I-PCR (i-polymerase chain reaction) yenye ye-in-vitro DNA amplification technologies, enembali engaphezu kweminyaka engama-30.

Iteknoloji yePCR yaqalwa nguKary Mullis waseCetus, eU.SA ngo-1983. U-Mullis wafaka isicelo selungelo elilodwa lomenzi we-PCR ngo-1985 waza wapapasha iphepha lokuqala lezifundo ze-PCR kwiSayensi kwangaloo nyaka.UMulis wawongwa ngembasa yeNobel kwichemistry ngo-1993 ngomsebenzi wakhe.

Imigaqo-siseko ye-PCR

I-PCR inokwandisa amaqhekeza e-DNA ekujoliswe kuyo ngamaxesha angaphezu kwesigidi esinye.Umgaqo uphantsi kwe-catalysis ye-DNA polymerase, usebenzisa i-DNA ye-strand yomzali njenge template kunye ne-primer ethile njengendawo yokuqala yokwandisa.Iphindwaphindwa kwi-vitro ngamanyathelo anje nge-denaturation, annealing, kunye nokwandiswa.Inkqubo yentombi strand DNA complementary kumzali strand template DNA.

Inkqubo yePCR eqhelekileyo yahlulwe yangamanyathelo amathathu:

1.I-Denaturation: Sebenzisa ubushushu obuphezulu ukwahlula imisonto ephindwe kabini yeDNA.Ibhondi ye-hydrogen phakathi kwe-DNA imisonto ephindwe kabini yaphulwa kubushushu obuphezulu (93-98℃).

I-2.Annealing: Emva kokuba i-DNA ephindwe kabini ihlukaniswe, yehlisa iqondo lokushisa ukuze i-primer ibophe kwi-DNA enye.

3.Ukwandiswa: I-DNA polymerase iqala ukudibanisa imicu ehambelanayo kunye nemigca ye-DNA evela kwii-primers eziboshwe xa ubushushu buhla.Xa ukwandiswa kugqityiwe, umjikelo ugqityiwe, kwaye inani leeqhekeza zeDNA liphindwe kabini

Ukubuyisela la manyathelo mathathu amaxesha angama-25-35, inani leeqhekeza ze-DNA liya kwanda ngokukhawuleza.

Ubuchule be-PCR kukuba ii-primers ezahlukeneyo zinokuyilwa kwiijene ezijoliswe kuzo ezahlukeneyo, ukwenzela ukuba iinqununu ezijoliswe kuzo zinokwandiswa ngexesha elifutshane.

Ukuza kuthi ga ngoku, i-PCR inokohlulwa ibe ngamacandelo amathathu, angala, i-PCR eqhelekileyo, i-fluorescent quantitative PCR kunye ne-PCR yedijithali.

Isizukulwana sokuqala sePCR eqhelekileyo

Sebenzisa isixhobo esiqhelekileyo sokukhulisa i-PCR ukukhulisa i-gene ekujoliswe kuyo, kwaye emva koko usebenzise i-agarose gel electrophoresis ukufumanisa imveliso, uhlalutyo lomgangatho kuphela olunokwenziwa.

Ezona zinto zingalunganga kwisizukulwana sokuqala sePCR:

I-1.I-prone kwi-amplification engacaciswanga kunye neziphumo ezingezizo zobuxoki.

2.Ukufumanisa kuthatha ixesha elide kwaye umsebenzi unzima.

3.Uvavanyo lomgangatho kuphela olunokwenziwa

Isizukulwana sesibini sexesha lokwenyani PCR

I-Real-Time PCR, ekwabizwa ngokuba yi-qPCR, isebenzisa iiprobes ze-fluorescent ezinokubonisa inkqubela yenkqubo yokusabela, kwaye ibeke iliso ekuqokeleleni kweemveliso ezandisiweyo ngokuqokelelwa kweempawu ze-fluorescent, kwaye igwebe iziphumo ngejika le-fluorescence.Inokulinganiswa ngoncedo lwexabiso leCq kunye nejika eliqhelekileyo.

