Izinto ezibangela ukuphazamiseka kwiimpendulo ze-PCR

Ngexesha lokuphendula kwe-PCR, kukho izinto ezithile eziphazamisayo ezidla ngokudibana nazo.
Ngenxa yokuba i-PCR inobuthathaka kakhulu, ungcoliseko luthathwa njengenye yezona zinto zibalulekileyo ezichaphazela iziphumo ze-PCR kwaye lunokuvelisa iziphumo ezingezizo ezilungileyo.
Imithombo eyahlukeneyo ekhokelela kwiziphumo ezingezizo ezilungileyo. Ukuba inxalenye enye okanye ezingaphezulu ezibalulekileyo zomxube we-PCR okanye i-amplification reaction ngokwayo iyathintelwa okanye iyaphazanyiswa, uvavanyo lokuxilonga lunokuthintela. Oku kunokukhokelela ekunciphiseni ukusebenza kakuhle kwaneziphumo ezingezizo ezilungileyo.
Ukongeza ekuthinteleni, ukulahleka kobunyani be-nucleic acid ekujoliswe kuyo kunokwenzeka ngenxa yeemeko zokuthunyelwa kunye/okanye zokugcina ngaphambi kokulungiswa kwesampuli. Ngokukodwa, amaqondo obushushu aphezulu okanye ukugcinwa okunganelanga kunokukhokelela ekonakaleni kweeseli kunye nee-nucleic acids. Ukuqina kweeseli kunye nezicubu kunye nokufakwa kweparafini zizizathu ezaziwayo zokuqhekeka kwe-DNA kunye nengxaki eqhubekayo (jonga iMifanekiso 1 kunye ne-2). Kwezi meko, nokuba kukwahlukaniswa kunye nokucocwa okufanelekileyo akuyi kunceda.

Umfanekiso 1 | Impembelelo yokungashukumi kwi-DNA epheleleyo
I-Agarose gel electrophoresis ibonise ukuba umgangatho we-DNA ehlukanisiweyo kwiindawo zeparafini zokuhlolwa kwemizimba yahluka kakhulu. I-DNA yobude obuhlukeneyo beenxalenye eziqhelekileyo yayikhona kwiincindi ngokuxhomekeke kwindlela yokulungiswa. I-DNA igcinwe kuphela xa ilungisiwe kwiisampuli zendalo ezikhenkcezisiweyo nakwi-buffered neutral formalin. Ukusetyenziswa kwe-formalin ene-asidi enamandla okanye engena-buffered, ene-formalin ene-formalin kwaphumela ekulahlekelweni okukhulu kwe-DNA. Inxalenye eseleyo yaqhekeka kakhulu.
Ngasekhohlo, ubude beziqwenga buchazwa ngee-kilobase pairs (kbp)

Umfanekiso 2 | Ukulahleka kokuthembeka kweethagethi ze-nucleic acid
(a) Umsantsa we-3′-5′ kuzo zombini iintambo uya kubangela ukuqhekeka kwi-DNA ekujoliswe kuyo. Ukwenziwa kwe-DNA kusaza kwenzeka kwiqhekeza elincinci. Nangona kunjalo, ukuba indawo yokutsalela i-primer ayikho kwiqhekeza le-DNA, kuphela ukwandiswa komgca okwenzekayo. Kwimeko efanelekileyo, iziqwenga zinokuphinda zigcwale, kodwa isivuno siya kuba sincinci kwaye singaphantsi kwamanqanaba okubhaqwa.
(b) Ukulahleka kweziseko, ikakhulu ngenxa yokususwa kwe-purination kunye nokwakheka kwe-thymidine dimer, kukhokelela ekunciphiseni inani lee-H-bonds kunye nokwehla kwe-Tm. Ngexesha lesigaba sokufudumala eside, ii-primers ziya kunyibilika kwi-matrix DNA kwaye aziyi kuphinda zinciphe naphantsi kweemeko ezinzima kangako.
(c) Iisiseko ze-thymine ezikufutshane zenza i-TT dimer.
Enye ingxaki eqhelekileyo edla ngokuvela kwi-molecular diagnostics kukukhululwa okungaphantsi kokulungeleyo kwee-nucleic acids ezijoliswe kuzo xa kuthelekiswa nokukhutshwa kwe-phenol-chloroform. Kwiimeko ezigqithisileyo, oku kunokunxulunyaniswa ne-false negatives. Ixesha elininzi linokongiwa ngokubilisa i-lysis okanye ukugaywa kwe-enzymatic kwee-debris zeseli, kodwa le ndlela idla ngokubangela ukuba i-PCR ibe novakalelo oluphantsi ngenxa yokukhululwa ngokwaneleyo kwe-nucleic acid.

