Umklomelo kaNobel waseMelika uKary Mullis washonela eCalifornia eneminyaka engama-74. Ngokusho kukankosikazi wakhe, ukushona kwenzeka ngo-Agasti 7. Imbangela ukwehluleka kwenhliziyo nokuphefumula ngenxa yenyumoniya.
UJames Watson ngokwakhe, owathola i-molecule ye-DNA, uzositshela ngeqhaza lakhe ku-biochemistry futhi athole ngayo uMklomelo KaNobel.
Kucashunwe encwadini kaJames Watson, Andrew Berry, Kevin Davis
I-DNA. Umlando Wezinguquko Zofuzo
Isahluko 7. I-genome yomuntu. Isimo sempilo
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I-polymerase chain reaction (PCR) yasungulwa ngo-1983 yisazi samakhemikhali ezinto eziphilayo u-Carey Mullis, owayesebenza kwa-Cetus. Ukutholakala kwalokhu kusabela kwakumangalisa kakhulu. Kamuva uMulis wakhumbula: “Ngobunye ubusuku bangoLwesihlanu ngo-April 1983, ngaba nomlando omubi. Ngangihamba ngesondo, ngihamba ngomgwaqo okhanya inyanga, omazombezombe eNyakatho California, izwe lamahlathi e-redwood.” Kuyamangaza ukuthi kwakusesimweni esinjalo lapho ugqozi lwamshaya khona. Futhi akukhona ukuthi inyakatho yeCalifornia inemigwaqo ekhethekile ekhuthaza ukuqonda; ukuthi nje umngane wakhe wake wabona u-Mullis egijima ngokunganaki emgwaqeni wezimoto ezimbili obandayo futhi akuzange kumkhathaze nakancane. Umngane watshela i- New York Times: “UMulis waba nombono wokuthi wayezofa ngokuphahlazeka esihlahleni se-redwood. Ngakho-ke akesabi lutho lapho eshayela, ngaphandle uma kunezihlahla ze-redwood ezimila emgwaqeni.” Ukuba khona kwe-redwoods eceleni komgwaqo kwaphoqa u-Mullis ukuba agxilise ingqondo futhi ... nakhu, ukuqonda. U-Mullis uthole uMklomelo KaNobel kuKhemistry ngokusungulwa kwakhe ngo-1993 futhi kusukela ngaleso sikhathi usephenduke umuntu ongamazi ezenzweni zakhe. Ngokwesibonelo, ungumsekeli wenkolelo-mbono ye-revisionist yokuthi ingculaza ayihlobene ne-HIV, eyathunaza kakhulu isithunzi sakhe futhi yagxambukela kodokotela.
I-PCR yindlela yokusabela elula. Ukuze sikwenze, sidinga ama-primers amabili ahlanganiswe ngamakhemikhali ahambisana nezinye iziphetho zemicu ehlukene yocezu oludingekayo lwe-DNA. Iziqalo ziyizingxenye ezimfushane ze-DNA enomucu owodwa, ngayinye icishe ibe ngamapheya angama-20 ubude. Okungavamile kwama-primers ukuthi ahambisana nezigaba ze-DNA ezidinga ukukhuliswa, okungukuthi, isifanekiso se-DNA.
(Isithombe siyachofozeka) u-Kary Mullis, umsunguli we-PCR
Ukucaciswa kwe-PCR kusekelwe ekwakhekeni kwezinkimbinkimbi ezihambisanayo phakathi kwesifanekiso nama-primer, ama-oligonucleotide amafushane okwenziwa. I-primer ngayinye ihambisana neyodwa yemicu yesifanekiso esinemicu ekabili futhi ikhawulela isiqalo nesiphetho sesifunda esikhulisiwe. Eqinisweni, “i-matrix” ewumphumela iwufuzo oluphelele, futhi umgomo wethu uwukuhlukanisa izingcezu esizithakaselayo kuwo. Ukwenza lokhu, isifanekiso se-DNA esinemicu ephindwe kabili sishiswa sibe ngu-95 °C imizuzu embalwa ukuze sihlukanise imicu ye-DNA. Lesi sigaba sibizwa ngokuthi i-denaturation ngoba amabhondi e-hydrogen phakathi kwemicu emibili ye-DNA aphukile. Uma izintambo sezihlukene, izinga lokushisa liyehliswa ukuze kuvunyelwe ama-primers ukuthi abophe isifanekiso esinentambo eyodwa. I-DNA polymerase iqala ukuphindaphinda kwe-DNA ngokubopha ochungechungeni lwe-nucleotide. I-enzyme DNA polymerase iphindaphinda umucu wesifanekiso isebenzisa i-primer njengesiqalo noma isibonelo sokukopisha. Njengomphumela womjikelezo wokuqala, sithola ukuphindaphindeka okuphindwe kabili okulandelanayo kwesigaba esithile se-DNA. Okulandelayo siphinda le nqubo. Ngemva komjikelezo ngamunye sithola indawo eqondiwe ngobuningi obuphindwe kabili. Ngemva kwemijikelezo ye-PCR engamashumi amabili nanhlanu (okungukuthi, ngaphansi kwamahora amabili), sinendawo ye-DNA esiyithakaselayo ngenani eliphindwe izikhathi ezingu-225 ngaphezu kokwasekuqaleni (okungukuthi, siyikhulise cishe izikhathi eziyizigidi ezingu-34). Eqinisweni, ekufakweni kwethu sithole ingxubevange yama-primers, i-template ye-DNA, i-DNA polymerase enzyme kanye nezisekelo zamahhala A, C, G no-T, inani lomkhiqizo othize wokusabela (olinganiselwe ngama-primers) likhula ngokushesha, futhi inani " ubude” amakhophi e-DNA anomugqa, ngakho kubusa imikhiqizo yokusabela.
