Imbasa yeNobel uKary Mullis, umyili weDNA polymerase chain reaction, uswelekile

Imbasa yeNobel yaseMelika uKary Mullis wasweleka eCalifornia eneminyaka engama-74. Ngokutsho kwenkosikazi yakhe, ukufa kwenzeka ngo-Agasti 7. Unobangela wokungaphumeleli kwentliziyo nokuphefumla ngenxa yenyumoniya.

UJames Watson ngokwakhe, umfumani we-molecule ye-DNA, uya kusixelela ngegalelo lakhe kwi-biochemistry kwaye wafumana ibhaso leNobel.

Isicatshulwa kwincwadi kaJames Watson, uAndrew Berry, uKevin Davis

DNA. Imbali yeNguqulelo yemfuza

Isahluko 7. I-genome yomntu. Imeko yobomi

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I-polymerase chain reaction (PCR) yasungulwa ngo-1983 yingcali yebhayokhemisti uCarey Mullis, owayesebenza kwa-Cetus. Ukufunyaniswa kwale ndlela yokusabela kwakumangalisa ngokwenene. Kamva uMulis ukhumbula oku: “Ngenye ingokuhlwa yangoLwesihlanu ngoAprili 1983, ndaba nembali engaqhelekanga. Ndandisemva kwevili, ndisihla kwindlela ekhanyiswe yinyanga, ejikajikayo yasentabeni kuMntla weKhalifoniya, ilizwe lamahlathi eredwood.” Kuyamangalisa ukuba wayekwimeko enjalo awathi wabethwa yimpembelelo. Kwaye akukhona ukuba umntla weCalifornia uneendlela ezikhethekileyo ezikhuthaza ukuqonda; yinto nje yokuba umhlobo wakhe wakhe wabona u-Mullis ebaleka ngokungakhathali kwindlela yeenqwelo ezimbini ezinomkhenkce kwaye ayizange imkhathaze kwaphela. Umhlobo waxelela iNew York Times oku: “UMulis wayenombono wokuba wayeza kufa ngokuntlitheka emthini obomvu. Ngoko ke, akoyiki nto xa eqhuba, ngaphandle kokuba kukho imithi yeredwood ekhula ecaleni kwendlela.” Ubukho be-redwoods ecaleni kwendlela kwanyanzela u-Mullis ukuba agxininise kwaye ... nantsi, ingqiqo. UMulis wafumana ibhaso leNobel kwiChemistry ngokuyila kwakhe ngo-1993 kwaye ukususela ngoko uye waba ngumntu ongaziwayo kwizenzo zakhe. Ngokomzekelo, ungumxhasi wengcamango yohlaziyo yokuba uGawulayo akanxulumananga neHIV, nto leyo eyalonakalisa ngokuphawulekayo isidima sakhe yaza yaphazamisana noogqirha.

I-PCR yindlela elula yokusabela. Ukuyiphumeza, sifuna iiprimers ezimbini ezenziwe ngokwekhemikhali ezihambelana neziphelo ezichaseneyo zemisonto eyahlukeneyo yesiqwenga esifunekayo seDNA. Iiprimers ngamacandelo amafutshane e-DNA enemisonto enye, ngalinye limalunga ne-20 isiseko sesibini ubude. I-peculiarity of primers kukuba ihambelana namacandelo e-DNA afuna ukunyuswa, oko kukuthi, i-template ye-DNA.

(Umfanekiso ucofa) Kary Mullis, umqambi wePCR

Ubume be-PCR busekelwe ekubunjweni kwee-complementary complexes phakathi kwetemplate kunye ne-primers, i-oligonucleotides emfutshane yokwenziwa. I-primer nganye ihambelana nenye yeentambo ze-template ephindwe kabini kwaye ikhawulela ukuqala kunye nokuphela kwendawo ekhulisiwe. Enyanisweni, isiphumo se-"matrix" yi-genome epheleleyo, kwaye injongo yethu kukuhlukanisa amaqhekeza anomdla kuthi kuyo. Ukwenza oku, itemplate ye-DNA ephindwe kabini ifudunyezwa kwi-95 ° C imizuzu emininzi ukuhlula imicu ye-DNA. Eli nqanaba libizwa ngokuba yi-denaturation kuba iibhondi zehydrogen phakathi kwemisonto emibini ye-DNA yophukile. Emva kokuba imicu ihlulwe, iqondo lokushisa liyancipha ukuvumela ii-primers ukuba zibophe kwi-template ene-stranded single. I-DNA polymerase iqala ukuphindaphinda kwe-DNA ngokuzibophelela kwikhonkco lenucleotide. I-enzyme ye-DNA polymerase iphindaphinda i-template strand isebenzisa i-primer njenge-primer okanye umzekelo wokukopisha. Njengomphumo womjikelo wokuqala, sifumana ukuphinda-phinda kabini okulandelelanayo kwecandelo elithile le-DNA. Okulandelayo siphinda le nkqubo. Emva komjikelo ngamnye sifumana indawo ekujoliswe kuyo ngobuninzi obuphindwe kabini. Emva kwemijikelo yePCR engamashumi amabini anesihlanu (oko kukuthi, ngaphantsi kweeyure ezimbini), sinommandla we-DNA onomdla kuthi kwisixa esiphindwe ngama-225 ngaphezu kweyokuqala (oko kukuthi, siye sandisa malunga nezigidi ezingama-34 amaxesha). Enyanisweni, kwigalelo safumana umxube we-primers, i-template DNA, i-DNA polymerase enzyme kunye neziseko zamahhala A, C, G kunye ne-T, inani lemveliso ethile yokusabela (elinganiselwe yi-primers) ikhula ngokukhawuleza, kunye nenani. Iikopi ze-DNA "ezinde" zinomgca, ngoko kwiimveliso zokusabela zilawula.

