Umshini we-PCR idivayisi yaselabhorethri esebenzisa ukuhamba ngebhayisikili okunembayo ukuze kube lula i-Polymerase Chain Reaction, okuvumela ukukhuliswa okusheshayo kwe-DNA ethile noma ukulandelana kwe-RNA ukuze kuhlaziywe. Ngokwenza ngokuzenzakalelayo izigaba zokushisisa nokupholisa ezidingekayo ukuze kuguqulwe umzimba, ukuhlanganisa, nokunwetshwa, umshini wesimanje we-PCR uhlinzeka ngokungaguquguquki nesivinini esidingekayo kuzicelo zomtholampilo nezocwaningo.
Ukuqonda ubuchwepheshe obuyisisekelo bale mishini kubalulekile kubaphathi baselabhorethri, abacwaningi, nochwepheshe bezokwelapha abafuna ukuthuthukisa ukugeleza komsebenzi wabo wokuxilonga. Kusukela ebunkingeni bezinto zokushisisa ze-Peltier kuya ku-optics ethuthukisiwe yezinhlelo zesikhathi sangempela, ubunjiniyela obusemuva kokukhulisa i-DNA buyinkimbinkimbi futhi buyamangalisa. Lesi sihloko sihlinzeka ngokuhlolwa okuphelele kobuchwepheshe be-PCR, izingxenye zayo eziwumshini, kanye nokusebenza kwazo okuhlukahlukene embonini yezokwelapha yomhlaba wonke.
Uhlolojikelele Lwesakhiwo Se-athikili
| Ingxenye |
Isifinyezo |
| Usebenza kanjani umshini we-PCR |
Incazelo yezinyathelo ezintathu zenqubo yokuhamba ngebhayisikili eshisayo (ukuguqula, ukuhlanganisa, nokunwetshwa) evumela ukukhula kwe-DNA ecacile. |
| Izingxenye Eziyinhloko ze-PCR Technology |
Ukuhlukaniswa kwezobuchwepheshe kwamabhulokhi okushisisa, ama-elementi e-Peltier, nezinzwa eziqinisekisa ukufana nokunemba okushisayo. |
| Izinhlobo zemishini ye-PCR kanye nokusetshenziswa kwayo |
Ukuqhathaniswa phakathi kwamabhayisikili ajwayelekile ashisayo, amasistimu we-Real-Time (qPCR), kanye nobuchwepheshe be-Digital PCR (dPCR). |
| Ukubaluleka Kokufana Kwe-Thermal |
Ukuhlaziywa kokuthi kungani ukungaguquguquki kwezinga lokushisa kuyo yonke imithombo kuyisici esibaluleke kakhulu semiphumela ethembekile yelabhorethri elungiselelwe i-SEO. |
| Izicelo ku-Modern Diagnostics |
Ubuchwepheshe be-PCR busetshenziswa kanjani ekuhlolweni kwezifo ezithathelwanayo, i-oncology, kanye nokuhlonza i-forensic. |
| Amathiphu Okugcina Nokuthuthukisa |
Izinqubo ezihamba phambili zokulinganisa nokugcina okokusebenza kwe-PCR ukuze kuqinisekiswe ukunemba nokusebenza kwesikhathi eside. |
Usebenza kanjani umshini we-PCR
Ukusebenza okuyisisekelo komshini we-PCR kuhilela ukuhamba ngebhayisikili ezindaweni ezintathu ezihlukene zokushisa ukuze kube lula ukushintshashintsha kwe-DNA, ukuhlanganisa i-primer annealing, kanye nokunwetshwa kwe-enzymatic, okuholela ekuphindaphindeni okuphawulekayo kwezinto zofuzo eziqondiwe.
