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METODE BIOLOGI MOLEKULER

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Tim Penulis: Miftahul Jannah, Nila Kartika Sari, Miftahul Mushlih, Muhammad Rifqi Hariri,
Priyambodo, Rina Hidayati Pratiwi, Dwi Sendi Priyono, Yana Rubiyana, Pratiwi Prananingrum, Dwi Anggorowati Rahayu, Sapto Andriyono, Mo Awwanah.
Desain Cover:
Proofreader: N. Rismawati
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Anggota IKAPI No. 360/JBA/2020
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Alhamdulillahirobbil’alamin dengan terselesaikannya penulisan dan penyusunan buku Metode Biologi Molekuler ini semoga dapat mendukung khasanah keilmuan dibidangnya. Perkembangan dan pemanfaatan biologi molekuler saat ini sangat pesat. Kajian biologi molekuler berperan penting dalam menjawab pertanyaan di berbagai bidang ilmu seperti pangan (pertanian dan perikanan), kesehatan (farmasi dan kedokteran), forensik, energi, lingkungan dan lain-lain termasuk dalam pengelolaan keanekaragaman hayati di Indonesia. Buku ini membahas beberapa metode dan aplikasinya di beberapa bidang melalui pendekatan pengembangan biologi molekuler. Buku ini terdiri dari 12 bab yang menjelaskan sejarah, pemanfaatan dan berbagai metode Biologi Molekuler terkini. Dua bab pertama mendeskripsikan tentang perkembangan Biologi Molekuler dan pemanfaatannya. Bab selanjutnya menjelaskan tentang berbagai metode terkini dalam bidang Biologi Molekuler, di antaranya: RAPD, RFLP, microsatellite, SNP, multipleks PCR, sekuensing, Reverse Transcription, real time-PCR, DNA barcode, e-DNA, dan CRISPR-Cas9. Setiap Bab dalam buku ini ditulis oleh para peneliti yang menguasai teknik tersebut dengan baik, sehingga dapat memberi gambaran yang komprehensif dan mendalam.
Indonesia merupakan salah satu negara yang memiliki Megabiodiversitas hayati di dunia, dengan demikian penelitian mengenai observasi dan inventarisasi keanekaragaman hayati sangatlah penting dalam konservasi dan pengelolaan sumber daya alam. Marka molekuler merupakan salah satu metode yang efektif dalam penelitian keanekaragaman genetik. Mikrosatelit atau simple sequence repeat (SSR), PCR-RAPD, PCR-RFLP adalah marka molekuler yang banyak diaplikasikan dalam identifikasi keanekaragaman genetik. Selain itu, teknologi DNA Barcoding memberikan alternatif cepat dan tepat dalam identifikasi makhluk hidup lokal dan Endemik Indonesia yang sudah maupun belum terdeskripsikan. Penelitian biodiversitas dalam skala besar dapat dilakukan
KATA PENGANTAR
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dengan aplikasi DNA lingkungan (eDNA). eDNA merupakan metode baru dalam penelitian keanekaragaman hayati. Sampel DNA dapat diambil dari lingkungan (air, tanah, dan udara) tanpa memerlukan tanda yang jelas dari sumber biologis. Kajian metabarcoding untuk menduga DNA yang tersebar di lingkungan (air, tanah dan udara) memberikan harapan kajian biodiversitas yang semakin efisien dan biaya yang relatif lebih rendah dibandingkan dengan survei tradisional. Aplikasi DNA lingkungan (eDNA) menggabungkan para ilmuwan ekologis, dan bioinformatik untuk bersama-sama terus mengembangkan metode yang lebih efektif dalam pengelolaan sumberdaya alam.
Penemuan PCR dan sekuensing berperan besar dalam perkembangan riset genetika dan biologi molekuler. Saat ini teknik PCR dan sekuensing telah berkembang pesat. Metode multipleks PCR memungkinkan amplifikasi beberapa sekuens menggunakan beberapa pasang primer dalam satu reaksi. Teknik multipleks PCR dapat menghemat waktu dan reagen. Aplikasi multipleks PCR dapat kita lihat secara nyata dalam diagnosis pasien Covid-19. Selain itu, revolusi metode deteksi urutan sekuen nukleotida melalui sekuensing memungkinkan deteksi mutasi virus Covid-19 yang sangat cepat. Awalnya sekuensing berkembang dari metode sanger dan maxam-gilbert, teknik ini telah berkembang menjadi next generation sequencing. Prinsip dan aplikasi teknologi sekuensing ini dapat dibaca dengan lebih jelas di buku ini.
Penemuan enzim reverse transkriptase oleh Termine dan Baltimore pada tahun 1970 merupakan cikal bakal berkembangnya reverse transcription secara in vitro. Teknik Reverse Transcription memanfaatkan enzim transkriptase untuk transkripsi balik RNA menjadi DNA. Aplikasi teknik reverse transcription dan real time-PCR dikenal luas di tengah mewabahnya virus SARS-CoV-2 saat ini. Namun demikian, aplikasi reverse transcription dan real time-PCR tidak terbatas pada analisis diagnosis Covid-19 saja, prinsip dan aplikasi reverse transcription dan real time-PCR dalam berbagai bidang dapat dibaca dalam buku ini.
Bab terakhir dari buku ini akan membahas secara detail tentang prinsip dan aplikasi CRISPR-Cas9, mulai dari sejarah penemuan dan klasifikasi sistem CRISPR, prinsip kerja CRISPR-Cas9 baik sebagai mekanisme imunitas adaptif pada organisme prokariotik maupun sebagai
v
alat dalam metode genome editing, serta aplikasi CRISPR-Cas9 khususnya pada pengeditan genom tumbuhan. Untuk memahami lebih detail tentang CRISPR-Cas9, pembaca dapat mempelajarinya pada bab terakhir dari buku ini.
Buku ini tidak akan hadir tanpa kontribusi dan komitmen berbagai pihak. Kami berterima kasih pada para reviewers karena telah memberikan kritik dan saran yang sangat berharga. Kami berterimakasih pada tim penerbit yang tidak lelah untuk mengkoordinasi dan menampung aspirasi kami. Terimakasih secara khusus pada guru-guru kami, mahasiswa kami, kolega kami, keluarga, dan orangtua kami yang selalu memberikan dukungan agar kami senantiasa berkontribusi positif bagi perkembangan pendidikan dan sains di Indonesia.
Kami berharap bahwa kita semua dapat menikmati membaca buku ini!
