PCR machine|Uyaqonda ngempela?

Ubuchwepheshe be-PCR obuzuze umklomelo kaNobel

Ngo-1993, usosayensi waseMelika uMulis wathola uMklomelo KaNobel kuKhemistry, futhi impumelelo yakhe kwaba ukusungulwa kobuchwepheshe be-PCR.Umlingo wobuchwepheshe be-PCR ukulezi zici ezilandelayo: Okokuqala, inani le-DNA okufanele likhuliswe lincane kakhulu, futhi ngokwembono i-molecule eyodwa ingasetshenziselwa ukukhulisa;okwesibili, ukusebenza kahle kwe-amplification kuphezulu, futhi inani lofuzo oluqondiwe liyachayeka.Ukukhulisa, izikhathi ezingaphezu kwezigidi eziyi-10 ngamahora ambalwa.Manje ithuluzi le-PCR selisetshenziswe kabanzi ocwaningweni lwesayensi yezempilo nezinye izici eziningi.

Amamodeli ahlukene nabakhiqizi bamabhayisikili ashisayo bangabonisa ukusebenza okuhlukile nokuphindaphinda.Lo mehluko akuthinti kuphela ukusebenza kahle kwe-PCR kodwa futhi nokunemba nokuvumelana kwedatha etholiwe.Ukuqonda izici zomshini we-PCR kungasisiza ukuthi sikhulise impumelelo yokuhlolwa kwethu.

Imojula yokushisa

Ukunemba kwezinga lokushisa lomjikelezo oshisayo kunganquma impumelelo noma ukwehluleka kwe-PCR.Ukungaguquguquki kwezinga lokushisa elisuka kahle liye emthonjeni endaweni yokushisa nakho kubalulekile ukuze uthole imiphumela ye-PCR ethembekile nephinde ikhiqizeke.

Enye indlela yokuqinisekisa ukunemba kokushisa ukuhlola njalo usebenzisa amakhithi okuqinisekisa izinga lokushisa futhi ulungise kabusha njengoba kudingwa uchwepheshe oqeqeshiwe.Ukuhlolwa kokuqinisekisa izinga lokushisa ngokuvamile kusetshenziselwa:

Ukunemba kahle kahle okuhlobene nokusetha izinga lokushisa kwimodi ye-isothermal

Ukunemba kahle-kuya emthonjeni okuhlobene nokusetha izinga lokushisa ngemuva kokuguqulwa kwezinga lokushisa

Ukunemba Kwezinga Lokushisa Lid

I-Primer Annealing Temperature Control

Ukulawulwa kwezinga lokushisa kwe-Gradient kuwumsebenzi wethuluzi le-PCR elisiza ukuthuthukiswa kokuhlanganisa i-primer ku-PCR.Injongo yokusetha kwegradient iwukuthola amazinga okushisa ahlukahlukene phakathi kwamamojula, futhi ngo-≥2°C izinga lokushisa likhuphuka liphinde lehle phakathi kwekholomu ngayinye, amazinga okushisa ahlukene angahlolwa ngesikhathi esisodwa ukuze kutholwe izinga lokushisa elilungile le-primer annealing.Ngokwetiyori, i-gradient yangempela ifinyelela izinga lokushisa lomugqa phakathi kwamamojula.

Kodwa-ke, amabhayisikili avamile we-gradient thermal cyclers ngokuvamile asebenzisa ibhulokhi eyodwa yokushisa kanye nokulawula izinga lokushisa ngokusebenzisa izinto ezimbili zokushisisa nokupholisa ezitholakala kuzo zombili iziphetho, ngokuvamile okuholela emikhawulweni elandelayo:

Amazinga okushisa amabili kuphela angasethwa: amazinga okushisa aphezulu naphansi e-primer annealing asethwa kuzo zombili iziphetho zemojuli ye-thermal, futhi ukusetha okunembile kwamanye amazinga okushisa akukwazi ukufinyelelwa phakathi kwe-modul.

Ngenxa yokushintshisana kokushisa phakathi kwamakholomu ahlukene, izinga lokushisa eliphakathi kwezifunda ezihlukene kumojuli kungenzeka lilandele ijika le-sigmoidal kunegradient yomugqa wangempela.

