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Teknikat molekulare t ё k ё rkimit

Teknikat molekulare t ё k ё rkimit. Teknikat m ё t ё p ё rdorura n ё nivel molekular q ё p ё rdoren n ё k ё rkime t ё bioteknologjis ё molekulare e t ё aplikuar : Sinteza kimike e ADN-s ё Teknikat e p ё rcaktimit t ё renditjes s ё nukleotideve t ё ADN-s ё ( sekuencimit )

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Teknikat molekulare t ё k ё rkimit

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  1. Teknikatmolekulare tё kёrkimit • Teknikat mё tё pёrdorura nёnivelmolekular qё pёrdoren nё kёrkime tёbioteknologjisёmolekulare e tёaplikuar: • Sintezakimike e ADN-sё • Teknikat e pёrcaktimit tёrenditjes sёnukleotideve tё ADN-sё (sekuencimit) • Teknika e PCR-sё (Polymerase Chain Reaction) • Metoda e fitimit tёantitrupavemonoklonalёsidhe pёrdorimet e tyre teknikatmolekulare tё kёrkimit

  2. Sintezakimike e ADN-sё - in vitro • Zinxhirёt polinukleotidikё, qёsintetizohenmund tё pёrdoren pёr qёllime tёshumta: • Pёr tёformuargjene tёplotaapofragmentetёtyre • Pёr tёamplifikuarfragmente tёcaktuara ADN-je • Pёr tёshkaktuarmutacionetё ADN- ja e izoluarparaprakisht • Si sonda pёr hibridizimnin e ADN-sё • Ku kryhetsintezakimike e oligonukleotideve? • Nёaparatura tёkompjuterizuara SINTETIZATORE automatik tё ADN-sё • E pёrbёrёnganjё sёrёpompashdhevalvulashqёprogramohen pёr tёfutur nё mёnyrёprecize tёgjithёreaktivёt kimikdhenukleotidet qёzgjasinzinxhirinoligoinukleotidik. teknikatmolekulare tё kёrkimit

  3. Sintezakimike e ADN-sё • Sinteza in vitro kryhet nёdrejtimin 3’-5’, cdo hap dhekohёzgjatjakontrollohen nё mёnyrёautomatike . • Metoda mё e njohur pёr sintetiziminkimikёshtёajo e fosforamiditit • Nucleoside phosphoramidites are derivatives of natural or synthetic nucleosides. They are used to synthesise short nucleic acid chains. • Nucleosides are glycosylamines consisting of a nucleobase (often referred to as simply base) bound to a ribose or deoxyribosesugar via a beta-glycosidic linkage. Examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine and inosine. teknikatmolekulare tё kёrkimit

  4. Sintezakimike e ADN-sё SINTETIZATORI OSE SINTIZAJZER teknikatmolekulare tё kёrkimit

  5. teknikat molekulare tё kёrkimit

  6. Sintezakimike e ADN-sё • Para fillimit tёreaksionit tёgjithanukleozidettrajtohenduke zёvendёsuaraminogrupet e bazave tёtyre me grupebenzoile, qёmos tёvije nёreaksione tё padёshiruara • Nukleozidi i parё qё do pёrbёjёskajin 3’ tёzinxhiritsintetizuesfillimishtlidhet me njёsuport tёngurtё (sferaxhami) (fiksim), me ndёrmjetёsin e njёgrupi qёquhetspejser. • Ndёrsa nёskajin 5’ tё kёtijnukleozidilidhetnjёgrup DMT (DIMETOKSITRITIL)., pёr tёmoslejuar qёky (nukleozidi) tёhyjё nёlidhje. Kykomponimquhetedhekomleksinismёtar i sintezёs kimike. teknikatmolekulare tё kёrkimit

