I-PCR, ezininzi I-PCR, Ikwi PCR, Buyela umva PCR, I-RT-PCR, qPCR (1)-I-PCR

Siza kulungisa iikhonsepthi, amanyathelo kunye neenkcukacha zePCR ezahlukeneyo

Ⅰ. I-PCR

I-Polymerase Chain Reaction, ekubhekiselwa kuyo njenge-PCR, bubuchwepheshe bebhayoloji yemolekyuli esetyenziselwa ukukhulisa amaqhekeza athile e-DNA.Inokuthi ithathwe njengento ekhethekileyo ye-DNA replication in vitro.I-DNA polymerase (i-DNA Polymerase I) yafunyanwa kwangoko ngo-1955, kwaye i-Klenow Fragment ye-E. Coli, enexabiso lokulinga kunye nokusebenza, yafunyanwa nguGqr H.I-enzymes esetyenziswayo namhlanje (ebizwa ngokuba yi-Taq polymerase), yahlukaniswa kwi-Thermus aquaticus, i-bacterium eshushu yasentwasahlobo ngo-1976.Ingqikelelo yoqobo yeprototype yantlandlolo yePCR iyafana nokulungiswa kofuzo kunye nokukopishwa, eyacetywa nguGqr. KJell Kleppe ngo-1971. Wapapasha ikopi yemfuza yokuqala elula neyexesha elifutshane (efana neempendulo zemijikelo emibini yokuqala ye-PCR).I-PCR ephuhliswe namhlanje yaphuhliswa nguGqr. Kary B. Mullis ngo-1983. UGqr. Mullis wakhonza iinkampani ze-PE ngaloo nyaka, ngoko ke i-PE inewonga elikhethekileyo kushishino lwe-PCR.UGqr. Mullis wapapasha ngokusemthethweni iphepha lokuqala elihambelanayo kunye noSaiki kunye nabanye kwi-1985. Ukususela ngoko, ukusetyenziswa kwe-PCR ngamawaka eekhilomitha ngosuku, kwaye umgangatho wamaphepha ahambelanayo unokuthiwa wenze ezinye iindlela ezininzi zophando zingathandeki.Emva koko, itekhnoloji ye-PCR isetyenziswa ngokubanzi kuphando lwezenzululwazi yebhayoloji kunye nokusetyenziswa kwezonyango, ibe yeyona teknoloji ibalulekileyo yophando lwebhayoloji yemolekyuli.UMulis uphinde waphumelela iBhaso leNobel kwiChemistry ngowe-1993.

I-PCRUmgaqo

Umgaqo osisiseko weteknoloji ye-PCR ufana nenkqubo yokuphindaphinda kwendalo ye-DNA, kwaye ukuchaneka kwayo kuxhomekeke kwi-oligonucleotide primer ehambelana neziphelo zombini zokulandelelana okujoliswe kuyo.I-PCR yenziwe nge-degeneration-annealing-eyandisa amanyathelo amathathu okusabela asisiseko: ①Ukwehla kwe-template ye-DNA: Emva kokuba itemplate ye-DNA ifudunyezwe malunga ne-93 ° C kangangexesha elithile, isisombululo se-DNA esiphindwe kabini se-DNA ye-double-chain chain eyenziwe yi-PCR yokukhulisa itemplate ye-template Ukushiya, ukwenzela ukuba ibe yingqungquthela elandelayo ye-reaction.②I-annealing (i-compound) ye-template ye-DNA kunye ne-primer: Emva kokuba i-template ye-DNA ifudunyeziwe kwaye yehliswe kwikhonkco elilodwa, ubushushu behla ukuya malunga ne-55 °C.Ulandelelwano oluhambelanayo lwe-primer kunye ne-template ye-DNA enye-chain.③Ukongezwa kwe-primer: itemplate ye-DNA-isibophelelo sokuqala sisekwe kwisenzo se-TaqDNA polymerase, kunye ne-dNTP njengento yokusabela ekrwada.Gcina umgaqo wokuphindaphinda, udibanise ikhonkco elitsha lekopi egciniweyo ehambelana netsheyini ye-template ye-DNA, kunye nokuphinda umjikelo wokuwohloka-annealing-extension iinkqubo ezintathu zinokufumana ngaphezulu "ikhonkco lekopi egciniweyo", kwaye eli khonkco litsha liyafumaneka kwakhona Yiba yitemplate kumjikelo olandelayo.Kuthatha i-2-4min ukugqiba i-loop, i-gene ekujoliswe kuyo inokwandiswa izihlandlo ezininzi zezigidi kwiiyure ezingama-2-3.

