METODE EKSTRAKSI DNA PADA Jatropha spp. TANPA MENGGUNAKAN NITROGEN CAIR / DNA Extraction Method of Jatropha spp. without Liquid Nitrogen

Kristianoto Nugroho, Rerenstradika T. Terryana, Habib Rijzaani, Puji Lestari

Abstract


Physic nut is one of potential plant which produces biofuel. It is necessary to do an intensive breeding program either conventionally or molecularly based to develop new varieties of physic nut. All this time the physic nut DNA extraction methods always used liquid nitrogen to destroy the plant tissue. Liquid nitrogen is not always available, especially in remote areas. Physic nut has high latex content which makes extraction process more difficult. The aim of this study was to find the technique of DNA extraction which can give a good result without using liquid nitrogen. This research was conducted in Molecular Biology Laboratory, ICABIOGRAD Bogor from November to Desember 2015. There were five Jatropha species from Thailand which used in this research e.g. Jatropha curcas, Jatropha podagrica, Jatropha gossypifolia, Jatropha multifida, and Baliospermum solanifolium. The extraction method used modification CTAB by addition of PVP, sodium metabisulphite, sucrose and ascorbic acid. The young leaves were used as the part to be extracted. The results showed that the modified method could produce a good quality and quantity of DNA. The banding pattern of DNA amplification clearly visible under UV light. This method can reduce the dependency of liquid nitrogen.

 

Abstrak

Ekstraksi DNA merupakan salah satu ta hap penting dalam kegiatan pemuliaan berbasis molekuler. Jarak merupakan salah satu tanaman dengan kandungan getah cukup tinggi sehingga proses ekstraksi DNA cukup sulit dilakukan. Selama ini metode ekstraksi yang dikembangkan umumnya menggunakan nitrogen cair untuk menghancurkan jaringan tanaman. Nitrogen cair merupakan senyawa yang cukup sulit untuk didistribusikan ke laboratorium yang letaknya jauh dari kota. Tujuan penelitian ini adalah untuk memperoleh teknik ekstraksi DNA jarak yang mampu menghasilkan DNA dengan kualitas dan kuantitas yang baik tanpa menggunakan nitrogen cair. Penelitian ini dilaksanakan di Laboratorium Biologi Molekuler, Balai Besar Penelitian dan Pengembangan Bioteknologi dan Sumberdaya Genetik Pertanian (BB Biogen), Bogor pada bulan November hingga Desember 2015. Terdapat lima spesies jarak asal Thailand yang digunakan pada penelitian ini yaitu Jatropha curcas, Jatropha podagrica, Jatropha gossypifolia, Jatropha multifida, dan Baliospermum solanifolium. Ekstraksi DNA dilakukan melalui modifikasi buffer CTAB melalui penambahan senyawa PVP, natrium metabisulfit, sukrosa dan asam askorbat. Bagian tanaman yang digunakan berupa daun yang masih muda. Hasil penelitian menunjukkan DNA jarak yang diekstraksi dengan metode ini mempunyai kualitas dan kuantitas yang baik serta mampu diamplifikasi dengan baik. Metode ini mampu mengurangi ketergantungan terhadap nitrogen cair yang ketersediaannya terbatas di laboratorium yang lokasinya jauh dari kota.

Kata kunci: Jatropha spp., ekstraksi, DNA, nitrogen cair


Keywords


Jatropha spp.;extraction;DNA;liquid nitrogen

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References


ACHTEN W.M.J., E. MATHIJS, L. VERCHOT, V.P. SINGH, R. AERTS, and B. MUYS. 2007. Jatropha biodiesel fueling sustainability?. Biofuels, Bioproducts and Biorefining 1(4): 283-291.

ANURADHA, H.J., K. V IJAYAN , C.V. NAIR, and A. MANJULA. 2013. A novel and efficient protocol for the isolation of genomic DNA from mulberry (Morus L.). Emir. J. Food Agric. 25(2):124-131.