Enhliziyweni yenqubo umjikelezo wokushisa. Phakathi nesigaba sokuqala, i-denaturation, umshini we-PCR ushisa ingxube yokusabela cishe ku-94 kuya ku-98 degrees Celsius. Lokhu kushisa okuphezulu kwephula izibopho ze-hydrogen phakathi kwemicu emibili ye-DNA double helix, okuholela ezifanekisweni ezimbili ze-DNA ezinomucu owodwa. Ukunemba kulesi sigaba kubalulekile, njengoba ukushisa okunganele kuzohluleka ukuhlukanisa izintambo, kuyilapho ukushisa okudlulele kungalimaza i-enzyme ye-polymerase ezinzile.
Isigaba sesibili i-annealing, lapho izinga lokushisa lehliswa libe phakathi kuka-50 no-65 degrees Celsius. Lokhu kuvumela ukulandelana kwe-DNA emifushane okubizwa ngokuthi ama-primers ukuthi abophe ukulandelana kwawo okuhambisanayo kuzifanekiso ze-DNA ezinomucu owodwa. Izinga lokushisa eliqondile lincike ekushiseni kokuncibilika kweziqalo ezisetshenzisiwe. wekhwalithi ephezulu Umshini we-PCR kufanele ukwazi ukufinyelela futhi uzinze kuleli zinga lokushisa elithile ngokushesha ukuze uvimbele ukubopha okungaqondile, okungaholela emiphumeleni engalungile noma 'umsindo' kudatha.
Ekugcineni, isigaba sokunwetshwa senzeka, ngokuvamile ku-72 degrees Celsius. Leli izinga lokushisa elilungile le-DNA polymerase (imvamisa i-Taq polymerase) ukuze ihlanganise umucu we-DNA entsha ngokwengeza ama-nucleotide kuma-primers. Njengoba umjikelezo uphindaphinda, inani lamakhophi e-DNA liphinda kabili isikhathi ngasinye. Ukuze uqonde kangcono ukutshalwa kwezimali okudingekayo kulobu buchwepheshe, ungahlola imininingwane mayelana Uyini Umshini we-PCR kanye Nezindleko Zawo zokuhlola isabelomali esidingekayo kumayunithi asebenza kahle kakhulu.
I-Mechanics of Exponential Growth
Umjikelezo 1: 2 amakhophi
Umjikelezo 10: amakhophi angu-1,024
Umjikelezo wama-30: Amakhophi angaphezu kwebhiliyoni elingu-1
Izingxenye Eziyinhloko ze-PCR Technology
Ubuhle bezobuchwepheshe bomshini we-PCR buncike ku-hardware yawo yangaphakathi, ngokuyinhloko izinto zokushisisa ze-Peltier, i-silver noma i-aluminium thermal block, kanye nesofthiwe yokulawula eyinkimbinkimbi elawula ukuguquka kwezinga lokushisa okusheshayo.
Ingxenye ebaluleke kakhulu i-elementi ye-Peltier, iphampu yokushisa esebenzayo yesimo esiqinile edlulisa ukushisa kusuka ohlangothini olulodwa lwedivayisi kuya kolunye ngokumelene ne-gradient yezinga lokushisa. Lokhu kuvumela kokubili ukushisisa nokupholisa okuvela emthonjeni owodwa, okwenza umshini we-PCR uguqule amazinga okushisa ngamasekhondi. Isivinini okwenzeka ngaso lezi zinguquko—esaziwa 'izinga lerempu' -sinquma ngokuqondile ukusebenza kahle kukonke kanye nokuphuma kwelabhorethri. Imishini ephezulu ivame ukufaka amabhlogo esiliva anegolide ukuze kwandiswe ukuqhutshwa kokushisa.
Izinzwa nama-microprocessors akha 'ubuchopho' bedivayisi. Lezi zingxenye ziqapha izinga lokushisa lokuvimba ngesikhathi sangempela, zilungisa amandla ku-elementi ye-Peltier ukuze kuvinjelwe izinga lokushisa elingaphezu kokudubula noma ukudubuleka ngaphansi. Amasistimu esimanje asebenzisa izinzwa eziningi kuyo yonke ibhulokhi ukuze aqinisekise ukuthi isampula ngayinye ihlangabezana nezimo ezishisayo ezifanayo ncamashi. Ukuze uthole izinsiza ezifuna ukuthuthukisa i-hardware yazo, ukukhetha okuthembekile Umhlinzeki womshini we-PCR uqinisekisa ukufinyelela kubuchwepheshe benzwa bakamuva nobunjiniyela obuqinile.