UmgangathoI-PCRIndlela yokusabela

Izinto ezintlanu zokusabela kwePCR

Kukho ubukhulu becala iintlobo ezintlanu zezinto ezibandakanyekayo kwi-PCR reaction, ezizezi, i-primer, i-enzyme, i-dNTP, itemplate kunye ne-buffer (iMg2+ iyafuneka).[inkqubo yePCR]

Inkqubo yePCR eqhelekileyo yahlulwe yangamanyathelo amathathu

1. I-DNA degeneration (90 ° C-96 ° C): Iitemplates ze-DNA ezimbini-chain phantsi kwesenzo se-thermal, i-hydrogen bonds break, yenza i-DNA-chain chain.

2. I-Annealing (25℃ -65℃): Iqondo lokushisa lenkqubo liyancitshiswa, i-primer idibaniswe ne-template ye-DNA ukwenza i-double-chain chain.

3. Ukwandiswa (70 ℃ -75 ℃): Ngaphantsi kwesenzo se-Taq enzyme (malunga ne-72 ° C, owona msebenzi ungcono kakhulu), i-dNTP isetyenziswe njengento eluhlaza, yandisa ukusuka kwi-5′ ekupheleni kwe-primer → i-3′ ekupheleni, i-synthesis kunye ne-template ihambelana ne-DNA chain.

Umjikelo ngamnye udityanisiwe, unqanyulwe kwaye wandiswa, uphinda kabini umxholo weDNA.Okwangoku, ngenxa yommandla omfutshane wokukhulisa, enye i-PCR inokuphinda iphindwe ngexesha elifutshane kakhulu nangona umsebenzi we-enzyme ye-Taq awulungile, ngoko unokutshintshwa ube ngamanyathelo amabini, oko kukuthi, i-annealing kunye nokwandiswa kunokwenziwa kwi-60 ° C-65 ° C ngexesha elifanayo.Ukuze kuncitshiswe inkqubo yokuphakamisa kunye nokupholisa kunye nokuphucula isantya sokuphendula.

Iimpawu ze-PCR Reaction

● Ingcaciso ePhakamileyo

Imiba ethile eqinisekileyo yempendulo ye-PCR zezi: ①Indibaniselwano ethile ye-primer kunye ne-template ye-DNA.②Umgaqo-siseko wokubhanqa.③Ukunyaniseka kwe-TaqDNA polymerase synthesis reaction.④Izinto ezithile kunye nolondolozo lwejini ekujoliswe kuyo.

Indibaniselwano echanekileyo yeeprimers kunye neetemplates ngundoqo.Ukubophelela kwe-primer kunye nethemplate kunye nokwandiswa kwekhonkco ye-primer kusekelwe kumgaqo wokulinganisa isiseko se-alkali.Ukunyaniseka kwe-polymerase synthesis reactions kunye nokumelana nobushushu obuphezulu be-Taq DNA polymerase ukwenza ukubopha (i-compound) ye-template kunye ne-primer kwi-reaction inokwenziwa kwiqondo lokushisa eliphezulu.Ukuchaneka kokudibanisa kwanda kakhulu.Ikliphu inokugcina iqondo eliphezulu lokuchaneka.Ngokukhetha indawo ekujoliswe kuyo yemfuza enokugcinwa okuphezulu kunye nokugcinwa okuphezulu, ukuchaneka kwayo kuphezulu.

● Uvakalelo Oluphezulu

Umthamo wemveliso yeemveliso ze-PCR unyuswe ngesalathisi, esinokwandisa itemplate yokuqala ye-Picker (PG = 10-12) ukunyusa izinga le-microcontroller kwinqanaba le-micrograms (μg = -6).Iiseli ekujoliswe kuzo zinokufunyanwa kwi-1 yezigidi zeeseli;ekufumaneni iintsholongwane, uvakalelo lwe-PCR lunokufikelela kwi-3 RFUs (iindawo ezingenanto ezenziwe iiyunithi);ubuncinci bokubona izinga kwisayensi yebhaktheriya yibhaktheriya ye-3.