  7. Adenozina → benzoil group teknikat molekulare tё kёrkimit

  8. Sintezakimike e ADN-sё • Cdonukleotidiri qё do ishtohetkompleksitfillestarmodifikohet duke lidhur nёskajin 5’ njёgrup DMT ndёrsa nё atё 3’ njёgrupdiizopropilamin (kripelitiumi). • Diisopropylamine is a secondary amine with the chemical formula (CH3)2HC-NH-CH(CH3)2 • Kjostrukturё e nukleotideve qёshtohen nёreaksionquhetnukleosid FOSFORAMIDIT, ngaemri I metodёs. • Procesi I zgjatjesbazohet nёalternimin e largimit tё DMT DIMETOKSITRITIL, duke lёnё tёlirёskajin 5’ dheshtimin e nukleozideve tёreja nё formёn e fosforamiditit. • Ngareaksione tёnjёpasnjёshme tё kёtijllojiformohetzinxhirii plotёi kёrkuarsipassekuencёs sё kёrkuarparaprakisht teknikatmolekulare tё kёrkimit

  9. The 5'-hydroxyl group is protected by an acid-labile DMT (dimethoxytrityl) group. cytidine, adenosine, guanosine, thymidine Bz (benzoyl) protection is used for A, dA, C, and dC, and G and dG are protected ёithisobutyrylgroup Ac (acetyl) group is often used to protect C and dC as shoёn in Figure teknikatmolekulare tё kёrkimit

  10. Metoda e FOSFORAMIDIT Largimi I DMT nukleozid Largimiidiizopropilaminitngafosforamiditi Orientimi 3’ 5’ fosforamiditi teknikatmolekulare tё kёrkimit

  11. Reaksionicapingdheoksidimit – nёnkuptonlargimin e grupevemetile qёjanё tёlidhura nёgrupinfosfat, largimi I grupevembrojtёse tёgrupitaminikdhelargimi I DMT-sё • Pas kёtyrereaksioneveologonukleotidetpurifikohendhe pёrdoren pёr qёllime e paracaktuara. APLIKIMI I OLIGONUKLEOTIDEVE Nё BIOTEKNOLOGJI teknikatmolekulare tё kёrkimit

  12. APLIKIMI/RёNDёSIA E OLIGONUKLEOTIDEVE Nё BIOTEKNOLOGJI • Ologonukleotidet (having feё, having little) qёsintetizohen nё mёnyrёsintetike pёrdorensi: • Sonda pёr eksperimentimin e hibridizimit tё ADN-sё • Si sekuencaprajmere pёr sekuencimin e ADN-sё • Pёr tёndryshuarrenditjen e gjeneve tёklonuara • Small interfering RNA teknikatmolekulare tё kёrkimit

  13. Produktet e sintezёs kimike tё ADN-sё ????? • Oligonukleotide me zinxhirё tёnjёfishtёprej 40-60 bazash, mund tёsintetizohenbazuar nёrenditjen e njёaminoacidi • Zinxhirё tёdyfishtё tё ADN-sё linker, (sekuencepalindromike qёmund tёmbyllet nёvetevete, me pozicionerestriktive, mund tё pёrdorensilidhёs me vektorё • Oligomere tё pёrbёrёr prej 17-24 nukleotidesh qёmund tё pёrdorensiprajmerё pёr reaksionet e sekuencimit tё ADN • Oligomerё me zinxhirё tёnjwfishtёmund tё pёrdoren pёr mutagjenzёn “in vitro” teknikatmolekulare tё kёrkimit

  14. Sinteza e gjeneve • Sinteza e gjenevenga 60-80 cb, fillimishtvargjetkomplementaresintetizohen nё formёn e segmenteve pёrbёrёse prejnjёzinxhiripor qёjanё tёmbivendoshёm njёrimbitjerin, ndёrhyhet me T4 ligazё • Gjenet mё tё mёdhanga 300 cb, kanёgabimegjatёsintetizimit in vitro, pёrdoretnjёstrategjitjetёr: edhe kёto formohenzinxhirёt e gjatё tёnjwfishtё POR FRAGMENTE JOTёPLOTA, kurkomlementohen me njёritjetrinngelinvende qёnukmbulojnё tёgjithёgjatёsine e dyvargjeve, NDёRHYHET ME ADN POLIMERAZё-I, qё “mbushё” hapёsirat e njefishta, ndёrsa T4 ligaza, ilidhёzonat e kёputura, kyprocesquhetasemblim in vitro teknikatmolekulare tё kёrkimit

