Ukuxilongwa kwamangqamuzana, ubuchwepheshe be-PCR obuvame ukusetshenziswa kanye nomgomo

I-PCR, i-polymerase chain reaction, ebhekisela ekwengezweni kwe-dNTP, i-Mg2+, izici zokwelula kanye nezici zokuthuthukisa ukukhulisa uhlelo ohlelweni ngaphansi kwe-catalysis ye-DNA polymerase, kusetshenziswa i-DNA yomzali njengesifanekiso neziqalo ezithile njengendawo yokuqala yesandiso , Ngezinyathelo zokuguqula, ukuhlanganisa, ukunwetshwa, njll., inqubo ye-in vitro iphindaphinda i-DNA yendodakazi yomucu ehambisana ne-DNA yesifanekiso somucu womzali ingakhulisa ngokushesha futhi ngokuqondile noma iyiphi i-DNA eqondiwe ku-vitro.

1. Hot Start PCR

Isikhathi sokuqala sokukhulisa ku-PCR evamile akukona ukufaka umshini we-PCR emshinini we-PCR, bese uhlelo luqala ukukhulisa.Lapho ukucushwa kwesistimu sekuqediwe, ukukhulisa i-amplification kuqala, okungase kubangele ukukhuliswa okungaqondile, futhi i-PCR yokuqala eshisayo ingaxazulula le nkinga.

Iyini i-PCR yokuqala eshisayo?Ngemva kokuba uhlelo lokusabela lulungisiwe, isishintshi se-enzyme sikhululwa ekushiseni okuphezulu (imvamisa ephakeme kuno-90°C) phakathi nesigaba sokushisa sokuqala sokusabela noma isigaba “sokuqala esishisayo,” ukuze i-DNA polymerase isebenze.Isikhathi esiqondile sokwenza kusebenze kanye nezinga lokushisa kuncike esimweni se-DNA polymerase kanye nesilungisi se-hot-start.Le ndlela ikakhulukazi isebenzisa izilungisi ezifana namasosha omzimba, i-affinity ligands, noma izishintshi zamakhemikhali ukuze zivimbele umsebenzi we-DNA polymerase.Njengoba umsebenzi we-DNA polymerase uvinjelwe ekamelweni lokushisa, ubuchwepheshe bokuqalisa okushisayo bunikeza lula kakhulu ukulungiselela amasistimu wokusabela we-PCR amaningi ekamelweni lokushisa ngaphandle kokudela ukucaciswa kokusabela kwe-PCR.

2. RT-PCR

I-RT-PCR (Reverse transcription PCR) iyindlela yokuhlola yokuhlehlisa ukuloba kusuka ku-mRNA kuya ku-cDNA futhi kusetshenziswe njengesifanekiso sokukhulisa.Inqubo yokuhlola iwukuba kukhishwe isamba se-RNA ezicutshini noma kumaseli kuqala, sebenzisa i-Oligo (dT) njengesiqalo, sebenzisa i-reverse transcriptase ukuze uhlanganise i-cDNA, bese usebenzisa i-cDNA njengesifanekiso sokukhulisa i-PCR ukuze kutholwe isakhi sofuzo esiqondiwe noma ukutholwa kofuzo.

3. I-Fluorescent quantitative PCR

I-Fluorescent quantitative PCR (Real-time Quantitative PCR,I-RT-qPCR) ibhekisela endleleni yokwengeza amaqembu e-fluorescent ohlelweni lokusabela kwe-PCR, kusetshenziswa ukunqwabelana kwamasignali e-fluorescent ukuze kuqashwe yonke inqubo ye-PCR ngesikhathi sangempela, futhi ekugcineni kusetshenziswa ijika elijwayelekile ukuze kuhlaziywe ngokwesilinganiso isifanekiso.Izindlela ezisetshenziswa kakhulu ze-qPCR zifaka i-SYBR Green I ne-TaqMan.

4. I-PCR efakiwe

I-Nested PCR isho ukusetshenziswa kwamasethi amabili we-PCR primers emizuliswaneni emibili yokukhulisa i-PCR, futhi umkhiqizo wokukhulisa umjikelezo wesibili ucezu lofuzo oluqondiwe.

Uma ukungafani kwepheya yokuqala yeziqalo (iziqalisi zangaphandle) kubangela ukuba umkhiqizo ongaqondile ukhuliswe, amathuba okuthi indawo engaqondile efanayo ibonwe ngababili besibili futhi ukuqhubeka nokukhulisa mancane kakhulu, ngakho-ke ukukhulisa ngepheya yesibili yeziqalo , ukucaciswa kwe-PCR kuthuthukisiwe.Enye inzuzo yokwenza imizuliswano emibili ye-PCR ukuthi isiza ukukhulisa umkhiqizo owanele kusukela ku-DNA yokuqala elinganiselwe.

