Izinyathelo Zenqubo Ye-Polymerase Chain Reaction (PCR).

I-Polymerase Chain Reaction, eyaziwa kabanzi nge-PCR, imele impumelelo ebaluleke kakhulu yezobuchwepheshe emlandweni webhayoloji yamangqamuzana. Yathuthukiswa ngeminyaka yawo-1980s, le ndlela isishintshile isuka enqubweni ekhethekile yaselabhorethri yaya ethuluzini eliyisisekelo elisetshenziswa ekuxilongweni kwezokwelapha, isayensi yezobunhloli, kanye nocwaningo lwezakhi zofuzo. Ngokuvumela ososayensi ukuthi bathathe isampula encane ye-DNA futhi bayikhulise ibe izigidi zamakhophi, i-PCR yenze kwaba nokwenzeka ukutadisha izakhi zofuzo ngokuningiliziwe, ukuthola amagciwane ngokunemba okwedlulele, nokuhlonza izimpawu zofuzo ebezingabonakali ngaphambili ezindleleni ezijwayelekile zokuhlaziya.

Inqubo ye-PCR iyindlela yaselabhorethri esetshenziselwa ukwenza amakhophi amaningi engxenye ethile ye-DNA ngomjikelezo wokushintsha kwezinga lokushisa, ohlanganisa ukuguquguquka kwe-denaturation, ukuhlanganisa, nokunwetshwa, okwenziwa umshini okhethekile we-PCR kanye ne-DNA polymerase ezinzile.

Ukuqonda ubunkimbinkimbi benqubo ye-PCR kubalulekile kochwepheshe baselabhorethri, abacwaningi bezokwelapha, kanye nabakhiqizi bezimboni ababambe iqhaza kumishini yokuxilonga. Njengoba isidingo sokuhlolwa kwamangqamuzana okusheshayo nokunembile siqhubeka sikhula emhlabeni wonke, ukwethembeka kwe Umshini we-PCR uba yisisekelo semiphumela yaselabhorethri ephumelelayo. Lesi sihloko sinikeza umhlahlandlela obanzi ezinyathelweni, amazinga okushisa, nezimfuneko zemishini zenqubo ye-PCR ukuze kuqinisekiswe ukukhuliswa kwekhwalithi ephezulu kwe-DNA kanye nemiphumela yokuhlola eqinile.

Ithebula lesifinyezo se-athikili

Ingxenye	Isifinyezo
Iyini i-PCR?	I-PCR iyindlela yebhayoloji yamangqamuzana esetshenziselwa ukukhulisa ngokucacile ukulandelana kwe-DNA ethile kwezinhlelo zokusebenza ezihlukahlukene eziya phansi.
Yini edingekayo ku-PCR?	I-PCR ephumelelayo idinga i-DNA yesifanekiso, ama-primers, ama-nucleotide, i-DNA polymerase ezinzile, kanye nomjikelezo oshisayo oshisa kakhulu.
Yiziphi izinyathelo ezi-4 ze-PCR?	Inqubo ilandela ukulandelana okunengqondo kokuqaliswa, ukukhishwa kwe-denaturation, ukuhlanganisa, kanye nesandiso sokuphinda kabili okuqukethwe kwe-DNA umjikelezo ngamunye.
Yiziphi izinyathelo zomshini we-PCR?	Izinto ezisetshenziswayo zizenzela ngokuzenzakalelayo ukuguquka kwezinga lokushisa okunembile, iqinisekisa ukuthi ukusabela kwamakhemikhali e-biochemical kwenzeka ngezikhathi ezidingekayo.
Liyini izinga lokushisa elisetshenziselwa isinyathelo sokuguqula isimo?	Amazinga okushisa aphezulu, ngokuvamile aphakathi kuka-94°C no-98°C, asetshenziselwa ukunqamula izibopho ze-hydrogen futhi ahlukanise i-DNA enemicu ekabili.
Kwenzekani ngesikhathi sokukhulelwa?	Phakathi nalesi sigaba, izinga lokushisa liyehliswa ukuze kuvunyelwe ama-primers ukuthi abophe ngokuqondile ukulandelana kwawo okuhlosiwe okuhambisanayo ku-DNA enomucu owodwa.
Lithini izinga lokushisa elisetshenziselwa isinyathelo sokunweba?	Lesi sinyathelo ngokuvamile senzeka ku-72°C, izinga lokushisa elilungile le-Taq polymerase ukuze ihlanganise umucu omusha we-DNA.
Kuyini ukugeleza kwezinga lokushisa kwe-PCR?	Ukugeleza kuhlanganisa iphethini yokugibela ibhayisikili esheshayo yamazinga okushisa aphezulu, aphansi, namaphakathi aphindayo kuze kufinyelelwe ekugxiliseni okufunayo.