● Ilula kwaye ikhawuleza

Ukubonakaliswa kwe-PCR kusebenzisa i-polymerase ye-Taq DNA ephezulu yokushisa, eyongeza isisombululo sokuphendula ngexesha elinye, oko kukuthi, i-degeneration-anneal-extension reaction on the DNA amplification solution kunye nembiza yokuhlamba amanzi.Ngokuqhelekileyo, i-amplification reaction igqitywe kwiiyure ezi-2 ukuya kwezi-4.Iimveliso ezongeziweyo zihlalutywa ngokubanzi ngekrele lombane, kwaye akufuneki zisebenzise i-isotopes, akukho ngcoliseko ye-radioactive, kunye nokukhuthaza okulula.

● Ukucoceka komzekelo kuphantsi

Akukho mfuneko yokwahlula iintsholongwane okanye iibhaktheriya kunye neeseli zenkcubeko.Iimveliso ezikrwada zeDNA kunye neRNA zinokusetyenziswa njengezandisi.Ukuchongwa kokukhulisa i-DNA kunokusetyenziswa ngokuthe ngqo kusetyenziswa imizekelo yeklinikhi efana negazi, ulwelo lomzimba, ulwelo lokuhlamba ukhohlokhohlo, iinwele, iiseli, kunye nezicubu eziphilayo.

I-PCRiingxaki eziqhelekileyo

● I-false negative, akukho bands eyonyusiweyo

Amanqanaba aphambili okusabela kwe-PCR abandakanya: ① ​​ukulungiswa kwetemplate ye-nucleic acid, ② umgangatho kunye nokucaciswa kweeprimers, ③ umgangatho wee-enzymes ④ iimeko zomjikelezo we-PCR.Ukufumana isizathu kufuneka kwakhona kuhlalutywe kwaye kufundwe kula makhonkco angentla.

Templates: ① Ithemplethi iqulathe iiprotheyini ezahlukeneyo, ② Itemplate iqulethe inhibitor ye-enzyme ye-Taq, ③ Iprotheyini ekwitemplate ayipheli, ingakumbi iprotein yeqela kwichromosome.⑤ I-deminer nucleic acid degeneration ayikho ngokucokisekileyo.Xa umgangatho wee-enzymes kunye ne-primers ulungile, akukho bhendi yokukhulisa, eyona nto inokwenzeka ukuba yonyango lokugaya imizekelo.Kukho into engalunganga ngenkqubo ye-template ye-nucleic acid extraction, ngoko ke ukulungiselela isisombululo esisebenzayo nesizinzileyo sokugaya, inkqubo yayo kufuneka ilungiswe kwaye ingatshintshwa ngokungqongqo.

Ukungasebenzi kwe-Enzyme: i-enzyme entsha okanye zombini i-enzymes endala kunye nentsha kufuneka isetyenziswe kunye ukuhlalutya ukuba umsebenzi we-enzyme ulahlekile okanye awunelanga, okukhokelela kwizinto ezingalunganga.Kufuneka kuqatshelwe ukuba i-Taq enzyme okanye i-ethidium bromide iyalityalwa ngamanye amaxesha.

