12/11/2009. Analiza genelor. ADN mut ARNm mut Proteină an Caracter an. Transcripţie. Transcripţie. Translaţie. Translaţie.
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1 Metode de analiză a genelor Expresia genelor ADN ARNm Proteină Caracter 5 -ATTGCAAGATTACCATGT-3 Catena codogenă (netranscrisă) 3 -TAACGTTCTAATGGTACA-5 Catena anticodogenă (transcrisă) Transcripţie 5 -AUUGCAAGAUUACCAUGU-3 ARNm Translaţie Leu Ala Arg Leu Pro Cys Maturizare Caracter fenotipic normal polipeptid Expresia genelor ADN mut ARNm mut Proteină an Caracter an 5 -ATTACAAGATTACCATGT-3 mutaţie G 4 A Transcripţie 5 -AUUACAAGAUUACCAUGU-3 ARNm mut Translaţie Leu Thr Arg Leu Pro Cys Caracter fenotipic anormal polipeptid an ADN Secvenţiere PCR Analiza genelor ARN Northern-blot RT PCR Proteine Western-blot Secvenţiere Utilizarea tehnicilor de analiză a acizilor nucleici în medicină Analiza ADN: depistarea mutaţiilor genice diagnostic precis al bolilor genetice (prenatal, postnatal precoce); identificarea polimorfismului individual dactiloscopie genomică (analiza filiaţiei); Southern-blot Hibridare in situ Analiza funcţiei depistarea ADN străin (bacterian, viral) diagnosticul bolilor infecţioase şi gradului de infectare. Hibridare in situ Analiza histochimică Analiza ARNm: analiza expresiei genice ţesut-specifică; evaluarea gradului de expresie a genei normale / patologice. 1
2 Obţinerea FR fragmentelor de restricţie prin acţiunea specifică a ER (enzimelor de restricţie) RFLPs polimorfismul FR la două persoane X şi Z - 12 kb 10 kb 10 kb 8 kb 2,5 kb 10 kb 6 kb 5 kb 6 kb 5 kb 2,5 kb + M 6 kb 4 kb 2 kb Tehnici de analiză a genelor PCR Southern - blot Secvenţierea ADN Principii de bază Etapele principale Componente necesare (la general) Indicaţii şi limite PCR reacţia ciclică de polimerizare Principiile PCR clonarea in vitro = amplificare Replicare semiconservativă, repetată Specificitatea primerilor sintetici pentru gena ţintă Hibridizarea ADN-ţintă primer complementar: Denaturare termică a ADN - de cercetat Modificarea ciclică a temperaturii: Denaturare Renaturare Sinteză 2
3 Clonarea in vitro PCR (reacţia ciclică de polimerizare a noilor copii de ADN) Componentele necesare pentru clonarea in vitro (PCR): - ADN de cercetat - Primeri specifici secvenţei de clonat complimentari secvenţelor flancante - Taq-polimeraza ADN-polimerază termostabilă - Dezoxiribonucleozidtrifosfaţi (dntp) - Instalaţie de modificare ciclică a temperaturii: - tº de denaturare ADN - tº de renaturare (ADN+primer) - tº de polimerizare (elongarea catenelor noi de ADN) Etapele tehnicii PCR A. Pregătirea amestecului cu toate componentele reactante B. Repetarea automată a procedurilor de denaturarerenaturare-elongare de ori Denaturarea ADN la 96 o C Răcirea până la temperatura de renaturare a primerilor Elongarea catenelor ADN la 72 o C C. Analiza produşilor PCR - electroforeza - colorarea - interpretarea rezultatelor Extragerea ADN Pregătirea componentelor pentru PCR Amplificarea ADN-ţintă Electroforeza produşilor PCR Colorarea şi vizualizarea produşilor PCR Interpretarea rezultatelor 3
4 Avantajele PCR Metodă rapidă Metodă ieftină Utilizarea cantităţilor mici de material analizat Nu necesită izotopi radioactivi Interpretarea uşoară a rezultatelor Dezavantajele PCR Necesitatea cunoaşterii secvenţei nucleotidice amplificate Necesitatea primerilor sintetici, specifici secvenţei analizate Utilizarea PCR Identificarea inserţiilor/deleţiilor nucleotidice Identificarea mutaţiilor punctiforme (substituţiile nucleotidice) Identificarea expresiei genice (RT-PCR) Dactiloscopia genomică (filiaţie) Identificarea agenţilor infecţioşi Identificarea gradului de infecţie (quantitative PCR) Identificarea inserţiilor / deleţiilor Identificarea mutaţiilor punctiforme Identificarea agenţilor infecţioşi 4