Iyini i-PCR?

I-PCR, noma i-Polymerase Chain Reaction, iyindlela eguquguqukayo yebhayoloji yamangqamuzana eklanyelwe ukukhiqiza ngokushesha izigidi kuya ezigidini zamakhophi esampula ethile ye-DNA.

Emgogodleni wayo, i-PCR isebenza 'njengesithwebuli sebhayoloji.' Ngaphambi kokusungulwa kwayo, i-DNA ekhulisayo kwakuyinqubo ehamba kancane futhi enzima ehlanganisa ukuhlanganiswa kwe-DNA ibe amagciwane. Ngokufika kwe Umshini we-PCR , abacwaningi manje sebengakwazi ukuhlukanisa isakhi sofuzo esithile noma ingxenye ethile ye-genome futhi bayenze ikhulise emahoreni ambalwa. Leli khono libalulekile ngoba ukuhlaziya okuningi kwamakhemikhali ezinto eziphilayo kudinga inani elibalulekile le-DNA ukuze kukhiqizwe isignali elinganisekayo, futhi amasampula emvelo ngokuvamile ahlinzeka ngamanani omkhondo kuphela.

Ukuhlukahluka kwalobu buchwepheshe kubonakala ezinhlobonhlobo zezinhlelo zokusebenza kuzo zonke izimboni ezahlukene. Ezilungiselelweni zomtholampilo, isetshenziselwa ukuthola inani legciwane, njengase-COVID-19 noma ekuhloleni i-HIV. Ku-forensics, ivumela abaphenyi ukuthi bakhombe abantu abavela kumasampula amancane wezinto eziphilayo. Emkhakheni wezimboni, i-PCR iqinisekisa ukuhlanzeka kwemikhiqizo yokudla kanye nokutholwa kwezinto eziphilayo eziguqulwe ngofuzo. Ukuqonda i imigomo nezindleko zobuchwepheshe be-PCR zibalulekile kumalebhu afuna ukuthuthukisa amakhono awo okuxilonga.

Yini edingekayo ku-PCR?

Ukusabela kwe-PCR okuphumelelayo kudinga izingxenye ezinhlanu ezibalulekile: ithempulethi ye-DNA, ama-primers athile, i-deoxynucleotide triphosphates (dNTPs), i-DNA polymerase ekwazi ukuzinza ekushiseni (njenge-Taq), kanye nesixazululo esikhethekile sebhafa.

Isifanekiso se-DNA sisebenza njengepulani yasekuqaleni ofisa ukuyikopisha. Iziqalo ziyizingcezu ezimfushane, zokwenziwa ze-DNA eziklanywe ngokwezifiso ukuze zifane nesiqalo nesiphetho sokulandelana okuqondiwe. Ngaphandle kwalokhu, i-DNA polymerase ibingeke yazi ukuthi ingaqala kuphi ukwakha umucu omusha. Ama-dNTP (A, T, C, kanye no-G) ​​ayizivimbeli zokwakha ezingavuthiwe ezisetshenziswa i-enzayimu ukuze kwakhiwe iketango elisha le-DNA.

Okubaluleke ngokufanayo indawo lapho ukusabela kwenzeka khona. Isigcinalwazi sihlinzeka ngendawo yamakhemikhali ezinzile, ikakhulukazi egxile ku-pH kanye nokugcwala kwama-ion e-magnesium, okungamacofactor abalulekile e-enzyme ye-DNA polymerase. Okokugcina, ukwenziwa komzimba kokusabela kudinga umjikelezo oshisayo osebenza kahle kakhulu, ovame ukubizwa ngokuthi umshini we-PCR , olawula ngokunembile izinguquko ezisheshayo ezidingekayo ukuze kuqalise isigaba ngasinye sokusabela.

Izingxenye Ezibalulekile Zomxube we-PCR

1. I-DNA yesifanekiso : Isampula equkethe ukulandelana okuqondiwe.

2. I-DNA Polymerase : Ngokuvamile i-Taq polymerase, ehlala isebenza emazingeni okushisa aphezulu.

3. Iziqalo : Dlulisela phambili uphinde uhlehlise imigqa echaza imingcele yokukhulisa.

4. I-dNTPs : Izisekelo ezine ze-nucleotide ezisebenza 'njengoyinki' womkopishi.

5. I-Buffer ne-Ions : Igcina ukusebenza kahle kwe-enzymatic nokuzinza.

Yiziphi izinyathelo ezi-4 ze-PCR?

