LAPORAN PRAKTIKUM BIOTEKNOLOGI “PERANCANGAN PRIMER”
Disusun oleh : Meranti Bekti Pertiwi 152210101117
BAGIAN BIOLOGI FARMASI FAKULTAS FARMASI UNIVERSITAS JEMBER 2017
i
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KATA PENGANTAR
Puji syukur kami panjatkan kehadirat Allah Subhanahuwata’ala yang telah melimpahkan segala rahmat-Nya sehingga saya bisa menyusun laporan Bioteknologi ini hingga selesai. . Tidak lupa saya juga mengucapkan banyak terima kasih atas bantuan dari semua pihak yang telah berkontribusi dengan memberikan sumbangan baik materi maupun pikirannya. Laporan ini disusun untuk memenuhi tugas pembuatan laporan praktikum Bioteknologi Farmasi dengan materi “Perancangan Primer”. Dan harapan saya semoga laporan ini dapat menambah pengetahuan dan pengalaman bagi para pembaca, untuk ke depannya dapat memperbaiki bentuk maupun menambah isi laporan agar menjadi lebih baik lagi. Karena keterbatasan pengetahuan maupun pengalaman saya, saya yakin masih banyak kekurangan dalam laporan ini. Oleh karena kare na itu, it u, saya sangat mengharapkan saran dan kritik yang membangun dari pembaca demi kesempurnaan laporan ini. Mudah-mudahan dengan dukungan kerja sama yang nyata dari semua pihak benar-benar mampu memberikan sekaligus meningkatkan informasi-informasi ang berkaitan dengan isi laporan. Akhirnya kepada semua pihak yang telah berpartisipasi dalam penyusunan program kerja ini disampaikan terimakasih.
Jember, 18 Mei 2017
Penyusun
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BAB I PENDAHULUAN
1.1
Latar Belakang
Penemuan yang berpengaruh terhadap perkembangan biologi molekuler salah saunya adalah PCR (Polymerase Chain Reaction). Teknik ini mempunyai fungsi utama untuk memperbanyak DNA target. Proses yang terjadi melibatkan suatu primer yan berfungsi sebagai cetakan untuk membuat DNA baru. Perancangan primer dilakukan untuk memperoleh primer yang dapat digunakan dalam amplifikasi DNA dengan metode Polymerase Chain Reaction (PCR). Keberhasilan amplifikasi DNA tergantung dari ketepatan primer yang digunakan. Primer yang digunakan dalam proses PCR harus dapat membatasi daerah yang akan diamplifikasi. Primer berfungsi sebagai pembatas fragmen DNA target yang akan diamplifikasi. Amplifikasi DNA dengan metode PCR terdiri dari tiga tahap. Tahap awal dari proses amplifikasi yaitu denaturasi untai DNA, selanjutnya dilakukan penempelan primer pada fragmen DNA target (annealing) dan tahap akhir merupakan proses extension yaitu proses pemanjangan sekuen DNA. Perancangan untuk memperoleh suatu primer yang memenuhi kriteria primer yang baik untuk amplifikasi dilakukan secara in silico, yaitu mErancang/mendesain primer dengan bantuan suatu program dalam komputer. Suatu keberhasilan reaksi PCR dipengaruhi oleh desain primer yang digunakan, desain primer dapat dibuat dengan menggunakan beberapa aplikasi, dalam pembuatan desain primer cukup sulit dilakukan sehingga dibutuhkan keterampilan dalam membuatnya. 1.2
1.3
Rumusan Masalah 1.2.1
Bagaimana cara melakukan perancangan primer secara online ?
1.2.2
Bagaimana cara menentukan spesifitas primer ?
Tujuan
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BAB II TINJAUAN PUSTAKA
2.1
Pengertian PCR (Polimerase Chain Reaction)
PCR ( polymerase chain reaction) reaction) merupakan suatu teknik atau metode perbanyakan (replikasi) DNA (replikasi) DNA secara enzimatik secara enzimatik tanpa menggunakan organisme. menggunakan organisme. Dengan teknik ini, DNA dapat dihasilkan dalam jumlah besar dengan waktu relatif singkat sehingga memudahkan berbagai teknik lain yang menggunakan DNA. Teknik ini dirintis oleh Kary Mullis pada Mullis pada tahun 1983 dan ia memperoleh hadiah memperoleh hadiah Nobel pada Nobel pada tahun 1994 berkat temuannya tersebut. Penerapan PCR banyak dilakukan di bidang biokimia bidang biokimia dan biologi molekular karena relatif murah dan hanya memerlukan jumlah sampel yang kecil. PCR (Polimerase Chain Reaction) atau reaksi berantai polimerase adalah suatu metode in vitro yang digunakan untuk mensintesis sekuens tertentu DNA dengan menggunakan dua primer oligonukleotida yang menghibridisasi pita yang berlawanan dan mengapit dua target DNA. Kesederhanaan dan tingginya tingkat kesuksesan amplifikasi sekuens DNA yang diperoleh menyebabkan teknik ini semakin luas penggunaannya. 2.2.