Ukwandiswa kwecandelo le-DNA elifunwayo: i-polymerase chain reaction

Kwiintsuku zokuqala ze-PCR, ingxaki enkulu yayiyilandelayo: emva komjikelo ngamnye wokufudumeza-ukupholisa, i-DNA polymerase kwafuneka yongezwe kumxube wokusabela, ekubeni yayingasebenzi kwiqondo lokushisa lama-95 ° C. Ke ngoko, bekuyimfuneko ukuba yongezwe kwakhona phambi komjikelo ngamnye we-25. Inkqubo yokusabela yayingasebenzi kakuhle, yayifuna ixesha elininzi kunye ne-enzyme ye-polymerase, kwaye izinto zazixabisa kakhulu. Ngethamsanqa, uMama weNdalo weza kuhlangula. Izilwanyana ezininzi ziziva zikhululekile kumaqondo obushushu angaphezu kwama-37 °C. Kwakutheni ukuze inani lama-37 °C libaluleke kuthi? Oku kwenzeka ngenxa yokuba obu bushushu bufanelekile kwi-E. coli, apho i-enzyme ye-polymerase ye-PCR yafunyanwa kuqala. Kwindalo kukho ii-microorganisms ezineeprotheyini, ngaphezu kwezigidi zeminyaka yokukhethwa kwendalo, ziye zamelana ngakumbi nokushisa okuphezulu. Kuye kwacetywa ukuba kusetyenziswe i-polymerases ye-DNA evela kwiibhaktheriya ze-thermophilic. Ezi enzymes zajika zakwazi ukufudumala kwaye zikwazi ukumelana nemijikelo emininzi yokusabela. Ukusetyenziswa kwabo kwenza ukuba kube lula kunye nokwenza i-PCR ngokuzenzekelayo. Enye yeepolymerase ze-DNA zokuqala ezishushu zaye zahlukaniswa kwibhaktheriya i-Thermus aquaticus, ehlala kwimithombo eshushu ye-Yellowstone National Park, kwaye kwathiwa yiTaq polymerase.

I-PCR yakhawuleza yaba lihashe lomsebenzi weProjekthi yeGenome yoMntu. Ngokubanzi, inkqubo ayifani naleyo iphuhliswe ngu-Mullis, isanda ngokuzenzekelayo. Besingasaxhomekekanga kwisihlwele sabafundi abaphumelele izidanga begalela amathontsi olwelo kwiityhubhu zovavanyo zeplastiki. Kwiilabhoratri zanamhlanje ezenza uphando lwe-molecular genetic, lo msebenzi wenziwa kwiirobhothi zokuhambisa. Iirobhothi zePCR ezibandakanyekayo kwiprojekthi yolandelelwano enkulu njengeGenome yoMntu isebenza ngokungayekiyo ngemithamo emikhulu yepolymerase ezinzileyo. Ezinye izazinzulu ezisebenza kwiProjekthi ye-Human Genome zacatshukiswa yintlawulo ephezulu ngokungekho ngqiqweni eyongezwe kwixabiso lezinto ezityiwayo ngumnini welungelo elilodwa lomenzi wechiza i-PCR, isigebenga samayeza saseYurophu uHoffmann-LaRoche.

Omnye "umgaqo wokuqhuba" yayiyindlela yokulandelelanisa iDNA ngokwayo. Isiseko semichiza yale ndlela sasingasekho sitsha ngelo xesha: IProjekthi ye-Interstate Human Genome (HGP) yamkela indlela ekrelekrele efanayo neyayiphuhliswe nguFred Sanger emva phayaa phakathi kwiminyaka yee-1970. Ukwenziwa kwezinto ezintsha kulele kwisikali kunye neqondo lokuzitshintsha oluthe ulandelelwano lwakwazi ukufikelela.