Penulis
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DAFTAR ISI
KATA PENGANTAR ············································································· iii DAFTAR ISI ························································································ vi BAB 1 PENGENALAN DAN PERKEMBANGAN BIOLOGI MOLEKULER ·······1
A. Pengenalan dan sejarah biologi molekuler ·································· 1 B. Prinsip kerja biologi molekuler ····················································· 3 C. Aplikasi biologi molekuler ·························································· 13 D. Rangkuman materi ····································································· 14
BAB 2 PEMANFAATAN BIOLOGI MOLEKULER ····································· 19 A. Pendahuluan ··············································································· 19 B. Pemanfaatan biologi molekuler dalam bidang kesehatan ······· 21 C. Pemanfaatan biologi molekuler dalam bidang pertanian ········· 24 D. Pemanfaatan biologi molekuler dalam bidang peternakan ······ 30 E. Pemanfaatan biologi molekuler dalam bidang perairan ··········· 33 F. Pemanfaatan biologi molekuler dalam bidang ekologi ············· 36 G. Rangkuman materi ····································································· 38
BAB 3 PRINSIP DAN APLIKASI PCR-RAPD DAN PCR-RFLP ····················· 43 A. Pengantar ·················································································· 43 B. Prinsip metode PCR-RAPD dan PCR-RFLP ··································· 45 C. Kelebihan PCR-RAPD dan PCR-RFLP ·········································· 50 D. Kekurangan PCR-RAPD dan PCR-RFLP ········································ 51 E. Pengembangan penggunaan PCR-RAPD dan PCR-RFLP ············· 52 F. Aplikasi PCR RAPD dan PCR-RFLP ··············································· 54 G. Rangkuman materi ····································································· 57
BAB 4 PRINSIP DAN APLIKASI METODE MICROSATELLITE ··················· 65 A. Pendahuluan ············································································· 65 B. Pengenalan metode microsatellite ············································· 66 C. Prinsip metode microsatellite ····················································· 67 D. Aplikasi metode microsatellite ··················································· 70 E. Rangkuman materi ····································································· 74
BAB 5 PRINSIP DAN APLIKASI METODE SNP ······································· 79 A. Pendahuluan ············································································· 79 B. Definisi single nucleotide polymorphisms (SNP) ························· 80
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C. Urgensi studi SNP pada bidang biologi ······································· 81 D. Metode analisis SNP ··································································· 82 E. Studi kasus SNP pada manusia ··················································· 84 F. Studi kasus SNP pada hewan ······················································ 85 G. Studi kasus SNP pada tumbuhan ················································ 86 H. Rangkuman materi ····································································· 87
BAB 6 PRINSIP DAN APLIKASI METODE MULTIPLEKS PCR ··················· 93 A. Pengertian metode multipleks PCR ············································ 93 B. Prinsip metode multipleks PCR ·················································· 95 C. Aplikasi metode multipleks PCR ················································· 99 D. Keunggulan dari metode multipleks PCR ································· 105 E. Rangkuman materi ··································································· 107
BAB 7 PRINSIP DAN APLIKASI METODE DNA SEQUENCING ··············· 111 A. Pendahuluan ············································································· 111 B. Prinsip dan metode-metode sekuensing DNA ························· 113 C. Penerapan sekuensing DNA ····················································· 130 D. Rangkuman materi ··································································· 132
BAB 8 PRINSIP DAN APLIKASI REVERSE TRANSCRIPTION ·················· 137 A. Pengenalan metode reverse transcription ······························ 137 B. Prinsip dasar reverse transcription ··········································· 138 C. Aplikasi reverse transcription ··················································· 147 D. Rangkuman materi ··································································· 152
BAB 9 PRINSIP DAN APLIKASI METODE REAL TIME POLYMERASE CHAIN REACTION (RT-PCR) ························································ 157 A. Pengenalan metode ·································································· 157 B. Prinsip kerja metode ································································ 160 C. Aplikasi metode ········································································ 171 D. Rangkuman materi ··································································· 173
BAB 10 PRINSIP DAN APLIKASI DNA BARCODE ································· 179 A. Pendahuluan ············································································ 179 B. Pengertian teknologi DNA barcoding dalam sistematika
Molekuler ················································································· 180 C. Prinsip kerja DNA barcoding ····················································· 184 D. Metode analisis DNA barcoding ··············································· 186 E. Studi kasus terkini DNA barcoding pada hewan ······················ 188
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F. Rangkuman materi ··································································· 194 BAB 11 PRINSIP DAN APLIKASI eDNA ··············································· 201
A. Pengenalan metode ·································································· 201 B. Sejarah singkat aplikasi eDNA dalam kegiatan konservasi ······· 202 C. Memahami ekologi DNA lingkungan ······································· 203 D. Kondisi eDNA ··········································································· 205 E. Dinamika perubahan eDNA secara fisik dalam lingkungan ······ 206 F. Faktor yang berperan terhadap keberadaan eDNA ················· 207 G. Tahap pengujian : metode dan prinsip kerja identifikasi
berbasis molekuler melalui DNA lingkungan ···························· 208 H. Aplikasi DNA lingkungan dalam pendugaan biodiversitas ikan 211 I. Rangkuman materi ··································································· 214
BAB 12 PRINSIP DAN APLIKASI CRISPR-Cas9 ····································· 221 A. Pengenalan CRISPR-Cas9 ·························································· 221 B. Prinsip kerja CRISPR-Cas9 ························································· 227 C. Aplikasi CRISPR-Cas9 pada pengeditan genom tumbuhan ······ 236 D. Rangkuman materi ··································································· 261
GLOSARIUM ··················································································· 275 PROFIL PENULIS ·············································································· 285
PENGENALAN DAN PERKEMBANGAN BIOLOGI MOLEKULER
Miftahul Jannah, S.Si., M.Sc Prodi Biologi, Fakultas Sains dan Teknologi, Universitas Islam As-Syafiiyah
A. PENGENALAN DAN SEJARAH BIOLOGI MOLEKULER Sejarah biologi molekuler dimulai pada tahun 1930-an dengan
pertemuan berbagai disiplin ilmu biologi dan fisika beserta cabang ilmu lainnya seperti biokimia, genetika, mikrobiologi, virologi dan fisika. Biologi molekuler berperan dalam memahami kehidupan pada tingkat yang paling mendasar.
Biologi molekuler adalah cabang ilmu biologi yang mempelajari dasar molekuler dari aktivitas biologi di dalam dan di antara sel, termasuk sintesis, modifikasi, mekanisme dan interaksi molekuler (Albert et al., 2014; Gannon, 2002). Dogma utama biologi molekuler menjelaskan proses di mana DNA ditranskripsikan menjadi RNA, kemudian diterjemahkan (translasi) menjadi protein (Michael, 2015).
Biologi molekuler mencoba menjelaskan fenomena kehidupan yang dimulai dari sifat makromolekul yang menghasilkannya. Dua kategori makromolekul khususnya yang menjadi fokus ahli biologi molekuler yaitu:
Pengenalan dan Perkembangan Biologi Molekuler | 15
DAFTAR PUSTAKA
Albert B, Johnson A, Lewis J, Raff M, Roberts K, and Walter P. (2008). Molecular Biology of the Cell. Garland Science, taylor & Francis Group. New York. p : 233-239.
Alberts B, Johnson A, Lewis J, Morgan D, Raff M, Roberts K, Walter P (2014). Molecular Biology of the Cell, Sixth Edition. Garland Science. pp. 1–10. ISBN 978-1-317-56375-4.
Avery, Oswald T.; Colin M. MacLeod; Maclyn McCarty (1944). "Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types: Induction of Transformation by a Desoxyribonucleic Acid Fraction Isolated from Pneumococcus Type III". Journal of Experimental Medicine. 79 (2): 137– 158. doi:10.1084/jem.79.2.137. PMC 2135445. PMID 19871359
Ausubel, F. M., Roger B., Robert E. K., David D. M., J.G. Seidman, John A. S., & Kevin S. (2003). Current Protocols in Molecular Biology. USA: John Wiley & Sons, Inc.
Brown, T.A. (2010). Gene cloning and DNA analysis: An introduction. Blackwell Publishing, Ltd. West Sussex.
Campbell, N.A., J.B. Reece, and L.G. Mitchell. (1999). Biologi. Edisi Kelima. Penerbit Erlangga. Jakarta. pp : 221-231.
Campbell, M. K. and Farrel, Shawn F. (2009). Biochemistry. USA: Thomson Brooks/Cole. pp. 459-467.
Dale, J. W. & von Schantz, M. (2007). From Genes to Genomes. John Wiley & Sons Ltd. West Sussex.
Gannon F (2002). "Molecular biology--what's in a name?". EMBO Reports. 3 (2): 101. doi:10.1093/embo- reports/kvf039. PMC 1083977. PMID 11839687
Gilbert, H. F. (2000). Basic Concepts in Biochemistry. USA: The McGraw- Hill Companies, Inc. pp 76-79.
Hershey, A.D. and Chase, M. (1952) "Independent functions of viral protein and nucleic acid in growth of bacteriophage" J Gen Physiol.
Holme, D. J. & Hazel P. (1998). Analytical biochemistry .England : Pearson Education Limited
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Jacob F, Monod J (1961). "Genetic regulatory mechanisms in the synthesis of proteins". J Mol Biol. 3 (3): 318–356. doi:10.1016/S0022- 2836(61)80072-7. PMID 13718526
Jeremy (2002). Biochemistry. Tymoczko, John L.; Stryer, Lubert (5th ed.). New York: W.H. Freeman. ISBN 0-7167-3051-0. OCLC 48055706
Kriman, M., J. Jakše, D. Barievi, B. Javornik, M. Prošek. (2006). Robust CTAB activated charcoal protocol for plant DNA extraction. Acta agriculturae Slovenica, 87 – 2.
Karp, Gerald. (2008). Cell and Molecular Biology. New York: John Willey & Sons, Inc. pp. 567-573.
Lodish H, Berk A, Zipursky SL, Matsudaira P, Baltimore D, Darnell J (2000). Molecular cell biology (4th ed.). New York: Scientific American Books. ISBN 978-0-7167-3136-8.
Lodish, H. et al. (2003). Molecular Cell Biology, 5th ed. WH Freeman & Co. Martin, Robin. (1996). Gel Electrophoresis: Nucleic Acids. UK: BIOS
Scientific Publishers Limited. pp. 11-27. McPherson, Michael J. and Simon G. Møller. (2006). PCR, 2nd ed. USA:
Taylor & Francis Group. pp. 1-22. Miesfeld, Roger L. (1999). Applied Molecular Genetics. New York: John
Willey & Sons, Inc.pp. 57-68. Moyo, M., Amoo, S.O., Bairu, M.W., Finnie, J.F., & Van Staden, J. (2008).
