PCR, ke polymerase chain reaction, e bolelang ho eketsoa ha dNTP, Mg2+, lintlha tsa bolelele le lintlha tsa ntlafatso ea amplification tsamaisong e tlas'a catalysis ea DNA polymerase, ho sebelisa DNA ea motsoali e le template le li-primers tse khethehileng e le qalo ea ho atolosa. Ka mehato ea denaturation, annealing, extension, joalo-joalo, ts'ebetso ea in vitro e etsisa morali oa DNA e tlatselletsang ho DNA ea template ea motsoali e ka holisa ka potlako le ka ho khetheha sepheo leha e le sefe sa DNA in vitro.
1. Hot Start PCR
Nako ea ho qala ea ho holisa ho PCR e tloaelehileng ha se ho kenya mochini oa PCR mochining oa PCR, ebe lenaneo le qala ho holisa.Ha tlhophiso ea sistimi e phethiloe, ho holisoa hoa qala, ho ka bakang amplification e sa tobang, mme PCR e qalang e chesang e ka rarolla bothata bona.
Hot Start PCR ke eng?Ka mor'a hore mokhoa oa ho itšoara o lokisoe, mofetoleli oa enzyme o lokolloa ka mocheso o phahameng (hangata o phahame ho feta 90 ° C) nakong ea mocheso oa pele oa ho futhumatsa kapa "ho qala ho chesang", e le hore DNA polymerase e sebetse.Nako e nepahetseng ea ts'ebetso le mocheso li ipapisitse le mofuta oa DNA polymerase le hot-start modifier.Mokhoa ona haholo-holo o sebelisa li-modifiers tse kang li-antibodies, affinity ligands, kapa li-chemical modifiers ho thibela mosebetsi oa DNA polymerase.Kaha ts'ebetso ea DNA polymerase e thibetsoe mocheso oa kamore, theknoloji ea ho qala e chesang e fana ka monyetla o moholo oa ho lokisetsa mekhoa e mengata ea ho itšoara ka PCR mocheso oa kamore ntle le ho tela ho khetholla ha PCR.
2. RT-PCR
RT-PCR (Reverse transcription PCR) ke mokhoa oa liteko oa ho fetolela ka morao ho tloha ho mRNA ho ea ho cDNA le ho e sebelisa e le template ea ho holisa.Mokhoa oa liteko ke ho ntša RNA e felletseng ka har'a lisele kapa liseleng pele, sebelisa Oligo (dT) joalo ka primer, sebelisa reverse transcriptase ho etsa cDNA, ebe o sebelisa cDNA joalo ka template ea ho holisa PCR ho fumana mofuta o lebisitsoeng kapa ho lemoha polelo ea lefutso.
3. Fluorescent quantitative PCR
Fluorescent quantitative PCR (Real-time Quantitative PCR,RT-qPCR) e bua ka mokhoa oa ho eketsa lihlopha tsa fluorescent ho PCR reaction system, ho sebelisa ho bokella lipontšo tsa fluorescent ho shebella ts'ebetso eohle ea PCR ka nako ea sebele, 'me qetellong ho sebelisa lekhalo le tloaelehileng ho hlahloba template ka bongata.Mekhoa e sebelisoang hangata ea qPCR e kenyelletsa SYBR Green I le TaqMan.
4. Nested PCR
Nested PCR e bua ka ts'ebeliso ea lihlopha tse peli tsa li-primers tsa PCR bakeng sa mekhahlelo e 'meli ea ntlafatso ea PCR,' me sehlahisoa sa amplification sa potoloho ea bobeli ke sekhechana sa lefutso.
Haeba ho se lumellane ha para ea pele ea li-primers (li-primers tsa ka ntle) ho etsa hore sehlahisoa se seng se khethehileng se holisoe, monyetla oa hore sebaka se tšoanang se sa khethehe se lemohuoe ke para ea bobeli ea li-primers le ho tsoela pele ho holisa ho nyane haholo, ka hona, ho holisoa ke li-primers tse peli, ho nepahala ha PCR ho ntlafalitsoe.Molemo o mong oa ho etsa mekhahlelo e 'meli ea PCR ke hore e thusa ho holisa sehlahisoa se lekaneng ho tloha ho DNA e lekanyelitsoeng ea ho qala.
5. Tobetsa PCR
Touchdown PCR ke mokhoa oa ho ntlafatsa sebopeho sa karabelo ea PCR ka ho lokisa liparamente tsa potoloho ea PCR.
