Uphuhliswe ngowe-1983, inkqubo ye-PCR yenza ukuba kwenzeke ukulandelelanisa i-DNA kunye nokuchonga umyalelo we-nucleotides kwizityalo zomzimba ngamnye.
Le ndlela isebenzisa ibhasi lokushisa okanye ukufudumala okuphindaphindiweyo nokupholisa ukuphendula kwe-DNA ukunyibilika nokuphindaphinda. Njengoko i-PCR iqhubeka, i-DNA "entsha" isetyenziselwa i-template yokuphindaphinda kwaye i-chain reaction responds, ngokucacileyo ikhulisa isifanekiso seDNA.
Amacandelo e-PCR asetyenziswa kwiindawo ezininzi ze-biotechnology kubandakanywa ubunjineli beprotein, i-cloning, i-forensics (i-DNA yeprint fingerprinting), ukuhlolwa kwezilwanyana, ukuxilongwa kwezityalo kunye / okanye izifo ezithintekayo, kunye nokuhlalutya kweesampuli zendalo.
Kwiingxelo zeengxelo, ngokukodwa, i-PCR ibaluleke ngakumbi kuba iphakamisa kwanomlinganiselo omncinci we-DNA ubungqina. I-PCR ingasetyenziselwa ukuhlalutya i-DNA eneminyaka eyizinkulungwane zeminyaka ubudala, kwaye ezi ndlela zisetyenziselwe ukuchonga zonke izinto ezisuka kuma-800,000 aneminyaka engama-800 ubudala ukuya kwimimemoni evela kwihlabathi jikelele.
Inkqubo ye-PCR ilandelayo:
- Ukuqalisa: Le nyathelo iyimfuneko kuphela kwi-DNA polymerases ezifuna i-PCR yokuqalisa. Impendulo ishushu iphakathi kwama-94 no-96 ° C kwaye ibanjwe imizuzu eyi-1-9.
- I-Denaturation: Ukuba le nkqubo ayifuni ukuqaliswa, i-denaturation yinyathelo lokuqala. Impendulo iyatshisa ukuya kuma-94-98 ° C malunga nemizuzwana engama-30 ukuya kweye-30. Iifomati ze-hydrogen ze-DNA ziphazamiseka kwaye i-molecule ye-DNA ene-DNA yodwa.
- I-Annealing: Iqondo lokushisa lokusabela liphantsi ukuya kuma-50 no-65 ° C kwaye ligcinwe imizuzwana engama-20-40. Iimpawu zokuqala ezifakelwa kwisifanekiso seDNA esisodwa. Ubushushu bubaluleke kakhulu ngeli nqanaba. Ukuba kushushu kakhulu, i-primer ayinakuyibopha. Ukuba kubanda kakhulu, i-primer ingazibopha ngokungaphelele. Ubunxulumano obuhle lubunjwa xa ulandelelwano lwe-primer luhambisana ngokulandelelana kwetemplate.
- Ukongezwa / Ulungelelaniso: Ubushushu ngexesha leli nyathelo liyahlukahluka kuxhomekeke kuhlobo lwe-polymerase. I-DNA polymerase idibanisa i-DNA entsha.
- Ukulinda kokugqibela: Eli nyathelo lenziwa ngo-70-74 ° C malunga nemizuzu emi-5-15 emva komjikelo wokugqibela we-PCR.
- Ibamba lokugqibela: Eli nyathelo likhethiweyo. Iqondo lokushisa ligcinwe ngo-4-15 ° C kwaye liphazamisa ukuphendula.
Inkqubo iyahlula zibe zigaba ezintathu:
- Ukuphakanyiswa kokuzimela: Kulo lonke umjikelezo, imveliso (iqhosha elithile le-DNA ephindaphindiweyo) liphindwe kabini.
- Isigaba sokumgangatho: Njengoko i-DNA polymerase ilahlekelwa ngumsebenzi kwaye idla izigulane, iyancipha.
- I-Plateau: Akukho mveliso iqokelela.