Optimising DNA isolation for medicinal plants. South African Journal of Botany 74: 771–775.
Reinhart, C.A. (2005). Molecular Genetics - Biology 495: Hybridization Experiment. http://bioweb.wku.edu/courses/biol495G/DNA1/Hybrid ization9.html. Diakses tanggal 2 Juni 2012.
Solomon. (2008). Biology 8th ed. USA: Thomson Brooks/Cole. pp. 261-265. Surzycki, S. (2000). Basic techniques in molecular biology. Springer-Verlag
Publishing. Berlin. Switzer. (1999). Experimental Biochemistry. Oxford: Blackwell Scientific
Pub. pp. 301-385. Varma, A., Padh, H., & Shrivastava, N. (2007). Plant genomic DNA
isolation: An art or a science. Biotechnology Journal 2: 386–392
Watson J.D.; Crick F.H.C. (1953). "A Structure for Deoxyribose Nucleic Acid" (PDF). Nature. 171 (4356): 737– 738. Bibcode:1953Natur.171..737W. doi:10.1038/171737a0. PMID 13054692. Retrieved 13 Feb 2007.
Widyatmoko, A.P.B.C. 2020. Aplikasi Genetika Molekuler Untuk Konservasi Genetik Tumbuhan Hutan Tropis Terancam Punah. Bogor: PT Penerbit IPB Press.
Wilson, Keith and Walker, John. (2010). Principles and Techniques of Biochemistry and Molecular Biology 7th ed. New York: Cambridge University Press. pp. 178-186.
Yuwono, Triwibowo. (2006). Teori dan Aplikasi Polymerase Chain Reaction. Yogyakarta: CV. Andi Offset. pp. 1-2.
A. PENDAHULUAN
Perkembangan awal Biologi molekuler dimulai dengan penemuan model struktur DNA oleh James Watson dan Francis Crick pada tahun 1953. DNA merupakan materi genetik yang tersusun dari gula pentose (deoksiribosa), gugus fosfat, dan basa nitrogen. Basa Nitrogen penyusun DNA terdiri atas dua macam, yaitu basa purin dan pirimidin. Basa Purin terdiri atas adenin (A) dan Guanin (G) yang memiliki struktur cincin ganda, sedangkan basa pirimidin terdiri atas sitosin (C) dan Timin (T) yang memiliki struktur cincin tunggal. Satu komponen pembangun (Building block) DNA terdiri atas satu gula pentose, satu gugus fosfat dan satu pasang basa yang disebut nukleotida (Fatchiyah, dkk. 2011).
Sifat organisme ditentukan oleh gen-gen yang dimilikinya. Gen dikode dalam materi genetik organisme, yang kita kenal sebagai molekul DNA, atau RNA pada beberapa virus. Terdapat dua jenis gen, yaitu gen struktural dan gen regulator. Gen-gen struktural mengkode urutan asam amino dalam protein, seperti enzim, yang menentukan kemampuan biokimia dari organisme pada reaksi katabolisme dan anabolisme, atau berperan sebagai komponen tetap pada struktur sel. Gen-gen regulator
Pemanfaatan Biologi Molekuler | 41
Fatchiyah., Arumingtyas, A.L., Widyarti, S., Rahayu, S. (2011). Basic Principles Of Molecular Biology Analysis. Jakarta: Erlangga
Ghaffar, Shabarni. (2007). Buku Ajar Bioteknologi Molekuler. Bandung: Unpad Press
Kalqutny, Septian Hary., Pakki, Syahrir., Muis., Amran. (2020). Potensi Pemanfaatan Teknik Molekuler Berbasis DNA dalam Penelitian Penyakit Bulai pada Jagung. Agrosainstek, (4)1, 17-27
Kline, M. C., Redman J. W., Butler, J. M. 2001. Training on STR Typing Using Commercial Kits and ABI 310/3100. National Institute of Standards and Technology.
Listyorini, dkk. (2020). Biologi Molekuler & Bioinformatika. Malang.UMM Press
Rahayu, Ayu. (2017). Aplikasi Biomolekuler di Dunia Perunggasan Khususnya Itik. Journal of Livestock Science and Production, (1)1, 13- 17
Santoso, Tri Joko. (2013). Aplikasi Teknik Molekuler Untuk Analisis Genetik Tomato Leaf Curl Virus. Jurnal Litbang, (32)4, 141-149
Sari, Nila Kartika. (2017). Penentuan Similaritas dan Variabilitas Genetik pada Keluarga Etnis Jawa dan Arab dengan DNA Fingerprint di Malang, Jawa Timur, Indonesia. Jurnal Ilmiah Sains, (17)1, 51-58
Suryaningtyas, Indyaswan Tegar. (2017). Aplikasi Bioteknologi Molekuler Dalam Budidaya Perairan. Oseana, (XLII)4, 13 – 24
Xu, J., W. Huang, C. Zhong, D. Luo, S. Li, Z. Zhu and W. Hu. (2011). Defining global gene expression changes of the hypothalamic pituitary- gonadal axis in female sGnRH antisense transgenic common carp (Cyprinus carpio). Plos-one 6(6): 1-12.
Yuda, Pramana. (2016). Aplikasi Teknik Molekuler pada Penelitian dan Konservasi Burung di Indonesia. Dipresentasikan pada Konferensi Peneliti dan Pemerhati Burung Indonesia II, UAJY, Yogyakarta
PRINSIP DAN APLIKASI PCR-RAPD DAN PCR-RFLP
Miftahul Mushlih, M.Sc Universitas Muhammadiyah Sidoarjo dan Indonesian Genetic and Biodiversity Community (IGBC)
A. PENGANTAR Perkembangan metode biologi molekuler untuk mendapatkan sebuah
variasi genetik berdasarkan karakteristik tertentu berkembang begitu cepat. Meskipun banyak metode yang baru, pada beberapa kasus Random Amplified Polymorphic DNA (RAPD) dan Restriction Fragment Length Polymorphism (RFLP) merupakan metode yang sampai saat ini digunakan dalam beberapa analisis molekuler dan masih banyak diminati. Metode RAPD dikembangkan pada tahun 1990 oleh Welsh dan McClelland pada tahun 1990 dengan menganalisis genom menggunakan primer acak. RAPD merupakan metode dimana amplifikasi dilakukan secara acak menggunakan primer tunggal, hasil potongan DNA dari proses amplifikasi juga berjalan acak sesuai dengan kecocokan basa nukleotida dengan DNA template/cetakan. Primer yang digunakan biasanya berkisar 9-12 bp saja. DNA template yang ada akan digandakan menghasilkan berjuta juta copy, band hasil analisis diamati mulai dari 100 bp - 3000 bp. Band yang muncul juga menunjukkan ketebalan yang berbeda.
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Adiningsih, M. W. et al. (2018) “Authentication of Sumateran Wild Boar (Sus scrofa vittatus) meat contamination by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) technique of Cytochrome b Gene,” Tropical Animal Science Journal, 41(3), hal. 157–164. doi: 10.5398/tasj.2018.41.3.157.
Ali, B. A. et al. (2004) “A review of random amplified polymorphic DNA (RAPD) markers in fish research,” Reviews in Fish Biology and Fisheries, 14(4), hal. 443–453. doi: 10.1007/s11160-005-0815-0.
Arif, I., Bakir, M.A., Khan, H., Farhan, A.H., Homaidan, A.A., Bahkali, A., Sadoon, M.A., & Shobrak, M. (2010). Application of RAPD for molecular characterization of plant species of medicinal value from an arid environment. Genetics and molecular research : GMR, 9 4, 2191-8 .
Asmelash, B., Diriba, S. dan Pal, S. K. (2017) “Molecular markers based characterization and conservation of wild animals,” Research Journal of Recent Sciences Res. J. Recent Sci, 6(7), hal. 53–62. Tersedia pada: http://www.isca.in/rjrs/archive/v6/i7/7.ISCA-RJRS- 2017-058.pdf.
Bardakci, F., Skibinski, D. Application of the RAPD technique in tilapia fish: species and subspecies identification. Heredity 73, 117–123 (1994). https://doi.org/10.1038/hdy.1994.110
Berg, H. (2012) “Restriction Fragment Length Polymorphism Analysis of PCR-Amplified Fragments (PCR-RFLP) and Gel Electrophoresis - Valuable Tool for Genotyping and Genetic Fingerprinting,” Gel Electrophoresis - Principles and Basics. doi: 10.5772/37724.
Borisov, A. Y. et al. (1999) “Identification of DNA amplification fingerprinting ( DAF ) markers close to the symbiosis-ineffective sym31 mutation of pea ( Pisum sativum L .),” (May 2014). doi: 10.1007/s001220051152.
