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Umatshini wePCR|Uyaqonda ngenene?

Iteknoloji yePCR ephumelele iBhaso leNobel

Ngo-1993, isazinzulu saseMelika uMulis wafumana ibhaso leNobel kwiKhemistry, kwaye impumelelo yakhe yaba kukuveliswa kobuchwepheshe bePCR.Ubugqi bobuchwepheshe be-PCR bukwezi mpawu zilandelayo: Okokuqala, umyinge we-DNA oza kunyuswa umncinci kakhulu, kwaye ngokwethiyori enye i-molecule ingasetyenziselwa ukukhulisa;okwesibini, ukusebenza kakuhle kwe-amplification kuphezulu, kwaye inani le-gene ekujoliswe kuyo liyi-exponential.Ukwandisa, ngaphezu kwezigidi ezili-10 amaxesha kwiiyure ezimbalwa.Ngoku isixhobo sePCR sisetyenziswe ngokubanzi kuphando lwesayensi yobomi kunye neminye imiba emininzi.

Iimodeli ezahlukeneyo kunye nabavelisi beebhayisikile ezishushu banokubonisa ukusebenza okwahlukileyo kunye nokuphindaphinda.Olu lwahluko aluchaphazeli nje kuphela ukusebenza kwe-PCR kodwa kunye nokuchaneka kunye nokuhambelana kwedatha efunyenweyo.Ukuqonda iimpawu zomatshini we-PCR kunokusinceda songeze impumelelo yovavanyo lwethu.

Imodyuli yokufudumeza

Ukuchaneka kobushushu bomjikelo we-thermal kunokugqiba ukuphumelela okanye ukungaphumeleli kwe-PCR.Ukuhambelana kweqondo lokushisa kakuhle kwibhloko yokufudumeza kukwabalulekile ekufumaneni iziphumo zePCR ezithembekileyo nezinokuphinda zenziwe kwakhona.

Enye indlela yokuqinisekisa ukuchaneka kobushushu kukuvavanya rhoqo usebenzisa iikhithi zokuqinisekisa ubushushu kwaye uhlengahlengise njengoko kufuneka yingcali eqeqeshiweyo.Iimvavanyo zokuqinisekisa ubushushu zidla ngokusetyenziswa:

Ukuchaneka kakuhle kakuhle ngokumalunga nokuseta ubushushu kwimowudi ye-isothermal

Ukuchaneka kakuhle kokuseta ubushushu emva kokuguqulwa kobushushu

Ukuchaneka koBubushushu beLid lobushushu

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I-Primer Annealing Control Control

Ulawulo lobushushu begradient ngumsebenzi wesixhobo se-PCR esiququzelela ukwenziwa kwe-primer annealing kwi-PCR.Injongo yokumisela i-gradient kukuphumeza amaqondo okushisa ahlukeneyo phakathi kweemodyuli, kunye ne-≥2 ° C ukunyuka kweqondo lokushisa kunye nokuwa phakathi kwekholomu nganye, amaqondo okushisa ahlukeneyo angavavanywa ngaxeshanye ukufumana ubushushu obuphezulu be-primer annealing.Ngokwethiyori, i-gradient yokwenyani ifezekisa ubushushu bomgca phakathi kweemodyuli.

Nangona kunjalo, iibhayisekile ze-thermal gradient ziqhele ukusebenzisa ibhloko enye ye-thermal kunye nokulawula ubushushu ngokusebenzisa izinto ezimbini zokufudumeza kunye nokupholisa ezibekwe kuzo zombini iziphelo, zihlala zikhokelela kwimida elandelayo:

Amaqondo obushushu amabini kuphela anokusetwa: amaqondo obushushu aphezulu naphantsi e-primer annealing asetwe kuzo zombini iziphelo zemodyuli ye-thermal, kwaye ukusetwa okuchanekileyo kwamanye amaqondo obushushu akunakufezekiswa phakathi kwemodyuli.

Ngenxa yokutshintshiselana kobushushu phakathi kweekholomu ezahlukeneyo, ubushushu phakathi kwemimandla eyahlukeneyo kwimodyuli kunokwenzeka ukuba ilandele ijika le-sigmoidal kunokuba i-gradient yomgca wenyani.