Ulandelelwano oluzenzekelayo lwaphuhliswa ekuqaleni kwilabhoratri ka-Lee Hood kwiZiko leTekhnoloji laseCalifornia. Ufunde kwisikolo samabanga aphakamileyo eMontana kwaye wadlala ibhola yasekholejini njengekota yokubuya; Enkosi kuHood, iqela liphumelele ubuntshatsheli bombuso ngaphezulu kwesinye. Izakhono zakhe zokusebenzisana nazo zeza luncedo kumsebenzi wakhe wesayensi. Ilabhoratri ye-Hood yayinabasebenzi be-motley crew of chemist, izazi zebhayoloji, kunye neenjineli, kunye nelebhu yakhe ngokukhawuleza yaba yinkokeli kwi-teknoloji entsha.

Enyanisweni, indlela yolandelelwano oluzenzekelayo yaqanjwa nguLloyd Smith noMike Hunkapiller. UMike Hunkapiller, ngoko esebenza kwilabhoratri yeHood, waya kuLloyd Smith ngesiphakamiso sendlela ephuculweyo yolandelelwano apho uhlobo ngalunye lwesiseko luya kuba nemibala eyahlukileyo. Umbono onjalo unokuphinda kane ukusebenza kakuhle kwenkqubo yeSanger. Kwi-Sanger, xa ulandelelanisa kwiityhubhu ezine (ngokwenani leziseko), kunye nokuthatha inxaxheba kwe-DNA polymerase, isethi ekhethekileyo ye-oligonucleotides yobude obuhlukeneyo iqulunqwa, kubandakanywa ukulandelelana kwe-primer. Emva koko, i-formamide yongezwa kwiityhubhu zokwahlula ikhonkco kunye ne-polyacrylamide gel electrophoresis yenziwa kwiindlela ezine. Kwinguqulelo kaSmith noHunkapiller, i-dieoxynucleotides ibhalwe ngeedayi ezine ezahlukeneyo kwaye iPCR yenziwa kwityhubhu enye. Emva koko, ngexesha le-polyacrylamide gel electrophoresis, i-laser beam kwindawo ethile kwi-gel ivuyisa umsebenzi weedayi, kwaye i-detector inquma ukuba yiyiphi i-nucleotide ehambayo ngoku kwi-gel. Ekuqaleni, uSmith wayengenathemba - wayesoyikela ukuba ukusebenzisa idosi yedayi ephantsi kakhulu kunokukhokelela ekubeni imimandla ye-nucleotide ingabonakali. Noko ke, ngenxa yokuqonda okugqwesileyo kobugcisa belaser, kungekudala wafumana indlela yokuphuma kuloo meko ngokusebenzisa idayi ezikhethekileyo ze-fluorochrome ezithi xa zichanabeke kwimitha yelaser.

(Uguqulelo olupheleleyo ngokucofa - 4,08 MB) Ushicilelo olucokisekileyo: Ulandelelwano lweDNA lulandelelwaniswa kusetyenziswa isilungelelanisi esizenzekelayo, esifunyenwe kumatshini ozenzekelayo. Umbala ngamnye uhambelana nesinye seziseko ezine

Kwinguqulo ye-classic yendlela ye-Sanger, enye yeentambo ze-DNA ehlalutyiweyo isebenza njenge template ye-synthesis ye-strand encedisayo nge-enzyme DNA polymerase, ngoko ukulandelelana kwamaqhekeza e-DNA kuhlelwe kwi-gel ngobukhulu. Isiqwenga ngasinye esifakwe kwi-DNA ngexesha lokudibanisa kwaye sivumela ukubonwa okulandelayo kweemveliso zokusabela kubhalwe ngedayi ye-fluorescent ehambelana nesiseko se-terminal (oku kwaxutyushwa kwi-p. 124); ke ngoko, i-fluorescence yesi siqwenga iya kuba sisazisi sesiseko esinikiweyo. Emva koko konke okuseleyo kukwenza ubhaqo kunye nombono weemveliso zokusabela. Iziphumo zihlalutywa yikhompyuter kwaye zinikezelwe njengolandelelwano lweencochoyi ezinemibala emininzi ehambelana neenucleotide ezine. Ulwazi ke lugqithiselwe ngqo kwinkqubo yolwazi lwekhompyutha, ukuphelisa inkqubo yokufaka idatha ethatha ixesha kwaye ngamanye amaxesha ibuhlungu eyenza ulandelelwano lube nzima kakhulu.

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