Bοusba, R. et al. (2020) “Genotypic diversity assessment of some durum wheat (Triticum durum) genotypes using rapd analysis,” Biodiversitas, 21(6), hal. 2696–2701. doi: 10.13057/biodiv/d210643.
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Daryono, B. S., Aristya, G. R. dan Kasiamdari, R. S. (2011) “Development of Random Amplified Polymorphism DNA Markers Linked to Powdery Mildew Resistance Gene in Melon,” Indonesian Journal of Biotechnology, 16(2), hal. 76–82. doi: 10.22146/ijbiotech.7837.
de Sousa, D. R. T. et al. (2015) “PCR-RFLP as a useful tool for diagnosis of invasivemycoses in a healthcare facility in the North of Brazil,” Electronic Journal of Biotechnology, 18(3), hal. 231–235. doi: 10.1016/j.ejbt.2015.03.012.
Fatemeh Keify (2012) “Exploitation of random amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) markers for genetic diversity of saffron collection,” Journal of Medicinal Plants Research, 6(14), hal. 2761–2768. doi: 10.5897/jmpr11.834.
Ferrito, V. dan Pappalardo, A. M. (2017) “Seafood species identification by DNA barcoding, a molecular tool for food traceability,” Biodiversity Journal, 8(1), hal. 65–72. Tersedia pada: http://www.biodiversityjournal.com/pdf/8(1)_65-72.pdf.
Govarthanan M. (2011) “Genetic variability among Coleus sp. studied by RAPD banding pattern analysis,” International Journal for Biotechnology and Molecular Biology Research, 2(12), hal. 202–208. doi: 10.5897/ijbmbr11.030.
Gurusubramanian, Guruswami & Kumar, N. S. (2016) “Random amplified polymorphic DNA ( RAPD ) markers and its applications Random amplified polymorphic DNA ( RAPD ) markers and its applications,” Mipograss, 11(3), hal. 116–124. Tersedia pada: www.usask.ca/.../pawlin/.
Gurusubramanian, Guruswami & Kumar, N. S. (2016) “Random amplified polymorphic DNA ( RAPD ) markers and its applications Random amplified polymorphic DNA ( RAPD ) markers and its applications,” Mipograss, 11(3), hal. 116–124. Tersedia pada: www.usask.ca/.../pawlin/.
Hadrys, H., Balick, M. Dan Schierwater, b. (1992) “applications of random amplified polymorphic dna (rapd) in molecular ecology,” molecular ecology, 1(1), hal. 55–63. doi: 10.1111/j.1365-294x.1992.tb00155.x.
Handayani, N. S. N. et al. (2021) “Splice-site and Frameshift Mutations of β-Globin Gene Found in Thalassemia Carrier Screening in Yogyakarta Special Region, Indonesia,” The Indonesian Biomedical Journal, 13(1), hal. 55–60. doi: 10.18585/inabj.v13i1.1406.
Hazlianda, C. P., Muis, K. dan Lubis, I. A. (2017) “Uji Diagnostik Tinea Kruris dengan Polymerase Chain Reaction Restriction Fragmented Length Polymorphism,” Periodical of Dermatology and Venereology, 29(2), hal. 158–163.
Hikmah, R., Retnoningsih, A. dan Habibah, N. (2016) “Keragaman Durian Berdasarkan Fragmen Internal Transcribed Pancers (ITS) DNA Ribosomal melalui analisis PCR RFLP,” 39(1), hal. 11–18.
Hiyagarajan, V. T., Au, S. L. dan Soi, M. T. (2010) “Monitoring Bacterial Biodiversity in Surface Sediment Using Terminal Restriction Fragment Length Polymorphism Analysis ( T-RFLP ): Application to Coastal Environment,” Coastal Environmental and Ecosystem Issues of the East China Sea, hal. 151–163.
https://www.goldbio.com/articles/article/Restriction-Enzyme-Cloning- Troubleshooting
Jahangir Tafrechi, R. S., van de Rijke, F. M., Allallou, A., Larsson, C., Sloos, W. C., van de Sande, M., Wählby, C., Janssen, G. M., & Raap, A. K. (2007). Single-cell A3243G mitochondrial DNA mutation load assays for segregation analysis. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society, 55(11), 1159–1166. https://doi.org/10.1369/jhc.7A7282.2007
Mhuka, C. et al. (2017) “Use of RAPD-PCR for breed/genotype identification in Zimbabwean cattle,” Journal of Cellular Biotechnology, 2(2), hal. 131–137. doi: 10.3233/jcb-15033.
Minarovi, T. et al. (2010) “Animal Species Identification by PCR – RFLP of Cytochrome b,” Animal Science and Biotechnologies, 43(1), hal. 296–299.
Minarovi, T. et al. (2010) “Animal Species Identification by PCR – RFLP of Cytochrome b,” Animal Science and Biotechnologies, 43(1), hal. 296–299.
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Mosa, K. A. et al. (2019) “The promise of molecular and genomic techniques for biodiversity research and DNA barcoding of the Arabian Peninsula Flora,” Frontiers in Plant Science, 9(January), hal. 1–19. doi: 10.3389/fpls.2018.01929.
Mushlih, M. et al. (2020) “Identification of molecular markers for type 2 Diabetes mellitus in Sidoarjo, Indonesia,” Jurnal Teknologi Laboratorium, 9(2), hal. 186–191. doi: 10.1525/9780520974166- 002.
Ozdemir, K. et al. (2014) “Identification of biodiversity of some Streptomyces species and determination of a restriction fragment length polymorphism (RFLP) profile of 16S rDNA gene region,” Journal of Animal and Veterinary Advances, 13(16), hal. 978–988. doi: 10.3923/javaa.2014.978.988.
Pal, P. (2015) “RAPD-PCR as a Molecular Discriminative Technique for Human Pathogenic Bacteria – A Review,” International Letters of Natural Sciences, 42, hal. 13–17. doi: 10.18052/www.scipress.com/ilns.42.13.
Pal, P. (2015) “RAPD-PCR as a Molecular Discriminative Technique for Human Pathogenic Bacteria – A Review,” International Letters of Natural Sciences, 42, hal. 13–17. doi: 10.18052/www.scipress.com/ilns.42.13.
Partis L, Wells RJ. Identification of fish species using random amplified polymorphic DNA (RAPD). Mol Cell Probes. 1996 Dec;10(6):435-41. doi: 10.1006/mcpr.1996.0060. PMID: 9025081.
Patrinos, G. P. dan Ansorge, W. J. (1986) Molecular Diagnostics: Past , Present , and Future. Second Edi, Molecular Diagnostics. Second Edi. Elsevier Ltd. doi: 10.1016/B978-0-12-374537-8.00001-8.
Prasad, M. P. (2014) “Molecular characterization and genetic diversity determination of Hibiscus species using RAPD molecular markers,” 4(3), hal. 50–56.
Prihatini, I. dan Faradilla, F. A. (2019) “Teknik Pcr Its RFLP Untuk Seleksi Isolat Jamur Pada Pengujian Agen Pengendali Hayati Pada Serangan Ganoderma Tanaman Acacia mangium PCR ITS-RFLP technique for selection of fungal isolates in biological control agent test against
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Ganoderma attack in Acacia,” Jurnal Pemuliaan Tanaman Hutan, 13(1), hal. 33–43.
Prystupa, A. et al. (2011) “Application of RFLP-PCR method for molecular diagnostics of hereditary non-polyposis colorectal cancer (HNPCC),” Journal of Pre-Clinical and Clinical Research, 5(2), hal. 70–73. Tersedia pada: www.jpccr.eu.
Ramella, M. S. et al. (2005) “Optimization of random amplified polymorphic DNA protocol for molecular identification of Lophius gastrophysus,” Ciência e Tecnologia de Alimentos, 25(4), hal. 733– 735. doi: 10.1590/s0101-20612005000400017.
Randriani, E. dan Tresniawati, C. (2012) “Pemanfaatan Teknik Random Amplified Polymorphic DNA ( RAPD ) Untuk Pengelompokan Secara Genetik Plasma Nutfah Jambu Mete (Annacardium occidentale L.),” Journal of Industrial and Beverage Crops, 3(1), hal. 1–6. doi: 10.21082/jtidp.v3n1.2012.p1-6.
Sheorey, R. R. dan Tiwari, A. (2011) “Random amplified polymorphic DNA ( RAPD ) for identification of herbal materials and medicines – A review.”
Tanaka, J. dan Taniguchi, F. (2002) “Emphasized-RAPD (e-RAPD): A simple and efficient technique to make RAPD bands clearer,” Breeding Science, 52(3), hal. 225–229. doi: 10.1270/jsbbs.52.225.
