{"id":37174,"date":"2019-10-31T22:16:11","date_gmt":"2019-10-31T19:16:11","guid":{"rendered":"https:\/\/prohoster.info\/blog\/umer-nobelevskij-laureat-keri-mullis-izobretatel-polimeraznoj-tsepnoj-reaktsii-dnk\/"},"modified":"2019-10-31T22:16:11","modified_gmt":"2019-10-31T19:16:11","slug":"umer-nobelevskij-laureat-keri-mullis-izobretatel-polimeraznoj-tsepnoj-reaktsii-dnk","status":"publish","type":"post","link":"https:\/\/prohoster.info\/sq\/blog\/news\/umer-nobelevskij-laureat-keri-mullis-izobretatel-polimeraznoj-tsepnoj-reaktsii-dnk","title":{"rendered":"Nd\u00ebrron jet\u00eb nobelisti Cary Mullis \u2014 shpik\u00ebsi i reaksionit t\u00eb zinxhirit t\u00eb polimeraz\u00ebs t\u00eb ADN-s\u00eb.","gt_translate_keys":[{"key":"rendered","format":"text"}]},"content":{"rendered":"

\"Nd\u00ebrron N\u00eb Kaliforni, n\u00eb mosh\u00ebn 74-vje\u00e7are vdiq amerikanin nobelist t\u00eb kimi Keri Mullis. Sipas bashk\u00ebshortes s\u00eb tij, vdekja ndodhi m\u00eb 7 gusht. Shkaku - d\u00ebshtimi kardiak dhe respirator p\u00ebr shkak t\u00eb pneumonis\u00eb.<\/p>\n

Rreth kontributit t\u00eb tij n\u00eb bio-kimie dhe arsyet pse mori \u00c7mimin Nobel do na flas\u00eb vet\u00eb Xhejms Votson - zbuluesi i molekul\u00ebs s\u00eb ADN-s\u00eb. <\/p>\n

Nj\u00eb fragment nga libri i Xhejms Votsonit, Endrju Beri, Kevin Devis <\/p>\n

ADN. Historia e revolucionit gjenetik<\/h2>\n

Kapitulli 7. Genomi i njeriut. Scenari i jet\u00ebs<\/h3>\n

\n<\/a><\/noindex>
\n\u2026
\nReaksioni i polimeraz\u00ebs s\u00eb zinxhirit (P\u00c7R) u shpik n\u00eb vitin 1983 nga bio-kimik Keri Mullis, i pun\u00ebsuar n\u00eb kompanin\u00eb Cetus. Zbulimi i k\u00ebtij reaksioni ishte mjaft dometh\u00ebn\u00ebs. M\u00eb von\u00eb, Mullis kujtoi: \u00abNj\u00eb her\u00eb, nj\u00eb t\u00eb premte n\u00eb mbr\u00ebmje n\u00eb prill 1983, ndjeva si t\u00eb m\u00eb kishte marr\u00eb fryma. Ishte duke drejtuar, duke kaluar nj\u00eb rrug\u00eb malore t\u00eb mbuluar me drit\u00eb h\u00ebne n\u00eb veri t\u00eb Kalifornis\u00eb, n\u00eb vendin e pyjeve t\u00eb sekvoja.\u00bb \u00cbsht\u00eb impresionuese se pik\u00ebrisht n\u00eb nj\u00eb situat\u00eb t\u00eb till\u00eb e vizitoi frym\u00ebzimi. Dhe nuk ka t\u00eb b\u00ebj\u00eb aspak me faktin se veriu i Kalifornis\u00eb ka rrug\u00eb t\u00eb ve\u00e7anta q\u00eb inkurajojn\u00eb frym\u00ebzimin; thjesht nj\u00eb mik i tij kishte par\u00eb nj\u00eb her\u00eb se Mullis po fluturonte pa kujdes n\u00eb rrug\u00ebn e akullt me dy kah\u00eb dhe kjo nuk e shqet\u00ebsonte aspak. Miku i tha New York Times: \u00abMullis kishte nj\u00eb vizion se do t\u00eb vdiste duke u p\u00ebrplasur me nj\u00eb sekvoin\u00eb. Prandaj ai nuk ka frik\u00eb nga drejtp\u00ebrdrejt\u00ebsia, p\u00ebr aq koh\u00eb sa p\u00ebrgjat\u00eb rrug\u00ebs nuk ka sekvoja.\u00bb Prania e sekvoive p\u00ebrgjat\u00eb rrug\u00ebs e b\u00ebnte Mullis t\u00eb p\u00ebrqendrohej dhe... ja, frym\u00ebzimi. P\u00ebr shpikjen e tij n\u00eb vitin 1993, Mullis fitoi \u00c7mimin Nobel n\u00eb kimi dhe q\u00eb nga at\u00ebher\u00eb b\u00ebhet edhe m\u00eb i \u00e7uditsh\u00ebm n\u00eb veprimet e tij. P\u00ebr shembull, ai \u00ebsht\u00eb nj\u00eb mb\u00ebshtet\u00ebs i teoris\u00eb revizioniste q\u00eb SIDA nuk \u00ebsht\u00eb e lidhur me HIV, \u00e7ka ndihmon n\u00eb d\u00ebmtimin e reputacionit t\u00eb tij dhe pengon mjek\u00ebt.<\/p>\n