Ukuthintela umsebenzi we-polymerase ngexesha lokukhulisa

Ngokubanzi, ukuthintela kusetyenziswa njengengcamango yesikhongozeli ukuchaza zonke izinto ezikhokelela kwiziphumo ze-PCR ezingagqibelelanga. Ngokwengqiqo ye-biochemical kuphela, ukuthintela kunqunyelwe kumsebenzi we-enzyme, oko kukuthi, kunciphisa okanye kuthintele ukuguqulwa kwe-substrate-product ngokusebenzisana nendawo esebenzayo ye-DNA polymerase okanye i-cofactor yayo (umz., i-Mg2+ ye-Taq DNA polymerase).
Izinto ezikwisampulu okanye kwii-buffers ezahlukeneyo kunye nezicatshulwa eziqulethe ii-reagents zinokuthintela ngokuthe ngqo i-enzyme okanye zibambe ii-cofactors zayo (umz. i-EDTA), ngaloo ndlela zingasebenzi i-polymerase kwaye oko kukhokelela kwiziphumo ze-PCR eziphantsi okanye ezingezizo.
Nangona kunjalo, ukusebenzisana okuninzi phakathi kwezinto ezisabelayo kunye nee-nucleic acids eziqulathe iithagethi nazo zibizwa ngokuba 'zii-PCR inhibitors'. Nje ukuba ukuthembeka kweseli kuphazanyiswe kukwahlukaniswa kwaye i-nucleic acid ikhutshiwe, ukusebenzisana phakathi kwesampuli kunye nesisombululo sayo esijikelezileyo kunye nesigaba esiqinileyo kunokwenzeka. Umzekelo, 'abahlaseli' banokubopha i-DNA enye okanye ezimbini ngokusebenzisa ukusebenzisana okungekho kwi-covalent kwaye baphazamise ukwahlukaniswa kunye nokuhlanjululwa ngokunciphisa inani leethagethi ezifikelela ekugqibeleni kwisitya se-PCR reaction.
Ngokubanzi, izithinteli ze-PCR zikhona kuninzi lolwelo lomzimba kunye nee-reagents ezisetyenziselwa uvavanyo lweklinikhi lokuxilonga (i-urea kumchamo, i-hemoglobin kunye ne-heparin egazini), izongezo zokutya (iinxalenye ze-organic, i-glycogen, amafutha, ii-Ca2+ ions) kunye neenxalenye ezikwindalo esingqongileyo (ii-phenols, ii-heavy metals)

Izithinteli ze-PCR ezixhaphakileyo zinokufumaneka kwiibhaktheriya nakwiiseli ze-eukaryotic, kwi-DNA engeyiyo ekujoliswe kuyo, kwiimacromolecules ezibopha i-DNA zeematrices zezicubu kunye nezixhobo zelebhu ezifana neeglavu kunye neeplastiki. Ukucocwa kwee-nucleic acids ngexesha okanye emva kokukhupha yindlela ekhethwayo yokususa izithinteli ze-PCR.
Namhlanje, izixhobo ezahlukeneyo zokukhupha ezizenzekelayo zingathatha indawo yeenkqubo ezininzi ezenziwe ngesandla, kodwa ukubuyiselwa kunye/okanye ukucocwa kweenjongo eziyi-100% akukaze kufezekiswe. Izithinteli ezinokubakho zisenokuba zikhona kwi-nucleic acids ezicociweyo okanye zisenokuba sele ziqalile ukusebenza. Kukho amaqhinga ahlukeneyo okunciphisa impembelelo yezithinteli. Ukukhethwa kwe-polymerase efanelekileyo kunokuba nefuthe elikhulu kumsebenzi wezithinteli. Ezinye iindlela eziqinisekisiweyo zokunciphisa ukuthintela kwe-PCR kukunyusa uxinano lwe-polymerase okanye ukusebenzisa izongezo ezifana ne-BSA.
Ukuthintela ii-PCR reactions kunokubonakaliswa ngokusebenzisa ulawulo lomgangatho wenkqubo yangaphakathi (IPC).
Kufuneka kuthathwe unonophelo ukususa zonke ii-reagents kunye nezinye izisombululo kwikhithi yokukhupha, ezifana ne-ethanol, i-EDTA, i-CETAB, i-LiCl, i-GuSCN, i-SDS, i-isopropanol kunye ne-phenol, kwi-nucleic acid isolate ngenyathelo lokuhlamba ngokupheleleyo. Ngokuxhomekeke kuxinzelelo lwazo, zinokwenza i-PCR isebenze okanye ithintele.