Inqubo ye-PCR iqukethe izigaba ezine zokusebenza eziyinhloko: Ukuqalisa, Ukukhishwa Kwemvelo, Ukunezela, kanye Nokwandiswa (okubuye kwaziwe ngokuthi I-Elongation).

Isigaba sokuqala, Ukuqalisa, isenzakalo sesikhathi esisodwa lapho igumbi lokusabela lishiselwa ezingeni lokushisa eliphezulu ukuze kuqinisekiswe ukuthi i-DNA polymerase yenziwe yasebenza ngokugcwele futhi noma yikuphi ukungcola akwenziwanga hlangothi. Ukulandela lokhu, umjikelezo we-Denaturation uyaqala, lapho i-DNA enemicu emibili ihlukaniswa khona. Lokhu kulandelwa i-Annealing, lapho ama-primers ethola khona okuhlosiwe, futhi ekugcineni Isandiso, lapho i-DNA entsha ihlanganiswa khona. Lo mjikelezo wezinyathelo ezintathu (Denaturation, Annealing, Extension) uphindwa izikhathi ezingama-25 kuye kwezingama-40.

Ngenxa yokuthi inani le-DNA liphinda kabili kuwo wonke umjikelezo ophumelelayo, ukukhula kuyakhula. Ngokwesibonelo, ngemva kwemijikelezo engu-30, i-molecule eyodwa ye-DNA ingashintshwa ibe amakhophi angaphezu kwezigidi eziyinkulungwane. Lokhu ukusebenza kahle yikho okwenza amabhayisikili ashisayo elabhorethri yesimanje abaluleke kakhulu kwisayensi yesimanje. Ngaphandle kwamabhulokhi okushisisa nokupholisa anesivinini esiphezulu atholakala emshinini we- PCR wekhwalithi ephezulu , inqubo izohamba kancane kakhulu ukuze isetshenziswe ngendlela engokoqobo ezindaweni zokuxilonga eziphezulu.

Yiziphi izinyathelo zomshini we-PCR?

Izinyathelo zomshini we-PCR zibandakanya ukuhamba ngebhayisikili okuzenzakalelayo kwamazinga okushisa ngokulawula okunembile kwe-elekthronikhi kwebhulokhi eshisayo, ukulawula izinga lerempu, isikhathi sokubamba, kanye nokupholisa kokugcina.

Umshini we-PCR usebenza ngokusebenzisa ama-elementi e-Peltier ukuze ushise ngokushesha futhi upholise ibhulokhi yensimbi ebambe amashubhu okusabela. 'izinyathelo' ngokombono womshini zifaka 'Irempu,' okuyijubane lokushintsha phakathi kwamazinga okushisa, kanye 'Bamba,' okuyisikhathi umshini ogcina ngaso izinga lokushisa elithile. Imishini ephezulu iklanyelwe ukuthi ibe namazinga erempu ashesha kakhulu ukuze kuncishiswe isikhathi esichithwa kuguquko, okunciphisa ubungozi bokubopha okungaqondile noma ukuwohloka kwe-enzyme.

Isoftware engaphakathi komshini ivumela abasebenzisi ukuthi bahlele amaphrothokholi ayinkimbinkimbi. Lokhu kuhlanganisa ukushisa kokuqala, amaluphu aphindayo ezigaba ezintathu eziyinhloko, kanye nesinyathelo sokugcina sokubamba ezingeni lokushisa elibandayo (ngokuvamile elingu-4°C) ukuze kulondolozwe amasampula kuze kube yilapho uchwepheshe ewathola. Ukuxhumana kwedijithali yesimanje emshinini we-PCR futhi kuvumela ukuqapha kwesikhathi sangempela kokusabela, kuqinisekiswe ukuthi iphrofayili eshisayo ilandelwa ngendlela ehlelelwe ukukhiqizwa kabusha okuphezulu.

Liyini izinga lokushisa elisetshenziselwa isinyathelo sokuguqula isimo?

Isinyathelo se-denaturation ngokuvamile sisebenzisa amazinga okushisa aphakathi kuka-94°C no-98°C ukuze kube lula ukugqashuka kwamabhondi e-hydrogen phakathi kwemicu ye-DNA.