Ukukhulisa ingxenye ye-DNA oyifunayo: ukusabela kwe-polymerase chain
Ezinsukwini zokuqala ze-PCR, inkinga enkulu yayilokhu okulandelayo: ngemva komjikelezo ngamunye wokushisisa-ukupholisa, i-DNA polymerase kwakudingeka ingezwe engxubeni yokusabela, njengoba yayingacushiwe ezingeni lokushisa elingu-95 ° C. Ngakho-ke, bekudingeka ukuthi kufakwe kabusha ngaphambi komjikelezo ngamunye we-25. Inqubo yokusabela yayingasebenzi kahle, idinga isikhathi esiningi kanye ne-enzyme ye-polymerase, futhi impahla yayibiza kakhulu. Ngenhlanhla, uMama Wendalo wasiza. Izilwane eziningi zizizwa zikhululekile emazingeni okushisa angaphezu kuka-37 °C. Kungani isibalo esingu-37 °C sibe sibalulekile kithi? Lokhu kwenzeke ngoba leli zinga lokushisa lilungele i-E. coli, okwatholwa kuyo i-enzyme ye-polymerase ye-PCR. Emvelweni kukhona ama-microorganisms amaprotheni, ngaphezu kwezigidi zeminyaka yokukhethwa kwemvelo, aye amelana kakhulu namazinga okushisa aphezulu. Kuye kwahlongozwa ukuthi kusetshenziswe i-DNA polymerase evela kubhaktheriya we-thermophilic. Lawa ma-enzyme atholakala ekwazi ukushibilika futhi akwazi ukumelana nemijikelezo eminingi yokusabela. Ukusebenzisa kwabo kwenza kwaba lula ukwenza i-PCR ibe lula. Enye ye-DNA polymerase yokuqala ekwazi ukushisisa yahlukaniswa ne-bacterium Thermus aquaticus, ehlala eziphethwini ezishisayo zase-Yellowstone National Park, futhi yaqanjwa ngokuthi i-Taq polymerase.
I-PCR ngokushesha yaba ihhashi lomsebenzi we-Human Genome Project. Ngokuvamile, le nqubo ayihlukile kuleyo eyakhiwe ngu-Mullis, isanda kwenziwa ngokuzenzakalelayo. Besingasancikile esixukwini sabafundi abaneziqu ezifiphele bethela amaconsi oketshezi kumashubhu okuhlola epulasitiki. Ezilabhorethri zesimanje enza ucwaningo lwezakhi zofuzo zamangqamuzana, lo msebenzi wenziwa kumarobhothi ahambisa izinto. Amarobhothi e-PCR abambe iqhaza kuphrojekthi yokulandelana enkulu njenge-Human Genome asebenza ngokungakhathali ngamavolumu amakhulu e-polymerase ekwazi ukumelana nokushisa. Abanye ososayensi abasebenza ku-Human Genome Project bacasulwa yizinkokhelo eziphezulu ngokungenangqondo ezingezwe ezindlekweni zezinto ezisetshenziswayo ngumnikazi welungelo lobunikazi le-PCR, isiqhwaga sezemithi yaseYurophu uHoffmann-LaRoche.
Enye "isimiso sokushayela" kwakuyindlela yokulandelana kwe-DNA ngokwayo. Isisekelo samakhemikhali sale ndlela sasingasesona sisha ngaleso sikhathi: I-Interstate Human Genome Project (HGP) yamukela indlela yobuhlakani efanayo naleyo uFred Sanger ayeyenzile emuva maphakathi nawo-1970. Ukuqamba okusha kulele esikalini kanye nezinga lokuzenzakalela lokho ukulandelana okukwazile ukukuzuza.