Isivalo esishisayo singenye ingxenye ebalulekile kodwa evame ukunganakwa. Ngokugcina isivalo ekushiseni okungaphezudlwana kwesilinganiso esiphezulu se-block (imvamisa sizungeze ama-degree Celsius angu-105), umshini uvimbela ukuhwamuka nokufiphala kwengxube yokusabela kumakepisi weshubhu. Lokhu kuqinisekisa ukuthi ukugxila kwama-reagents kuhlala kungashintshi kuyo yonke imijikelezo engama-30 kuye kwengama-40, okubalulekile ebhalansi yamakhemikhali yokusabela.
Izici Zekhompuyutha Ezibalulekile
I-Thermal Block: Ivamise ukuphatha imithombo engama-96 noma engama-384.
I-Ramp Rate: Isivinini sokushintsha kwezinga lokushisa (kukalwa ngo-°C/s).
Ukunemba: Kusondele kangakanani izinga lokushisa le-block endaweni yokubeka.
Ukufana: Ukungaguquguquki kwezinga lokushisa kuyo yonke imithombo.
Izinhlobo zemishini ye-PCR kanye nokusetshenziswa kwayo
Ibhayoloji yamangqamuzana yesimanje isebenzisa izigaba ezintathu eziyinhloko zobuchwepheshe be-PCR: I-Gradient PCR yokwenza kahle, i-Real-Time PCR (qPCR) yokulinganisa, kanye ne-Digital PCR ngokunemba okuphelele ekutholweni kwe-allele engavamile.
Ama-Standard Thermal Cyclers awuhlobo oluvame kakhulu olusetshenziswa ocwaningweni oluyisisekelo nasemfundweni. Le mishini yakhelwe 'iphoyinti lokugcina' i-PCR, okusho ukuthi imiphumela ihlaziywa ngemva kokuba imijikelezo isiqediwe, ngokuvamile ngejeli electrophoresis. Okuhlukile kwalokhu umshini we-Gradient PCR , ovumela umsebenzisi ukuthi asethe amazinga okushisa ahlukene kuyo yonke ibhulokhi phakathi nokugijima okukodwa. Lokhu kuwusizo ngendlela emangalisayo ekunqumeni izinga lokushisa elilungile le-anneal kuma-primers amasha, ukonga amahora okuhlola amaphutha.
Imishini ye-PCR yesikhathi sangempela (qPCR) imele ukweqa okubalulekile kobuchwepheshe. Lawa madivaysi ahlanganisa isistimu yokubona edidiyelwe (ama-laser noma ama-LED nezitholi) ukuze kuqashwe i-fluorescence yamasampula e-DNA phakathi nenqubo yokukhulisa. Lokhu kuvumela ukuhlaziya 'kobuningi', lapho abacwaningi benganquma khona ukuthi ingakanani i-DNA elandelanayo eyayikhona kusampula yoqobo. Lobu buchwepheshe buyizinga eligolide lokuhlolwa komthamo wegciwane egazini kanye nezifundo zesisho sofuzo. Ukuqonda i intengo kanye nokucaciswa kwamayunithi e-PCR kubalulekile lapho unquma phakathi komjikelezo ojwayelekile nohlelo oluthuthukisiwe lwe-qPCR.
I-Digital PCR (dPCR) iwukuziphendukela kwemvelo kwakamuva, enikeza ukulinganisa okuphelele ngaphandle kwesidingo sejika elijwayelekile. Isampula ihlukaniswe yaba izinkulungwane zamaconsi amancane, futhi ukusabela kwe-PCR kwenzeka kulelo nalelo. Ngokubala inani lamaconsi 'okuhle' kuqhathaniswa 'negative', umshini uhlinzeka ngokubala okunembe kakhulu kwama-molecule e-DNA. Lokhu kuwusizo ikakhulukazi ekutholeni ukuguqulwa kwezakhi zofuzo ezingavamile kuma-liquid biopsies noma amasampula emvelo lapho i-DNA eqondiwe ikhona ekugxilweni okuphansi kakhulu.
