Aplikasi Klinis Aplikasi Klinis Biologi Molekuler Biologi Molekuler S. Yudha Patria S. Yudha Patria Pediatrics Dept, Fac. Medicine, Pediatrics Dept, Fac. Medicine, Gadjah Mada University Gadjah Mada University


Embed Size (px)

Citation preview

  • Aplikasi Klinis Biologi MolekulerS. Yudha PatriaPediatrics Dept, Fac. Medicine,Gadjah Mada University

  • Introduksi

  • Techniques Approach in Molecular Genetic StudiesPCR based classic PCR, RACE, SSCP, RT-PCR, insitu-PCR, qPCR,etc

    Cloning subcloning

    Enzym digestion RFLP, haplotyping

    Hybridization Southern, Northern, Western bloting

    Sequensing enzymatic, radioactive

  • Methods in Molecular Genetic StudiesMutation exploration and analysis of gene structr. PCR, Southern blot, enzyme digestion, RFLP, sequensing, homology analysis

    Gene expression analysis (gene function analysis) PCR of cDNA, Western blot, recombinant DNA, Transgenic mice, Regenerative medicine, etc

    Gene engineering recombinant DNA, cloning

  • Methods .

    Finger printing PCR, RFLP, enzyme digestion, sequensing, microsatelit analysis, etc

    Molecular genetic epidemiology Methods Variation analysis, Polymorphism analysis

    Gene therapy methods Cell therapy, Vector-mediated gene transfer, Lipid carrier systems, Antisense oligonucleotides, Cloning modification

  • How to enter molecular-biological research ?Pattern of inheritance ?Simple or Complex disease ?Gold standard clinical diagnosis ?Unknown or well-known mechanism ?What the purposes of research ?

  • An example case : DMDiabetes is a chronic metabolic disorder that is inability to manufacture and use insulin failed the conversion of food into energy.

    DM greatly increases the risk of blindness, heart disease, kidney failure, neurological disease, and other conditions

    Type 1, or juvenile onset diabetes, is the more severe form of the illness.

  • In Type 1 diabetes, the body's immune system assault on its own insulin and the pancreatic cells, however, the mechanism of how this happens is not yet understood.

    About 10 loci in the human genome have been found to confer susceptibility to IDDM

    Among these are: 1) a gene at the locus IDDM2 on chromosome 11 2) the gene for glucokinase (GCK), an enzyme of glucose metabolism which helps modulate insulin secretion, on chromosome 7.

  • At DM2 involving many genes(race, environment, lifestyle, etc)

    Calpain 10 NEUROT1 (2q32), HNF4A DM2 late onsetGLUT4, GLUT2GPD2dll

  • 2. ObesityObesity is an excess of body fat that known as risk factor for chronic diseases including heart disease, diabetes, high blood pressure, stroke and some forms of cancer

    The body weight that more than 25% body fat in a man and women with more than 30% are obese.

    Evidence, suggests that obesity has more than one cause: genetic, environmental, psychological and other factors may all play a part.

  • The hormone leptin, is act as a lipostat

    Most overweight people have high levels of leptin in their bloodstream.

  • Clinical Application

  • Molecular AnalysisCytogenetics

    DNA level

    mRNA level

    Expressivity (transcription, translation)

  • 1. Prevensi Polimorfisme variasi nukleotid tunggal / ulangan pada sekuens DNA

    Konsekuensi Polimorfisme- Netral (fenotipe normal): tidak menunjukkan perbedaan fenotip atau berbeda bentuk/struktur protein (isozym) yang baru tampak aktif pada kondisi tertentu (misalnya pada tekanan udara atau suhu atau pH tertentu)

    - Penyakit genetik multifaktorial seseorang mempunyai kecenderungan untuk mempunyai penyakit tertentu dipengaruhi oleh lingkungan tertentu

  • Contoh analisis polimorfisme

    Penentuan alel T235 gena Angiotensinogen Alel T235 sangat berhubungan dg tingginya kejadian hipertensi

    Alel T235 terletak pada nukleotid 704 ekson 2 gena angitensinogen transisi C T (Thr Met)

  • Teknik analisis alel T235 pada pre-eklamsiPCR ekson 2 + RE (Tth111-I GAC NNGTC)Prediksi hasil : 246 bp

    1 = AA 2 = Aa 3 = aa M 1 2 3

  • Frekuensi Alel

  • 2. Diagnosis mencari perbedaan susunan gena (mutasi)

    membandingkan susunan gena orang normal dan gena pasien

  • M C P C P C P C PExon 45 46 53 54M: Marker C: Normal control DNA P: Patient DNA DNA Analysis of Dystrophin Gene

  • Diagnosis Cytogenetic Analysis to see the chromosomal abnormality

  • FISH to see the specific chromosomal abnormality

  • Teknik Pemeriksaan RNASuryono Yudha PatriaPediatrics Dept, Fac. Medicine,Gadjah Mada University

  • Control of gene expression

  • Struktur RNA - DNA

  • Struktur RNA & DNA

  • Comparison with DNA

    RNA is a single-stranded molecule

    Much shorter chain of nucleotides

    Contains ribose (two hydroxyl groups attached to the pentose ring) hydroxyl groups make RNA less stable than DNA because it is more prone to hydrolysis.