Tattikota, S. G. et al. (2013) “Argonaute2 regulates the pancreatic β-cell secretome.,” Molecular & cellular proteomics: MCP, 12(5), hal. 1214–25. Tersedia pada: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=36503 33&tool=pmcentrez&rendertype=abstract.
Thiyagarajan, V., Lau, S.C., Tsoi, M., Zhang, W., & Qian, P. (2010). Monitoring Bacterial Biodiversity in Surface Sediment Using Terminal Restriction Fragment Length Polymorphism Analysis (T- RFLP): Application to Coastal Environment.
Tingey, S. (2003) “Random Amplified Polymorphic DNA (RAPDs),” Nucleic Acid Protocols Handbook, The, 25, hal. 675–677. doi: 10.1385/1- 59259-038-1:675.
VI, K. (2017) “Using RAPD PCR Method for Studying DNA Polymorphism of Agricultural Importance Arthropods,” Agricultural Research & Technology: Open Access Journal, 9(4), hal. 106–107. doi: 10.19080/artoaj.2017.09.555769.
Ylmaz, R. et al. (2015) “Genetic differentiation of lactobacillus delbrueckii subsp. Bulgaricus and streptococcus thermophilus strains isolated from raw milk samples collected from different regions of Turkey,” Food Biotechnology, 29(4), hal. 336–355. doi: 10.1080/08905436.2015.1092091.
Yongjun, F. et al. (2014) “Application of random amplified polymorphic DNA (RAPD) markers to identify Taxus chinensis var. mairei cultivars associated with parthenogenesis,” African Journal of Biotechnology, 13(24), hal. 2385–2393. doi: 10.5897/ajb2014.13646.
Zahid, R. A., Sulaiman, B. K. dan Abd, Ahmed, B. (2011) “Molecular Investigation of Genetic Polymorphisms,” Iraqi Journal of Cancer and Medical Genetics, (2), hal. 47–54.
PRINSIP DAN APLIKASI METODE MICROSATELLITE
Muhammad Rifqi Hariri, M.Si Pusat Penelitian Konservasi Tumbuhan dan Kebun Raya – Lembaga Ilmu Pengetahuan Indonesia & Indonesian Genetic and Biodiversity Community
A. PENDAHULUAN Marka molekuler merupakan sebuah pendekatan yang cukup efektif
dan memiliki peran sentral penting dalam analisis keragaman genetik dan asesmen kekerabatan, baik di dalam (intra) maupun antar (inter) spesies tumbuhan (Kumar et al., 2009). Hal tersebut menunjukkan potensi yang dimiliki oleh marka molekuler dalam mendeteksi keragaman genetik dan membantu dalam pengelolaan sumber daya genetik tumbuhan (Harikumar & Sheela, 2019). Mikrosatelit merupakan salah satu jenis marka molekuler dengan sebutan yang bervariasi, seperti Simple Sequence Repeats (SSR), Short Tandem Repeats (STRs), atau Simple Sequence Length Polymorphism (SSLP) (Litt & Luty, 1989; Tautz, 1989; Edwards et al., 1991; Jacob et al., 1991).
Marka ini diperkenalkan pertama kali oleh Condit & Hubbell (1991) yang mendeteksi adanya pengulangan nukleotida (AC)n dan (AG)n pada tumbuhan. Target marka ini adalah nukleotida yang berulang secara
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Brassicaceae memberikan kerangka kerja yang sangat penting dalam penelitian genomik komparatif. Pemetaan gen komparatif dan keterkaitannya terhadap kromosom pada kerabat-kerabat dekat Arabidopsis menyimpulkan adanya ilustrasi kariotipe leluhur spesies tumbuhan pada famili ini. Selain itu, pemetaan komparatif terhadap genus Brassica menunjukkan adanya blok genom yang telah dipertahankan sejak terjadinya divergensi garis keturunan Arabidopsis dan Brassica (Schranz et al., 2007).
E. RANGKUMAN MATERI Marka mikrosatelit tidak hanya digunakan dalam studi keragaman
genetik, genetika populasi, dan studi evolusi, tetapi juga digunakan dalam penelitian fundamental seperti analisis genom, pemetaan gen, maupun marker assisted selection. Pemetaan asosiasi berbasis marka SSR sangat menjanjikan untuk mengejawantahkan keragaman genetik, mengkarakterisasi variasi fenotipik, dan mengaitkan penanda terhadap sifat-sifat yang ada pada plasma nutfah tumbuhan. Marka mikrosatelit yang terkait dengan fenotipe yang jelas dapat digunakan dalam program pemuliaan tumbuhan untuk mempercepat proses pemuliaan. Penanda mikrosatelit dapat memfasilitasi pemetaan komparatif dan membantu mengidentifikasi 'blok keterkaitan', sinkronisasi gen utama, penataan ulang kromosom, dan sinkronisasi mikro antar spesies.
DAFTAR PUSTAKA
Breseghello, F., & Sorrells, M. E. (2006a). Association mapping of kernel size and milling quality in wheat (Triticum aestivum L.) cultivars. Genetics, 172(2), 1165-1177.
Breseghello, F., & Sorrells, M. E. (2006b). Association analysis as a strategy for improvement of quantitative traits in plants. Crop Science, 46(3), 1323-1330.
Condit, R., & Hubbell, S. P. (1991). Abundance and DNA sequence of two- base repeat regions in tropical tree genomes. Genome, 34(1), 66-71.
Prinsip dan Aplikasi Metode Microsatellite | 75
Dorar, N., & Akkaya, M. S. (2001). Optimization of PCR amplification of wheat simple sequence repeat DNA markers. Turkish Journal of Biology, 25(2), 153-158.
Edwards, A., Civitello, A., Hammond, H. A., & Caskey, C. T. (1991). DNA typing and genetic mapping with trimeric and tetrameric tandem repeats. American journal of human genetics, 49(4), 746.
Fraser, L. G., Tsang, G. K., Datson, P. M., De Silva, H. N., Harvey, C. F., Gill, G. P., ... & McNeilage, M. A. (2009). A gene-rich linkage map in the dioecious species Actinidia chinensis (kiwifruit) reveals putative X/Y sex-determining chromosomes. BMC genomics, 10(1), 1-15.
Grover, A., & Sharma, P. C. (2016). Development and use of molecular markers: past and present. Critical reviews in biotechnology, 36(2), 290-302.
Harikumar, P., & Sheela, M. N. (2019). Simple Sequence Repeat (SSR) Marker Based Genetic Diversity Analysis in White Yam (Dioscorea rotundata Poir.), Indian journal of pure and applied biosciences, 7(5), 259-264.
Heywood, V. H., & Iriondo, J. M. (2003). Plant conservation: old problems, new perspectives. Biological conservation, 113(3), 321-335.
Hiroe, M. (1958). Umbelliferae of Japan. Univ. Calif. Publ. Bot., 30, 1-444. Jacob, H. J., Lindpaintner, K., Lincoln, S. E., Kusumi, K., Bunker, R. K., Mao,
Y. P., ... & Lander, E. S. (1991). Genetic mapping of a gene causing hypertension in the stroke-prone spontaneously hypertensive rat. Cell, 67(1), 213-224.
Jakse, J., Stajner, N., Kozjak, P., Cerenak, A., & Javornik, B. (2008). Trinucleotide microsatellite repeat is tightly linked to male sex in hop (Humulus lupulus L.). Molecular Breeding, 21(2), 139-148.
Jin, L., Lu, Y., Xiao, P., Sun, M., Corke, H., & Bao, J. (2010). Genetic diversity and population structure of a diverse set of rice germplasm for association mapping. Theoretical and Applied Genetics, 121(3), 475- 487.
Jones, A. G., Small, C. M., Paczolt, K. A., & Ratterman, N. L. (2010). A practical guide to methods of parentage analysis. Molecular ecology resources, 10(1), 6-30.
76 | Metode Biologi Molekuler
Joshi, S. P., Ranjekar, P. K., & Gupta, V. S. (1999). Molecular markers in plant genome analysis. Current Science, 230-240.
Kalia, R. K., Rai, M. K., Kalia, S., Singh, R., & Dhawan, A. K. (2011). Microsatellite markers: an overview of the recent progress in plants. Euphytica, 177(3), 309-334.
Kelkar, Y. D., Strubczewski, N., Hile, S. E., Chiaromonte, F., Eckert, K. A., & Makova, K. D. (2010). What is a microsatellite: a computational and experimental definition based upon repeat mutational behavior at A/T and GT/AC repeats. Genome biology and evolution, 2, 620-635.