Komponen
Selain DNA template yang akan digandakan dan enzim DNA polymerase, komponen lain yang dibutuhkan adalah : a. Primer
Primer adalah sepasang DNA utas tunggal atau oligonukleotida pendek yang menginisiasi sekaligus membatasi reaksi pemanjangan rantai atau polimerisasi DNA. PCR hanya mampu menggandakan DNA pada daerah tertentu sepanjang maksimum 10000 bp saja, dan dengan teknik tertentu bisa sampai 40000 bp. Primer dirancang untuk memiliki sekuen yang komplemen dengan DNA template, jadi dirancang agar menempel mengapit daerah tertentu yang kita inginkan. b. dNTP (deoxynucleoside triphosphate)
dNTP alias building blocks sebagai ‘batu bata’ penyusun DNA yang baru. dNTP terdiri atas 4 macam sesuai dengan basa penyusun DNA, yaitu dATP, dCTP, dGTP dan dTTP.
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d. Ion Logam -
Ion logam bivalen, umumnya Mg++, fungsinya sebagai kofaktor bagi enzim DNA polymerase. Tanpa ion ini enzim DNA polymerase polymerase tidak dapat bekerja.
-
Ion logam monovalen, kalsium (K+).
2.3 Prinsip Kerja
Secara prinsip, PCR merupakan proses yang diulang-ulang antara 20 – 30 30 kali siklus. Setiap siklus terdiri atas tiga tahap. Berikut adalah tiga tahap bekerjanya PCR dalam satu siklus: 1. Tahap peleburan (melting ( melting ) atau denaturasi. Pada tahap ini (berlangsung pada suhu tinggi, 94 – 96 96 °C) ikatan hidrogen DNA terputus te rputus (denaturasi) dan DNA menjadi berberkas tunggal. Biasanya pada tahap awal PCR tahap ini dilakukan agak lama (sampai 5 menit) untuk memastikan semua berkas DNA terpisah. Pemisahan ini menyebabkan DNA DNA tidak stabil dan siap menjadi templat (“patokan”) bagi primer. Durasi tahap ini 1 – 2 menit. 2. Tahap penempelan atau annealing . Primer menempel pada bagian DNA templat yang komplementer urutan basanya. Ini dilakukan pada suhu antara 45 – 45 – 60 60 °C. Penempelan ini bersifat spesifik. Suhu yang tidak tepat menyebabkan tidak terjadinya penempelan atau primer menempel di sembarang tempat. Durasi tahap ini 1 – 1 – 2 menit. 3. Tahap pemanjangan atau elongasi. Suhu untuk proses ini tergantung dari jenis DNA polimerase yang dipakai. Dengan Taq-polimerase, proses ini biasanya dilakukan pada suhu 76 °C. Durasi tahap ini biasanya 1 menit. Setelah melewati tahap 3, siklus diulang kembali mulai tahap 1. Akibat denaturasi dan renaturasi, beberapa berkas baru (berwarna hijau) menjadi templat bagi primer lain. Akhirnya terdapat berkas DNA yang panjangnya dibatasi oleh primer yang dipakai. Jumlah DNA yang dihasilkan berlimpah karena penambahan terjadi secara eksponensial. Pada tahap denaturasi, pasangan untai DNA templat dipisahkan satu sama lain sehingga menjadi untai tunggal. Pada tahap selanjutnya, masing-masing untai tunggal akan ditempeli oleh primer. Jadi, ada dua buah primer yang masing-masing menempel pada untai tunggal DNA templat. Biasanya, kedua primer tersebut dinamakan primer maju (forward primer) dan primer) dan primer mundur (reverse (reverse
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putaran reaksi pertama akan diperoleh dua pasang untai DNA jika DNA templat awalnya berupa sepasang untai DNA. Pasangan-pasangan untai DNA yang diperoleh pada suatu akhir putaran reaksi akan menjadi templat pada putaran reaksi berikutnya. Begitu seterusnya hingga pada putaran yang ke n diharapkan akan diperoleh fragmen DNA pendek sebanyak 2 n – 2n. Fragmen DNA pendek yang dimaksudkan adalah fragmen yang ukurannya sama dengan jarak antara kedua tempat penempelan primer. Fragmen pendek inilah yang merupakan urutan target yang memang dikehendaki untuk digandakan (diamplifikasi). Bisa kita bayangkan seandainya PCR dilakukan dalam 20 putaran saja, maka pada akhir reaksi akan diperoleh fragmen urutan target sebanyak 2 20 – 2.20 = 1.048576 – 40 = 1.048536. Jumlah ini masih dengan asumsi bahwa DNA templat awalnya hanya satu untai ganda. Padahal kenyataannya, hampir tidak mungkin DNA templat awal hanya berupa satu untai ganda. Jika DNA templat awal terdiri atas 20 untai ganda saja, maka jumlah tadi tinggal dikalikan 20 menjadi 20.970.720, suatu jumlah yang sangat cukup bila akan digunakan sebagai fragmen pelacak. 2.4 Perancangan Primer