5 Stabilirea paternităţii (filiaţiei) Tehnica Southern-blot Bazată pe RFLPs Hibridizare cu sonde marcate Cu transferul FR din gel-electroforeză pe un suport solid (pe membrane de nitroceluloză) Hibridarea acizilor nucleici Unirea complementară a fragmentelor (moleculelor) de acizi nucleici: ADN de cercetat cu sonda oligonucleotidică În reacţie participă molecule monocatenare Moleculele ADN-ţintă sunt fixate Moleculele-sonde sunt marcate radioactiv sau fluorescent Identificarea complexelor hibride se realizează prin intermediul autoradiografiei Tehnica Southern-blot Extragerea ADN Denaturarea şi transferul ADN pe membrane Restriţia ADN Hibridare cu sonda Electroforeza ADN Autoradiografia Vizualizarea şi interpretarea rezultatelor Utilizarea tehnicii Southern-blot Identificarea inserţiilor / deleţiilor mari Identificarea mutaţiilor punctiforme ce afectează SR Dactiloscopia genomică RFLPs RFLPs şi analiza genotipului Alela Normală cu 3 SR Alela mutantă cu 2 SR, mutaţie în SR2 C (mm) B (Nm) A (NN) Electroforeza FR a 3 persoane A,B şi C 5
6 Tehnica Northern-blot Extragerea ARN din ţesutul ţintă Electroforeza ARN în condiţii de denaturare Transfer pe membrană de nailon Hibridarea cu sonda marcată Autoradiografia Tehnica Northern-blot Utilizarea tehnicii Northern-blot Extragerea ARN Transferul ARN pe membrane Hibridare cu sonda Electroforeza ARN Autoradiografia Vizualizarea şi interpretarea rezultatelor Identificarea expresiei genice în anumite ţesuturi, anumite condiţii Identificarea variantei de splicing a proarnm Identificarea nivelului de expresie Identificarea agenţilor infecţioşi (ribovirusuri) Identificarea nivelului de expresie şi a variantei de splicing 6
7 Secvenţierea ADN Stabilirea secvenţei de nucleotide Metoda chimică (tehnica Maxam-Gilbert, 1973) Metoda dideoxi (tehnica Sanger, 1975) Metoda automată (cu utilizarea agenţilor fluorescenţi) ADN mut ARNm mut Proteină an Caracter an 5 -ATTACAAGATTACCATGT-3 mutaţie G 4 A Transcripţie 5 -AUUACAAGAUUACCAUGU-3 ARNm mut Translaţie Leu Thr Arg Leu Pro Cys polipeptid an Caracter fenotipic anormal 5 -ATTACAAGATTACCATGT-3 catena codogenă catena anticodogenă (matriţă) Tehnica dideoxi 5 -ATTAC-3 5 -ATTACA-3 5 -ATTACAA-3 5 -ATTACAAG-3 5 -ATTACAAGA-3 5 -ATTACAAGAT-3 5 -ATTACAAGATT-3 5 -ATTA -- primer marcat P 32 pentru ADN-polimerază A, T, C, T dntp (2 -dntp) pentru sinteza noilor catene A, T, C, T ddntp (2,3 -ddntp) pentru stoparea sintezei Sinteza enzimatică a ADN Utilizarea în paralel a nucleotidelor ce conţin deoxiriboză şi dideoxiriboză Stoparea sintezei în cazul incorporării ddntp A T G C 5 -ATTACA-3 5 -ATTACAA-3 5 -ATTACAAGA-3 5 -ATTACAAGATTA-3 5 -ATTACAAGATTACCA-3 5 -ATTACAAGAT-3 5 -ATTACAAGATT-3 5 -ATTACAAGATTACCAT-3 5 -ATTACAAGATTACCATGT-3 5 -ATTACAAG-3 5 -ATTACAAGATTACCAT GATTACCATG-3 5 -ATTAC-3 5 -ATTACAAGATTAC-3 5 -ATTACAAGATTACC-3 A T C G 5 -ATTACAAGATTACCATGT Extragerea ADN Denaturarea ADN Sinteza catenelor complementare utilizând primeri marcaţi cu 32 P în prezenţa dntp şi ddntp Electroforeza în condiţii de denaturare Vizualizarea fragmentelor marcate prin autoradiografie Tehnica dideoxi 7
8 - Metoda automată (+) 5' TAAATGGCTA...3'(-) + Pregătirea preparatelor de cromozomi Hibridarea in situ Hibridarea in situ pentru depistarea secvenţelor ADN Denaturarea ADN Hibridarea cu sonda marcată Vizualizarea la microscopul optic Hibridarea in situ cu utilizarea preparatelor de cromozomi metafazici Hibridarea in situ cu utilizarea preparatelor cu nuclei interfazici Hibridarea in situ pentru depistarea secvenţelor ARN Hibridarea in situ cu utilizarea probei sens (stânga) şi antisens (dreapta) Analiza histochimică identificarea proteinelor în ţesut 8
9 Rolul practic al tehnicilor de analiză a acizilor nucleici: 1. Cercetare secvenţierea ADN, formarea bibliotecilor de gene, studiul expresiei genice, studiul mecanismelor de reglare, studiul consecinţelor mutaţiilor. 2. Depistarea persoanelor purtătoare de mutaţii patologice - prenatal sau postnatal, cu scop de prevenire a naşterii copiilor cu anomalii genetice sau manifestării unor patologii severe. 3. Terapie genică (alegerea genei ţintă, construirea vectorilor de gene etc.). 4. Depistarea ADN străin (viral sau bacterian) în diagnosticul bolilor infecţioase. 5. Identificarea polimorfismelor individuale în analiza filiaţiei, în criminalistică. 9
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