Kulokhu kushisa okukhulu, ukwakheka kwe-double-helix ye-DNA kuba nokungazinzi. Amabhondi e-hydrogen abamba amapheya e-adenine-thymine ne-cytosine-guanine ndawonye ayancibilika, okuphumele ekubeni kube nemicu emibili emele ye-DNA. Lokhu kuyimfuneko ebalulekile ezinyathelweni ezilandelayo, njengoba ama-primer kanye ne-enzyme ye-DNA polymerase ingasebenzisana kuphela nezifanekiso ezinomucu owodwa. Uma izinga lokushisa liphansi kakhulu, i-DNA ngeke ihlukane ngokugcwele, okuholela ekukhulisweni okuhlulekile noma okungasebenzi kahle.

Nokho, ukulondoloza lokhu kushisa kudinga i-DNA polymerase enamandla kakhulu. Kungakho ukutholakala kwe-Taq polymerase, ehlukanisiwe ne-bacterium ethanda ukushisa i-Thermus aquaticus , kwaba nezinguquko ezingaka. Ama-enzyme ajwayelekile azobhujiswa ku-95°C, kodwa i-Taq isasebenza. Amalabhorethri kufanele aqinisekise ukuthi umshini wabo we-PCR unikeza ukufudumeza okufanayo kuyo yonke imithombo ukuvimbela 'izindawo ezibandayo' lapho ukuguqulwa kwe-denaturation kungase kuhluleke, okuyisici esibalulekile imishini yebhayoloji yamangqamuzana esezingeni eliphezulu.

Kwenzekani phakathi nesinyathelo sokukhipha i-PCR?

Phakathi nesinyathelo sokukhipha i-anneal, izinga lokushisa liyehliselwa phakathi kuka-50°C no-65°C, okuvumela ama-primers e-DNA ukuthi abophe ukulandelana kwawo okuhambisanayo kuzifanekiso ze-DNA ezinomucu owodwa.

Lesi sinyathelo ngokungangabazeki siyingxenye ebucayi kakhulu yenqubo ye-PCR. Izinga lokushisa elithile elisetshenzisiwe lincike emazingeni okushisa ancibilikayo (Tm) weziqalo ezisetshenziswayo. Uma izinga lokushisa liphezulu kakhulu, ama-primers ngeke abophe isifanekiso. Uma iphansi kakhulu, ama-primers angase abophezele ekulandeleni okufanayo 'ingxenye' kuphela, okuholela ekukhuliseni okungaqondile kanye nemiphumela engcolile. Umshini we-PCR kufanele ukwazi ukushaya lelizinga lokushisa eliqondiwe ngezinga eliphezulu lokunemba (ngokuvamile ngaphakathi kuka-0.1°C).

Ubude besinyathelo se-anneal ngokuvamile amasekhondi angu-20 kuya kwangu-40. Phakathi naleli windi elifushane, ama-primers azulazula ekuxubeni kokusabela ngokunyakaza kwamamolekyuli futhi adlulele kusayithi eliqondiwe. Lapho ama-primer eseqedile, anikeza indawo yokuqala ukuze i-DNA polymerase iqale ukwengeza ama-nucleotide. Lokhu kuhlanganisa okunembayo yikho okuvumela ukutholwa koguquko oluthile lwezakhi zofuzo noma amagciwane kusampula yebhayoloji eyinkimbinkimbi, okwenza ukutshalwa kwezimali emishinini yokuxilonga esezingeni eliphezulu kubaluleke kakhulu kumalebhu omtholampilo.

Lithini izinga lokushisa elisetshenziselwa isinyathelo sokunweba?

Isinyathelo sokunweba ngokuvamile senziwa ku-72°C, okuyizinga lokushisa elilungile lokusebenza le-DNA polymerase ezinzile ukuze kuhlanganiswe umucu omusha we-DNA.

Ku-72°C, i-enzyme ye-DNA polymerase isezingeni eliphezulu. Iqala esizeni sokuqala futhi iqala ukwengeza ama-dNTP ekugcineni okungu-3' kwe-primer, ihamba ngomucu wesifanekiso. I-enzyme 'ifunda' isifanekiso bese ibeka isisekelo esihambisanayo kumucu omusha. Isibonelo, uma ithempulethi ine-Adenine, i-polymerase yengeza i-Thymine. Ijubane lalokhu kusabela liyamangalisa; I-Taq polymerase ingangeza cishe amapheya ayisisekelo ayi-1,000 ngomzuzu.