I-Primer: umgangatho we-primer, ukuxinwa kwe-primer, kunye nokuba ukuxinwa kwee-primers ezimbini ku-symmetrical.Sisizathu esiqhelekileyo sokungaphumeleli kwe-PCR okanye ibhendi ekhulayo ayifanelekanga kwaye ilungele ukusabalalisa.Kukho iingxaki ngomgangatho wee-primers zamanani athile ebhetshi.Iiprimers ezimbini zinogxininiso oluphezulu kunye nogxininiso oluphantsi, olubangela ukunyusa okuphantsi kwe-asymmetric.Imilinganiselo yokuchasana yile: ① Khetha iprimer elungileyo ukwenza iiyunithi.② Ukuxinwa kwe-primer akuxhomeki kuphela kwixabiso le-OD, kodwa kwakhona kunika ingqalelo kwi-primer's original liquid ukwenza i-agar sugar gel electrophoresis.Kufuneka kubekho i-primer strip zone, kwaye ukukhanya kweeprimer ezimbini kufuneka kufane ngokuqhelekileyo.Ibhanti, i-PCR inokusilela ngeli xesha, kwaye kufuneka isonjululwe ngeyunithi ye-primer synthesis.Ukuba i-primer iphezulu, ukukhanya kuphantsi, kwaye ukugxininiswa kwayo kufuneka kulinganiswe xa kuhlanjululwe.③ I-primer kufuneka ihlawulwe kwaye igcinwe kwindawo ephakamileyo ukukhusela iindawo ezininzi zokukhenkceza okanye ixesha elide lefriji yefriji, eya kubangela ukuba i-primer yonakalise kwaye ihlahle.④ Uyilo lwe-primer alunangqiqo, njengokuba ubude be-primer abunelanga, kwaye i-di cluster yenziwa phakathi kwee-primers.

Ugxininiso lweMg2 +: Ugxininiso lweMg2 + ion lunempembelelo enkulu kwimpumelelo yokukhulisa iPCR.Ukugxininiswa okugqithisileyo kunokunciphisa isini esahlukileyo sokukhulisa i-PCR.Ukuba ugxininiso luphantsi kakhulu, imveliso yokukhulisa iPCR iya kwenza ukungaphumeleli kokukhulisa iPCR ngaphandle kwebhendi yokwandisa.

Ukutshintsha umthamo wokuphendula: Umthamo osetyenziswe kwi-PCR yokukhulisa i-20ul, i-30ul, kunye ne-50ul okanye i-100uL, umthamo omkhulu wesicelo sokukhulisa i-PCR ibekwe ngokweenjongo ezahlukeneyo zophando lwezenzululwazi kunye novavanyo lweklinikhi.Emva kokwenza imiqulu emincinci efana ne-20ul, kuyimfuneko ukwenza imeko yentambo xa usenza ubungakanani, ngaphandle koko kuya kuphumelela.

Izizathu zenyama: Utshintsho lubaluleke kakhulu kwi-PCR yokukhulisa.Ukuba ukushisa kweqondo lokushisa liphantsi, ixesha lokunciphisa lifutshane, kunokwenzeka ukuba kwenzeke kwizinto ezimbi ezingalunganga;iqondo lobushushu elisezantsi kakhulu le-annealing linokubangela ukukhulisa okungangqaliyo kunye nokunciphisa ukusebenza kakuhle kokukhulisa ukukhulisa.Ichaphazela kakhulu indibaniselwano yee-primers kunye neetemplates ukunciphisa ukusebenza kakuhle kokukhulisa i-PCR.Ngamanye amaxesha kuyimfuneko ukusebenzisa i-thermometers esemgangathweni ukubona ukuguquguquka, ukuxhamla kunye nokushisa okwandisiweyo kwi-extension okanye i-cooker e-soluble yamanzi, enye yezizathu zokungaphumeleli kwe-PCR.

Uluhlu lolandelelwano lwethagethi: Ukuba ulandelelwano lwethagethi lwenzeka, ukuguqulwa okanye ukususwa, indibaniselwano yeprototype kunye nethemplethi kudityanisiwe, okanye ngenxa yokunqongophala kolandelelwano lwethagethi, i-primer kunye nethemplethi ziya kulahlekelwa lulandelelwano oluncedisayo, kwaye ukukhulisa i-PCR yayo akuyi kuphumelela.

● Ubuxoki

Ibhendi yokukhulisa iPCR ibonakala ihambelana nebhendi yolandelelwano ekujoliswe kuyo, kwaye ngamanye amaxesha ibhendi yayo icocekile kwaye iphezulu.

Uyilo lwe-Primer alufanelekanga: ukulandelelana kokukhulisa okukhethiweyo kunye nolandelelwano lwe-amplification olungelona njongo lune-homologous, ngoko xa i-PCR yokukhulisa, iimveliso ze-PCR ezinyusiweyo azikho ulandelelwano olungenanjongo.Ulandelelwano ekujoliswe kulo lufutshane kakhulu okanye i-primer imfutshane kakhulu, kwaye ithandeka kwi-positive positive.Kufuneka uyilwe ngokutsha.

