QUANTIFICATION OF DNA IN

SELECTED SEA WEEDS OF CHENNAI USING NANO VUE SPECTROPHOTOMETER Tamil selvi, A. Vanitha, A. Prem kumar and S. Chandra PG and Research Dept. of Botany, Queen Marys College, Chennai, TN, India Hindustan college of Arts & Science, Chennai, TN, India

chan1958@gmail.com

Quantification of DNA in selected sea weeds of Chennai using Nano vue spectrophotometer ABSTRACT It is for the first time an attempt has been made to isolate pure nuclear DNA in the red alga Bryocladia thwaitesii (Harvey ex.J.Agardh) Detoni and it has been compared with a green alga Ulva covelongenesis V. Krishnamurthy & H. Joshi from Covelong Chennai, Tamil Nadu. Best method of isolation of DNA in these sea weeds has been identified (Remi A. Wattier et al (2000) modification of Dellaporta et al (1983) . Quantification analysis ( purity and concentration ) was made using Nano vue UV Spectrophotometer as a pioneering effort in Bryocladia thwaitesii and Ulva covelongenesis. Qualitative analysis of DNA was also performed in these two species of algae by agarose gel electrophoresis. Purity, quantity and yield of DNA were found to on the higher side in these two taxa. Nano vue UV Spectro photometer used in the present study is for reliable measurement to check purity and concentration of DNA with OD range 260:280 ratio, which might reveal satisfactory DNA purity for PCR based applications also and would be a great research tool for restriction endonuclease based (RE) studies. The DNA isolation protocol identified in the present investigation in these sea weeds would become well suited for PCR amplification and genomic library construction. .

Present investigation a pioneering research

Much work on molecular level is to be done in sea weeds. In the present study an attempt has been made to quantify DNA of a red alga Bryocladia thwaitesii and a green alga Ulva covelongenesis collected from Covelong shores, Chennai, TamilNadu., with the help of Nano vue UV spectrophotometer. Out of four methods of trial for the isolation of DNA, a Protocol deviced be Remi A. Wattier et al (2000) modification of Dellaporta et al (1983) has been identified as the best one in these selected sea weeds in the present work , which is well suited for PCR amplification and genomic library construction It is a pioneering attempt in respect of quantification of DNA in the red sea weed , Bryocladia thwaitesii. Nano vue UV Spectrometer has been proved to be a great research tool for restriction endonuclease (RE) based studies.

Bryocladia thwaitesii

Ulva covelongenesis

Material and Methods

Red sea weed Bryocladia thwaitesii and a green sea weedalga Ulva covelongenesis collected from Covelong shores, Chennai, TamilNadu Epiphytes and sand removed by repeated washing with sea water.

Four methods of DNA isolation were tried.

Ist Method (Wing Y. Chung et al, !993) IInd Method ( Kanto E A, Hirata MH et al 2005) IIIrd Method (Yolaine Joubert & Joel Fleurence 2005) IVth method (Remi A.Wattier, et al, 2000 ( modification of Dellaporta et al 1983).

IVth Method of DNA isolation

In the IVth method, In the first step 20% SDS is added in complete extraction buffer Only in this method , both proteinase k and Ribonuclease A is added.

Quality evaluation of isolated DNA by Agarose gel Electrophoresis

Reagents: Agarose, TAE Buffer, loading dye;- Glycerol, Bromophenol blue, xylene cyanol. And lastly Ethidium bromide solution Procedure: 0.8% agarose prepared by dissolving 0.24gm in 25 ml of water 1x TAE buffer. Solution boiled Gel poured in gel casting tray Tray left for 20 minutes for solidification of the gel 10mul of DNA sample was taken mixed with 3mul of the gel loading dye. The mixture loaded carefully on the corresponding lane in the gel. Electrophoresis performed at 50 volts for 1hr. Gel removed from tank transferred into Ethidium bromide solution for staining for 20 minutes Gel was destained in distiled water for 20 minutes. Gel was visualized under UV- trans illuminator.

Quantity evaluation of isolated genomic DNA by Nano vue uv Spectrophotometer

Nano vue spectrophotometer is easy to use and reliable instrument for the measurement of nucleic acid and protein samples. Samples of 0.5 mul to 2mul pipetted directly on to a novel sample plate for measurement, and then simply recovered using a pipette. This novel instrument provides a range of prestored methods for the determination of RNA , DNA and oligo nucleotide concentrations and purity, plus wave length scanning to detect impurities. Concentration measurements can be displayed in choice of units (umg/ml, ng/mul pmol/mul). Oligo nucloeotide primers can be characterized by keying in the base sequence ( upto 66-mer) to obtain parameters such as conversion factor (mug/ml), molecular weight, therotical absorbance(AU/mumol), and theoritical Tm. Relevant parameters are calculated automatically and displayed at the touch of a button on a large display.

Nano vue spectro photometer

 Up to 90 user defined methods can be stored with optional passward protection. Data out put is via standard USB and PVC (print via Computer) software, allowing data to be transferred to a PC or with the integrated printer or wireless bluetooth options.

 Results and discussion

conclusion
It is a pioneering investigation to extract and quantify DNA in red alga Bryocladia thwaitesii (Harvey ex J.Agardh) De Toni, as so far no report has been traced in this species. It has also been compared with Ulva covelongensis V. Krishnamurthy & H. Joshi. A total genomic isolation protcol (Dellaporta et al (1983) modified by(Remi A. Wattier et al (2000) ) has been identified in these sea weeds for the first time to minimise polysaccharide co-isolation and to simplify the procedure for processing large number of samples The DNA isolated from the two sea weeds by the above protocol was of good quality, because both purity as well as concentration were very high i.e. from 1.763-1.772 and 569.5-594.5 ng/mul respectively in the red and green sea weed. Favourable elimination of high molecular weight polysaccharides ie. 1. centrifugation after the lysis step at 37 c . (Patwarry and Vander Meer 1994) 2. cold incubation at -20c over night.

 This protocol applied to these two sea weeds is time effective with a dozen samples processed in 4-5 hrs of labour. The procedure also includes three 30 min. incubation periods enough time to do all the handwork for the second setof samples. This protocol requires very small amout of material a major advantage when working with field collected material. It is cheaper also. It is user friendly not involving organic solvents or hazardous chemicals. Beta-mercatoethanol used in the other protocol was substituted by proteinase K later in the same study. This report is well In accordance with Remi A. Wattier etal (2000).

 The quality of DNA by agarose gel Electrophoresis in the two sea weeds was clearly indicated by band formation. Use of Nano vue uv spectrophotometer for quantification of DNA is an added success in the present study . Further study is recommended on these areas in sea weeds.

Thank You