P\u00c7R \u00ebsht\u00eb nj\u00eb reaksion mjaft i thjesht\u00eb. P\u00ebr ta realizuar at\u00eb, na duhen dy primer\u00eb t\u00eb sintetizuar kimikisht, q\u00eb jan\u00eb komplementar\u00eb me skajet e kund\u00ebrta t\u00eb zinxhir\u00ebve t\u00eb ndrysh\u00ebm t\u00eb fraksionit t\u00eb k\u00ebrkuar t\u00eb ADN-s\u00eb. Primer\u00ebt jan\u00eb pjes\u00eb t\u00eb shkurtra t\u00eb ADN-s\u00eb nj\u00ebnj\u00ebshe, secila rreth 20 \u00e7ift\u00eb bazash n\u00eb gjat\u00ebsi. Karakteristika e primer\u00ebve \u00ebsht\u00eb se ata p\u00ebrputhen me pjes\u00ebt e ADN-s\u00eb q\u00eb duhet t\u00eb amplifikohen, dmth ADN-n\u00eb matric\u00eb. <\/p>\n

\"Nd\u00ebrron<\/a><\/noindex>
\n(Imazhi \u00ebsht\u00eb i klikuesh\u00ebm) Keri Mullis, shpik\u00ebsi i P\u00c7R<\/i><\/p>\n

Specifiku e PCR-s\u00eb bazohet n\u00eb formimin e komplekseve komplementar\u00eb midis matric\u00ebs dhe primer\u00ebve, oligonukleotideve t\u00eb shkurt\u00eb sintetike. \u00c7do primer \u00ebsht\u00eb komplementar me nj\u00eb nga zinxhir\u00ebt e matric\u00ebs dyzan\u00ebshe dhe kufizon fillimin dhe p\u00ebrfundimin e seksionit q\u00eb do t\u00eb amplifikohet. N\u00eb fakt, 'matrica' e marr\u00eb p\u00ebrfaq\u00ebson nj\u00eb genom t\u00eb plot\u00eb, dhe q\u00ebllimi yn\u00eb \u00ebsht\u00eb t\u00eb nxjerrim pjes\u00ebt q\u00eb na interesojn\u00eb prej saj. P\u00ebr k\u00ebt\u00eb, matrica e ADN-s\u00eb dyzan\u00ebshe ngrohet deri n\u00eb 95 \u00b0C p\u00ebr disa minuta, n\u00eb m\u00ebnyr\u00eb q\u00eb zinxhir\u00ebt e ADN-s\u00eb t\u00eb ndahen. Ky hap quhet denaturim, pasi lidhjet hidrogjenore midis dy zinxhir\u00ebve t\u00eb ADN-s\u00eb prishen. Kur zinxhir\u00ebt jan\u00eb ndar\u00eb, temperatura ulet, n\u00eb m\u00ebnyr\u00eb q\u00eb primer\u00ebt t\u00eb mund t\u00eb lidh\u00ebzen me matric\u00ebn nj\u00ebzan\u00ebshe. ADN-polimeraza fillon replikimin e ADN-s\u00eb, duke u lidhur me nj\u00eb segment t\u00eb zinxhirit t\u00eb nucleotideve. Enzima ADN-polimeraza replikon zinxhirin matricor duke p\u00ebrdorur primerin si nj\u00eb fillim ose shembull p\u00ebr kopjim. Si rezultat i ciklit t\u00eb par\u00eb, marrim nj\u00eb shum\u00ebfishim t\u00eb vazhduesh\u00ebm t\u00eb nj\u00eb seksioni t\u00eb caktuar t\u00eb ADN-s\u00eb. Pastaj e p\u00ebrs\u00ebrisim k\u00ebt\u00eb procedur\u00eb. Pas \u00e7do cikli, marrim nj\u00eb segment q\u00ebllim n\u00eb nj\u00eb sasi dyfish. Pasi kalojm\u00eb nj\u00ebzet e pes\u00eb cikle PCR (dmth m\u00eb pak se dy or\u00eb), kemi seksionin e ADN-s\u00eb q\u00eb na intereson n\u00eb nj\u00eb sasi q\u00eb \u00ebsht\u00eb 225 her\u00eb m\u00eb e madhe se origjinali (dmth e kemi amplifikuar at\u00eb rreth 34 milion her\u00eb). N\u00eb fakt, n\u00eb hyrje kishim nj\u00eb p\u00ebrzierje t\u00eb primer\u00ebve, ADN-s\u00eb matricore, enzimes ADN-polimeraza dhe bazave t\u00eb lira A, C, G dhe T, sasia e produktit specifik t\u00eb reaksionit (e kufizuar nga primer\u00ebt) rritet n\u00eb m\u00ebnyr\u00eb eksponenciale, nd\u00ebrsa sasia e kopjeve 't\u00eb gjata' t\u00eb ADN-s\u00eb rritet n\u00eb m\u00ebnyr\u00eb lineare, prandaj n\u00eb produktet e reaksionit dominon.<\/p>\n

