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Current Trends in Biotechnology and PharmacyVol. 5 (2) 1163-1172 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1165ground with liquid nitrogen to fine powder.Extraction buffer containing CTAB 2% (w/v),NaCl 1.4 M, EDTA 20mM, Tris-HCl (pH 8)100mM, PVP 1% (w/v) and betamercaptoethanol 0.2% (v/v) (pH 7.5 to 8.0) wasadded to the powder. The slurry was incubatedat 60°C in a water bath for 60 – 90 min followedby extraction using chloroform: isoamyl alcohol(24:1) (v/v) and centrifugated at 9000 rpm for 10min at 4°C. The chloroform: isomyl alcoholextraction was repeated twice with the aqueousphase. Finally, the aqueous phase was separatedand DNA was precipitated with ice cold ethanol.Nucleic acid was recovered by centrifugation at8,000 rpm for 15 min at 4°C and the pellet wasdissolved in TE buffer (Tris-HCl 10 mM, EDTA1mM; pH 8). The contaminating RNA wasremoved by treating with RNase A (20 µg/µL)for 30 min at 37°C. The quantity and purity ofDNA was checked using UV spectrophotometerby calculating the A 260/A 280ratio (20) (Shimadzu,Tokyo, Japan). The DNA stock was maintainedat a concentration of 30 – 50 ng / µL.Amplification of C. fenestratum ITS region:ITS1 and ITS2 regions of C. fenestratum wereamplified using the universal primers (21). Detailsof the primers and the region amplified are givenin Table 2. PCR reaction contained approximately60 ng genomic DNA, 2.5 µL 10X Taq buffer,1.5 mM MgCl 2, 0.4 mM dNTP mixture, 30 pM ofeach primer, 1U Taq DNA polymerase and madeup to 25 µL with sterile distilled water.Amplification reaction was optimized at 95°C for3 min followed by 35 cycles of 95°C for 35sec,60°C for 30 sec, 72°C for 45sec with a finalextension step at 72°C for 3min. PCR productswere resolved in 1.2% agarose, 0.5X TBE gelsprestained with ethidium bromide (0.5 µg/ml).Simultaneously, 100 bp DNA ladder or LambdaDNA EcoRI/HindIII double digest (Fermentas,Burlington, Canada) was loaded to confirm thesize of the amplicon. The gel was visualized underUV radiation on a transilluminator (Reprostar,CAMAG, Muttenz, Switzerland) and digitalphotographed (Canon, Tokyo, Japan). Eachexperiment was repeated at least three times withall available accessions of each species to confirmits reproducibility and repeatability.Direct sequencing of C. fenestratum ITS1 andITS2 region: PCR product of C. fenestratumITS1 and ITS2 amplification was purified fromagarose gel using QIAquick gel extraction kit(Qiagen, Hilden, Germany) followingmanufacturer’s instruction. Sequencing wasperformed in an automated ABI 3100 GeneticAnalyser (Applied Biosystems, CF, USA) byBangalore Genei (Bangalore, India). Forward andreverse sequencing were done individually withprimers ITS1and ITS2 respectively. Sequenceresults were submitted to GenBank, NationalCentre for Biotechnology Information (NCBI),Bethesda MD, USAHomology analysis: Sequence of ITS1 and ITS2region for B. aristata, B. asiatica, B. lycium andM. umbellata was adopted from NCBInucleotide sequence database, GenBank (http://www.ncbi.nlm.nih.gov/nucleotide/). The details ofthe accession numbers used in this study are givenin the Table 3. Homology analysis was performedby employing CLUSTALW (www.ebi.ac.uk.clustalw/). The homology percentage in theITS regions of C. fenestratum with its substitutespecies was calculated.Development of species-specific marker forC. fenestratum: Variation in the ITS1 and ITS2sequences of C. fenestratum, B. aristata, B.asiatica, B. lycium, and M. umbellata was usedfor developing species-specific marker for C.fenestratum. DNA primers capable of givingspecific amplification with C. fenestratum ITS1region was developed using NCBI primer blasttool (http://www.ncbi.nlm.nih.gov/tools/primerblast/).The oligonucleotides were custom-Molecular Marker to distinguish Coscinium fenestratum
Current Trends in Biotechnology and PharmacyVol. 5 (2) 1163-1172 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1166synthesized by Bioserve biotechnologies(Hyderabad, India). PCR reaction with thespecies specific primer set (CF1F and CF1R)contained 30 ng of genomic DNA, 2.5 µl of 10xassay buffer, 1.5 mM MgCl 2, 0.3mM dNTP mix,30pM of each primer, 1U of Taq DNApolymerase (Bangalore Genei, India) and thevolume was made up to 25 µl with sterile distilledwater. PCR programme conditions optimized forthe species-specific primer set was as follows:94°C for 4 min, followed by 35 cycles of 30 secat 94°C, 30 sec at 60°C and 40 sec at 72°C witha final extension step for 2 min at 72°C.ResultsDNA extraction and Quality checking: Theprotocol described in the Materials and Methodsyielded a good quality and quantity of highmolecular weight genomic DNA from dried stemsamples of the test species studied. The yield wasabout 400-600 ng of DNA per 100 mg of tissue.An absorbance (A 260/A 280) ratio of 1.6-1.8indicated insignificant levels of contaminatingproteins and polysaccharides.Amplification and Sequencing of C.fenestratum ITS1 and ITS2 region: PrimersITS1 and ITS2 amplified a 245 bp (Figure 1)sequence of the ITS1 region while a 386 bp ITS2region (Figure 2) was amplified with the primersITS3 and ITS4 for the C. fenestratum samples(Table 2). Homology searches were performedwithin GenBank’s non-redundant database usingthe BLAST (Basic Local Alignment Search Tool)algorithm at http://www.ncbi.nlm.nih.gov/BLAST/ of the National Center for BiotechnologyInformation (NCBI), with the program BLASTN.BLAST results revealed that the sequence didnot show complete similarity with any knownnucleotide sequences. The sequences weresubmitted to GenBank (Accession numbers:GQ496588 for ITS1 and GQ496589 for ITS2)(Figure 3).Fig. 1. PCR amplification of the C. fenestratum ITS1sequence (245 bp) Lane M- 100 bp ladder; Lanes 1 to5- different C. fenestratum accessions used in thisstudyFig. 2. PCR amplification of C. fenestratum ITS2 sequence(386 bp) Lane 1 – 100 bp ladder; Lane 2 – C.fenestratum L/06/09/031; Lane 3 – C. fenestratum L/07/02/033; Lane 4 – C. fenestratum L/07/08/010; Lane5 – Lambda DNA EcoRI/HindIII double digestSequence analysis : Homology analysis of theC. fenestratum ITS1 and ITS2 regions wasperformed with the corresponding regions of thesubstitute plant species B. aristata, B. asiatica,B. lycium and M. umbellata. Details of the ITSsequence employed for each adulterant speciesBalasubramani and Venkatasubramanian
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