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