Tahapan PCR yang paling menentukan adalah penempelan primer. Sepasang primer oligonukleotida (primer maju dan primer mundur) yang akan dipolimerisasi masing-masing harus menempel pada sekuens target, tepatnya pada kedua ujung fragmen yang akan diamplifikasi. Untuk itu urutan basanya harus komplementer atau setidak-tidaknya memiliki homologi cukup tinggi dengan urutan basa kedua daerah ujung fragmen yang akan diamplifikasi itu. Padahal, kita belum mengetahui dengan pasti urutan basa sekuens target. Oleh karena itu, diperlukan cara tertentu untuk merancang urutan basa kedua primer yang akan digunakan. Dasar yang digunakan adalah urutan basa yang diduga mempunyai kemiripan dengan urutan basa sekuens target. Urutan ini adalah urutan serupa dari sejumlah spesies/strain organisme lainnya yang telah diketahui/dipublikasikan. Daerah lestari juga dapat ditentukan melalui penjajaran urutan asam amino pada
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basa primer yang disusun dapat merupakan kombinasi beberapa kemungkinan. Primer dengan urutan basa semacam ini dinamakan primer degenerate. degenerate. Selain itu, primer yang disusun melalui penjajaran urutan basa DNA pun dapat merupakan primer degenerate karena degenerate karena urutan basa pada daerah lestari di tingkat DNA pun tidak selamanya memperlihatkan homologi sempurna (100%). Urutan basa pasangan primer yang telah disusun kemudian dianalisis menggunakan program komputer untuk mengetahui kemungkinan terjadinya primer-dimer akibat homologi sendiri(self-homology) sendiri(self-homology) atau homologi silang (cross-homology). (cross-homology). Selain itu, juga perlu dilihat kemungkinan terjadinya salah tempel(mispriming) tempel (mispriming),, yaitu penempelan primer di luar sekuens target. Analisis juga dilakukan untuk mengetahui titik leleh (T ( T m) masing-masing primer dan kandungan GC-nya. Sepasang primer yang baik harus mempunyai T m yang relatif sama dengan kandungan GC yang cukup tinggi. Pemilihan primer yang akan digunakan pada PCR harus memenuhi kriteria atau parameter tertentu agar primer berfungsi maksimal, parameter tersebut diantaranya adalah : 1. Panjang primer 18-24 nukleotida, jika kurang dari 15 akan menurunkan spesifitas hibridisasi dengan DNA target, jika lebih dari 40 akan menurunkan aktifitas DNA polymerase 2. Suhu leleh (Tm) primer 50-60°C dengan selisih Tm antara primer kurang dari 5°C 3. Urutan nukleotida yang sama tidak boleh lebih dari 4 karena akan menurunkan spesifitas 4. Kandungan GC sebaiknya 40-60% 5. Ujung 3’ kedua primer tidak saling berkomplemen Berikut adalah rumus untuk menghitung Tm dari primer : Tm = 4 (G+C) + 2 (A+T)
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RNA, RNA kemudian diterjemahkan untuk menghasilkan rantai asam amino alias protein. Dari sekian panjang DNA genome, bagian yang menyandikan protein inilah yang disebut gen, sisanya tidak menyandik an an protein atau disebut ‘junk DNA’, DNA ‘sampah’ yang fungsinya belum diketahui dengan baik. Kembali ke pembahasan isolasi gen, para ahli seringkali membutuhkan gen tertentu untuk diisolasi. Sebagai contoh, dulu kita harus mengekstrak insulin langsung dari pankreas sapi atau babi, kemudian menjadikannya obat diabetes, proses yang rumit dan tentu saja mahal serta memiliki efek samping karena insulin dari sapi atau babi tidak benar-benar sama dengan insulin manusia. Berkat teknologi rekayasa genetik, kini mereka dapat mengisolasi gen penghasil insulin dari DNA genome manusia, lalu menyisipkannya ke sel bakteri (dalam hal ini E. coli) agar bakteri dapat memproduksi insulin juga. Hasilnya insulin yang sama persis dengan yang dihasilkan dalam tubuh manusia, dan sekarang insulin tinggal diekstrak dari bakteri, lebih cepat, mudah, dan tentunya lebih murah ketimbang cara konvensional yang harus ‘mengorbankan’ sapi atau babi. Untuk mengisolasi gen, diperlukan DNA pencari atau dikenal dengan nama ‘probe’ yang memiliki urutan basa nukleotida sama dengan gen yang kita inginkan. Probe ini bisa dibuat dengan teknik PCR menggunakan primer yang sesuai dengan gen tersebut. b. DNA Sequencing