\"Nd\u00ebrron
\nAmplifikimi i seksionit t\u00eb nevojsh\u00ebm t\u00eb ADN-s\u00eb: reaksioni i zinxhirit polimeraz\u00eb<\/i><\/p>\n

N\u00eb fillim t\u00eb p\u00ebrdorimit t\u00eb PCR, problemi kryesor ishte ky: pas \u00e7do cikli ngrohje-ftohjeje, duhej t\u00eb shtohej p\u00ebrs\u00ebri ADN-polymeraza n\u00eb p\u00ebrzierjen reaktive, sepse ajo inaktivizohej n\u00eb temperatur\u00ebn 95 \u00b0C. Prandaj, duhej ta rishkurtoshim at\u00eb para \u00e7do nj\u00ebrit nga 25 ciklet. Procedura e zhvillimit t\u00eb reaksionit ishte relativisht joefikase, k\u00ebrkonte shum\u00eb koh\u00eb dhe fermentin e polymeraz\u00ebs, nd\u00ebrsa materiali ishte mjaft i shtrenjt\u00eb. Me fat, natyra erdhi n\u00eb ndihm\u00eb. Shum\u00eb kafsh\u00eb ndihen rehat n\u00eb temperatura shum\u00eb m\u00eb t\u00eb larta se 37 \u00b0C. Pse p\u00ebr ne \u00ebsht\u00eb e r\u00ebnd\u00ebsishme numri 37 \u00b0C? Kjo ndodhi sepse kjo temperatur\u00eb \u00ebsht\u00eb optimale p\u00ebr E. coli, nga e cila fillimisht u mor fermenti polymeraz\u00eb p\u00ebr PCR. N\u00eb natyr\u00eb, ekzistojn\u00eb mikroorganizma, proteinat e t\u00eb cil\u00ebve p\u00ebr miliona vjet seleksionimi natyror jan\u00eb b\u00ebr\u00eb m\u00eb t\u00eb forta ndaj veprimit t\u00eb temperaturave t\u00eb larta. U propozua t\u00eb p\u00ebrdoren ADN-polymerazat nga bakteret termofile. K\u00ebto enzima rezultuan t\u00eb ishin termostabile dhe ishin n\u00eb gjendje t\u00eb p\u00ebrballonin shum\u00eb cikle t\u00eb reaksionit. P\u00ebrdorimi i tyre e leht\u00ebsoi dhe automatizoi zhvillimin e PCR. Nj\u00eb nga ADN-polymerazat e para termostabile u izolua nga bakteria Thermus aquaticus, q\u00eb jeton n\u00eb burimet e nxehta t\u00eb Parkut Komb\u00ebtar Yellowstone, dhe u quajt Taq-polymeraza.<\/p>\n

