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TRANSKRIPTOMIK, PROTEOMIK DAN METABOLOMIK DR. REFLI, MSc Jurusan Biologi FST Universitas Nusa Cendan
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GENOM
• Mengadung informasi genetika –Infromasi genetika pada • organisme eukariotik
DNA
• Sebagian virus mengandung informasi genetik
RNA
GENOM ; khusus pada manusia
• Genom pada manusia terdiri atas ; –Genom Nukleus –Genom Mitokondria Mitokondria
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GENOM ; khusus pada manusia • Genom Nukleus; – pada manusia: – tersusun sekitar 3,2 x 109 nukleotida yang terkandung dalam 24 untaian DNA
– setiap untaian terkandung dalam kromosom yang berbeda • 46 kromosom : 44 autosom dan 2 genosom (X dan Y)
– Sekitar 35.000 gen terdapat di genom nukleus
GENOM ; khusus pada manusia
• Genom Mitokondria; – Merupakan untaian DNA berbentuk cincin
– tersusun sekitar 16.569 nukleotida – Mengandung sekitar 37 gen
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EKSPRESI GENOM • Genom; kumpulan informasi genetik yang terkandung di dalam tubuh suatu organisme
• Informasi genetik (genom) tersebut dapat diekspresi melalui tahapan reaksi secara berurutan; – Transkripsi produk transkripsi (Transkriptome); RNA
– Translasi
produk translasi (proteome); protein
TAHAPAN EKSPRESI GENOM
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Mengukur Ekspresi genom RNA
Microarrays Monitors the level of each gene: Is it turned on or off in a particular biological condition? Is this on/off state different between two biological conditions? Microarray is a rectangular grid of spots printed on a glass microscope slide, where each spot contains DNA for a different gene
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Two-color DNA microarray design
Reverse Transcription
cDNA-chip of brain glioblastoma
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Types of microarrays •
Spotted (cDNA) – Robotic transfer of cDNA clones or PCR products – Spotting on nylon membranes or glass slides coated with poly-lysine
•
Synthetic (oligo) – Direct oligo synthesis on solid microarray substrate – Uses photolithography (Affymetrix) or ink-jet printing (Agilent)
•
All configurations assume the DNA on the array is in excess of the hybridized sample—thus the kinetics are linear and the spot intensity reflects that amount of hybridized sample.
•
Labeling can be radioactive, fluorescent (one-color), or two-color
Microarray Spotter
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Affymetrix High Density Arrays
Microarrays (continued)
Imaging – Radioactive 32P labeling: Autoradiography or phosphorimager – Fluorescent labeling: Confocal microscope (invented by Marvin Minsky!!)
Feature density – Nylon membrane macroarrays 100-1000 features – Glass slide spotted array 5,000 features / cm2 – Synthesized arrays 50,000 features / cm 2
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• Collects sharply defined optical sections from which 3D renderings can be created • The key is spatial filtering to eliminate out-of-focus light or glare in specimens whose thickness exceeds the immediate plane of focus. • Two lasers for excitation • Two color scan in less than 10 minutes • High resolution, 10 micron pixel size
Microarray confocal scanner
cDNA / EST sequencing projects • cDNA = complementary or copy DNA • EST = Expressed Sequence Tag • The microarray could be described as a “closed system” because information about RNAs is limited by the targets available for hybridization. RNAs not represented on the array are not interrogated. • Direct sequencing of cDNAs (yielding ESTs) overcomes this problem by large-scale random sampling of sequences from a whole-cell RNA extract • Statistical counting of distinct sequences provides an estimate of expression level • Conversely, cDNA library can be normalized to capture rare messages • Requires large scale sequencing to get statistical significance
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cDNA / EST Sequencing: Preparation of a cDNA library in phage l vector
SAGE Technology
Serial Analysis of Gene Expression >50X more efficient than cDNA sequencing because many RNAs are represented in a single sequencing run
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Steps to SAGE • Copy mRNA ds cDNA using biotinylated (dT) • Cleave with anchoring enzyme (AE) which cleaves • • • • •
within ~250bp of poly-A tail at 3’ end. Capture this segment on streptavidin beads Ligate to linkers containing a type IIs restriction site, which cleave DNA 14 bp away from this site. Ligate sequences to each other and PCR amplify Cleave with AE to remove linkers Concatenate, clone, and sequence
Velculescu et al. Science (1995)
WHY DI-TAGS?