Kumar, P., Gupta, V. K., Misra, A. K., Modi, D. R., & Pandey, B. K. (2009). Potential of molecular markers in plant biotechnology. Plant omics, 2(4), 141-162.
Lee, J., Joh, H. J., Kim, N. H., Lee, S. C., Jang, W., Choi, B. S., ... & Yang, T. J. (2017). High-throughput development of polymorphic simple sequence repeat markers using two whole genome sequence data in Peucedanum japonicum. Plant Breeding and Biotechnology, 5(2), 134-142.
Lieckfeldt, E., Meyer, W., & Börner, T. (1993). Rapid identification and differentiation of yeasts by DNA and PCR fingerprinting. Journal of Basic Microbiology, 33(6), 413-425.
Litt, M., & Luty, J. A. (1989). A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene. American journal of human genetics, 44(3), 397.
Ma, H., Yin, Y., Guo, Z., Chen, L., Zhang, L., Zhong, M., & Shao, G. (2011). Establishment of DNA fingerprinting of Liaojing series of Japonica rice. Middle East Journal of Scientific Research, 8(2), 384-392.
Meng, W. A. N. G., Fei, X. U. E., Peng, Y. A. N. G., Duan, X. Y., Zhou, Y. L., Shen, C. Y., ... & Wang, B. T. (2014). Development of SSR markers for a phytopathogenic fungus, Blumeria graminis f. sp. tritici, using a FIASCO protocol. Journal of Integrative Agriculture, 13(1), 100-104.
Meyer, W., Lieckfeldt, E., Kuhls, K., Freedman, E. Z., Börner, T., & Mitchell, T. G. (1993). DNA-and PCR-fingerprinting in fungi. In DNA fingerprinting: State of the Science (pp. 311-320). Birkhäuser, Basel.
Prinsip dan Aplikasi Metode Microsatellite | 77
Mittal, N., & Dubey, A. (2009). Microsatellite markers-A new practice of DNA based markers in molecular genetics. Pharmacognosy Reviews, 3(6), 235.
Morgante, M., & Olivieri, A. M. (1993). PCRamplified microsatellites as markers in plant genetics. The plant journal, 3(1), 175-182.
Neeraja, C. N., Maghirang-Rodriguez, R., Pamplona, A., Heuer, S., Collard, B. C., Septiningsih, E. M., ... & Mackill, D. J. (2007). A marker-assisted backcross approach for developing submergence-tolerant rice cultivars. Theoretical and Applied Genetics, 115(6), 767-776.
Parasnis, A. S., Ramakrishna, W., Chowdari, K. V., Gupta, V. S., & Ranjekar, P. K. (1999). Microsatellite (GATA) n reveals sex-specific differences in papaya. Theoretical and applied genetics, 99(6), 1047-1052.
Powell, W., Machray, G. C., & Provan, J. (1996). Polymorphism revealed by simple sequence repeats. Trends in plant science, 1(7), 215-222.
Rahman, M., Malik, T. A., Aslam, N., Asif, M., Ahmad, R., Khan, I. A., & Zafar, Y. (2002). Optimization of PCR conditions to amplify microsatellite loci in cotton Gossypium hirsutum L. genomic DNA. Intern J Agriculture Biol, 4(2), 282-284.
Rahman, M., Iqbal, M.A., & Shaheen, N. (2014). Microsatellites: Methods & Protocols. In Miransari, M (ed.) Stress and Plant Biotechnology. New Delhi: Studium Press, pp 316.
Rode, J., InChol, K., Saal, B., Flachowsky, H., Kriese, U., & Weber, W. E. (2005). Sexlinked SSR markers in hemp. Plant Breeding, 124(2), 167-170.
Romero, G., Adeva, C., & Battad, Z. (2009). Genetic fingerprinting: advancing the frontiers of crop biology research. Philipp. Sci. Lett, 2, 8-13.
Rosenblum, E. B., Belfiore, N. M., & Moritz, C. (2007). Anonymous nuclear markers for the eastern fence lizard, Sceloporus undulatus. Molecular Ecology Notes, 7(1), 113-116.
Schranz, M. E., Song, B. H., Windsor, A. J., & Mitchell-Olds, T. (2007). Comparative genomics in the Brassicaceae: a family-wide perspective. Current opinion in plant biology, 10(2), 168-175.
78 | Metode Biologi Molekuler
Senan, S., Kizhakayil, D., SASIKUMAR, B., & SHEEJA, T. E. (2014). Methods for development of microsatellite markers: an overview. Notulae Scientia Biologicae, 6(1), 1-13.
Spigler, R. B., Lewers, K. S., Main, D. S., & Ashman, T. L. (2008). Genetic mapping of sex determination in a wild strawberry, Fragaria virginiana, reveals earliest form of sex chromosome. Heredity, 101(6), 507-517.
Silva, P. I., Martins, A. M., Gouvea, E. G., Pessoa-Filho, M., & Ferreira, M. E. (2013). Development and validation of microsatellite markers for Brachiaria ruziziensis obtained by partial genome assembly of Illumina single-end reads. Bmc Genomics, 14(1), 1-9.
Simko, I., Costanzo, S., Haynes, K. G., Christ, B. J., & Jones, R. W. (2004). Linkage disequilibrium mapping of a Verticillium dahliae resistance quantitative trait locus in tetraploid potato (Solanum tuberosum) through a candidate gene approach. Theoretical and Applied Genetics, 108(2), 217-224.
Suotang, P., Jieyun, Z., Qichuan, Y., & Kangle, Z. (2003). SSR markers selection and purity detection of major hybrid rice combinations and their parents in China. Zhongguo shuidao kexue, 17(1), 1-5.
Tautz, D. (1989). Hypervariability of simple sequences as a general source for polymorphic DNA markers. Nucleic acids research, 17(16), 6463- 6471.
Venter, J. C., Adams, M. D., Myers, E. W., Li, P. W., Mural, R. J., Sutton, G. G., ... & Kalush, F. (2001). The sequence of the human genome. science, 291(5507), 1304-1351.
Wang, M. L., Barkley, N. A., & Jenkins, T. M. (2009). Microsatellite markers in plants and insects. Part I: Applications of biotechnology.
Xiao, X. Y., Wang, Y. P., Zhang, J. Y., Li, S. G., & Rong, T. Z. (2006). SSR marker-based genetic diversity fingerprinting of hybrid rice in Sichuan, China. Chinese J Rice Sci, 20(1), 1-7.
Yue, X. Y., Liu, G. Q., Zong, Y., Teng, Y. W., & Cai, D. Y. (2014). Development of genic SSR markers from transcriptome sequencing of pear buds. Journal of Zhejiang University Science B, 15(4), 303-312.
PRINSIP DAN APLIKASI METODE SNP
Priyambodo, S.Pd., M.Sc Universitas Lampung
A. PENDAHULUAN Dunia merupakan hal yang begitu dinamis, termasuk ilmu
pengetahuan dan teknologi, termasuk bidang biologi molekuler. Seiring bergantinya bilangan tahun, perkembangan dalam bidang biologi molekuler dan aplikasinya turut terus berubah dan berkembang. Salah satu perkembangan studi biologi molekuler adalah kajian terkait Single Nucleotide Polymorphism (SNP). Fenomena SNP menjadi bahan kajian yang penting dalam kaitan biologi molekuler dengan studi genetika populasi, ekologi, dan evolusi. Peran SNP dalam membentuk keragaman individu dalam populasi menjadi pijakan penting dalam lahirnya variasi populasi yang merupakan modal dasar pembahasan seleksi alam dan konsep evolusi.
Pada bab ini akan dibahas terkait definisi SNP dan urgensi SNP dalam perkembangan dunia biologi molekuler, serta metode analisis SNP. Lebih lanjut, dipaparkan contoh-contoh dalam studi kasus SNP yang terjadi pada manusia, hewan dan tumbuhan.
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DAFTAR PUSTAKA
Bryant, D., Bouckaert, R., Felsenstein, J., Rosenberg, N. A., & RoyChoudhury, A. (2012). Inferring species trees directly from biallelic genetic markers: bypassing gene trees in a full coalescent analysis. Molecular biology and evolution, 29(8), 1917–1932. https://doi.org/10.1093/molbev/mss086.
Christos, B., Dulger, A.O., Uncu, A.T., Spaniolas, S., Spanos, T., Kalaitzis, P. (2012) A SNP-based PCR-RFLP capillary electrophoresis analysis for the identification of the varietal origin of olive oils. Food Chemistry 134(4):2411-8
Collins, FS. Single Nucleotide Polymorphisms (SNPs). https://www.genome.gov/genetics-glossary/Single-Nucleotide- Polymorphisms
Franssila, S., Davis, C.E., LeVasseur, M.K., Cao, Z., Yobas, l. (2020). Chapter 22 - Microfluidics and bioMEMS in silicon. Handbook of Silicon Based MEMS Materials and Technologies (Third Edition). Elsevier. pp 547-563.
