Molecole Cellule Tessuti Organismi Sistemi Esseri viventi animali e umani BIOTECNOLOGIE

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1 Biotecnologie i e strumentazione t - 1 Le biotecnologie trattano i principi, i fenomeni e i processi per studiare la materia vivente e ricavare, dalla conoscenza, beni e prodotti utilizzabili anche a scopo commerciale. La strumentazione è di supporto a questa dinamica scientifico-tecnica. Molecole Cellule Tessuti Organismi Sistemi Esseri viventi animali e umani In questa lezione trattiamo solo strumentazione coinvolta in aspetti computazionali e bioinformatici relativi a tre molecole fondamentali, che costituiscono i blocchi fondamentali delle cellule e dei tessuti BIOTECNOLOGIE DNA RNA PROTEINE 42/68

2 Biotecnologie i e strumentazione t - 2 Sui pazienti si interviene quasi esclusivamente per riparare un danno acquisito (patologia). Su materia vivente, dalle molecole agli organismi, si interviene anche con lo scopo di trasformarne le caratteristiche per un loro miglioramento o adeguamento a particolari necessità. In questo caso si incontrano altri tipi di problematiche. Complessità dimensionale delle tre molecole - 1 Archivio d informazione posto 1. DNA ACGTTAGGCCTTA... TTAGCGGCA... TCCTTGAACGTAC...TGAATCGA... in una sequenza, in una sola caratteri totali Contenuto d informazione i del DNA: esempio dimensione, di 4 caratteri A, C, G, T, rappresentanti le basi del DNA, alfabeto sorgente. Consideriamo una sequenza di basi; esistono possibili combinazioni di A,C,G,T, tutte a priori equiprobabili. Tenendo conto che = , allora la probabilità di ciascuna è p= , e dalla definizione d informazione, ognuna contiene la quantità I d informazione I = log p = log 2 = bit 25Kbyte 2 2 = Elettronica: occorrono 4 nm 3 per DNA: la stringa di basi è lunga nm, con memorizzare 1 bit nella RAM; spessore di 2 nm e contiene 25KB d informazione; serve un volume di nm 3 per serve una superficie di 10 3 nm 2 per memorizzare 25KB memorizzare 25KB (NO bit errori) (spazio elettronica)/(spazio DNA) = !!! 43/68

3 Biotecnologie i e strumentazione t - 3 Complessità dimensionale delle tre molecole - 2 The relatively static nature and low dimensionality of DNA allow it to serve as a reference and anchor for most other biologic computing databases. 2. mrna 3. PROTEINE mrna (messanger RNA) and protein are more highly dimensional, with dynamic range, modifications, 3D patterns of expression and interactions with other mrnas and proteins dictating normal and pathologic conditions. Much of current and future efforts in biologic computing are toward recording changes of mrna and protein patterns as a function of a specific variable, with the goal of assembling changes into biologically relevant networks and pathways. The long-term goal is to understand and predict the responses of cells, tissues, and the patient to environmental and pathologic conditions; however, the very high dimensionality makes this a formidable task. 44/68

4 Biotecnologie i e strumentazione t - 4 Basi di Dati -1 Sono lo strumento fondamentale della Genomica e della Proteomica Campi per la scelta della specie e dei dati sul gene da indagare Link a genomi più frequentemente indagati 45/68

5 Biotecnologie i e strumentazione t - 5 Basi di Dati -2 DataBase pubblico Accesso al cariotipo umano: si indaga sui geni contenuti nei cromosomi Dettagli sui geni contenuti nel cromosoma 7 46/68

6 Biotecnologie i e strumentazione t - 6 La strumentazione usata in campo biotecnologico è molto diversa da quella utilizzata in campo biomedico. Trattiamo i quattro tipi di strumentazione più frequentemente utilizzata nelle biotecnologie Spettrometria di massa Elettroforesi Micro array Real Time PCR 47/68