PCR shpejt u b\u00eb kali pun\u00ebtor kryesor i projektit \"Gjenomi i Njeriut\". N\u00eb thelb, procesi nuk ndryshon nga ai i zhvilluar nga Mullis, thjesht ai u automatizua. Ne nuk varej m\u00eb nga turma e asistent\u00ebve t\u00eb verb\u00ebr, q\u00eb derdhnin kujdessh\u00ebm pika l\u00ebngu n\u00eb tubat plastik\u00eb. N\u00eb laborator\u00ebt moderne q\u00eb kryejn\u00eb k\u00ebrkime molekulare-gjenetike, kjo pun\u00eb kryhet n\u00eb linjat robotike. Robot\u00ebt e PCR-s\u00eb, q\u00eb jan\u00eb t\u00eb angazhuar n\u00eb nj\u00eb projekt t\u00eb till\u00eb t\u00eb gjer\u00eb p\u00ebr sekvencimin si \"Gjenomi i Njeriut\", punojn\u00eb pa ndalim me sasi t\u00eb m\u00ebdha t\u00eb polymeraz\u00ebs rezistente ndaj nxeht\u00ebsis\u00eb. Disa shkenc\u00ebtar\u00eb t\u00eb angazhuar n\u00eb projektin \"Gjenomi i Njeriut\" ishin t\u00eb shqet\u00ebsuar p\u00ebr tarifat e tep\u00ebrta q\u00eb shtohen n\u00eb koston e materialeve shpenzuese nga pronari i patent\u00ebs p\u00ebr PCR, gjiganti industrial-farmaceutik evropian Hoffmann-LaRoche.<\/p>\n

Nj\u00eb tjet\u00ebr \"nxit\u00ebs\" ishte vet\u00eb metoda e sekvencimit t\u00eb ADN-s\u00eb. Bazat kimike t\u00eb k\u00ebsaj metode at\u00ebher\u00eb nuk ishin m\u00eb nj\u00eb novitet: Projekti Nd\u00ebrqeveritar \"Gjeni i Njeriut\" (HGP) mori n\u00eb p\u00ebrdorim t\u00eb nj\u00ebjt\u00ebn metod\u00eb t\u00eb nd\u00ebrlikuar q\u00eb Fred Sanger e zhvilloi n\u00eb mesin e viteve 1970. Inovacioni q\u00ebndronte n\u00eb shkall\u00ebn dhe nivelin e automatizimit q\u00eb arrit\u00ebn gjat\u00eb sekvencimit.<\/p>\n

Sekvencimi automatik fillimisht u zhvillua n\u00eb laboratorin e Lee Hood n\u00eb Institutin e Teknologjis\u00eb t\u00eb Kalifornis\u00eb. Ai kishte mbaruar shkoll\u00ebn n\u00eb shtetin Montana dhe luante futboll amerikan si sulmues; fal\u00eb Hudit, ekipi fitoi disa her\u00eb kampionatin e shtetit. Aft\u00ebsit\u00eb e tij p\u00ebr pun\u00eb n\u00eb ekip i sh\u00ebrbyen edhe n\u00eb karrier\u00ebn shkencore. N\u00eb laboratorin e Hudit punonte nj\u00eb grup t\u00eb larmish\u00ebm kimist\u00ebsh, biolog\u00ebsh dhe inxhinier\u00ebsh, dhe s\u00eb shpejti laboratorit t\u00eb tij i dha p\u00ebrpar\u00ebsi n\u00eb inovacionet teknologjike.<\/p>\n

Faktikisht, metoda e automatik\u00ebs s\u00eb sekvencimit u shpik nga Lloyd Smith dhe Mike Hunkapiller. Mike Hunkapiller, i cili at\u00ebkoh\u00eb punonte n\u00eb laboratorin e Hoodit, i drejtoi Lloyd Smithit nj\u00eb propozim p\u00ebr nj\u00eb metod\u00eb t\u00eb avancuar sekvencimi, ku baza e \u00e7do tipi do t\u00eb ngjyrosej n\u00eb nj\u00eb ngjyr\u00eb t\u00eb ve\u00e7ant\u00eb. Kjo ide mund t\u00eb rrit\u00eb kat\u00ebrfish efikasitetin e procesit Sanger. N\u00eb metod\u00ebn Sanger, gjat\u00eb sekvencimit n\u00eb secil\u00ebn nga kat\u00ebr t\u00fcp (n\u00eb numrin e bazave), me pjes\u00ebmarrjen e ADN-polimeraz\u00ebs krijohet nj\u00eb set unik oligonukleotidash me gjat\u00ebsi t\u00eb ndryshme, duke p\u00ebrfshir\u00eb sekuenc\u00ebn e primerit. M\u00eb pas n\u00eb t\u00fcp shtohet formamid p\u00ebr t\u00eb ndar\u00eb zinxhir\u00ebt dhe kryhet elektroforeza n\u00eb nj\u00eb xhel poliakrilamid n\u00eb kat\u00ebr korsi. N\u00eb variantin e Smithit dhe Hunkapillerit, dideoksinukleotid\u00ebt ngjyrosen me kat\u00ebr ngjyra t\u00eb ndryshme dhe PCR kryhet n\u00eb nj\u00eb t\u00fcp. Pastaj, gjat\u00eb elektroforez\u00ebs n\u00eb xhel poliakrilamid, nj\u00eb rreze laseri n\u00eb nj\u00eb pik\u00eb t\u00eb caktuar t\u00eb xhelit aktivizon aktivitetin e ngjyrave, dhe detektori p\u00ebrcakton se cilin nukleotid po migradon n\u00eb at\u00eb moment n\u00ebp\u00ebr xhel. Fillimisht, Smith kishte nj\u00eb qasje pesimiste - kishte frik\u00eb se p\u00ebrdorimi i dozave t\u00eb ultrat t\u00eb vogla t\u00eb ngjyr\u00ebs do t\u00eb sillte q\u00eb pjes\u00ebt nukleotidike t\u00eb ishin t\u00eb path\u00ebnshme. Megjithat\u00eb, duke qen\u00eb mjesht\u00ebr n\u00eb teknologjit\u00eb laserike, ai shpejt gjeti nj\u00eb zgjidhje duke p\u00ebrdorur spiciale ngjyrues me fluorokrom\u00eb q\u00eb fluorescojn\u00eb n\u00ebn veprimin e rrezatimit laserik.<\/p>\n