Ukuqhathaniswa kwe-PCR Technologies
| Isici |
I-PCR ejwayelekile |
I-PCR yesikhathi sangempela (qPCR) |
I-PCR yedijithali (dPCR) |
| Ukutholwa |
Iphoyinti lokugcina |
Isikhathi sangempela |
Iphoyinti lokugcina (Absolute) |
| Quantification |
Ikhwalithi |
Isihlobo/Ubuningi |
Impela |
| Ukuzwela |
Kuphakathi |
Phezulu |
I-Ultra-High |
| Inkimbinkimbi |
Phansi |
Kuphakathi |
Phezulu |
Ukubaluleka Kokufana Kwe-Thermal
Ukufana okushisayo kubhekisela ekuhambisaneni kwezinga lokushisa kuyo yonke imithombo ye-PCR block, isici esibalulekile esiqinisekisa ukuthi yonke isampula kupuleti lomthombo we-96 ikhula ngesilinganiso esifanayo futhi iveza imiphumela ephindaphindekayo.
Uma ikhona elilodwa lebhulokhi eshisayo lipholile ngisho nangu-0.5 degrees Celsius kunesikhungo, i-DNA kuleyo mithombo ingase ingashintshi ngokuphelele, noma ama-primers angase abophe ngokungaqondile. Lokhu kuholela 'ekushintsheni kokuhamba kahle,' okungase konakalise konke ukuhlola noma, okubi nakakhulu, kuholele ekubeni negethivu engamanga esimweni sokuxilonga emtholampilo. Abakhiqizi bamamodeli yekhwalithi ephezulu emishini ye-PCR batshala imali eningi emshinini onembayo kanye nokushisisa izindawo eziningi ukuze kuncishiswe lezi zingozi futhi kunikeze ukufana komngcele ukuya kowokuphela.
Ama-algorithms esofthiwe athuthukisiwe asetshenziselwa ukunxephezela 'umphumela onqenqemeni,' lapho imithombo ezungezwe yibhulokhi ilahlekelwa ukushisa ngokushesha kunaleyo emaphakathi. Ngokulawula izindawo ezihlukene zePeltier ngokuzimela, umshini ungampompa ukushisa okwengeziwe emaphethelweni ukuze ugcine iphrofayili eshisayo eyisicaba. Lapho ucwaninga imishini, kunenzuzo ukubheka a ikhathalogi yomshini we-PCR wekhwalithi ephezulu ukuze uqhathanise ukuthi amabhrendi ahlukene aphatha kanjani ukuphatha okushisayo kanye nokulinganiswa kwebhulokhi.
Ukukhiqiza kabusha kuyisisekelo sesayensi. Noma ngabe ukuhlolwa kwenziwa e-London noma e-Shanghai, umshini we-PCR kufanele ulethe ukusebenza okufanayo okushisayo. Kungakho izindinganiso zamazwe ngamazwe kanye nokulinganisa okuvamile kuyisibopho kumalebhu omtholampilo. Umshini onokufana okungekuhle uzokhiqiza amanani 'Ct' (umkhawulo womjikelezo) angahambisani ku-qPCR, okwenza kungenzeki ukulinganisa ngokunembile inani legciwane egazini noma izimpawu zofuzo kuwo wonke ama-run noma amalabhorethri ahlukene.
Izici Ezithinta Ukufana
I-Block Material: Isiliva liphakeme kune-aluminium yokusabalalisa ukushisa.
Ikhwalithi yePeltier: Ama-Peltier ebanga eliphezulu anikeza ukuphila okude kanye nokushisisa okungaguquki.
Ukubekwa Kwenzwa: Izinzwa eziningi zinikeza impendulo engcono yeluphu yokulawula.