Ungcoliseko olunqamlezayo lokulandelelana okujoliswe kuko okanye iimveliso zokukhulisa: Kukho izizathu ezibini zolu ngcoliseko: Okokuqala, ukungcoliseka komnqamlezo we-genome yonke okanye izahlulo ezinkulu, ezikhokelela kwiimpawu zobuxoki.Olu hlobo lobuxoki lunokusonjululwa ngezi ndlela zilandelayo: Qaphela kwaye ube mnene ngexesha lokusebenza ukukhusela ulandelelwano olujoliswe kuyo ukuba lufakwe kwisampulu yompu okanye ukutshiza ngaphandle kwetyhubhu ye-centrifugal.Ngaphandle kwee-enzymes kunye nezinto ezingenakukwazi ukumelana nokushisa okuphezulu, zonke ii-reagents okanye izixhobo kufuneka zihlanjululwe nge-disinfected ngoxinzelelo oluphezulu.Imibhobho ye-centrifugal kunye neesampuli kufuneka zisetyenziswe ngexesha elinye.Xa kuyimfuneko, ngaphambi kokuba ungeze iisampuli, ityhubhu yokusabela kunye ne-reagent ibonakaliswe kwimisebe ye-ultraviolet ukutshabalalisa i-nucleic acid ekhoyo.Okwesibini, amaqhekeza amancinci kungcoliseko lomoya.Ezi ziqwenga zincinci zifutshane kunolandelelwano ekujoliswe kulo, kodwa zine-homology ethile.Inokudityaniswa kunye.Emva kokuncedisa i-primers, imveliso ye-PCR inokwandiswa, eya kubangela imveliso yobuxoki.Ingasetyenziselwa ukunciphisa okanye ukuphelisa indlela ye-PCR yendlwane.

● Ibhendi yokwandisa engachazwanga

Iibhendi ezivele emva kokwandiswa kwe-PCR azihambelani nobungakanani obulindelekileyo, okanye obukhulu okanye obuncinci, okanye ngexesha elifanayo, okanye ngexesha elifanayo, iibhendi ezithile zokukhulisa kunye neebhendi ezingezona ezikhethekileyo.Ukuvela kweebhendi ezingezizo ezona zinto zilandelayo: Okokuqala, iiprimers azigqibekanga zihambelana nokulandelelana okujoliswe kuko, okanye i-polymerization ye-primer ukwenza i-di cluster.Okwesibini kukuba i-concentration ye-MG2 + ion iphezulu kakhulu, ukushisa kwe-annealing kuphantsi kakhulu, kwaye inani lemijikelo ye-PCR inxulumene.Okwesibini, umgangatho kunye nesixa se-enzymes.Rhoqo, ii-enzymes zeminye imithombo ziqhelekile kwiibhendi ezingezizo ezodwa kwaye ii-enzymes zomnye umthombo azenzekanga.Ngamanye amaxesha i-amplification engangqalanga yee-enzyme nayo iyenzeka.Amanyathelo okuchasa ngala: imitsalane eyilwe ngokutsha ukuba kuyimfuneko.Ukunciphisa inani le-enzyme okanye endaweni ye-enzyme yomnye umthombo.Ukunciphisa inani leprayimari, ukwandisa inani leetemplates ngokufanelekileyo, kwaye unciphise inani lemijikelezo.Yandisa ngokufanelekileyo iqondo lobushushu le-anneal okanye sebenzisa indlela yobushushu obumbini (93°C degeneration, annealing and extended about 65°C).

● Ukubonakala kwe tow ethambileyo okanye iteyiphu yesmear

Ukwandiswa kwe-PCR ngamanye amaxesha kubonakala kufakwe okanye kufakwe i-shelled okanye ibhanti elifana nekhaphethi.Ngesizathu, ngenxa yobuninzi obugqithisileyo bee-enzymes okanye umgangatho ombi we-enzyme, i-dNTP yoxinaniso iphezulu kakhulu, i-concentration ye-Mg2 + iphezulu kakhulu, ukushisa kwe-annealing kuphantsi kakhulu, kwaye inani lemijikelo lininzi kakhulu.Imilinganiselo yokuchasana yile: ①Nciphisa umthamo wee-enzymes, okanye utshintshe i-enzyme yomnye umthombo.②Ukunciphisa ukuxinana kwe-dNTP ③Nciphisa ngokufanelekileyo i-Mg2+ yoxinaniso.④Yandisa ubungakanani betemplates kwaye unciphise inani lemijikelo.

