSA (400 ng/ul) showed best amplification. The amplified strand did not consist of unnecessary noise in it compared with that of the control. The addition of DMSO was a little bit effective while formamide had no impact on amplification. Higher percent glycerol could help in amplification.
In the case of RPI primer-9, BSA addition was showing good amplification. Formamide at 2.5 percent concentration did not amplify DNA in two of the best samples. Twenty percent of glycerol could amplify DNA in one sample and did not amplify DNA in another while 15 percent glycerol gave best results in both the samples. BSA addition could amplify the DNA very well in the case of PCR done with RPI primer-10.
Pramod Sairkar, Shweta Chouhan, Nitin Batav, Rajesh Sharma. Optimization of DNA isolation process and enhancement of RAPD PCR for low quality genomic DNA of Terminalia arjuna. Journal of Genetic Engineering and Biotechnology (2013) 11, 17-24.
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