Izicelo ku-Modern Diagnostics
Ukusetshenziswa kobuchwepheshe be-PCR kudlulela kukho konke ukuhlonza izifo ezithathelwanayo, i-oncology yokuthola umaka womdlavuza, kanye nesayensi yezobunhloli, lapho kusetshenziswa khona ukwakha amaphrofayili e-DNA kusuka kumasampula ebhayoloji amaminithi amancane.
Ezimweni zezifo ezithathelwanayo, umshini we-PCR usuyithuluzi elibalulekile. Ivumela ukutholwa kwamagciwane afana ne-HIV, i-Hepatitis, namagciwane okuphefumula anokuzwela okuphezulu kakhulu kanye nesivinini kunezindlela zesiko lendabuko. Ngenxa yokuthi umshini ukwazi ukubona ukuba khona kwe-genome yegciwane ngokuqondile, ungakwazi ukubona ukutheleleka kudala ngaphambi kokuba isiguli sibe nokusabela komzimba noma izimpawu, okuvumela ukungenelela kwangaphambi kwesikhathi kanye nokuphathwa okungcono kwezempilo yomphakathi.
I-Oncology ngomunye umkhakha ozuza ngokujulile kulobu buchwepheshe. I-PCR isetshenziselwa ukuthola ukuguqulwa kofuzo okuthile okuhlobene nezinhlobo ezithile zomdlavuza. Lokhu kuvumela 'umuthi womuntu siqu,' lapho odokotela bengakhetha khona ukwelashwa okuphumelelayo ngokusekelwe kuphrofayili yofuzo yesimila. Kulabo abafuna ukwazi ngeqhaza lobuchwepheshe ekunakekelweni kwezempilo, ukufunda mayelana izindleko kanye nokusetshenziswa kwezinhlelo ze-PCR kunikeza ukuqonda kokuthi izibhedlela zibhalansisa kanjani ubuchwepheshe obusezingeni eliphezulu nezabelomali zokusebenza.
Isayensi yezobunhloli incike ku-PCR ukuze ikhulise i-DNA ezigcawini zobugebengu, njengezinwele ezilodwa noma iconsi legazi. Ngokuqondisa izindawo ezithile zofuzo lomuntu ezihluka phakathi kwabantu (STRs), ososayensi bezobunhloli bangakha isigxivizo somunwe se-DNA esiyingqayizivele. Lobu buchwepheshe buye benza izinguquko ezinhle ohlelweni lwezomthetho, banikeza ubufakazi obunamandla kukho kokubili ukugwetshwa kanye nokukhululwa kwabasolwa ngokungafanele.
Imikhakha Emikhulu Yezicelo
Ukuxilongwa Komtholampilo: Ukuhlolwa kwe-pathogen kanye nokuhlolwa kokubeletha.
Ukuphepha Kokudla: Ukuthola ama-GMO noma amagciwane atholakala ekudleni njenge-Salmonella.
Isayensi Yezemvelo: Ukuqapha isibalo samagciwane enhlabathini noma emanzini.
Ucwaningo: I-Cloning, ukulandelana, nokuhlaziywa kwenkulumo yofuzo.
Amathiphu Okugcina Nokuthuthukisa
Ukuqinisekisa ukuphila isikhathi eside nokunemba komshini we-PCR, amalabhorethri kufanele asebenzise izimiso eziqinile zokunakekela, okuhlanganisa ukuhlanzwa kwamabhulokhi okuvamile, ukulinganisa kwezivalo, nokuqinisekiswa kokushisa okusebenzayo.
Ukungcola kuyisitha esikhulu se-PCR. Ngisho ne-molecule eyodwa ye-DNA kusukela ekugijimeni kwangaphambilini ingakhuliswa, okuholela emiphumeleni engalungile. Ngakho-ke, i-thermal block yomshini we-PCR kufanele ihlanzwe njalo ngezixazululo ezikhethekile zokukhipha i-DNA noma i-bleach engu-10% elandelwa yi-ethanol. Kubalulekile futhi ukuqinisekisa ukuthi amashubhu avaliwe ngendlela efanele ukuze kuvinjelwe ukuvuza kwesampula emithonjeni, okungagqwalisa indawo evimbayo futhi kuphazamise ukudluliswa kokushisa.