7 Biotecnologie i e strumentazione t - 7 The complexity of protein networks is much larger than that of RNA: 20 amino acids as building blocks instead of 4 bases for RNA and DNA; once a protein is translated from a gene, a long list of variables influences activity and function of the protein (e.g. phosphorylation); folding of the protein dictates activity, as do interactions with additional copies of itself or other proteins; subcellular localization and local concentration of substrates and cofactors can dramatically influence protein function. Technical problems add complexity when trying to sequence the proteins amino acids string: It is considerably more difficult to purify and sequence proteins, compared to the cloning and automated sequencing technologies available for nucleic acids (DNA, RNA). Also, the exquisitely sensitive and specific sequence-specific hybridization used to query nucleic acids is not available for proteins. 48/68

8 Biotecnologie i e strumentazione t - 8 The fundamentals of proteomics are as follows: In Mass Spectrometry technique, ionized protein fragments (peptides) moving through a vacuum have a specific mass over charge (m/z) ratio. The secondary structure (amino acid sequence) of proteins can be predicted by techniques in silico (translation of genes - transcript units both known and predicted). A protein sequence can be fragmented in silico into predicted patterns of m/z fragments. Mass Spectrometry assumes major importance! 49/68

9 Biotecnologie i e strumentazione t - 9 MASS SPECTROMETRY ionization Scope: pick a single protein from a complex solution. Imparts charge to protein fragments, to be driven through space by electromagnetic forces within a vacuum. The two most frequently used methods matrix assisted laser desorption ionization (MALDI ) which analyzes samples as dry solids co-precipitated with an ultraviolet laser-absorbing matrix ti on a probe Electrospray ionization (ESI), a solution-based method sprayed through a narrow stainless steel capillary. Vacuum units that manipulate the charged polypeptides in such a way that the m/z ratio is directly related to time at detection by the detector. Most common mass analyzers: time-of-flight flight (TOF) where e the m/z ratio is related to a linear flight path from the ion source to the detector; ion traps and quadrupoles have a series of electromagnetic forces that modulate ion frequency in a nonlinear manner. Finally, the detector is where the charged particles are transduced into electrical signals via a photon diode and electron multiplier. 50/68

10 Biotecnologie i e strumentazione t - 10 GLI ALTRI STRUMENTI PER LE BIOTECNOLOGIE O OG ELETTROFORESI PCR MICRO ARRAY 51/68

11 ELETTROFORESI 1 - Glossario Biotecnologie i e strumentazione t - 11 Gel Electrophoresis is used to compare the similarity of DNA, PROTEIN, RNA Micropipet i instrument t used to measure millionths of fliters. Electrophoresis the use of electricity to pull apart molecules. Agarose a sugar used to make lab gel. Buffer solution of water and ions which help to conduct electricity. Gel box box designed to conduct electricity across/through a gel. Gel lab jello made from agar and buffer solutions Gels Purpose 1) To hold the samples 2) To help separate the samples based on charge and Gels Process size. 1) Pour gel 2) Load the samples 3) Cover the gel with buffer (The buffer helps to conduct electricity) 4) Connect power cords 5) Turn on the power source and set to 100 volts for 15 minutes. 52/68

12 ELETTROFORESI 2 (DNA) Enzymes molecules which cut DNA. DNA Electrophoresis steps 1) Cut DNA with enzymes (Restriction Enzymes) Biotecnologie i e strumentazione t ) Run the DNA 3) Identify the bands by comparison Practical Applications: Genetic Research, Crime Scene, Forensic Science, DNA is negatively charged. When placed in an electrical field, DNA will migrate toward the positive pole (anode). An agarose gel (porous) is used to slow the movement of DNA and separate by size. 53/68

13 Biotecnologie i e strumentazione t - 13 ELETTROFORESI 3 (DNA) DNA Comparing DNA using gel electrophoresis - large small + Restriction Enzymes 54/68

14 PCR: polymerase chain reaction -1 Biotecnologie i e strumentazione t - 14 Technique to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. PCR is a cyclic process controlled by the temperature of the reaction mixture. First, the temperature t is raised to 95 C, causing the template DNA to separate (denature). Second, the temperature is then decreased to around 50 C (122 F), allowing short primer pieces of DNA to hybridise to each strand of the template at opposite ends of the sequence to be amplified. 55/68