  15. teknikatmolekulare tё kёrkimit

  16. Teknikat e sekuencimit tё ADN-sё • The term DNA sequencing refers to sequencing methods for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA. • Shfrytёzohet nё: biotechnology, forensic biology and biological systematics • Teknika mё e njohurёshtёMaxamdhe Gilbert (1976) dhemetodasipas Sanger • Sipas MG, zinxhirit tё ADN-sё qё kёrkohetrenditjashёnohet nё mёnyrёradioaktive nёskajin 5’, mё pas ADN-jatrajtohetkimisht nё mёnyrё qё tёprishenlidhjet midis bazave. teknikatmolekulare tё kёrkimit

  17. Teknikat e sekuencimit tё ADN-sё • Krijohenkushte tёtilla qё nёkatёr reaksione tёndryshme pёr, A,G, C,T tёnxitetprerja nёnjёrёn ngabazat. • Pas elektroforezёs nё 4 shirita, vec pёr cdoreaksiondhe pas autoradiografisё , mund tё pёrcaktohetrenditja e bazave tёzinxhirit tёanalizuar nёdrejtimin 3’-5’ teknikatmolekulare tё kёrkimit

  18. chain-terminator method, Sanger • Because the chain-terminator method (or Sanger method after its developer Frederick Sanger) is more efficient and uses feёer toxic chemicals and loёer amounts of radioactivity than the method of Maxam and Gilbert, it rapidly became the method of choice. The key principle of the Sanger method ёas the use of dideoxynucleotidetriphosphates (ddNTPs) as DNA chain terminators • Dideoksinukleotidetjanёnukleotide tёsintetizura nёrrugёkimikekumungojnёkarbonet 2’ dhe 3’ tёpentozёs teknikatmolekulare tё kёrkimit

  19. teknikatmolekulare tё kёrkimit The classical chain-termination method requires a single-stranded DNA template, a DNA primer, a DNA polymerase, radioactively or fluorescently labeled nucleotides, and modified nucleotides that terminate DNA strand elongation. The DNA sample is divided into four separate sequencing reactions, containing all four of the standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase. To each reaction is added only one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP) ёhich are the chain-terminating nucleotides, lacking a 3'-OH group required for the formation of a phosphodiester bond betёeen tёo nucleotides, thus terminating DNA strand extension and resulting in DNA fragments of varying length.

  20. The neёly synthesized and labeled DNA fragments are heat denatured, and separated by size (ёith a resolution of just one nucleotide) by gel electrophoresis on a denaturing polyacrylamide-urea gel ёith each of the four reactions run in one of four individual lanes (lanes A, T, G, C); • the DNA bands are then visualized by autoradiography or UV light, and the DNA sequence can be directly read off the X-ray film or gel image. • In the image on the right, X-ray film ёas exposed to the gel, and the dark bands correspond to DNA fragments of different lengths. A dark band in a lane indicates a DNA fragment that is the result of chain termination after incorporation of a dideoxynucleotide (ddATP, ddGTP, ddCTP, or ddTTP). The relative positions of the different bands among the four lanes are then used to read (from bottom to top) the DNA sequence teknikatmolekulare tё kёrkimit

  21. teknikatmolekulare tё kёrkimit

  22. Sintezakimike e AND-sё DNA SEKUENCATORI teknikatmolekulare tё kёrkimit

  23. Metoda e dideoksinukleotideve(Sanger) • Prasinteza do tёndalet nёnivelin e cdobaze tёatijlloji, • Prapritet qё p.sh nёtubin e reaksionit pёr adeninёn do formohenzinxhirё me gjatёsiprogresivesinteza e tёcilёvendalet nёcdobazёadeninё qёajo has. • Ndahetzinxhiri I dyfishte me FORMAMID, dhe mё pas nёzhelpolakrilamid (aftesi tёlartёfraksionuesedhjetrabaza), dhebandat bёhen tёdukshmeose me antё tёradiografisёose me anё tёngjyrosjesspecifike. • Sot, nёmetodёn Sanger shfrytёzohetshenuesfluoreshent nё vend radioaktivё, pёr cdobazё. Kjo ka bёrё tёmundurautomatizimin e kёsajmetode. teknikatmolekulare tё kёrkimit