\"Nd\u00ebrron
\n(Versioni i plot\u00eb me klikim<\/a><\/noindex> \u2014 4,08 MB) Me shkronja t\u00eb vogla: sekuenca e ADN-s\u00eb, e dekoduara duke p\u00ebrdorur nj\u00eb sekvencues automatik, e marr\u00eb nga aparati p\u00ebr sekvencimin automatik. \u00c7do ngjyr\u00eb korrespondon me nj\u00eb nga kat\u00ebr bazat.<\/i> <\/p>\n

N\u00eb variantin klasik t\u00eb metod\u00ebs s\u00eb Sanger, nj\u00eb nga zinxhir\u00ebt e ADN-s\u00eb q\u00eb po analizohet sh\u00ebrben si matrice p\u00ebr sintez\u00ebn e nj\u00eb zinxhiri komplementar nga enzima ADN-polimeraza, pastaj sekuenca e fragmenteve t\u00eb ADN-s\u00eb klasifikohet n\u00eb xhel sipas madh\u00ebsis\u00eb. Secili fragment q\u00eb p\u00ebrfshihet n\u00eb p\u00ebrb\u00ebrjen e ADN-s\u00eb gjat\u00eb sintez\u00ebs dhe q\u00eb lejon m\u00eb pas vizualizimin e produkteve t\u00eb reaksionit, tregohet me nj\u00eb boj\u00eb fluorescente q\u00eb p\u00ebrputhet me baz\u00ebn terminale (kjo u p\u00ebrmend n\u00eb fq. 124); p\u00ebr rrjedhoj\u00eb, fluorescenca e k\u00ebtij fragmenti do t\u00eb jet\u00eb identifikuesi p\u00ebr k\u00ebt\u00eb baz\u00eb. M\u00eb pas, mbetet vet\u00ebm t\u00eb b\u00ebhet detektimi dhe vizualizimi i produkteve t\u00eb reaksionit. Rezultatet analizohen me ndihm\u00ebn e nj\u00eb kompjuteri dhe paraqiten si nj\u00eb sekuenc\u00eb majiko-ngjyrash, q\u00eb p\u00ebrputhen me kat\u00ebr nukeotidet. Informacioni p\u00ebrshkruhet drejtp\u00ebrdrejt n\u00eb sistemin informatik t\u00eb kompjuterit, gj\u00eb q\u00eb p\u00ebrjashton nj\u00eb proces t\u00eb shpenzuesh\u00ebm n\u00eb koh\u00eb dhe ndonj\u00ebher\u00eb t\u00eb mundimsh\u00ebm t\u00eb hyrjes s\u00eb t\u00eb dh\u00ebnave, q\u00eb v\u00ebshtir\u00ebsonte shum\u00eb sekvencionimin.<\/p>\n

\u00bb M\u00eb shum\u00eb informacion mbi librin mund t\u00eb gjendet n\u00eb faqen e botuesit<\/a><\/noindex>
\n\u00bb P\u00ebrmbajtja<\/a><\/noindex>
\n\u00bb Pjesa<\/a><\/noindex><\/p>\n

P\u00ebr an\u00ebtar\u00ebt e Habra ka nj\u00eb zbritje prej 25% me kodin \u2014 PCR<\/b>
\n
Burimi: habr.com<\/a><\/p>","protected":false,"gt_translate_keys":[{"key":"rendered","format":"html"}]},"excerpt":{"rendered":"

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