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Ubushushu besampuli

Ikhono lomjikelo we-thermal ukulawula ubushushu besampulu kubaluleke kakhulu ukuchaneka kweziphumo zePCR.Iiparamitha ezithe ngqo kwisixhobo ezifana nemilinganiselo yerempu, amaxesha okubamba, kunye ne-algorithms zibalulekile ukuqikelela ubushushu besampulu.

Izinga lokufudumeza kunye nokupholisa umatshini we-PCR kuthetha ukuba ubushushu butshintsha phakathi kwamanyathelo e-PCR okwenzeka kwixesha elithile.Ekubeni kuthatha ixesha elithile ukuba ubushushu butshintshe ukusuka kwimodyuli ukuya kwisampuli, ukufudumala kwangempela kunye nesantya sokupholisa isampuli kuya kuba kancinci.Ngoko ke, inkcazo yesantya sokutshintsha kweqondo lokushisa kufuneka yahlulwe kwaye iqondwe.

Ubuninzi okanye incopho yemodyuli yerampu imele utshintsho olukhawulezayo lobushushu olunokufikelela kwimodyuli ngexesha elincinci kakhulu ngexesha lerempu.

Umyinge wesantya serempu yebhloko imele isantya sokutshintsha kobushushu ngexesha elide kwaye iya kubonelela ngomlinganiselo omele ngakumbi wesantya somatshini wePCR.

Ubuninzi besampulu yokufudumeza kunye nesantya sokupholisa kunye nesampulu ephakathi yokufudumeza kunye nezinga lokupholisa libonisa ubushushu obufunyenwe yisampuli.Izinga lokufudumeza isampuli kunye nokupholisa kuya kunika uthelekiso oluchanekileyo ngakumbi lokusebenza komatshini we-PCR kunye nefuthe elinokubakho kwiziphumo ze-PCR.

Xa usenza ukutshintshwa kwebhayisikile, kuyacetyiswa ukuba kusetyenziswe isixhobo esinenkqubo yesantya serampu esilinganisa imowudi yangaphambili ukuze kube lula ukutshintshwa kunye nefuthe elincinci lokuphindaphinda kwe-PCR.

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Umjikelo we-thermal kufuneka uyilwe kumanyathelo exesha kuphela emva kokuba isampuli ifikelele kubushushu obumiselweyo.Ngale ndlela, ixesha apho isampuli igcinwa kwiqondo lokushisa elimiselweyo liya kugcinwa ngokuchanekileyo kunye neemeko zomjikelezo ohambelanayo ezifunekayo kwinkqubo yokusebenza.

Iibhayisekile ze-Thermal zihlala zisebenzisa i-algorithms zemathematika ezintsonkothileyo zokuqinisekisa ukuba iisampulu zinokukhawuleza ukufikelela kwiqondo lobushushu elibekiweyo ngokwenkqubo esetwe kwangaphambili.Ngokusekelwe kumthamo wenkqubo yokusabela kunye nobukhulu beeplastiki zePCR ezisetyenzisiweyo, i-algorithm inokuqikelela ubushushu besampulu kunye nexesha eliza kuthatha ukufikelela kwiqondo lokushisa elibekiwe.Ukuxhomekeka kwezi algorithms, ngexesha lokufudumeza okanye ukupholisa inkqubo ye-thermal cycler, ukushisa kwebhloko ngokuqhelekileyo kuya kudlula ixabiso elimiselweyo ngenkqubo ebizwa ngokuba yi-thermal block overshoot okanye i-undershoot.Ukuseta okunjalo kuqinisekisa ukuba isampuli ifikelela kwiqondo lobushushu elisetiweyo ngokukhawuleza kangangoko ngaphandle overshooting okanye undershooting ngokwayo.

Ukwenziwa kovavanyo

Izinto ezinokonyusa ukuphuma komjikelo we-thermal zibandakanya amaxabiso e-ramp, ukucwangciswa kwebhloko ye-thermal, kunye nokuhlanganiswa kweeplatifomu ezizenzekelayo.

Izinga lokufudumeza kunye nokupholisa kwe-thermal cycler limele isantya apho ifikelela kwiqondo lokushisa elibekiwe.Ukukhawuleza ukunyuka kweqondo lokushisa kunye nokuwa, ngokukhawuleza i-PCR iya kuqhuba, oku kuthetha ukuba ezinye iimvavanyo zingagqitywa ngexesha elithile.Ukongeza, iimvavanyo zinokukhawuleziswa ngokusebenzisa iipolymera zeDNA ezikhawulezayo.

