Itekhnoloji yokuxilonga imolekyuli isebenzisa iindlela zebhayoloji yemolekyuli ukufumanisa intetho kunye nokwakheka kwemathiriyeli yemfuza yomzimba womntu kunye neepathogens ezahlukeneyo, ukuze kufezekiswe injongo yokuqikelela kunye nokufumanisa izifo.

Kwiminyaka yakutshanje, ngokuphuculwa kunye nokuphindaphinda itekhnoloji yoxilongo lwe-molekyuli, ukusetyenziswa kweklinikhi yokuxilongwa kweemolekyuli kuye kwanda kakhulu kwaye kunzulu, kwaye imakethi yokuxilongwa kweemolekyuli ingene kwixesha lophuhliso olukhawulezayo.

Umbhali ushwankathela itekhnoloji yokuxilongwa kweemolekyuli eziqhelekileyo kwimarike, kwaye yahlulwe yangamacandelo amathathu: inxalenye yokuqala yazisa itekhnoloji yePCR, icandelo lesibini lazisa itekhnoloji ye-nucleic acid isothermal amplification, kwaye inxalenye yesibini yazisa itekhnoloji yokulandelelana.

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Icandelo I: PCR Technology

Itekhnoloji yePCR

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

Itekhnoloji yePCR yaqalwa ngo-1983 nguKary Mullis waseCetus, eU.SA.U-Mullis wenza isicelo se-PCR patent ngo-1985 waza wapapasha iphepha lokuqala le-PCR lezifundo zeSayensi kwangaloo nyaka.UMulis waphumelela ibhaso leNobel kwiChemistry ngo-1993.

Imigaqo-siseko ye-PCR

I-PCR inokwandisa amaqhekeza e-DNA ekujoliswe kuyo ngamaxesha angaphezu kwesigidi esinye.Umgaqo-siseko kukuba phantsi kwe-catalysis ye-DNA polymerase, i-DNA ye-strand yomzali isetyenziswe njenge template, kwaye i-primer ethile isetyenziswe 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.Iibhondi ze-hydrogen phakathi kwe-DNA imisonto ephindwe kabini yaphuka kumaqondo obushushu aphezulu (93-98°C).

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

3. Ulwandiso: I-DNA polymerase iqala ukudibanisa imicu ehambelanayo ecaleni kwemisonto ye-DNA ukusuka 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 zinokuthi zenziwe 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:

-Kuxhomekeke ekukhuliseni okungaqhelekanga kunye neziphumo ezilungileyo ezingeyonyani.

-Ukufumanisa kuthatha ixesha elide kwaye umsebenzi unzima.

-Kuphela uvavanyo lomgangatho onokuthi lwenziwe.

Isizukulwana sesibini se-fluorescence quantitative PCR

I-Fluorescence quantitative PCR (i-PCR yeXesha lokwenyani), ekwabizwa ngokuba yi-qPCR, isetyenziselwa ukubeka esweni ukuqokelelana kweemveliso ezandisiweyo ngokuqokelelwa kwemiqondiso ye-fluorescent ngokudibanisa i-fluorescent probes enokubonisa inkqubela yenkqubo yokusabela, kunye nokugweba iziphumo ngokusebenzisa ijika le-fluorescence elinomgangatho we-curve kunye noncedo lwe-curve.

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 ezisetyenziswe kwixesha lokwenyani lobungakanani befluorescent quantitative PCR inokwahlulwa ibe: TaqMan fluorescent probes, iibhakhoni zemolekyuli kunye nedayi efluorescent.

1) TaqMan fluorescent probe:

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

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 zefluorescent zeSYBR:

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) Iibhikoni zeemolekyuli

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, ayiyi kuvelisa i-fluorescence.

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 sisesichengeni sokuphazamiseka.

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-endpoint 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: i-microfluidic, i-chip kunye ne-droplet systems.