Ngenxa yokuba itekhnoloji ye-qPCR iqhutyelwa kwinkqubo evaliweyo, amathuba ongcoliseko ayancitshiswa, kwaye isignali ye-fluorescence inokubekwa iliso ukuze ibonwe ubungakanani, ngoko ke yeyona nto isetyenziswa kakhulu kwiklinikhi kwaye iye yaba yeyona teknoloji ibalaseleyo kwi-PCR.

Izinto zefluorescent ezisetyenziswa ngexesha lokwenyani lobungakanani befluorescent quantitative PCR zinokwahlulwa zibe: TaqMan fluorescent probe, iibhikhoni zemolekyuli kunye nedayi yefluorescent.

1) TaqMan fluorescent probe:

Ngethuba lokukhulisa i-PCR, i-probe ethile ye-fluorescent yongezwa ngelixa idibanisa i-primer.I-probe yi-oligonucleotide, kwaye zombini iziphelo zibhalwe kunye neqela le-reporter fluorescent kunye neqela le-fluorescent ye-quencher.

Xa i-probe ilungile, isibonakaliso se-fluorescent esikhutshwe liqela leengxelo lixutywe liqela lokucima;ngexesha lokwandiswa kwe-PCR, umsebenzi we-5′-3′ we-exonuclease we-enzyme ye-Taq iqhekeza kwaye ithoba i-probe, yenza i-reporter fluorescent group kunye ne-quencher Iqela le-fluorescent lahluliwe, ukuze inkqubo yokubeka iliso ye-fluorescence ikwazi ukufumana umqondiso we-fluorescence, oko kukuthi, lonke ixesha i-DNA strand is acculation of the Umqondiso ungqamaniswa ngokupheleleyo kunye nokwakheka kwemveliso yePCR.

2) Idayi ye-fluorescent yeSYBR:

Kwinkqubo yokusabela kwe-PCR, ubuninzi bedayi ye-SYBR ye-fluorescent yongezwa.Emva kokuba idayi ye-fluorescent ye-SYBR ingabandakanywanga ngokukodwa kwi-DNA ephindwe kabini, ikhupha isignali ye-fluorescent.I-molecule yedayi ye-SYBR engabandakanywanga kwikhonkco ayiyi kukhupha naluphi na uphawu lwe-fluorescent, ngaloo ndlela iqinisekisa uphawu lwe-fluorescent Ukwanda kweemveliso ze-PCR kulungelelaniswa ngokupheleleyo nokunyuka kweemveliso ze-PCR.I-SYBR ibophelela kuphela kwi-DNA enemisonto ephindwe kabini, ngoko ke igophe lokunyibilika lingasetyenziselwa ukufumanisa ukuba i-PCR isabela ngokuthe ngqo.

3) Ibhakana yemolekyuli:

I-stem-loop ebhalwe kabini i-oligonucleotide probe eyenza i-hairpin structure malunga neziseko ezi-8 kwi-5 kunye ne-3 ekupheleni.Ukulandelelana kwe-asidi ye-nucleic kuzo zombini iziphelo kudityaniswa ngokuhambelanayo, kubangela ukuba iqela le-fluorescent kunye neqela lokucima libe lukhuni.Vala, akukho fluorescence iya kuveliswa.

Emva kokuba imveliso ye-PCR iveliswe, ngexesha lenkqubo ye-annealing, inxalenye ephakathi ye-molecular beacon idityaniswe ngokulandelelana kwe-DNA ethile, kwaye i-fluorescent gene ihlukaniswe kwi-quencher gene ukuvelisa i-fluorescence.

Ezona zinto zingalunganga zePCR yesizukulwana sesibini:

Uvakalelo lusanqongophele, kwaye ukuchongwa kwemizekelo enekopi ephantsi ayichanekanga.

Kukho impembelelo yexabiso elingasemva, kwaye isiphumo sisengozini yokuphazamiseka.