Ukulinganisa okuvamile nakho kubalulekile. Ngokuhamba kwesikhathi, ama-elementi e-Peltier angehlisa izinga, okuholela emazingeni erempu ehamba kancane noma ukuncipha kokufana kokushisa. Izinsizakalo zokulinganisa ezichwepheshile zisebenzisa isistimu 'multiprobe' ukukala izinga lokushisa lemithombo eminingana ngesikhathi esisodwa ngesikhathi sokugijima. Lokhu kuqinisekisa ukuthi umshini usasebenza ngaphakathi kokubekezelela okucacisiwe komkhiqizi. Kumalebhu afuna okokusebenza okuqinile okunamaphrofayili okulungisa alula, ukuxhumana nabakhethekile Umhlinzeki wemishini ye-PCR angasiza ekukhetheni amamodeli aklanyelwe ukusetshenziswa komsebenzi osindayo.
Izibuyekezo zesofthiwe akufanele zizitshwe. Abakhiqizi bavame ukukhulula izibuyekezo ze-firmware ezithuthukisa ama-algorithms okulawula izinga lokushisa noma ezengeze izici ezintsha kusixhumi esibonakalayo somsebenzisi. Ukugcina isofthiwe yomshini isesikhathini samanje kuqinisekisa ukuthi uyazuza kusukela ekuthuthukisweni kwakamuva ekuphathweni kokushisa. Ukwengeza, sebenzisa njalo izinto ezisetshenziswayo ze-PCR ezisezingeni eliphezulu (amashubhu namapuleti), njengoba amapulasitiki anezindonga ezincane eklanyelwe ukwenza lula ukudluliswa kokushisa okushesha kusuka kubhulokhi kuya engxubeni yokusabela.
Uhlu Lokuhlola Isondlo
Nsuku zonke: Sula ingaphandle futhi uhlole isivalo esishisayo.
Maviki onke: Hlola imithombo ukuthi akukho yini imfucumfucu noma ukuvuza; hlanza ibhulokhi.
Nyanga zonke: Yenza ukuhlolwa kokuzihlola uma kutholakala kusofthiwe.
Minyaka yonke: Ukuqinisekiswa kokushisa okusebenzayo kanye nokulinganisa.
Isiphetho
Ubuchwepheshe bomshini we-PCR buyizinga eliphezulu kwezobunjiniyela obunembayo kanye nekhemikhali yamangqamuzana. Ngokuhlinzeka ngendawo elawulwayo yokukhulisa i-DNA, lawa madivayisi avule izimfihlo zekhodi yofuzo futhi ahlinzeka ngamathuluzi adingekayo ukulwa nobhadane lomhlaba wonke, ukwelapha umdlavuza, nokuxazulula ubugebengu. Njengoba siqhubekela kumasistimu adidiyelwe kakhulu nawedijithali, imigomo ewumongo yebhayisikili elishisayo isalokhu isebenza njengakuqala.
Kumalabhorethri nezindawo zezokwelapha, ukukhetha ubuchwepheshe obufanele be-PCR ibhalansi yesivinini, ukunemba, kanye nokusebenza kahle kwezindleko. Kungakhathaliseki ukuthi umjikelezo oshisayo oyisisekelo wezinjongo zemfundo noma isistimu yesikhathi sangempela ethuthukisiwe yokuxilongwa okubucayi, ukuqonda izinsimbi ezingemuva komshini kuyisinyathelo sokuqala sokuthola imiphumela ethembekile nenomthelela. Ikusasa lobuchwepheshe be-PCR lithembisa imiphumela eshesha nakakhulu, ukuphatheka okukhulu, nokuzwela okuphezulu, ukuqhubeka nendima yayo njengetshe legumbi lenqubekelaphambili yesayensi.