  • Comparison with DNA Biologically active RNAs (tRNA, rRNA, snRNAs and other non-coding RNAs) are extensively base paired to form double stranded helices (secondary-, tertiary- structure)

    RNA can achieve chemical catalysis, like enzymes

    RNA function as active form of gene expression

  • Struktur sekunder RNA

  • Three major types of RNA

    messenger RNA (mRNA) has an information storage function, like DNA. Carries information from DNA to the ribosome sites of protein synthesis in the cell

    transfer RNA (tRNA) a small RNA chain of about 74-95 nucleotides that transfers a specific amino acid to a growing polypeptide chain at the ribosomal site

    ribosomal RNA (rRNA) rRNA and ribozymes have catalytic functions, like proteins, 80% of the RNA molecules found in a typical eukaryotic cell

  • tRNA

  • rRNA

  • Other types of RNA noncoding RNAA number of other types of RNA identified until now, present in smaller quanitites

    small nuclear RNA (snRNA)small nucleolar RNA (snoRNA) the 4.5S signal recognition particle (SRP) RNA

  • snRNA

  • RNA Synthesis (Transcription)Using DNA as a template Catalyzed by an enzyme - RNA polymeraseInitiation of synthesis begins with the binding of the enzyme to a promoter sequence in the DNA (usually found "upstream" of a gene)The DNA double helix is unwound by the helicase enzyme The progresses along the template strand in the 3 -> 5 direction, synthesizing a complementary RNA molecule with elongation occurring in the 5 -> 3 direction

  • Transcription mRNA maturation

  • Simpulan (from introduction)RNA is very similar to DNA, but differs in: nucleotides contain ribose sugars and RNA uses predominantly uracil instead of thymine present in DNA. RNA is transcribed (synthesized) from DNA by enzymes called RNA polymerases and further processed by other enzymes. RNA serves as the template for translation of genes into proteins, transferring amino acids to the ribosome to form proteins, and also translating the transcript into proteins.

    RNA is the only biological polymer that serves as both a catalyst (like proteins) and as information storage (like DNA)

  • Aplikasi Pemeriksaan RNA Penelitian biologi molekuler secara umum (PCR, kloning/subkloning, dsb) Diagnostic tool Genetic expression study Microarray application (to understand processes as diverse as the mechanisms of toxicity, embryogenesis and disease progression)

  • Contoh Aplikasi Pemeriksaan RNADNA LevelmRNA LevelM C PC P C P C P MExon 71 72 73 74Exon 65-74 74-79 70-75M:Marker C: Normal Control P:Patient

  • Contoh aplikasi pemeriksaan RNA Ekspresi genLymphoblast mRNA C P C P C P MM C PM: Marker C : Normal Control P: PatientMuscle mRNA

  • Isolasi RNAPengambilan materi RNA untuk keperluan pemeriksaan

    Isolasi RNA isolasi RNA total

  • Isolasi RNA.The successful isolation of intact RNA requires four steps: 1) effective disruption of cells or tissue 2) denaturation of nucleoprotein complexes 3) inactivation of endogenous ribonucleases (RNases) 4) removal of contaminating DNA and protein

    The most important step is the immediate inactivation of endogenous RNases that are released from membrane-bound organelles upon cell disruption

  • Contoh Langkah Isolasi RNAThe Maxwell 16 Total RNA Purification Kit combines the disruptive and protective properties of guanidine thiocyanate (GTC) to lyse samples, denature nucleoprotein complexes and inactivate ribonucleases.

    Adding -mercaptoethanol (BME) is strongly recommended for samples containing a high level of nucleases (e.g., spleen or pancreas tissue).

    The genomic DNA is removed from the sample lysate using the Clearing Agent.

    The total RNA is captured from the cleared sample lysate using MagneSil PMPs and is further purified from contaminating salts, proteins and cellular impurities by ethanol washes.

    Finally, total RNA is eluted in Nuclease-Free Water.

  • Evaluasi Hasil Isolasi RNA

  • RT-PCR (Reverse Transcriptase-PCR)Pengertian Konversi RNA DNA (cDNA)

    AlasannyaRNA tidak stabil/mudah rusakRNA membentuk struktur sekunder atau tersier

  • The Principle of RT-PCR

    An RNA could be converted into DNA, allowing manipulation and cloning

    Use of reverse transcriptases to perform first-strand cDNA synthesis

    Application of PCR to amplify DNA. It does not take long before first strand cDNA synthesis

  • Microarray

  • Microarray also known as gene or genome chip, DNA/RNA chip, or gene array

    is a collection of microscopic DNA spots, commonly representing single genes, arrayed on a solid surface by covalent attachment to chemically suitable matrices

  • How does DNA microarray technology work?

  • Skema cara kerja microarray

  • DNA microarrays are commonly used for:

    Expression profiling i.e., monitoring expression levels of thousands of genes simultaneously, or for comparative genomic hybridization

    Comparative genomic hybridization (Array CGH) Assessing large genomic rearrangements

    SNP detection arrays Looking for Single nucleotide polymorphism in the genome of populations

    Chromatin immunoprecipitation (chIP) studies Determining protein binding site occupancy throughout the genome, employing ChIP-on-chip technology

  • Aplikasi klinis yg lain

  • Transgenic Mice

  • 3. Analisis Hubungan Kekerabatan

  • 4. Therapy Some recombinant DNA products used in human therapyinsulin for diabetics factor VIII for males suffering from hemophilia A factor IX for hemophilia B human growth hormone (GH) erythropoietin (EPO) for treating anemia Interferons several interleukins granulocyte-macrophage colony-stimulating factor (GM-CSF) for stimulating the bone marrow after a bone marrow transplant tissue plasminogen activator (TPA) for dissolving blood clots adenosine deaminase (ADA) for treating some forms of severe combined immunodeficiency (SCID) angiostatin and endostatin for trials as anti-cancer drugs parathyroid hormone

  • Gene Therapy

    Modulation of gene expressionGene Replacement Therapy: 1. Cell-mediated gene transfer (cell therapy) 2. Vector-mediated gene transfer (gene therapy) 3. Combine techniques