Ubude besikhathi salesi sinyathelo buncike kubude bengxenye ye-DNA ekopishwayo. Uma ukulandelana okuqondiwe kuyi-1,000 base pair ubude, isinyathelo sokunweba singasethwa iminithi elilodwa. Uma okuqondiwe kukufushane, isikhathi singancishiswa ukuze kongiwe sonke isikhathi sokucubungula. Ukuqinisekisa ukuthi umshini we-PCR ugcina u-72°C ozinzile kuso sonke lesi sigaba kubalulekile ukuze kuqedwe imicu ye-DNA yobude obugcwele.

Kuyini ukugeleza kwezinga lokushisa kwe-PCR?

Ukugeleza kwezinga lokushisa kwe-PCR kulandela umjikelezo ophindaphindayo wokushintshashintsha kokushisa okuphezulu, ukuncishiswa kokushisa okuphansi, kanye nesandiso sokushisa okusesilinganisweni, kudala 'sawtooth' iphrofayela eshisayo.

Lokhu kugeleza kuklanyelwe ukukhulisa ukuqhubeka kwejometri yobuningi be-DNA. Ngokugijima okuvamile, umshini uqala ku-95 ° C imizuzu engu-2 (I-Denaturation Yokuqala), bese ungena ku-loop: 95 ° C imizuzwana engu-30, 55 ° C imizuzwana engu-30, futhi 72 ° C imizuzwana engu-60. Le loop iphinda izikhathi ezingama-30. Ekugcineni, kukhona 'Isandiso Sokugcina' ku-72°C imizuzu engu-5-10 ukuqinisekisa ukuthi yonke i-DNA enomucu owodwa iboshwe ngokugcwele ngaphambi kokuba umshini uphole ufike ku-4°C ukuze ugcinwe.

Ukunemba kwalokhu kugeleza kwezinga lokushisa kuthinta ngokuqondile isivuno nobumsulwa bomkhiqizo we-PCR. Uma ukugeleza kungahambisani, i-enzyme ingase ilahlekelwe umsebenzi noma ama-primer angakha 'ama-primer dimers,' okungama-artifact angenamsebenzi wokusabela. Ngenxa yalokhu, ukulinganisa kanye nokufana okushisayo komshini we-PCR yizinto ezibaluleke kakhulu kunoma iyiphi ilabhorethri eyenza ukuhlonza amangqamuzana noma ucwaningo.

Ithebula Elifingqiwe Lokugeleza Kwezinga lokushisa

Isigaba	Izinga Lokushisa Elijwayelekile	Inhloso
Ukuqaliswa	94°C – 96°C	Ivula i-enzyme, iguqule i-DNA eyinkimbinkimbi.
I-Denaturation	94°C – 98°C	Ihlukanisa i-DNA enemicu ekabili ibe imicu eyodwa.
Anealing	50°C – 65°C	Ivumela ama-primers ukuthi abophe ukulandelana okuqondiwe.
Isandiso	72°C	I-DNA polymerase ihlanganisa imicu emisha ye-DNA.
Ukubamba Kokugcina	4°C – 10°C	Ukugcinwa kwesikhashana komkhiqizo okhulisiwe.

Isiphetho

I-Polymerase Chain Reaction iyithuluzi elihle nelinamandla eliguqule isimo sesayensi yezinto eziphilayo. Ngokulandela izinyathelo ezicophelelayo zokuguqula isimo, ukuhlanganisa, nokunwetshwa, ososayensi bangavula izimfihlo ezigcinwe ngaphakathi kwe-DNA, banikeze izimpendulo zemibuzo eyinkimbinkimbi yezokwelapha neye-forensic. Impumelelo yale nqubo incike kakhulu kwikhwalithi yama-reagents kanye nokunemba komshini we -PCR osetshenziselwa ukwenza imijikelezo yokushisa.

Kunoma iyiphi ilabhorethri efuna ukuzuza imiphumela engaguquki nethembekile, ukuqonda ama-nuances okulawula izinga lokushisa nokuphathwa komjikelezo kubalulekile. Noma ngabe wenza ucwaningo oluyisisekelo noma ukuxilongwa komtholampilo kwenani eliphezulu, ukukhetha kwezinto zokusebenza kanye nokubambelela kuzimiso eziyisisekelo kuzochaza ukunemba komsebenzi wakho. Kulabo abanentshisekelo ohlangothini lwezokuthutha lokusetha ilebhu ye-molecular, ukuhlola izindleko kanye nemininingwane yobuchwepheshe yezinhlelo zesimanje ze-PCR isinyathelo esilandelayo esiphusile sokuthuthukisa amakhono akho okuxilonga.