Izinto eziphazamisayo kwiimpendulo ze-PCR

Ngexesha lokuphendula kwe-PCR, ezinye izinto eziphazamisayo zihlala zidibana.
Ngenxa yovakalelo oluphezulu kakhulu lwe-PCR, ukungcoliseka kuthathwa njengenye yezona zinto zibalulekileyo ezichaphazela iziphumo ze-PCR kwaye zinokuvelisa iziphumo ezingezizo zobuxoki.
Okubaluleke ngokulinganayo yimithombo eyahlukeneyo ekhokelela kwiziphumo ezingezizo ezingalunganga. Ukuba enye okanye iindawo ezibaluleke kakhulu zomxube we-PCR okanye i-amplification reaction ngokwayo iyathintelwa okanye iphazamisekile, uvavanyo lokuxilonga lunokuthintelwa. Oku kunokukhokelela ekunciphiseni ukusebenza kakuhle kunye neziphumo ezingezizo ezingezizo.
Ukongeza kwi-inhibition, ilahleko yengqibelelo ye-nucleic acid ekujoliswe kuyo inokuthi yenzeke ngenxa yokuthumela kunye / okanye iimeko zokugcina ngaphambi kokulungiswa kwesampuli. Ngokukodwa, ubushushu obuphezulu okanye ukugcinwa okungonelanga kunokukhokelela ekonakaleni kweeseli kunye ne-nucleic acids. Ukulungiswa kweeseli kunye nezicubu kunye nokufakela iparafini zizizathu ezaziwayo zokuqhekeka kwe-DNA kunye nengxaki eqhubekayo (jonga iMifanekiso 1 kunye ne-2). Kwezi meko, kwanokwahluka ngokupheleleyo kunye nokuhlanjululwa akuyi kunceda.

Umzobo 1 | Impembelelo yokunyanzeliswa kwingqibelelo ye-DNA
I-Agarose gel electrophoresis ibonise ukuba umgangatho we-DNA obekwe wodwa kumacandelo eparafini ovavanyo lwesidumbu wahluka kakhulu. I-DNA ye-avareji ye-fragment ubude obuhlukeneyo yayikhona kwiincindi ngokuxhomekeke kwindlela yokulungisa. I-DNA yagcinwa kuphela xa ilungisiwe kwiisampulu zemveli ezikhenkcezisiweyo kunye ne-buffered neutral formalin. Ukusetyenziswa kwe-Bouin ene-acidic eqinile okanye engathintekiyo, i-formalic acid-equlethe i-formalin ibangele ilahleko enkulu ye-DNA. Iqhekeza eliseleyo lahlulwe kakhulu.
Ekhohlo, ubude bamaqhekeza bubonakaliswe kwiikhilobase pairs (kbp)

Umzobo 2 | Ukulahleka kwengqibelelo yeethagethi ze-nucleic acid
(a) I-3'-5′ i-gap kwimicu yomibini iya kubangela ikhefu kwi-DNA ekujoliswe kuyo. Ukwenziwa kwe-DNA kuseza kwenzeka kwiqhekeza elincinci. Nangona kunjalo, ukuba indawo yokudibanisa i-primer ilahlekile kwiqhekeza le-DNA, kukwandiswa komgca kuphela okwenzekayo. Kwimeko efanelekileyo kakhulu, amaqhekeza angaphinda azalise enye kwenye, kodwa isivuno siya kuba sincinci kwaye singaphantsi kwamanqanaba okufumanisa.
(b) Ukulahlekelwa kweziseko, ngokukodwa ngenxa yokuchithwa kunye nokwakheka kwe-thymidine dimer, kukhokelela ekunciphiseni inani le-H-bond kunye nokuncipha kwe-Tm. Ngexesha lokufudumala kwenqanaba elide, iiprimers ziya kunyibilika zisuka kwi-matrix DNA kwaye aziyi kubamba naphantsi kweemeko ezingqongqo.
(c) Iziseko zethymine ezikufutshane zenza i-TT dimer.
Enye ingxaki eqhelekileyo ehlala isenzeka kwi-molecular diagnostics kukukhutshwa okungaphantsi kweyona nto ilungileyo ye-nucleic acid ekujoliswe kuyo xa kuthelekiswa ne-phenol-chloroform extraction. Kwiimeko ezigqithiseleyo, oku kunokudibaniswa nezinto ezingalunganga. Ixesha elininzi linokugcinwa ngokubilisa i-lysis okanye i-enzymatic digestion ye-debris yeseli, kodwa le ndlela ihlala ibangela uvakalelo oluphantsi lwe-PCR ngenxa yokungonelanga kokukhutshwa kwe-nucleic acid.