Isampula lokushisa

Ikhono le-thermal cycler ukulawula izinga lokushisa lesampula libaluleke kakhulu ekunembeni kwemiphumela ye-PCR.Amapharamitha aqondene nezinsimbi ezifana nezilinganiso zerempu, izikhathi zokubamba, nama-algorithms abalulekile ekubikezeleni izinga lokushisa lesampula.

Izinga lokushisisa nokupholisa lomshini we-PCR lisho ukuthi izinga lokushisa liyashintsha phakathi kwezinyathelo ze-PCR ezenzeka esikhathini esithile.Njengoba kuthatha isikhathi esithile ukuze ukushisa kudluliselwe kusuka kumojula kuya kusampula, izinga langempela lokushisa nokupholisa lesampula lizohamba kancane.Ngakho-ke, incazelo yejubane lokushintsha kwezinga lokushisa idinga ukuhlukaniswa futhi iqondwe.

Izinga lerempu yemojuli ephezulu noma ephezulu limelela ukuguqulwa kwezinga lokushisa okushesha kakhulu okungafinyelelwa yimojuli ngesikhathi esincane kakhulu phakathi kwerempu.

Isilinganiso serempu yebhulokhi emaphakathi simele izinga lokushisa lokushintsha esikhathini eside futhi sizonikeza isilinganiso esimele kakhulu sejubane lomshini we-PCR.

Isilinganiso esiphezulu sesampula sokushisisa nokupholisa kanye nesilinganiso esimaphakathi sesampula sokushisisa nokupholisa sibonisa izinga lokushisa langempela elitholwe isampula.Izinga lokushisisa nokupholisa kwesampula lizohlinzeka ngokuqhathanisa okunembe kakhudlwana kokusebenza komshini we-PCR kanye nomthelela wawo ongaba khona emiphumeleni ye-PCR.

Uma wenza ukumiselela kwamabhayisikili, kuyanconywa ukusebenzisa ithuluzi elinohlelo lwerempu elilingisa imodi yangaphambilini ukuze kube lula ukumiselela kanye nomthelela omncane ekuphindaphindeni kwe-PCR.

I-thermal cycler kufanele yakhelwe ukwenza izinyathelo zesikhathi kuphela ngemva kokuba isampuli ifinyelele izinga lokushisa elimisiwe.Ngale ndlela, isikhathi lapho isampula sigcinwa ekushiseni okusethiwe sizogcinwa ngokunembe kakhulu nezimo zomjikelezo ohambisanayo ezidingekayo kwinqubo yokusebenza.

Amabhayisikili ashisayo ngokuvamile asebenzisa ama-algorithms ezibalo ayinkimbinkimbi ukuze aqinisekise ukuthi amasampuli angafinyelela ngokushesha izinga lokushisa elimisiwe ngokohlelo olusethiwe.Ngokusekelwe kumthamo wesistimu yokusabela kanye nogqinsi lwamapulasitiki e-PCR asetshenzisiwe, i-algorithm ingabikezela izinga lokushisa lesampula kanye nesikhathi esizosithatha ukuze kufinyelelwe izinga lokushisa elimisiwe.Ngokuncika kulawa ma-algorithms, phakathi nenqubo yokushisisa noma yokupholisa yomjikelezo oshisayo, izinga lokushisa lokuvimba ngokuvamile lizodlula inani elimisiwe ngenqubo ebizwa ngokuthi i-thermal block overshoot noma i-undershoot.Ukusetha okunjalo kuqinisekisa ukuthi isampuli ifinyelela izinga lokushisa elimisiwe ngokushesha ngangokunokwenzeka ngaphandle kokuzidubula ngokweqile noma ukuzidubula ngaphansi kwaso.

Ukusebenza kokuhlola

Izinto ezingakhuphula ukuphuma komshini oshisayo zifaka amanani erempu, ukulungiselelwa kwebhulokhi eshisayo, nokuhlanganiswa kwamaplathifomu ezishintshayo.

Izinga lokushisa nokupholisa le-thermal cycler limelela isivinini lapho ifinyelela izinga lokushisa elimisiwe.Lapho izinga lokushisa likhuphuka futhi lehla ngokushesha, i-PCR izosebenza ngokushesha, okusho ukuthi ukuhlolwa okwengeziwe kungaqedwa ngesikhathi esithile.Ngaphezu kwalokho, ukuhlola kungasheshiswa ngokusebenzisa ama-polymerase e-DNA asheshayo.

