Izinto zokuphazamiseka kwimicimbi yePRR

Ngexesha lokuphendula kwe-PCR, ezinye izinto eziphazamisayo zihlala zidibana.
Ngenxa yovakalelo olude kakhulu lwe-PCR, ungcoliseko luthathwa njengenye yezona zinto zibalulekileyo zichaphazela iziphumo ze-PCR kwaye zinokuvelisa iziphumo ezilungileyo.
Ngokulinganayo kubaluleke kakhulu yimithombo eyahlukeneyo ekhokelela kwiziphumo ezibi. Ukuba enye okanye ngakumbi inqaku elibalulekileyo lomxube we-PCR okanye indlela yokuphendula ithintelwe okanye iphazamisekile, i-associcy ye-divistic ingathintelwa. Oku kunokukhokelela ekunciphiseni ukusebenza kakuhle kunye neziphumo ezibi.
Ukongeza kwi-infinimition, ukuphulukana nokungaqwalaselwa kwethagethi ye-acid ye-acid ye-acid ye-acid inokwenzeka ngenxa yokuthumela kunye / okanye iimeko zokugcina ngaphambi kolungiselelo lwesampulu. Ngokukodwa, amaqondo obushushu aphezulu okanye ukugcinwa okungaphelelanga kunokukhokelela ekutyeni kweeseli kunye neeseli ze-nucleic acid. Iseli kunye ne-tishu yezicubu kunye neparafiningding zinobangela we-DNA yokuhluza kunye nengxaki eqhubekayo (jonga amanani 1 kunye 2). Kwezi meko, nokuba nokubekwa kwedwa kwaye ukucocwa akuyi kunceda.

Umzobo 1 | Iziphumo zokungasebenziyo kwi-DNA ingqibelelo
I-Agarose Gel Electrophoressis ibonise ukuba umgangatho we-DNA uhlolwe ngokwamacandelo eparafini kwii-autopsies ezahlukeneyo. I-DNA yovavanyo ophakathi lwenqanaba lokuqhekeka kobude belikho kwizicatshulwa ezixhomekeke kwindlela yokulungisa. I-DNA igcinwe kuphela xa igqitywe kwiisampulu zemveli eziqengqelekayo nakwindlela engathathi cala kwezopolitiki. Ukusetyenziswa kwe-acidic bouin ye-acidic bouin okanye engadibaniyo, i-formic acid-enemifanekiso iphumela kwi-DNA ebalulekileyo yeDNA. Iqhezu elishiyekileyo liqhekekile.
Ngasekhohlo, ubude beziqwengana ezichazwe kwi-kilobase abalindi (i-KBP)

Umzobo 2 | Ukuphulukana kwentembeko yeethagethi ze-nucleic
. I-Synthesis yeDNA iza kwenzeka kwisiqwengana esincinci. Nangona kunjalo, ukuba indawo ye-primer ilahlekile kwisiqwengana seDNA, kuphela kukwenza kuphela i-pharm. Kwimeko entle, amaqhekeza anokukhulisa omnye komnye, kodwa izivuno ziya kuba mncinci kwaye zingaphantsi kokufunyanwa.
. Ngexesha lesigaba sokufudumeza, i-primers iya kunyibilika kude kwi-darix ye-darix kwaye ayiyi kuba ne-anneal kwangaphantsi kweemeko ezincinci.
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Enye ingxaki eqhelekileyo ehlala yenzeka kwi-molecular diaclostics kukukhutshwa okungaphantsi kwe-acids ye-nucleic ekujolise kuyo xa kuthelekiswa nokukhutshwa kwe-phenol-chlorofor. Kwiimeko ezigqithisileyo, oku kunokunxulunyaniswa neempawu zobuxoki. Ixesha elininzi linokugcinwa ngokubila i-lysis okanye ukugaya okukhohlakeleyo kwenkunkuma, kodwa le ndlela isebenza ikhokelela kwiimvakalelo eziphantsi ze-prc ngenxa yokukhutshwa kwe-nucleid engaphelelanga.