Ukuvinjelwa komsebenzi we-polymerase ngexesha lokukhulisa

Ngokubanzi, isithintelo sisetyenziswa njengengqikelelo yesikhongozeli ukuchaza zonke izinto ezikhokelela kwiziphumo ezisezantsi zePCR. Ngokwengqiqo engqongqo ye-biochemical, inhibition inqunyelwe kumsebenzi we-enzyme, oko kukuthi, iyanciphisa okanye ithintele ukuguqulwa kwemveliso ye-substrate ngokusebenzisana nendawo esebenzayo ye-DNA polymerase okanye i-cofactor yayo (umzekelo, i-Mg2 + ye-Taq DNA polymerase).
Amalungu kwisampulu okanye izithinteli ezahlukeneyo kunye nezicatshulwa eziqulethe i-reagents zinokunqanda ngokuthe ngqo i-enzyme okanye zibambise iicofactors zayo (umz. i-EDTA), ngaloo ndlela ingasebenzi i-polymerase kwaye ikhokelela ekuncipheni okanye kwiziphumo ezingalunganga zePCR.
Nangona kunjalo, unxibelelwano oluninzi phakathi kwamacandelo okusabela kunye nethagethi-equlethe i-nucleic acids zikwabizwa ngokuba yi-'PCR inhibitors'. Emva kokuba ingqibelelo yeseli iphazamisekile ngokuzimeleyo kwaye i-nucleic acid ikhutshwe, ukusebenzisana phakathi kwesampuli kunye nesisombululo sayo esijikelezileyo kunye nesigaba esiqinileyo singenzeka. Ngokomzekelo, 'abahlaseli' banokubopha i-DNA enye okanye i-double-stranded DNA ngokusebenzisa i-non-covalent interactions kunye nokuphazamisa ukuhlukaniswa kunye nokuhlanjululwa ngokunciphisa inani leethagethi ezithi ekugqibeleni zifike kwisitya sokusabela kwe-PCR.
Ngokuqhelekileyo, i-PCR inhibitors ikhona kumanzi amaninzi omzimba kunye nee-reagents ezisetyenziselwa iimvavanyo zokuxilonga ikliniki (i-urea kumchamo, i-hemoglobin kunye ne-heparin egazini), izongezo zokutya (i-organic components, i-glycogen, i-fat, i-Ca2 + ion) kunye namacandelo kwindawo (phenols). , iintsimbi ezinzima)

I-PCR inhibitors exhaphakileyo inokufumaneka kwiibhaktheriya kunye neeseli ze-eukaryotic, i-DNA engajoliswanga, i-DNA-binding macromolecules ye-tissue matrices kunye nezixhobo zelabhoratri ezifana neeglavu kunye neeplastiki. Ukucocwa kwe-nucleic acids ngexesha okanye emva kokukhutshwa yindlela ekhethiweyo yokususa i-PCR inhibitors.
Namhlanje, izixhobo ezahlukeneyo zokutsalwa kwe-automated zinokuthatha indawo yeprotocol ezininzi, kodwa i-100% yokubuyisela kunye / okanye ukucocwa kweethagethi akuzange kufezekiswe. Ii-inhibitors ezinokwenzeka zisenokuba zikhona kwi-nucleic acids ecocekileyo okanye mhlawumbi sele isebenze. Kukho izicwangciso ezahlukeneyo zokunciphisa impembelelo ye-inhibitors. Ukukhethwa kwepolymerase efanelekileyo kunokuba nefuthe elibonakalayo kumsebenzi we-inhibitor. Ezinye iindlela eziqinisekisiweyo zokunciphisa inhibition ye-PCR kukwandisa ugxininiso lwe-polymerase okanye ukusebenzisa izongezo ezifana ne-BSA.
Ukuvinjelwa kweempendulo ze-PCR kunokubonakaliswa ngokusetyenziswa kwenkqubo yokulawula umgangatho wangaphakathi (IPC).
Ukunyamekela kufuneka kuthathwe ukususa zonke ii-reagents kunye nezinye izisombululo kwi-kit extraction, njenge-ethanol, i-EDTA, i-CETAB, i-LiCl, i-GuSCN, i-SDS, i-isopropanol kunye ne-phenol, kwi-nucleic acid yodwa ngesinyathelo sokuhlamba ngokucokisekileyo. Ngokuxhomekeke ekugxininiseni kwabo, banokuvula okanye bavimbele i-PCR.