15 PCR: polymerase chain reaction -2 Biotecnologie e strumentazione - 15 Third, the thermostable enzyme Taq - Termophilus aquaticus (a DNA polymerase that is active at high temperatures) t then binds to and extends the primers at an intermediate temperature of 72 C (162 F), so that two new doublestranded sta dedcopeso copies of the etemplate pateare made. 72 C (162 F) This cyclic process of separating DNA strands, copying and reannealing the daughter strands is repeated multiple times to increase the numbers of DNA product. Amplicons 56/68

16 Real-time PCR - 1 Biotecnologie e strumentazione - 16 Real-time PCR using double-stranded DNA dyes A DNA-binding dye binds to all double-stranded (ds)dna in PCR, causing fluorescence of the dye. An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity and is measured at each cycle, thus allowing DNA concentrations to be quantified. However, dsdna dyes such as SYBR Green will bind to all dsdna PCR products, including nonspecific PCR products (such as Primer dimer. This can potentially interfere with or prevent accurate quantification of the intended target t sequence. 1. The reaction is prepared as usual, with the addition of fluorescent dsdna dye. 2. The reaction eacto is run in a thermocycler, e and after each cyce, cycle, the levels es of fluorescence are measured with a detector; the dye only fluoresces when bound to the dsdna (i.e., the PCR product). With reference to a standard dilution, the dsdna concentration in the PCR can be determined. Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production at each PCR cycle (in real time) as opposed to the endpoint detection. 57/68

17 Biotecnologie e strumentazione - 17 Real-time PCR /68

18 Biotecnologie e strumentazione - 18 Microarrays - 1 A microarray is a multiplex lab-on-a-chip. It is a 2D array on a solid substrate (usually a glass slide or silicon thin-film cell) that assays large amounts of biological material using high-throughput screening (statistical image analysis) methods. Types of microarrays include: DNA microarrays, such as cdna microarrays, oligonucleotide microarrays and SNP microarrays MMChips, for surveillance of microrna populations Protein microarrays Tissue microarrays Cellular microarrays (also called transfection microarrays) Chemical compound microarrays Antibody microarrays Carbohydrate arrays (glycoarrays) 59/68

19 Biotecnologie e strumentazione - 19 Microarrays - 2 Microarrays provide a platform for a genome-wide view of a biological sample. Microarray analysis makes use of the vast amounts of data that the microarray platform provides. La progettazione di questi dispositivi si basa su una conoscenza acquisita relativa alla corrispondenza nota tra molte sequenze di DNA e i corrispondenti geni. It is through the intelligent combination of mathematical algorithms and clinical validation that microarray analysis provides a real opportunity to realize the goal of targeted personalized medicine. The array is prepared on a small solid base such as a piece of glass or nylon divided into a grid of tiny squares. To each square is attached a different and specific piece of DNA, typically a short DNA sequence that can act as a probe for a particular gene. Every chip corresponds to a particular set of sequences. 60/68

20 Microarrays - 3 Biotecnologie e strumentazione - 20 A DNA "chip" or microarray is, generally speaking, a matrix of short oligonucleotide probes attached to a hard surface for the purpose of selectively hybridizing with DNA in a solution. Sequenza (target) da analizzare, viene distribuita sul chip in soluzione A DNA microarray is a multiplex technology used in molecular biology and in medicine. It consists of an arrayed series of thousands of microscopic spots of DNA oligonucleotides, called features, each containing picomoles of a specific DNA sequence, known as probes (or reporters). Target is the unknown sequence. Probe-target hybridization is usually detected and quantified by detection of fluorophore-, silver-, or chemiluminescence-labeled targets to determine relative abundance of nucleic acid sequences in the target. 61/68

21 Biotecnologie e strumentazione - 21 Microarrays - 4 Confronto tra sequenze The design, selection, length, construction, and attachment of these probes vary depending on experimental design, and can be very diverse depending on the application. Avviene l interazione tra probe e sequenza ignota. e la conseguente colorazione delle singole celle sulla base di opportuna attivazione della fluorescenza 62/68

22 Biotecnologie e strumentazione - 22 Microarrays - 5 Fare attenzione al singolo filamento, al cdna e al doppio filamento ibridizzato Example of an approximately 40,000 probe spotted oligo microarray with enlarged inset to show detail. This result is submitted to statistical image analysis 63/68