Iyini i-PCR futhi Kungani Ibalulekile?

I-PCR, noma i-polymerase chain reaction, iyindlela esetshenziselwa ukukhulisa ukulandelana kwe-DNA.Yasungulwa okokuqala ngeminyaka yawo-1980s nguKary Mullis, owaklonyeliswa ngeNobel Prize in Chemistry ngo-1993 ngomsebenzi wakhe.I-PCR iguqule ibhayoloji yamangqamuzana, ivumela abacwaningi ukuthi bakhulise i-DNA kumasampula amancane futhi bayifunde kabanzi.

I-PCR inqubo enezinyathelo ezintathu eyenzeka kumjikelezo oshisayo, umshini ongashintsha ngokushesha izinga lokushisa lengxube yokusabela.Izinyathelo ezintathu ziyi-denaturation, annealing, kanye nesandiso.
 
Esinyathelweni sokuqala, i-denaturation, i-DNA enemicu ephindwe kabili ishiselwa ekushiseni okuphezulu (ngokuvamile okungaba ngu-95°C) ukuze kunqamuke amabhondi e-hydrogen abamba imicu emibili ndawonye.Lokhu kubangela amangqamuzana amabili e-DNA anomucu owodwa.
 
Esinyathelweni sesibili, i-annealing, izinga lokushisa liyehliswa lifinyelele cishe ku-55°C ukuze kuvunyelwe ama-primers ukuthi ahambisane nokulandelana okuhambisanayo ku-DNA enomucu owodwa.Iziqalo ziyizingcezu ezimfushane ze-DNA eziklanyelwe ukufanisa ukulandelana kwesithakazelo ku-DNA eqondiwe.
 
Esinyathelweni sesithathu, isandiso, izinga lokushisa liyakhuphuka lifinyelele cishe ku-72°C ukuze kuvunyelwe i-Taq polymerase (uhlobo lwe-DNA polymerase) ukuthi ihlanganise umucu omusha we-DNA osuka kuma-primers.I-Taq polymerase isuselwa kubhaktheriya elihlala eziphethwini ezishisayo futhi likwazi ukumelana namazinga okushisa aphezulu asetshenziswa ku-PCR.

Ngemva komjikelezo owodwa we-PCR, umphumela uba amakhophi amabili okulandelana kwe-DNA okuqondiwe.Ngokuphinda izinyathelo ezintathu zenani lemijikelezo (imvamisa engu-30-40), inani lamakhophi okulandelana kwe-DNA okuhlosiwe lingakhuliswa ngokushesha.Lokhu kusho ukuthi ngisho nenani elincane le-DNA yokuqala lingakhuliswa ukuze likhiqize izigidi noma izigidigidi zamakhophi.

 
I-PCR inezinhlelo zokusebenza eziningi ocwaningweni nasekuxilongweni.Isetshenziswa kuzakhi zofuzo ukutadisha umsebenzi wezakhi zofuzo nokuguqulwa kwezakhi zofuzo, kuma-forensics ukuhlaziya ubufakazi be-DNA, ekuxilongweni kwezifo ezithathelwanayo ukuze kutholwe ukuba khona kwamagciwane, nasekuxilongweni kwangaphambi kokubeletha ukuze kuhlolwe ukuphazamiseka kofuzo ezinganeni.
 
I-PCR iphinde yashintshwa ukuze isetshenziswe ezinhlobonhlobo eziningi, njenge-quantitative PCR (qPCR), evumela inani le-DNA ukuthi lilinganiswe futhi lihlehliselwe ukubhalwa kwe-PCR (RT-PCR), engasetshenziswa ukukhulisa ukulandelana kwe-RNA.

Ngaphandle kwezinhlelo zayo eziningi zokusebenza, i-PCR ayinakho ukulinganiselwa.Idinga ulwazi lokulandelana okuqondiwe kanye nokwakheka kweziqalo ezifanele, futhi ingathambekela ekwenzeni iphutha uma izimo zokusabela zingalungiswanga kahle.Kodwa-ke, ngomklamo wokuhlola wokuhlola nokusebenza, i-PCR ihlala ingelinye lamathuluzi anamandla kubhayoloji yamangqamuzana.