  24. Metoda e dideoksinukleotideve(Sanger) • Dideoksinukleotidetjanёnukleotide tёsintetizura nёrrugёkimikekumungpjnёkarbonet 2’ dhe 3’ tёpentozёs • Dezoksiribonukleotidetnatyralegrupinhidroksil e kanё nёpozcionin 3’ (kujto 5’-3’), pragrupi 3’OH ёshtёgrupireaktiv qёmerpjesё nёlidhjen midis nukleotideve nёsintesёn normale tё AND-sёin vivo. Kjo do tёthotё se nёsёinkoroporohetnjёdideoksinukleotid , zinxhirinukёshtё mё nёgjёndje tёzgjatet. • Nё kёtёmetodёfillimishtndahenzinxhirёt, njёringavargjet I nёnshtrohetreaksioneve tёsekuencimit. • Fillimisht, nёzinxhirin e reaksionevenjёritvarg (zinxhirittanjfishtё) tё ADN-sёI shtohetnjёoligonukleotid qё do tёshёrbejёsiprajmer pёr tёvazhduarsintetizimin. teknikatmolekulare tё kёrkimit

  25. Metoda e dideoksinukleotidevechain termination (Sanger) • Nёcdo tub reaksionishtohen tёgjithadezoksirbonukleotidtrifosfatet (dNTP), dATP, dTTP,dCTP, dhedGTP, ku vetёm njёriёshtё I shёnuar nёsecilinprejreaksioneve me lёndёradioaktiveosejoradioaktive, dhe nёsecilin tub ne vecantishtohetnganjёriprejdideoksiribonukleotidtrifosfateveddATP, ddTTP, ddGTP, ddCTP, nё pёrqёndrime tёtilla qё tёmund tёinkorporohen nёzinxhirin nёrritje nё tёgjithavendet e mundshme pёr to. • Pas reaksioneve nёsecilinprejtubave nёprani tё ADN-polimerazёs, do tёndodhёprocesiizgjatjes sёzinxhirёve tёrinjderisareaksioniinkorporonnje dd. • PёRPARёSI: Sekuencohetedhe ARN, revesrztranskriptaza pёr tёkthyer ne cDNAkomplementare., mё pas behetsekuencimisi mёlartё. teknikatmolekulare tё kёrkimit

  26. Sekuencimi I fragementeve mё tё mёdha • Me metodёn: • Pёrdorimi I fagut M13, sekuencohenderi nё 500 cb, • Dhe primer ёalking, disamijёra CB , prajmer qёecin, sintetizohenprajmer qёdrejtojnёsintezёn nё 200-250 nukleotide. teknikatmolekulare tё kёrkimit

  27. ReaksionizinxhiripolimerazёsPCR – polymerase chain reaction • Metoda e reaksionitzinxhirё tёpolimerazёs, pёrdoret pёr amplifikimin (shumimin) e fragmenteve tёinteresit tё ADN-sё, deri nёmiliona kopje in vitro . • Zbuluesii kёsajmetodeKary Mullis, fituesiçmimit Nobёl me dr. Micheal Smith, nёkiminё e acidevenukleike. • Parakushtikryesor pёr shumimin e fragmentit tё ADN-sёёshtёnjohja e sёpakunjёpjese tёvogёl tёsekuencёs nukleotide qёmundёson sintezёn e sekuencaveoligonukleotidevekomplementarenjёzinxhirore, pra sic njihenndryshe “primer” me 3OH fund. teknikatmolekulare tё kёrkimit

  28. ReaksionizinxhiripolimerazёsPCR – polymerase chain reaction • Çfarduhet pёr reaksioninfillestar tё PCR-sё? • Molekulё e ADN-sё me njёgjenosesekuencёn target tёcilёn duam tёamplifikojmё • Tёkatёr deoksiribonukleotidtrifosfatet (dATP, dGTP, dCTP, dTTP) • Sekuencatfillestare tёoligonukleotideve (prajmerёt) • dhe ADN polimeraza (zakonishtTaqpolimeraza) teknikatmolekulare tё kёrkimit

  29. teknikatmolekulare tё kёrkimit

  30. ReaksionizinxhiripolimerazёsPCR – polymerase chain reactionpёrvojangapuna e magjistraturёs Njё pёrzierje e pёr PCR nё 25 uLkonsiston nё: • QH2O 15,5 μL • Master mix 8,0 μL (5 x puffer TaqMaster, 10 pufer, dNTP (10mM) • Primer ngasecili 0,5 μL • ADN nga 0.5 μL • PolimerzaTaq 0.5 μL teknikatmolekulare tё kёrkimit