5. Thinta phansi i-PCR

I-Touchdown PCR iyindlela yokuthuthukisa ukucaciswa kokusabela kwe-PCR ngokulungisa amapharamitha omjikelezo we-PCR.

Ku-PCR yokuthinta, izinga lokushisa le-anneal emijikelezweni embalwa yokuqala lisethwa ngamadigri ambalwa ngaphezu kwezinga lokushisa eliphezulu lokukhipha (Tm) lama-primers.Izinga lokushisa eliphakeme le-annealing linganciphisa ngempumelelo ukukhuliswa okungaqondile, kodwa ngesikhathi esifanayo, izinga lokushisa eliphakeme le-annealing lizokwandisa ukuhlukaniswa kwama-primers nokulandelana okuqondisiwe, okuholela ekunciphiseni kwesivuno se-PCR.Ngakho-ke, emijikelezweni embalwa yokuqala, izinga lokushisa le-anneal ngokuvamile lihlehliswa ngo-1°C umjikelezo ngamunye ukuze kwandiswe okuqukethwe kofuzo oluqondiwe ohlelweni.Uma izinga lokushisa le-anneal lehliselwa ezingeni lokushisa elilungile, izinga lokushisa le-anneal liyagcinwa emijikelezweni esele.

6. I-PCR eqondile

I-Direct PCR isho ukukhuliswa kwe-DNA eqondiwe ngqo kusuka kusampula ngaphandle kwesidingo sokuhlukaniswa nokuhlanzwa kwe-nucleic acid.

Kunezinhlobo ezimbili ze-PCR eqondile:

indlela eqondile: thatha inani elincane lesampula bese ulengeza ngokuqondile ku-PCR Master Mix ukuze uthole ukuhlonza i-PCR;

indlela yokuqhekeka: ngemva kokusampula isampula, yengeze ku-lysate, i-lyse ukuze ukhulule i-genome, thatha inani elincane le-lysed supernatant bese uyifaka ku-PCR Master Mix, yenza ukuhlonza i-PCR.Le ndlela yenza ukuhamba komsebenzi kube lula, inciphisa ukusebenza ngesikhathi, futhi igwema ukulahleka kwe-DNA phakathi nezinyathelo zokuhlanza.

7. I-SOE PCR

I-Gene splicing by overlap extension PCR (SOE PCR) isebenzisa ama-primer aneziphetho ezihambisanayo ukwenza imikhiqizo ye-PCR yakhe amaketanga agqagqene, ukuze ekuphenduleni okulandelayo kokukhulisa, ngokunwetshwa kwamaketango agqagqene, imithombo ehlukene yesu A lapho izingcezu ezikhulisiwe zigqigqana khona. futhi ihlanganiswe ndawonye.Lobu buchwepheshe njengamanje bunezikhombisi-ndlela ezimbili eziyinhloko zohlelo lokusebenza: ukwakhiwa kwezakhi zofuzo ezixubile;ukuguqulwa kwesayithi okuqondiswe kofuzo.

8. IPCR

I-Inverse PCR (IPCR) isebenzisa ama-primer ahambisanayo ahlehlayo ukuze ikhulise izingcezu ze-DNA ngaphandle kwama-primers amabili, futhi ikhulise ukulandelana okungaziwa kuzo zombili izinhlangothi zocezu olwaziwayo lwe-DNA.

I-IPCR ekuqaleni yayiklanyelwe ukunquma ukulandelana kwezifunda eziseduze ezingaziwa, futhi isetshenziswa kakhulu ukutadisha ukulandelana komgqugquzeli wofuzo;ukuhlelwa kabusha kwe-chromosomal ye-oncogenic, njengokuhlanganiswa kwezakhi zofuzo, ukudluliselwa kanye nokuguqulwa;kanye nokuhlanganiswa kwezakhi zofuzo zegciwane, nazo zivame ukusetshenziswa manje Ukuze uthole i-mutagenesis eqondiswe kusayithi, kopisha i-plasmid enoguquko olufunayo.

9. dPCR

I-Digital PCR (dPCR) iyindlela yokulinganisa okuphelele kwama-nucleic acid molecule.

Njengamanje kunezindlela ezintathu zokulinganisa ama-nucleic acid molecule.I-Photometry isekelwe ekumunceni kwama-nucleic acid molecule;I-PCR yesikhathi sangempela ye-fluorescent quantitative (Isikhathi Sangempela PCR) isekelwe kunani le-Ct, futhi inani le-Ct libhekisela enambeni yomjikelezo ehambisana nevelu ye-fluorescence engatholwa;I-PCR yedijithali iwubuchwepheshe bakamuva be-Quantitative obusekelwe endleleni ye-PCR ye-molecule eyodwa yokubala ukulinganisa kwe-nucleic acid kuyindlela yobuningi obuphelele.