Iimveliso ezinxulumeneyo

PCR Heroᵀᴹ (Ngedayi)

◮ Ukunyaniseka okuphezulu: amaxesha ama-6 kune-enzyme yeTaq eqhelekileyo;

◮ Isantya sokukhulisa ngokukhawuleza

◮ Ukulungelelaniswa ngakumbi kwetemplate

◮ Ukwandiswa kobuchule obuphezulu

◮ Ukunyamezela kokusingqongileyo kunamandla: kubekwe kuma-37°C kangangeveki, kugcinwe umsebenzi ongaphezu kwama-90%;

◮ Ino-5'→3' we-DNA polymerase umsebenzi kunye no-5'→3' we-exonuclease, ngaphandle kwe-3'→5' ye-exonuclease.

PCR Easyᵀᴹ (Ngedayi)

Inkqubo eyodwa yokusabela kunye nokusebenza okuphezulu kwe-Taq DNA Polymerase yenza ukuba i-PCR iphendule ibe nomgangatho ophezulu wokukhulisa, ukuchaneka kunye novakalelo.

RT-qPCR Easyᵀᴹ (Inyathelo elinye)-SYBR Green I

◮ Ikiti enenyathelo elinye yenza uguqulelo oluguqulelweyo kunye ne-qPCR iimpendulo ezimbini kwityhubhu enye, kufuneka kuphela ukongeza i-template ye-RNA, iiprimers ezithile zePCR kunye ne-RNase-Free ddH₂O.

◮ Ikhithi inokucazulula ngokukhawuleza nangokufanelekileyo ngokobungakanani bentsholongwane ye-RNA okanye ilandele i-RNA.

◮ Ikhithi isebenzisa i-reagent eyodwa ye-Foregene reverse transcription kunye ne-Foregene HotStar Taq DNA Polymerase edityaniswe nenkqubo yokusabela ekhethekileyo ukuze kuphuculwe ngokufanelekileyo ukusebenza kakuhle kokukhulisa kunye neenkcukacha zokusabela.

◮ Inkqubo yokusabela ephuculweyo yenza impendulo ibenovakalelo oluphezulu lokuqonda, uzinzo olomeleleyo lwe-thermal, kunye nokunyamezela okungcono.

◮ RT-qPCR Easy^TM(Inyathelo elinye) -SYBR Green I kit iza ne-ROX yedayi yesalathiso yangaphakathi, engasetyenziselwa ukuphelisa imvelaphi yomqondiso kunye neempazamo zesignali phakathi kwamaqula, ekulungele ukuba abathengi basebenzise kwiimodeli ezahlukeneyo zezixhobo ze-PCR zobungakanani.

RT Easy^TMII (I-Master Premix ye yokuqala-strand cDNA synthesis forIxesha lokwenyani PCR)

-Ikhono elisebenzayo lokususa i-gDNA, enokususa i-gDNA kwi-template ngaphakathi kwemizuzu emi-2.

-Inkqubo esebenzayo yokukhuphela umva, kuthatha imizuzu eyi-15 kuphela ukugqiba udityaniso lwe-cDNA yomtya wokuqala.

-Iitemplates eziyinkimbinkimbi: iitemplates ezinomxholo ophezulu weGC kunye nesakhiwo sesibini esiyinkimbinkimbi sinokuphinda sitshintshwe ngokusebenza okuphezulu.

-I-High-sensitivity reverse transcription system, i-pg-level templates inokufumana i-cDNA ephezulu.

-Isistim yokukhutshelwa umva inozinzo oluphezulu lwe-thermal, elona qondo lobushushu lokusabela liphezulu yi-42℃, kwaye isenomsebenzi omhle wokukhutshelwa umva kwi-50℃.