Ukulandelana okuzenzakalelayo kwasungulwa ekuqaleni elabhorethri ka-Lee Hood e-California Institute of Technology. Ufunde esikoleni samabanga aphezulu e-Montana futhi wadlala ibhola lasekolishi njenge-quarterback; Ngenxa ye-Hood, iqembu liwine ubuqhawe bombuso izikhathi ezingaphezu kwesisodwa. Amakhono akhe okusebenzisana nawo aba usizo emsebenzini wakhe wesayensi. Ilabhorethri ye-Hood yayinabasebenzi be-motley bamakhemikhali, izazi zezinto eziphilayo, nonjiniyela, futhi i-laboratory yakhe ngokushesha yaba umholi emisha yezobuchwepheshe.
Eqinisweni, indlela yokulandelana ezenzakalelayo yasungulwa nguLloyd Smith noMike Hunkapiller. UMike Hunkapiller, ngaleso sikhathi owayesebenza elabhorethri ye-Hood, waya ku-Lloyd Smith ngesiphakamiso sendlela yokulandelana ethuthukisiwe lapho uhlobo ngalunye lwesisekelo luzoba nemibala ehlukile. Umbono onjalo ungaphinda kabili ukusebenza kahle kwenqubo ye-Sanger. Ku-Sanger, lapho kulandelwa ishubhu ngalinye kwamane (ngokwenani lezisekelo), ngokubamba iqhaza kwe-DNA polymerase, kwakhiwa isethi eyingqayizivele yama-oligonucleotide yobude obuhlukene, kuhlanganise nokulandelana kwe-primer. Okulandelayo, i-formamide yengezwa kumashubhu okuhlukanisa amaketango futhi i-polyacrylamide gel electrophoresis yenziwa emizilanjeni emine. Enguqulweni kaSmith noHunkapiller, i-dieoxynucleotides ibhalwe ngodayi abane abahlukene futhi i-PCR yenziwa ngeshubhu elilodwa. Khona-ke, phakathi ne-polyacrylamide gel electrophoresis, i-laser beam endaweni ethize kujeli ijabulisa umsebenzi wodayi, futhi umtshina unquma ukuthi iyiphi i-nucleotide efudukayo njengamanje kujeli. Ekuqaleni, uSmith wayengenathemba - wayesaba ukuthi ukusebenzisa imithamo ephansi kadayi kuzoholela ekutheni izifunda ze-nucleotide zingabonakali. Nokho, ngenxa yokuqonda okuhle kakhulu ngobuchwepheshe be-laser, ngokushesha wathola indlela yokuphuma kulesi simo ngokusebenzisa odayi abakhethekile be-fluorochrome abacwebezelayo lapho bechayeke emisebeni ye-laser.
(Inguqulo egcwele - 4,08 MB) Ukuphrinta kahle: Ukulandelana kwe-DNA kusetshenziswa umshini wokulandelanisa ozenzakalelayo, otholakala emshinini wokulandelanisa ozenzakalelayo. Umbala ngamunye uhambisana nesisekelo esisodwa kwezine
Kunguqulo yakudala yendlela ye-Sanger, enye yezintambo ze-DNA ehlaziyiwe isebenza njengesifanekiso sokuhlanganiswa komucu ohambisanayo nge-enzyme DNA polymerase, bese ukulandelana kwezingcezu ze-DNA kuhlelwa ngejeli ngosayizi. Ucezu ngalunye, olufakwe ku-DNA ngesikhathi sokuhlanganiswa futhi luvumela ukuboniswa okulandelayo kwemikhiqizo yokusabela, lubhalwe ngodayi we-fluorescent ohambisana nesisekelo setheminali (lokhu kwaxoxwa ngakho ekhasini 124); ngakho-ke, i-fluorescence yalesi siqeshana izoba isihlonzi sesisekelo esinikeziwe. Khona-ke okusele nje ukwenza ukuthola nokubona ngeso lengqondo imikhiqizo yokusabela. Imiphumela ihlaziywa ngekhompyutha futhi yethulwe njengokulandelana kweziqongo ezinemibala eminingi ehambisana nama-nucleotide amane. Ulwazi lube seludluliselwa ngokuqondile ohlelweni lolwazi lwekhompiyutha, kuqedwe inqubo yokufaka idatha edla isikhathi futhi ngezinye izikhathi ebuhlungu eyenza ukulandelana kube nzima kakhulu.
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Ngesaphulelo esingu-25% se-Khabrozhiteley usebenzisa isigqebhezana - I-PCR
Source: www.habr.com