Urutan basa suatu DNA dapat ditentukan dengan teknik DNA Sequencing, metode yang umum digunakan saat ini adalah metode Sanger Sa nger (chain termination method) yang sudah dimodifikasi menggunakan dye-dideoxy terminator, dimana proses awalnya adalah reaksi PCR dengan pereaksi yang agak berbeda, yaitu hanya menggunakan satu primer (PCR biasa menggunakan 2 primer) dan adanya tambahan dideoxynucleotide yang dilabel fluorescent. Karena warna fluorescent untuk setiap basa berbeda, maka
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dengan DNA sidik jari keluarganya yang memiliki pertalian darah, misalnya ibu atau bapak kandung. Jika memiliki kecocokan yang sangat tinggi maka bisa dipastikan identitas orang yang dimaksud. Konon banyak kalangan tertentu yang memanfaatkan pengujian ini untuk menelusuri orang tua ‘sesungguhnya’ ‘sesungguhn ya’ dari seorang anak jika sang orang tua merasa ragu. d. Diagnosa Penyakit
Penyakit berbahaya seperti infeksi virus memerlukan diagnosa yang cepat dan akurat. PCR merupakan teknik yang sering digunakan. Teknologi saat ini memungkinkan diagnosa dalam hitungan jam dengan hasil akurat. Disebut akurat karena PCR dapat mengamplifikasi daerah tertentu DNA yang merupakan ciri khas virus penginfeksi yang tidak dimiliki oleh virus atau makhluk lainnya.
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BAB III METODOLOGI 3.1 Waktu dan Tempat
Praktikum dilakukan pada hari Jum’at tanggal 12 Mei 12 Mei 2017 pukul 07.00 – 09.40 09.40 WIB, bertempat di Ruang Kuliah 5 Fakultas Farmasi Universitas Jember. 3.2 Alat dan Bahan
Alat
:
Seperangkat lengkap komputer dan assesorinya yang terkoneksi dengan internet. Bahan : CDS gen dari organisme tertentu sebagai molekul DNA target. 3.3 Prosedur Kerja
1.
Mencari CDS gen target dari situs http://www.ncbi.nlm.nih.gov http://www.ncbi.nlm.nih.gov.. Dibuka situs http://www.ncbi.nlm.nih.gov http://www.ncbi.nlm.nih.gov ketikkan gen yang akan dicari sekuensnya pada kolom search. pilih nucleotide pada pilihan all databases. tekan tombol search pilih complete genom
klik Fasta, maka akan muncul tampilan yang menunjukkan CDS genome
2. Mencari Primer Buka kembali situs http://www.ncbi.nlm.nih.gov http://www.ncbi.nlm.nih.gov di tab yang berbeda
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3. Menentukan spesifitas primer Buka kembali situs http://www.ncbi.nlm.nih.gov http://www.ncbi.nlm.nih.gov di tab yang berbeda
Pilih BLAST
Pilih nucleotide BLAST Masukkan primer yang ingin dicek pada kolom enter accession number (s), gi (s), or FASTA sequence. Pilih nucleotide pada pilihan database, lalu klik BLAST
Tunggu Tunggu beberapa sat, akan diperoleh presentasi kesamannya kesamannya
3.4 Pengamatan
1. Mencari CDS gen target dari situs http://www.ncbi.nlm.nih.gov http://www.ncbi.nlm.nih.gov.. a. Buka laman tersebut
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c. tampilannya akan seperti berikut ini :
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d. Pilih homo sapiens. Tampilan sebagai berikut :
e. Klik FASTA, akan muncul tampilan seperti berikut :
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b. Klik DNA & RNA
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c. Klik Primer – Primer – BLAST. BLAST. Hasil tampilan Primer-BLAST :
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3. Menentukan spesifitas primer a. Buka kembali laman http://www.ncbi.nlm.nih.gov di tab yang berbeda
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Pilih nucleotide blast
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d. Masukkan primer yang ingin dicek pada kolom enter accession number(s), gi(s), or FASTA sequence. Pilih nucleotide pada pilihan database. Lalu klik BLAST.
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