Ho touchdown PCR, mocheso o kenang bakeng sa lipotoloho tse 'maloa tsa pele o behiloe ka likhato tse' maloa ka holimo ho mocheso o phahameng oa annealing (Tm) oa li-primers.Thempereichara e phahameng ea annealing e ka fokotsa matlafatso e sa khetheheng, empa ka nako e ts'oanang, mocheso o phahameng oa annealing o tla mpefatsa karohano ea li-primers le tatellano ea sepheo, e leng se tla fella ka ho fokotseha ha chai ea PCR.Ka hona, lipotolohong tse 'maloa tsa pele, mocheso oa anneal hangata o behoa hore o fokotsehe ka 1 ° C ka potoloho ho eketsa likahare tsa lefutso le lebisitsoeng tsamaisong.Ha mocheso oa annealing o theoleloa ho mocheso o nepahetseng, mocheso oa annealing o bolokoa bakeng sa lipotoloho tse setseng.
6. PCR e tobileng
Direct PCR e bua ka ho holisoa ha sepheo sa DNA ka kotloloho ho tsoa sampoleng ntle le tlhoko ea ho itšehla thajana le tlhoekiso ea nucleic acid.
Ho na le mefuta e 'meli ea PCR e tobileng:
mokhoa o tobileng: nka sampuli e nyenyane 'me u e kenye ka ho toba ho PCR Master Mix bakeng sa boitsebiso ba PCR;
mokhoa oa ho senya: ka mor'a ho etsa mohlala oa sampuli, o e kenye ho lysate, lyse ho lokolla genome, nka tekanyo e nyenyane ea lysed supernatant 'me u e kenye ho PCR Master Mix, etsa boitsebiso ba PCR.Mokhoa ona o nolofatsa mosebetsi oa liteko, o fokotsa nako ea matsoho, 'me o qoba tahlehelo ea DNA nakong ea mehato ea ho hloekisa.
7. SOE PCR
Gene splicing by overlap extension PCR (SOE PCR) e sebelisa li-primers tse nang le lipheletso tse tlatselletsang ho etsa hore lihlahisoa tsa PCR li thehe liketane tse holimo, e le hore karabelong e latelang ea ho holisa, ka katoloso ea liketane tse fetang, mehloli e fapaneng ea mokhoa oa A moo likhechana tse holisitsoeng li kopanngoang. 'me li kopantsoe hammoho.Theknoloji ena hajoale e na le litaelo tse peli tse kholo tsa ts'ebeliso: kaho ea liphatsa tsa lefutso tsa fusion;phetoho e lebisitsoeng sebakeng sa lefutso.
8. IPCR
Inverse PCR (IPCR) e sebelisa li-primers tse tlatsetsang ka morao ho holisa likhechana tsa DNA ntle le li-primers tse peli, le ho holisa tatellano e sa tsejoeng mahlakoreng ka bobeli a sekhechana se tsebahalang sa DNA.
IPCR qalong e ne e etselitsoe ho fumana tatellano ea libaka tse haufi tse sa tsejoeng, 'me e sebelisoa haholo ho ithuta tatelano ea liphatsa tsa lefutso;li-arrangements tsa chromosomal tsa oncogenic, tse kang fusion gene, translocation le transposition;le ho kopanya liphatsa tsa lefutso tsa kokoana-hloko, li boetse li sebelisoa ka tloaelo hona joale Bakeng sa mutagenesis e lebisitsoeng sebakeng, kopitsa plasmid ka phetoho e lakatsehang.
9. dPCR
Digital PCR (dPCR) ke mokhoa oa ho lekanya palo e felletseng ea limolek'hule tsa nucleic acid.
Hona joale ho na le mekhoa e meraro ea quantification ea limolek'hule tsa nucleic acid.Photometry e itšetlehile ka ho monya ha limolek'hule tsa nucleic acid;PCR ea nako ea sebele ea fluorescent quantitative (Real Time PCR) e itšetlehile ka boleng ba Ct, 'me boleng ba Ct bo bolela palo ea potoloho e tsamaellanang le boleng ba fluorescence bo ka fumanoang;digital PCR ke theknoloji ea morao-rao ea Quantitative e ipapisitseng le mokhoa oa PCR oa molek'hule o le mong oa ho bala nucleic acid quantification ke mokhoa o felletseng oa bongata.
Nako ea poso: Jun-13-2023