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Uyilo lwemodyuli yebhayisekile eshushu lukwabalulekile kwimifuniselo yePCR.Ngokomzekelo, iimodyuli ezithathelwanayo zivumela ukuguquguquka kwinani leesampuli ngokubaleka.Ukongezelela, iimodyuli zokufudumeza ezineemodyuli ezilawulwa ngabanye zilungele ukuqhuba iinkqubo ezahlukeneyo ze-PCR ngaxeshanye kwibhayisikili enye ye-thermal.

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Kwi-PCR ye-automated high-throughput, isofthiwe elawula inkqubo yokuphatha imibhobho kufuneka icwangciswe kwaye ihambelane.Iisistim ezizisebenzelayo zifanelekile ukwenza iimpendulo zePCR eziphezulu kuba zinokuqhutywa ngokuqhubekayo ngongenelelo oluncinane lomntu, ngokwenjenjalo kuncitshiswa ixesha elifunekayo kulungiselelo lovavanyo lwezandla nokwandisa inani leempendulo kwixesha elinikiweyo.

Ukuthembeka, ukuqina kunye nokuqinisekiswa komgangatho wee-Cyclers ze-Thermal

Ukongeza ekusebenzeni kunye nobuchule bokuphuma, umatshini wePCR kufuneka kwakhona ukwazi ukumelana nokusetyenziswa okuphindaphindiweyo, uxinzelelo lwendalo, kunye neemeko zokuthumela.Abanye abavelisi banokunika ingxelo malunga nendlela isixhobo esenza ngayo ukuthembeka kunye novavanyo lokuqina.Ukufunyanwa kwesixhobo sePCR esihambelanayo kubandakanya:

Ukuthembeka: Izixhobo zoomatshini zisetyenziselwa ukwenza iimvavanyo eziphindaphindiweyo kwizinto zesixhobo ezisetyenziswa rhoqo ezifana nezivalo ze-thermal, iipaneli zokulawula / ii-touchscreens, kunye neemodyuli zokuhamba ngebhayisikili.

Uxinzelelo lwe-Ambient: Amagumbi okusingqongileyo angasetyenziselwa ukulinganisa iimeko ezahlukeneyo zovavanyo lwesiqhelo, olufana nobushushu, ukufuma.

Uvavanyo lokuthumela: Ukutshatyalaliswa okukhulu kunye novavanyo lokungcangcazela lunokwenziwa ngokwemigangatho ye-International Safety Shipping Association ukuqinisekisa ukuba isixhobo sinokufika kwiimeko zokusebenza ezingonakali.

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Iwaranti kunye nenkonzo yokugcina umatshini wePCR

Ngaphandle kokuthembeka okungqongqo kunye novavanyo lokuqina, iibhayisekile ezishushu ngokuqinisekileyo zinemiba yobuchwephesha kubomi besixhobo.Ukufumana uxolo lwengqondo, iwaranti yomenzi, inkonzo kunye nokugcinwa kufuneka kuthathelwe ingqalelo xa kuthengwa isixhobo.

Ukuguquguquka kweenkonzo ezifana nokugcinwa kwendawo / ukubuyiswa kwefektri, iinkonzo zokubeka iliso kude, kunye nezixhobo zokutshintsha kwinkqubo yokugcinwa, njl., ukunciphisa impembelelo ekusebenzeni kakuhle.

Ubude bexesha lewaranti, ixesha lokuguqulwa kwenkonzo, ukufikeleleka kwenkxaso yobugcisa, kunye nezakhono zabasebenzi benkxaso yobuchwephesha.

Ukuba nokwenzeka kofakelo lwesixhobo, ukusebenza, intsebenziswano, kunye nokuqinisekisa ukuhlangabezana neelebhu kunye neemfuno zolawulo ezinxulumeneyo.Iinkonzo zokugcina ezifana nokuqinisekiswa kweqondo lokushisa, ukuvavanya, kunye nokulinganisa ziyafumaneka ukuze kuqinisekiswe ukuba isixhobo senza ngokufanelekileyo kunye neeparitha ezihambelanayo.

Iimveliso ezinxulumeneyo:

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Ixesha lokuposa: Oct-18-2022