ARDIANA, D.W. 2009. Teknik isolasi DNA genom tanaman pepaya dan jeruk dengan menggunakan modifikasi bufer CTAB. Buletin Teknik Pertanian 14 (1): 12-16.

BAHAGIAWATI . 2011. Peran Markah Molekuler dalam Pemuliaan Tanaman. Sinar Tani Edisi 16-22 Maret 2011 No. 3397 Tahun XLI.

BECKER, K., and H.P.S. MAKKAR. 2008. Jatropha curcas: A potential source for tomorrow’s oil and biodiesel. Lipid Technology 20 (5): 104-107.

BEKESIOVA, I., J.P.NAP , and L. MLYNAROVA. 1999. Isolation of high quality DNA and RNA from leaves of the carnivorous plant Drosera rotundifolia. Plant Molecular Biology Reporter. 17: 269-277.

BORSE, T., P. JOSHI, and S. CHAPHALKAR. 2011. Biochemical role of ascorbic acid during the extraction of nucleic acids in polyphenol rich medicinal plant tissues. J. Plant. Mol. Biol. Biotechnol. 2(2): 1-7.

DEVAPPA, R.K., H.P.S. MAKKAR, and K. BECKER. 2011. Jatropha diterpenes: a review. J Am Oil Chem Soc.88:301-322. DOYLE, J.J. and J.L.

DOYLE. 1990. Isolation of plant DNA from fresh tissue. Focus. 12: 13-15.

ERYTHRINA. 2007. Jarak tanam dan pemupukan fosfat pada tanaman jarak pagar (Jatropha curcas L.) di Propinsi Lampung. Prosiding Lokakarya-II Status Teknologi Tanaman Jarak Pagar Jatropha curcas L. Pusat Penelitian dan Pengembangan Perkebunan Bogor. p. 43-49.

FATCHIYAH. 2011. Uji kuantitatif dan kualitatif. Dalam: Fatchiyah, Arumingtyas E.L., Widyarti S., dan Rahayu S. (ed). Biologi Molekular Prinsip Dasar Analisis. Jakarta: Penerbit Erlangga. p. 33-37.

FATCHIYAH, S. RAHAYU , dan E.L. ARUMINGTYAS. 2011. Isolasi DNA. Dalam: Fatch iyah, Arumingtyas E.L., Widyarti S., dan Rahayu S. (ed.). Biologi Molekular Prinsip Dasar Analisis. Jakarta: Penerbit Erlangga. p. 21-32.

FANG, G., S. HAMMAR, and R. GR UMET. 1992. A quick and inexpensive method for removing polysaccharides from plant genomic DNA. Biotechniques.13: 52-54.

FLEISCHMANN, A. and G. HEUBL. 2009. Overcoming DNA extraction problems from carnivorous plants. Anales del Jardin Botάnico de Madrid.66(2): 209-215.

GUILLEMAUT, P. and L. MARECHAL-DROUARD. 1992. Isolation of plant DNA: a fast, inexpensive, and reliable method. Plant Molecular Biology Reporter. 10(1): 60-65.

HAMBALI, E. 2007. Dive rsifikasi produk olahan jarak pagar (Jatropha curcas L.). Prosiding Lokakarya-II Status Teknologi Tanaman Jarak Pagar Jatropha curcas L.Pusat Penelitian dan Pengembangan Perkebunan Bogor. hlm. 181-194.

KRISHNAN, P.R. and M. PARAMATHMA. 2009. Potentials and Jatropha species wealth of India. Current Science. 97: 1000-1004.

KUMAR, R.S., K.T. PARTHIBAN, P.HEMALATHA, T. KALAISELVI, and M.G. RAO. 2009. Investigation on crosscompatibility barriers in the biofuel crop Jatropha curcas L. with wild Jatropha species. Crop Science. 49: 1667-1674.