Gaudet, M., Fara, AG., Sabatti, M. et al. Single-reaction for SNP Genotyping on Agarose Gel by Allele-specific PCR in Black Poplar (Populus nigra L.). Plant Mol Biol Rep 25, 1–9 (2007). https://doi.org/10.1007/s11105-007-0003-6
Guo, CY., Xu, XF., Wu, JY., & Liu, SF. 2008. PCR-SSCP-DNA Sequencing Method in Detecting PTEN Gene Mutation and its Significance in Human Gastric Cancer. World Journal of Gastroenterology, 14(24): 3804 – 3811.
Han W, Yip SP, Wang J, Yap MKH. (2004). Using denaturing HPLC for SNP discovery and genotyping, and establishing the linkage disequilibrium pattern for the all-trans-retinol dehydrogenase (RDH8) gene. J Hum Genet. 49(1):16-23.
Hahner, S., Kostrzewa, M., Wenzel, T., Fröhlich, T. (2003). Strategies for SNP genotyping by mass spectrometry. International Congress Series. 1239, 11-16. https://doi.org/10.1016/S0531-5131(02)00286- 8.
Inazuka, M., Wenz, HM., & Sakabe, M. 1997. A Steamlined Mutation Detection System: Multicolor Post-PCR Fluorescence Labeling and Single-Stand Conformational Polymorphism Analysis by Capilary Electrophoresis. Genome Research CSH Press. Genome Research 7: 10094 – 1103.
Kurniawati, S., Hartati, N.S., Hartati, Sudamonowati, E. (2020). Motif Single Nucleotide Polymorphism (SNP) Gen Phytoene Synthase (PSY) Penyandi Karotenoid Ubi Kayu Berumbi Kuning. Jurnal Ilmu Dasar. 21(1):19-26.
Lonetti A, Fontana MC, Martinelli G, and Iacobucci I. (2016). Single Nucleotide Polymorphisms as Genomic Markers for High- Throughput Pharmacogenomic Studies, In Microarray Technology (pp. 143- 159). Humana Press, New York, NY.
Nakajima, E., Matsumoto, T., Yamada, R., Kawakami, K., Takeda, K., Ohnishi, A., & Komatsu, M. 1996. Technical Note: Use of A PCR- Single Strand Conformation Polymorphism (PCR-SSCP) for Detection of a Point Mutation in the Swine Ryanodine Receptor (RYR1) Gene. Journal of Animal Science. 74: 2904-2906.
Prihandini, P.W., Hariyono, D.N.H., Tribudi, Y.A. (2021). Gen Myostatin sebaga Marka Genetik untuk Sifat Pertumbuhan dan Karkas Sapi Potong. Wartazoa. 31(1): 37-42.
Priyambodo. (2014). Deteksi Molekuler Pembawa Sifat β-thalassemia di Daerah Istimewa Yogyakarta. Thesis. Yogyakarta: Universitas Gadjah Mada.
Rahmah, S.F., Widodo, Putri, J.F., Lukitasari, M, Rahman, M.S. (2013). Analisis Single Nucleotide Polymorphism (SNP) -217 Gen Human Angiotensinogen (hAGT) pada Penderita Hipertensi di Rumah Sakit Syaiful Anwar secara PCR-Sekuensing. Biotropika. 1 (2): 71-74.
Rajatileka, S., Luyt, K., Williams, M., Harding, D., Odd, D., Molnár, E., & Váradi, A. (2014). Detection of three closely located single nucleotide polymorphisms in the EAAT2 promoter: comparison of single-strand conformational polymorphism (SSCP), pyrosequencing and Sanger sequencing. BMC genetics, 15, 80. https://doi.org/10.1186/1471-2156-15-80
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Raut, AA., Kumar, A., Kala, SN., Chhokar, V., Rana, N., Beniwal, V., Jaglan, S., Samuchiwal, S.K., Singh, J.K., & Mishra, A.. 2012. Identification of Novel Single Nucleotide Polymorphisms in the DGAT1 Gene of Buffaloes by PCR-SSCP. Genetics and Molecular Biology, 35(3): 610- 613.
Sadewa, AH. (2015). Peran Single Nucleotide Polymorphisms (Snps) Pada Metabolisme Mikronutrien Dan Enzim Antioksidan Sebagai Predisposisi Terhadap Kanker,Prosiding Anual Scientific Meetng, 1- 11.
Salwati, E., Handayani, S., Jekti, R.P. (2014). Identifikasi Single Nucleotide Polymorphism (SNP) Gen pvmdr1 pada Penderita Malaria Vivaks di Minahasa Tenggara (Sulawesi Utara). Jurnal Biotek Medisiana Indonesia. 3 (2): 49-57.
Singh, R.P., Singh, P.K., Gupta, R., & Singh, L. (2018). Biotechnological Tools to Enhance Sustainable Production. Biotechnology for Sustainable Agriculture. Woodhead Publishing. 19-66. https://doi.org/10.1016/B978-0-12-812160-3.00002-7.
Twyman, RD. (2005). Single Nucleotide Polymorphism (SNP) Genotyping Techniques-An Overvies, Encyclopedia of Diagnostic Genomic.
Zhang, J., Zhang, J., Tao, R., Yang, Z., Zhang, S.., Li, C. (2019). Mass spectrometry-based SNP genotyping as a potential tool for ancestry inference and human identification in Chinese Han and Uygur populations. Science & Justice. 59 (3). 228-233. https://doi.org/10.1016/j.scijus.2019.01.006.
Dr. Rina Hidayati Pratiwi, M.Si Universitas Indraprasta PGRI, Jakarta
A. PENGERTIAN METODE MULTIPLEKS PCR
Gambar 6.1 Jenis dan Variasi Teknik PCR
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10. Dapat menghemat volume DNA yang digunakan, waktu pengerjaan yang lebih efektif serta mengurangi biaya untuk amplifikasi DNA (Sambrook & Russel, 2001).
11. Deteksi bakteri Escherichia coli O157:H7 menggunakan multipleks PCR lebih cepat dibandingkan dengan menggunakan metode kultur secara konvensional (mikrobiologi) pada media sorbitol Mac Conkey agar (SMAC), selain itu penggunaan PCR juga menghilangkan kemungkinan kesalahan deteksi ada atau tidaknya bakteri Escherichia coli O157:H7 dalam suatu sampel karena menggunakan berbagai macam primer yang spesifik terhadap gen tertentu.
12. Multipleks PCR memungkinkan amplifikasi beberapa target berbeda dalam satu tube PSC secara bersamaan sehingga menghemat waktu, reagent dan sampel & dapat membandingkan beberapa amplikon secara simultan.
E. RANGKUMAN MATERI PCR multipleks merupakan teknik Biologi Molekuler, adaptasi dari
teknik PCR yang memungkinkan amplifikasi secara simultan dari berbagai sekuen gen. Dalam pengujian multipleks, lebih dari satu urutan target dapat diperkuat dengan menggunakan beberapa pasangan primer dalam campuran reaksi. Dengan metode PCR multipleks dapat mengamplifikasi dua atau lebih sekuen target (multi target) secara simultan dengan satu kali reaksi PCR sehingga dapat menghemat alat, biaya, waktu pengerjaan, dan reagent yang digunakan. PCR multipleks banyak digunakan untuk berbagai kepentingan yang paling umum diantaranya dapat digunakan untuk identifikasi patogen, deteksi GMOs (Genetically Modified Organisms), analisis polimorfisme, analisis mutasi, analisis delesi gen, deteksi RNA, penelitian mengenai forensik dan lain sebagainya. Pada dasarnya, prosedur dan komponen dalam reaksi multiplex PCR sama dengan PCR reguler.
DAFTAR PUSTAKA
Borah P. 2011. Primer Designing for PCR. Science Vision 11(3): 134-136. Chamberlain, J. S., Gibbs, R. A., Ranier, J. E., Nguyen, P. N., dan Caskey, C.
T. 1988. Deletion Screening of the Duchenne Muscular Dystrophy
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Locus Via Multiplex DNA Amplification. Nucleid Acid Research. 16(23): 11141-11156.
De las Rivas B, Marcobal A, Muñoz R. 2005. Improved multiplex-PCR method for the simultaneous detection of food bacteria producing biogenic amines. Federation of European Microbiology Societies. 244:367- 372.
