Path: Top S2-Theses Biology 1998

Analisis Urutan Nukleotida 0,3 Kb Gen ND2 dan Gen COI
DNA Mitokondria (MtDNA) Manusia Indonesia

Nucleotide Sequences Analysis of 0.3 kilo-base fragments of ND2 and COI genes of Indonesian human mtDNA

Master Theses from JBPTITBPP / 2001-08-25 12:18:00
Oleh : I Wayan Suama, S2 - Biology (Adi@bi.itb.ac.id)
Dibuat : 2001-04-30, dengan 1 file

Keyword : Urutan nukleotida, DNA mitokondria, Manusia, ND2, COI, Homologi Sekuens, Substitusi nukleotida

Organisasi genom mtDNA manusia sangat sederhana (16.569 kb yang tersusun dalam 37 gen) dan memiliki keunikan tersendiri yang salah satunya ditunjukkan dengan tingkat polimorfisme yang tinggi. Berkaitan dengan hal tersebut, telah dilakukan penelitian mengenai analisis urutan nukleotida mtDNA manusia Indonesia normal khususnya pada posisi nukleotida 5.042-5.341 (0,3 kb gen ND2) dan posisi nukleotida 6.085-6.384 (0,3 kb gen COI) sebagai bagian dari penelitian hibah tim proyek URGE tentang “Normal Variant Data Base of the Indonesian Human Mitochondrial DNA and Translational Products” oleh Noer et al., (1999/2000). Permasalahan utama pada penelitian ini difokuskan pada bagaimanakah urutan nukleotida mtDNA manusia Indonesia normal khususnya pada posisi nukleotida 5.042-5.341 dan posisi nukleotida 6.085-6.384 bila dibandingkan dengan urutan nukleotida dari “Cambridge sequence” sebagai rujukan standar. Metode yang dikembangkan adalah melalui teknik lisis sel epitel rongga mulut tanpa melakukan isolasi mtDNA. Amplifikasi templat mtDNA dilakukan melalui PCR dengan menggunakan sepasang primer X1 dan X2 yang diikuti dengan aplikasi kloning ke dalam sel E.coli JM109 melalui vektor pGEM-T dan sekuensing dengan metode “dye terminator labeling”. Total basa hasil sekuensing dari 1,3 kb panjang fragmen PCR yang telah diklon adalah 600 basa untuk masing-masing sampel dari 9 sampel manusia Indonesia normal. Analisis hasil dilakukan dengan cara homologi melalui teknik “pairwise alignment sequence” dengan bantuan program komputer. Hasil yang diperoleh menunjukkan bahwa ada 11 substitusi nukleotida varian normal pada 10 posisi nukleotida polimorfik dibandingkan terhadap “Cambridge sequence”. Keseluruhan substitusi nukleotida varian normal tersebut merupakan varian baru yang belum pernah dipublikasikan oleh peneliti lain. 5 dari 11 substitusi nukleotida varian normal tersebut tergolong substitusi transisi dan 6 substitusi nukleotida varian normal yang lainnya tergolong substitusi transversi. Semua substitusi transversi plus 1 dari 5 substitusi transisi, tergolong substitusi “nonsynonymous” dan 4 substitusi transisi lainnya tergolong substitusi “synonymous” atau substitusi “silent”. Perkiraan rata-rata nilai “divergence” urutan nukleotida 0,3 kb gen ND2 dan 0,3 kb gen COI mtDNA manusia Indonesia normal masing-masing adalah sebesar 0,11% dan 0,73% lebih kecil dibandingkan terhadap perkiraan nilai “divergence” pada daerah D-loop mtDNA manusia Indonesia normal (Gaffar, 1998).

Deskripsi Alternatif :

Human mtDNA genome organization is very simple (16,569 kb with a total of 37 genes), however it is unique in having a high polymorphism compared to nuclear DNA. In this context, we have performed research work about nucleotide sequences analysis of Indonesian human mtDNA especially at nucleotide sites 5,042–5,341 (0.3 kb of ND2 gene) and at nucleotide sites 6,085–6,384 (0.3 kb of COI gene). The research is a part of URGE project about “Normal Variant Data Base of the Indonesian Human Mitochondrial DNA and Translational Products” by Noer et al., (1999/2000). The problem of the research has focused on how nucleotide sequences of Indonesian human mtDNA especially nucleotide sites 5,042–5,341 and 6,085–6,384 compared to the Cambridge sequence as a standard reference. A Method has been developed in this research for mouth epithelial cells lysis without mtDNA isolation. Amplification of mtDNA template by PCR with a pair primers of X1 and X2 was to performed amplify a 1.3 kb PCR fragment of mtDNA at nucleotide sites 5,042–6,384. Then the PCR fragment was cloned in to E.coli JM109 cells and sequenced with dye terminator labeling method. The total bases that were sequenced was 600 base of each sample from nine of normal Indonesian human. The results of homology analysis with pairwise aligned sequence were eleven substitutions of normal nucleotide variants in ten nucleotide polymorphic sites compared to the Cambridge sequence as a standard reference. All the substitutions were new variants that never published by other researchers. Five of them were transition substitutions and six of the others were transversion substitutions. All of transversion substitutions plus one of transition substitutions were nonsynonymous substitutions that caused seven variants of amino acid translational products and four transition substitutions of the others were synonymous substitutions or silent substitutions. Average divergence estimated were 0,11% and 0,73% for 0.3 kb of ND2 and 0.3 kb of COI genes, respectively. These were smaller than estimate value of divergence of D-loop of normal Indonesian human mtDNA (Gaffar, 1998).

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