LEFORT, F. and G.C. DOUGLAS. 1999. An efficient micromethod of DNA isolation from mature leaves of four hardwood tree species Acer, Fraxinus, Prunus, and Quercus. Ann. For. Sci. 56: 259-263.

LODHI, M.A., G.N.YE, N.F. WEEDEN, and B.I. REISCH. 1994. A simple and efficient method for DNA extraction from grapevine cultivars, Vitis species and Ampelopsis. Plant Molecular Biology Reporter.12(1): 6-13.

MICHIELS, A., W. VAN DEN ENDE, M.TUCKER, L. VAN RIET , and A. VAN LAERE. 2003. Extraction of high-quality genomic DNA from latex-containing plants. Analytical Biochemistry. 315: 85-89.

MOORE, E., A. ARNSCHEIDT, A. KRUGER, C. STROMPL, and M. Mau. 2004. Simplified protocols for the preparation of genomic DNA from bacterial cultures. Molecular Microbial Ecology Manual. 1(01): 3-18.

MULADNO. 2010. Teknologi Rekayasa Genetika. Bogor: IPB Press. 130 hlm.

RANJAN, S., G. KISHORE, V.S. JADON, J.P. BHATT, and S. GUPTA. 2010. Standardization of extraction of genomic DNA and PCR-RFLP conditions of Allium stracheyi: a high altitude plant. Academia Arena. 2(7): 11-14.

ROGERS, S.O. and A.J. BENDICH. 1989. Extraction of DNA from plant tissues. In: Gelvin, S.B., Schilperoort, R.A., and Verma, D.P.S (eds.). Plant Molecular Biology Manual. Kluwer Academic Publishers. Dordrecht. p. 73-83. JURNAL LITTRI VOL. 22 NO. 4, DESEMBER 2016: 159 - 166 166.

SAHU, S.K., M. THANGARAJ, and K. KATHIRESAN. 2012. DNA extraction protocol for plants with high levels of secondary metabolites and polysaccharides without using liquid nitrogen and phenol. IS RN Molecular Biology p. 1-7.

SAMBROOK, J. and D.W. RUSSELL. 1989. Molecular Cloning: A Laboratory Manual 2nd e dition. N ew York: Cold Spring Harbor Laboratory Press. p. 165. SHARMA, P., N. JOSHI, and A.

SHARMA. 2010. I solation of genomic DNA from medicinal plants without liquid nitrogen. Indian J. Exp. Biol. 48: 610-614.

SYAFARUDDIN dan T.J. SANTOSO. 2011. Optimasi teknik isolasi dan purifikasi DNA yang efisien dan efektif pada kemiri sunan (Reutalis trisperma (Blanco) Airy Shaw). Jurnal Littri. 17(1): 11-17.

SYAFARUDDIN, E. RANDRIANI, dan T.J. SANTOSO. 2011. Efektivitas dan efisiensi teknik isolasi dan purifikasi DNA pada jambu mete. Buletin RISTRI 2(2): 151- 160.

SYUKUR, M., S. SUJIPRIHATI, dan R. YUNIANTI. 2012. Teknik Pemuliaan Tanaman. Jakarta: Penebar Swadaya. p. 348 hlm.

TAI, T.H. and S.D. TANKSLEY. 1990. A rapid and inexpensive method for isolation of total DNA from dehydrated plant tissue. Plant Molecular Biology Reporter 8 (4): 297-303.

TANYA, P., P. TAEPRAYOON, Y. HADKAM , and P. SRINIVES. 2011. Genetic diversity among Jatropha and Jatropharelated species based on ISSR markers. Plant Mol Biol Rep. 29: 252-264.

VARMA, A., H. PADH, and N . SHRIVASTAVA . 2007. Plant genomic DNA isolation: an art or a science. Biotechnol. J. 2: 386-392.




DOI: http://dx.doi.org/10.21082/littri.v22n4.2016.159-166

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