Yintoni i-PCR kwaye kutheni ibalulekile?

I-PCR, okanye i-polymerase chain reaction, yindlela esetyenziswa ukukhulisa ulandelelwano lwe-DNA.Yaqala ukuphuhliswa ngeminyaka yoo-1980 nguKary Mullis, owawongwa ngeBhaso leNobel kwiChemistry ngo-1993 ngomsebenzi wakhe.I-PCR iguqule ibhayoloji yemolekyuli, ivumela abaphandi ukuba bakhulise i-DNA kwiisampulu ezincinci kwaye bayifunde ngokweenkcukacha.

I-PCR yinkqubo yamanyathelo amathathu eyenzeka kumjikelo we-thermal, umatshini onokutshintsha ngokukhawuleza ubushushu bomxube wokuphendula.La manyathelo mathathu kukudinaturation, ukunyibilikisa, kunye nokwandiswa.
 
Kwinqanaba lokuqala, i-denaturation, i-DNA enemisonto ephindwe kabini ifudunyezwa kwiqondo lobushushu eliphezulu (elidla ngokumalunga ne-95°C) ukuze kuqhawulwe iibhondi zehydrogen ezibambe le misonto mibini kunye.Oku kubangela iimolekyuli zeDNA ezinemisonto enye.
 
Kwinqanaba lesibini, i-annealing, iqondo lobushushu liyehliswa ukuya kutsho kuma-55°C ukuze kuvumeleke iiprimers ukuba zilandele ulandelelwano oluhambelanayo kwi-DNA enomsonto omnye.Iiprimers ziingceba ezimfutshane ze-DNA ezenzelwe ukufanisa ukulandelelana komdla kwi-DNA ekujoliswe kuyo.
 
Kwinqanaba lesithathu, ulwandiso, iqondo lokushisa liphakanyiswe malunga ne-72 ° C ukuvumela i-Taq polymerase (uhlobo lwe-DNA polymerase) ukuba yenze i-strand entsha ye-DNA evela kwii-primers.I-Taq polymerase ivela kwibhaktheriya ehlala kwimithombo eshushu kwaye iyakwazi ukumelana nobushushu obuphezulu obusetyenziswa kwi-PCR.

Emva komjikelo omnye we-PCR, isiphumo ziikopi ezimbini zokulandelelana kwe-DNA ekujoliswe kuyo.Ngokuphinda amanyathelo amathathu kwinani lemijikelezo (ngokuqhelekileyo i-30-40), inani leekopi ze-DNA ekujoliswe kuyo linokunyuswa ngokukhawuleza.Oku kuthetha ukuba kwanomlinganiselo omncinci wokuqalisa i-DNA unokwandiswa ukuvelisa izigidi okanye iibhiliyoni zeekopi.

 
I-PCR inezicelo ezininzi kuphando kunye noxilongo.Isetyenziswa kufuzo ukufunda umsebenzi wemfuza kunye notshintsho, kwizifundo zangaphambili zokuhlalutya ubungqina be-DNA, ekuxilongweni kwezifo ezosulelayo ukubona ubukho bentsholongwane, kunye noxilongo lwaphambi kokubeleka ukuze kuhlolwe ukuphazamiseka kwemfuza kubantwana abangekazalwa.
 
I-PCR iphinde yatshintshwa ukuze isetyenziswe kwinani leenguqu, ezifana ne-quantitative PCR (qPCR), evumela ukuba inani le-DNA lilinganiswe kwaye liguqule i-PCR ye-transcription (RT-PCR), engasetyenziselwa ukukhulisa ukulandelelana kwe-RNA.

Ngaphandle kwezicelo ezininzi, iPCR ayinayo imida.Ifuna ulwazi lolandelelwano olujoliswe kuyo kunye noyilo lwee-primers ezifanelekileyo, kwaye inokuthambekela kwimpazamo ukuba iimeko zokusabela azilungiswanga ngokuchanekileyo.Nangona kunjalo, ngoyilo lovavanyo olunyamekileyo kunye nokwenziwa, i-PCR ihlala isesona sixhobo sinamandla kwibhayoloji yemolekyuli.