De las Rivas B, Marcobal A, Carrascosa AV, Muñoz R. 2006. PCR detection of foodborne bacteria producing the biogenic amines histamine, tyramine, putrescine, and cadaverine. Journal of Food Protection. 69(10): 2509-2514.
Edwards MC, Gibbs RA. 1994. Multiplex PCR: Advantages, Development, and Applications. Cold Spring Harbor Laboratory 3: 65-75.
Elnifro, E.M., Ashshi, A.M., Cooper, R. J., Klapper, P.E. (2000). Multiplex PCR: optimization and application in diagnostic virology. Clinical Microbiology Reviews, 13(4), p. 559–570.
Gopinath K, Singh S. 2009. Multiplex PCR Assay for Simultaneous Detection and Differentiation of Mycobacterium tuberculosis, Mycobacterium avium complexes and Other Mycobacterial Species Directly from Clinical Specimens. Journal of Applied Microbiology 107: 425-435.
Handoyo D, Rudiretna A. 2001. Prinsip Umum dan Pelaksanaan Polymerase Chain Reaction (PCR). Unitas 9(1): 17-29.
Hayden, M. J., Nguyen, T. M., Waterman, A., dan Chalmers, K. J. 2008. Multiplex-ready PCR: A New Methode for Multiplexed SSR and SNP Genotyping. BMC Genomics. 9: 80.
Hwang CC, Lee YC, Huang YR, Lin CM, Shiau CY, Hwang DF, Tsai YH. 2010. Biogenic amines content, histamineforming bacteria and adulteration of bonito in tuna candy products. Food control. 21: 845-850.
Irine, Nuraini H, Sumantri C. 2013. Species authentication of dog, cat and tiger using cytochrome β gene. J Anim Sci Tech. 36:171- 78.
Kemalaputri DW, Jannah SN, Budiharjo A. 2017. Deteksi MRSA (Methicillin Resistant Staphylococcus aureus) pada Pasien Rumah Sakit dengan Metode Maldi-TOF MS dan Multiplex PCR. Jurnal Biologi (6): 4.
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Kitano, et al. (2020). The impact analysis of a multiplex PCR respiratory panel for hospitalized pediatric respiratory infections in Japan. J. Infect Chemoteraphy, 26, pp 82-85.
Lanzilao I, Burgalassi F, Fancelli S, Settimelli M, Fani R. 2005. Polymerase chain reactionrestriction fragment length polymorphism analysis of mitochondrial cyt b gene from species of dairy interest. J AOAC Int. 88:128- 135.
Maksum IP, Suhaili, Amalia R , Kamara DS, Rachman SD, Rachman RW. 2018. PCR Multipleks untuk Identifikasi Mycobacterium tuberculosis Resisten terhadap Isoniazid dan Rifampisin pada Galur Lokal Balai Laboratorium Kesehatan Provinsi Jawa Barat. Jurnal Kimia VALENSI: Jurnal Penelitian dan Pengembangan Ilmu Kimia, 4(2), 107-118.
Markoulatos P, Siafakas N, Moncany M. 2002. Multiplex Polymerase Chain Reaction: A Practical Approach. Journal of Clinical Laboratory Analysis 16: 47-51.
Martin I, Garcia T, Fajardo V, Rojas M, Hernandez PE, Gonzalez I, Martin R. 2007. Technical Note: detection of cat, dog, and rat or mouse tissues in food and animal feed using speciesspecific polymerase chain reaction. J Anim Sci. 85:2734-2739.
Matsunaga T, Chikuni K, Tanabe R, Muroya S, Shibata K, Yamada J, Shinmura Y. 1999. A quick and simple method for the identification of meat species and meat products by PCR assay. Meat Sci. 51:143- 148.
Muladno. 2010. Teknologi Rekayasa Genetika. Edisi Ke-2. Penerbit IPB Press. Bogor.
Nuraini H, Primasari A, Andreas E, Sumantri C. 2012. The use of cytochrome b gene as a specific marker of the rat meat (Rattus norvegicus) on meat and meat products. J Anim Sci Tech. 35:15-20.
Nuraini H, Furqon A, Sari SA, Dewi MK, Zainatha FM, Novianti WT, Sumantri C. 2013. Deteksi kandungan gelatin babi pada produk permen jelly meggunakan penanda gen sitokrom b. J Ilmu Produksi dan Teknologi Hasil Peternakan. 1:2303-2227.
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Rajalakshmi, S. 2017. Different types of PCR technique and its applications. International Journal of Pharmaceuticals, Chemical and Biological Science, 7 (3), 285-292.
Rodriguez PH, Ramirez AG. 2012. Polymerase Chain Reaction. Croatia: Dalam: Tech Europe. Hlm. 159.
Sambrook, J. & D.W. Russell. 2001. Molecular cloning a laboratory manual 3rd Ed. Cold Spring Harbor Laboratory Press, New York.
Sulistyaningsih, E. 2007. Polymerase Chain Reaction (PCR): Era Baru Diagnosis dan Managemen Penyakit Infeksi. Biomedis. 1(2)
Suryadi TP, Ratnayani K, Yowani SC. 2014. Desain Primer untuk Amplifikasi Gen katG Multidrug Resistance Tuberculosis (MDRTB) dengan Metode Polymerase Chain Reaction (PCR). Jurnal Kimia 8(1): 77-82.
Toma C, Lu Y, Higa N, Nakasone N, Chinen I, Baschkier A, Rivas M, Iwanaga M. 2003. Multiplex PCR Assay for Identification of Human Diarrheagenic Escherichia coli. Journal of Clinical Microbiology 41(6): 2669–2671.
Verma P, Yadav AN, Kazy SK, Saxena AK, Suman A. 2014. Evaluating the diversity and phylogeny of plant growth promoting bacteria associated with wheat (Triticum aestivum) growing in central zone of India. International Journal off Current Microbiology Applied Science. 3(5): 432- 447.
Wu, Xiaojing., et al. (2020). Co-infection with SARS-CoV-2 and Influenza A Virus in Patient with Pneumonia, China. Emerging Infectious Diseases. Vol. 26, (6).
Yang L, Wang C, Wang L, Xu C, Chen K. 2013. An Efficient Multiplex PCR Assay for Early Detection of Agrobacterium tumefaciens in Transgenic Plant Material. Turk J Agric For 37: 157-162.
Yusuf, Zuhriana. 2010. PCR. Makalah. FIKK, Universitas Negeri Gorontalo, Gorontalo.
Yuwono T. 2006. Teori dan aplikasi polymerase chain reaction. Yogyakarta (ID): Penerbit Andi Yogyakarta.
PRINSIP DAN APLIKASI METODE DNA SEQUENCING
Dr. Dwi Sendi Priyono, S.Si., M.Si Fakultas Biologi – UGM
A. PENDAHULUAN Bayangkan Anda memiliki sebuah perpustakaan dengan koleksi 20.000
buku dan buku-buku ini memiliki sumber pengetahuan yang luar biasa, seperti penyakit yang langka, segala bentuk instruksi yang mengatur bentuk tubuh dan tingkah laku, dan bahkan kemampuan berpikir manusia. Menariknya, semua buku itu ditulis dengan bahasa yang terdiri dari 4 huruf dengan pola-pola misterius. Buku-buku itu adalah gambaran dari gen-gen yang membawa informasi penting, dan perpustakaan yang menyimpan buku-buku itu adalah genom manusia. Sekuensing DNA atau DNA sequencing adalah suatu proses penentuan urutan basa nukleotida pada bagian atau seluruh molekul DNA (deoxyribonucleic acid). Ibarat Anda sedang membaca buku-buku itu yang tersusun dari 4 huruf basa (C, G, A, dan T). Runutan basa tersebut sangat penting untuk memahami bahasa DNA. Dengan memahami bahasa DNA dalam suatu molekul DNA, akan menyediakan banyak informasi tentang bagaimana struktur dan suatu gen itu berfungsi.
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Ansorge, W. J. (2009). Next-generation DNA sequencing techniques. New Biotechnology, 25(4), 195–203.
Bentley, D. R., Balasubramanian, S., Swerdlow, H. P., Smith, G. P., Milton, J., Brown, C. G., Hall, K. P., Evers, D. J., Barnes, C. L., & Bignell, H. R. (2008). Accurate whole human genome sequencing using reversible terminator chemistry. Nature, 456(7218), 53–59.
Callaway, E. (2021). Million-year-old mammoth genomes shatter record for oldest ancient DNA. Nature.
Castro, C. J., Marine, R. L., Ramos, E., & Ng, T. F. F. (2020). The effect of variant interference on de novo assembly for viral deep sequenc