Idizayini yemojuli ye-thermal cycler nayo ibalulekile ezivivinyweni ze-PCR.Isibonelo, amamojula ashintshekayo avumela ukuguquguquka kwenani lamasampuli ngokugijima ngakunye.Ukwengeza, amamojula okushisa anamamojula alawulekayo alungele ukusebenzisa izinhlelo ezihlukene ze-PCR ngesikhathi esisodwa kumjikelezo owodwa oshisayo.

Ku-PCR esebenza ngokuzenzakalelayo ephezulu, isofthiwe elawula isistimu yokubamba amapayipi kufanele ihleleke futhi ihambisane.Amasistimu azenzakalelayo alungele ukwenza ukusabela kwe-PCR ephezulu ngenxa yokuthi angaqhutshwa ngokuqhubekayo ngokungenelela okuncane komuntu, ngaleyo ndlela kuncishiswe isikhathi esidingekayo sokusethwa kokuhlolwa okwenziwa ngesandla futhi kwandise inani lokusabela esikhathini esithile esinikeziwe.

Ukuthembeka, Ukuqina kanye Nokuqinisekiswa Kwekhwalithi Yama-Thermal Cyclers

Ngokungeziwe ekusebenzeni namandla okuphuma, umshini we-PCR kufanele futhi ukwazi ukumelana nokusetshenziswa okuthile okuphindaphindiwe, ukucindezeleka kwemvelo, nezimo zokuthumela.Abanye abakhiqizi bangase babike ukuthi ithuluzi lenza kanjani ukuhlolwa kokwethembeka nokuqina.Ukutholwa kwethuluzi le-PCR elihambisanayo kuhlanganisa:

Ukuthembeka: Izinsimbi zemishini zisetshenziselwa ukwenza izivivinyo eziphindaphindiwe ezintweni zensimbi ezisetshenziswa njalo njengezivalo ezishisayo, amaphaneli okulawula/izikrini zokuthinta, namamojula wokuhamba ngebhayisikili.

Ingcindezi ye-ambient: Amakamelo emvelo angasetshenziswa ukulingisa izimo ezihlukene zokuhlolwa okujwayelekile, njengezinga lokushisa, umswakama.

Ukuhlolwa Kokuthunyelwa Kwemikhumbi: Ukuhlolwa kokushaqeka okukhulu nokudlidliza kungenziwa ngokuvumelana nezindinganiso ze-International Safety Shipping Association ukuze kuqinisekiswe ukuthi ithuluzi lingafika ngaphansi kwezimo zokusebenza ezingonakalisiwe.

Iwaranti nesevisi yokugcina umshini we-PCR

Ngaphandle kokuthembeka okuqinile nokuhlola ukuqina, amabhayisikili ashisayo anezinkinga zobuchwepheshe empilweni yonke yethuluzi.Ukuze uthole ukuthula kwengqondo, iwaranti yomkhiqizi, isevisi, nokunakekelwa kufanele kucatshangelwe lapho kuthengwa ithuluzi.

Ukuvumelana nezimo kwezinsizakalo ezifana nokulungiswa kwendawo/ukubuyisela embonini, izinsizakalo zokuqapha kude, namathuluzi okushintsha enqubweni yokulungisa, njll., ukuze kuncishiswe umthelela ekusebenzeni kahle komsebenzi.

Ubude besikhathi sewaranti, isikhathi sokushintsha isevisi, ukufinyeleleka kosekelo lobuchwepheshe, namakhono abasebenzi abangochwepheshe bosekelo.

Ukuba nokwenzeka kokufakwa kwensimbi, ukusebenza, ukubambisana, nokuqinisekisa ukuze kuhlangatshezwane nezidingo zelabhorethri nezihlobene nokulawula.Izinsizakalo zokunakekela ezifana nokuqinisekisa izinga lokushisa, ukuhlola, nokulinganisa kuyatholakala ukuze kuqinisekiswe ukuthi ithuluzi lisebenza kahle namapharamitha ahambisanayo.

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