  31. ReaksionizinxhiripolimerazёsPCR – polymerase chain reaction GeneAmp PCR System 2400 (Applied Biosysytems) teknikatmolekulare tё kёrkimit

  32. Reaksionizinxhiripolimerazёssiduketnjё program i PCR-sё ? t° denaturimit t° e elongacionit t° e hibridizimit teknikatmolekulare tё kёrkimit

  33. Pёrdorimii PCR-sё • Pёr detektimin e sasive tёvogla tё AND-sё nёkampionesifloku, sasishumё tёvoglagjaku, kockashumё tёvjetra, pjesёindesh tёdegjeneruara, nёdiagnostifikimin e sёmundjevegjentike, identifikimin e mutacioneve, detektimi I baktereve, viruseve, nёindustrinёushёimore, sondavemolekulare , revolucion nёbilogjinёeksperimentale • These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases. teknikatmolekulare tё kёrkimit

  34. Second part next week, • Second part next week teknikat molekulare tё kёrkimit

  35. Antitrupatmonoklonalё • Monoclonal antibodies (mAb or moAb) are monospecific antibodies (proteinaspecifike) that are the same because they are made by identical immune cells that are all clones of a unique parent cell. • Si formohen nёrrugё in vitro: qelizat e mielomёs sёmiut (tumorale qёshumohen nё mёnyrё tёpakontrolluar, pornukprodhojnёantitrupa) u shkrinё me qelizanormale tёshpretkёs tёmarranganjё mi iimunizuarndajnjёantigjeni tё dhёnё. Kёto tёfunditprodhojnёantitrupatspecifikndajantigjenitspecifik, pornukritenshpejt. • Ngafuzionimi e kёtyreqelizave u fituanqelizahibridesomatike (HIBRIDOMAT) kuishin tёkombinuaracilёsitёpozitive tё tёdyjaqelizave. teknikatmolekulare tё kёrkimit

  36. Formimiihibridomavedheantitrupavemonoklonaledhe pёrzgjedhja me HGPRT-hipoksantine-guaninё-fosforibozil-transferaza) (hipoksantinё-aminopterine-timidinё) teknikatmolekulare tё kёrkimit

  37. Si identifikohenhibridёt e dёshiruar • Detektimiiantitrupave tёcliruarngaqelizatprodhuese • Lidhja e antitrupit me antigjenin me tёcilinёshtёinokuluarkafshangajanёnxjerrёqelizatfillestare, • Lidhjaantigjen-antitrupёshtё e qёndrueshmedhe pas lidhjes me njёantitrupsekondarvihet nёdukjeprania e antitrupit tёdeshiruar. Antitrupisekondarlidhet mёnjёenzimё, qёshndronnjёsubstrat pa ngjyrё nёsubstrat tёngjyrosur. Praklonet qёshfaqinsubstrta tёngjyrosurjanёlinjathibrideantitrupprodhuese tё dёshiruara. teknikatmolekulare tё kёrkimit

  38. Pёrdorimiiantitrupavemonoklonalё 1. Pёrdoren pёr ndarjen e interferoneve (Interferons (IFNs) are proteins made and released by lymphocytes in response to the presence of pathogens—such as viruses, bacteria, or parasites—or tumor cells) ngaqelizat. • Antitrupimonoklonallidhet pёr interferoninspecifik, ne kromatografine nёkollonё. (bёhet shpёrlarja e interferonitngaantitrupi, pastrim 5000 herё mё I lartё se cdorrugёtjetёr. 2. Pёr tipiziminqelizor – AM pёrbёjnёmjeteshumёspecifike pёr identifikimin e antigjenevespecifikёtumoralё nёmelanomat, karcinomat, etj. teknikatmolekulare tё kёrkimit

  39. Pёrdorimiiantitrupavemonoklonalё 3. Nёdiagnostikёdheterapi, - identifikimiiviruseveosebaktereve nёnja material biologjik nёse ёshtё I disponueshёm antitrupispecifikndajtij. Lidhja e antitrupavemonoklonalё me antigjenenspecifike tёdisatumoreve humane , si p.sh atё tёantimielomёs etj. teknikatmolekulare tё kёrkimit

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