Ditags are used to detect bias in the PCR amplification step.
B B B
A A A
PrimerA
PrimerA
The probability of any two tags being coupled in the same ditag is small.
PrimerB
PrimerB
Biased amplification can be detected as many ditags always having the same 2 tags present.
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SAGE (continued) Example of a concatemer:
CATGACCCACGAGCAGGGTACGATGATACATGGAAACCTATGCACCTTGGGTAGCACATG TAG1
TAG2
TAG3
TAG4
Tag Sequence Tag Sequence
Counting the tags:
Count
Count
GC GATATTGT
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ATC TG AGTTC
10 75
TACGTTTCCA
66
GCGCAGA CTT
125
TCCCGTACAT
66
TCCC CGTACA
112
TCCCTATTAA
66
TAGGAC GAGG
92
GGATCAC AAT
55
GCGATGGCGG
91
AAGGTTCTGG
54
TAGCCCAGAT
83
CAGAAC CGCG
50
GCCTTGTTTA
80
GGAC CGCCC C
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PROTEOMIC
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Pengertian • Proteomic; Ilmu yang mengkaji protein secara keseluruhan dalam sel atau organisme; plasma, sel dan jaringan • Proteome; kumpulan protein yang terkandung di dalam organisme; contoh hemeglobin dan insulin
Fungsi Proteomik • Mengkaji modifikasi pada akhir translasi protein • Indentifikasi target protein baru untuk obat • Analisis tumor atau kanker • Membandingkan antara jaringan normal dan jaringan sakit • Membandingkan antara jaringan sakit dan jaringan yang mendapatkan pengobatan
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Bidang Kijang Proteomik • Area proteomik dibagi ke dalam dua kajian utama; – Proteomik fungsional • Mengidentifikasi protein yang belum dikeltahui fungsi biologinya di dalam tubuh
– Proteomik ekspresi • Mengkaji secara kuantitatif dan kualitatif tingkat ekpresi protein yang dipengaruhi oleh faktor internal atau eksternal tubuh; penyakit seluler, fase pertumbuhan, stres
GENOM & PROTEOME
Genom antara larva dan kupu dewasa indek namun proteiom berbeda pada ke duanya
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GENOM & PROTEOME
Genom antara Berudu dan katak dewasa indek namun proteiom berbeda pada ke duanya
Pemahaman yang baik tentang Proteome akan memudahkan untuk
• Karakterisasi jenis protein • Mengetahui pola interaksi antar protein atau protein dengan non protein • Mengindentifikasi biomarka suatu penyakit
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Biomarka • Indikator suatu penyakit • Berfungsi untuk diagnosis, prognosis dan mengetahui respons tubuh terhadap terapi • Mengindentifikasi biomarka suatu penyakit
Biomarka
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Biomarka
Alir Kerja Proteomik
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Tehnik Proteomik; 2D electrophoreses
Tehnik Proteomik; 2D electrophoreses
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Tehnik Proteomik; 2D electrophoreses
Tehnik Proteomik; Mass Spectrometry
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Tehnik Proteomik; Micro Array
METABOLOMIC
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Pengertian • Metabolomic ; kajian secara simultan dan komprehensif tentang senyawa metabolit yang dihasilkan akibat pengaruh stimulus
• Metabolome; kumpulan senyawa metabolit yang berukuran kecil
yang bersifat
dinamis
• Metabolit; produk metabolisme – Metabolit primer; asam amino, monosakarrida, asam lemak, mononukleotida, polipeptida, polisakarida, lipida da polinuklotida – Metabolit sekunder; plavonoid, terpenoid, alkaloid dan steroid
Empat hal utama dalam Analisis metabolome
• Efesiensi • Pemisahan metabolit • Deteksi • Identifikasi dan quantifikasi
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Tehnik Pemisahan & Deteksi Metabolome
• Tehnik pemisahan –Gas Kromatografi –Elektroforesis kapiler
–HPLC –UPLC
Tehnik Pemisahan & Deteksi Metabolome
• Tehnik deteksi – NMR (Nuclear Magnetic Resonance Spectroscopy ) – Spektrometetri massa
• Tehnik pemisahan-deteksi – GC-MS – HPLC-MS
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Aplikasi metabolomic
• Evaluasi obat • Toksikologi klinik • Nutrigenomic • Genom fungsional
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Resea metabolomic
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