Ukuthintela umsebenzi we-polymerase ngexesha lokwahlukana

Ngokubanzi, ukuthintela kusetyenziswa njengengcinga yesikhongozeli ukuchaza zonke izinto ezikhokelela kwi-ProPlaptis PCR iziphumo. Kwingqondo ye-biochemical ngokungqongqo, isithintelo silinganiselwe kwimisebenzi ye-enzyme, oko kukuthi, inciphisa okanye ithintela ukuguqulwa kwemveliso ye-DNA ye-DNA Polymerase okanye i-SFOCT (MG2 ye-TAQ Polymerase).
Izinto ezikwisampulu okanye i-buffers eyahlukeneyo kunye nezicatshulwa eziqukethe i-Remegents zithintele ngokuthe ngqo i-enzyme okanye i-edtators yayo (umz. I-EDTA), ngaloo ndlela ikhupha i-polymerase kwaye ikhokelela ekunciphiseni iziphumo ze-PCR.
Nangona kunjalo, uninzi lonxibelelwano phakathi kwamacandelo okuphendula kunye nethagethi-ene-acid yenyukliya ichongelwe njenge 'i-prc inhibitors'. Nje ukuba ingqibelelo yeseli iphazanyiswa ngokuthi yedwa kunye ne-acic acid ikhutshiwe, ukunxibelelana phakathi kwesampulu kunye nesisombululo esijikelezileyo kunye nesigaba esiqinileyo sinokwenzeka. Umzekelo, 'ukutshayela' kunokukubopha i-DNA okanye i-DNA-phinda iduthwe i-DNA malunga nokunxibelelana okungafaniyo kwaye iphazamiseni ngokuhlanjwa kunye nokucocwa ngokunciphisa inani leethagethi eziza kufika ekugqibeleni kwinqanawa ye-PRR.
Ngokubanzi, i-PRC i-Intenitors ikhona kwiimvavanyo zomzimba ezisetyenziselwa uvavanyo lweklinikhi (i-Urea e-Urine, i-hemoglobin kunye ne-heparin egazini), i-Dhemory, i-Dhenols, i-phenols, i-phenos, izinyithi ezinzima)

I-PRCR exhaphakileyo i-PRCR inokuthi ifumaneke kwiiseli zeBacteria kunye ne-Eukhartatic yeDNA, i-DNA-Bing-Bind-Bind-Izixhobo zeLabhoratri ezifana neeglosti kunye neeplastiki. Ukuhlanjululwa kwee-acid zenyukliya ngexesha okanye emva kokukhupha le ndlela ikhethwayo yokususa ii-prc inhibitors.
Namhlanje, izixhobo ezahlukeneyo zokukhupha ezenzekelayo zinokutshintsha iiprotocol ezininzi, kodwa i-100% yokubuyisela kunye / okanye ukucocwa kweethagethi akuzange kufezekiswe. Izithinteli ezinokubakho zisenokubakho kwii-asidi ezihlanjululweyo ze-nucleic okanye zisenokuba zenze ukusebenza. Izicwangciso ezahlukeneyo zikhona ukunciphisa ifuthe lezinto zokugcina. Ukukhetha kwe-polymerase efanelekileyo kunokuba nefuthe elibonakalayo kwimisebenzi engafakwanga. Ezinye iindlela eziqinisekisiweyo zokunciphisa ukuthintelwa kwe-prc kukhulisa uxinzelelo lwePolymerase okanye ukusebenzisa izongezo ezinjenge-BSA.
Ukuthintelwa kwendlela yePRR kunokuboniswa ngokusetyenziswa kwenkqubo yenkqubo yangaphakathi yolawulo lwenkqubo (iPC).
Kufuneka kuthathelwe ingqalelo ukususa zonke ii-regegers kunye nezinye izisombululo kwikhithi yokukhupha, e-Edta, i-gitab, i-giscropanol kunye ne-funol acid ye-nucleid inyathelo. Kuxhomekeka kuxinzelelo lwabo, banokuvula okanye basebenze okanye bathintele i-pcc.