Xa kukho i-PCR inhibitors kwinkqubo yokusabela, iziphumo zokufumanisa ziyakwazi ukuphazamiseka.

Isizukulwana sesithathu se-PCR yedijithali

I-PCR yeDijithali (i-DigitalPCR, i-dPCR, i-Dig-PCR) ibala inani lekopi yokulandelelana okujoliswe kuyo ngokuchongwa kwendawo yokugqibela, kwaye inokwenza ukufumanisa ubuninzi obuchanekileyo obuchanekileyo ngaphandle kokusebenzisa ulawulo lwangaphakathi kunye neegophe eziqhelekileyo.

I-PCR ye-Digital isebenzisa ukufumanisa i-end-point point kwaye ayixhomekeke kwixabiso le-Ct (umda wokujikeleza), ngoko i-PCR ye-digital reaction ayichaphazeli kangako ukusebenza kwe-amplification, kwaye ukunyamezela kwi-PCR reaction inhibitors kuphuculwe, ngokuchaneka okuphezulu kunye nokuveliswa kwakhona.

Ngenxa yeempawu zobuntununtunu obuphezulu kunye nokuchaneka okuphezulu, ayiphazanyiswa lula yi-PCR reaction inhibitors, kwaye inokufikelela kwi-quantification yokwenyani ngaphandle kweemveliso eziqhelekileyo, eziye zaba luphando kunye ne-hotspot yesicelo.

Ngokweendlela ezahlukeneyo zeyunithi yokusabela, inokwahlulwa ibe ziindidi ezintathu eziphambili: i-microfluidic, i-chip kunye ne-droplet systems.

1) Microfluidic digital PCR, mdPCR:

Ngokusekelwe kwi-teknoloji ye-microfluidic, i-template ye-DNA ihlukaniswe.Itekhnoloji ye-microfluidic inokuqonda isampula ye-nano-upgrade okanye isizukulwana samathontsi amancinci, kodwa amathontsi adinga indlela ekhethekileyo ye-adsorption emva koko idityaniswe nenkqubo yokusabela kwe-PCR.mdPCR iye yamkelwa ngokuthe ngcembe zezinye iindlela endaweni.

2) IPR yedijithali esekwe kwiDroplet, ddPCR:

Sebenzisa itekhnoloji yokuvelisa i-water-in-oyile ukwenza isampuli ibe ngamathontsi, kwaye yahlule inkqubo yokusabela equlathe iimolekyuli ze-nucleic acid ibe ngamawaka amathontsi e-nanoscale, ngalinye lingaqulathanga imolekyuli ye-nucleic acid ekujoliswe kuyo ukuba ibhaqwe, okanye Iqulethe enye ukuya kwiimolekyuli ezininzi ekujoliswe kuzo ze-nucleic acid eziza kuvavanywa.

3) I-PCR yedijithali esekwe kwiChip, cdPCR:

Sebenzisa iteknoloji yendlela yolwelo ehlanganisiweyo ukukrola ii-microtubes ezininzi kunye ne-microcavities kwii-silicon wafers okanye iglasi yequartz, kwaye ulawule ukuhamba kwesisombululo ngeevalvu zolawulo ezahlukeneyo, kwaye wahlule ulwelo lwesampulu kwiinanometers zobungakanani obufanayo kumaqula okusabela kwi-PCR yedijithali Reaction ukuphumeza ubungakanani obupheleleyo.

Ezona zinto zingalunganga zePCR yesizukulwana sesithathu:

Izixhobo kunye nee-reagents ziyabiza.

Iimfuno zomgangatho wetemplate ziphezulu.Ukuba ubungakanani betemplate budlula ubuninzi be-microsystem, akuyi kuba nzima ukulinganisa, kwaye ukuba incinci kakhulu, ukuchaneka kobungakanani kuya kuncitshiswa.

Ii-positives ezingeyonyani zisenokwenziwa xa kukho ulwandiso olungachanekanga.