Zvinhu zvinokanganisa maitiro ePCR

Munguva yePCR reaction, zvimwe zvinhu zvinopindira zvinowanzo sangana nazvo.
Nekuda kwekunyanya kubatwa nePCR, kusvibiswa kunoonekwa sechimwe chezvinhu zvinonyanya kukanganisa mhedzisiro yePCR uye kunogona kuburitsa mhedzisiro yenhema.
Zvimwe zvinhu zvakakoshawo ndizvo zvinokonzera mhedzisiro yenhema-negative. Kana chimwe kana zvimwe zvikamu zvakakosha zvemusanganiswa wePCR kana amplification reaction pachayo zvikadziviswa kana kukanganiswa, kuongororwa kwechirwere kunogona kudziviswa. Izvi zvinogona kukonzera kudzikira kwekushanda zvakanaka uye kunyange mhedzisiro yenhema-negative.
Pamusoro pekudziviswa, kurasikirwa kwehunhu hwe nucleic acid inogona kuitika nekuda kwemamiriro ekutumira uye/kana kuchengetwa kwesampuro isati yagadzirwa. Kunyanya, tembiricha yakakwira kana kusakwana kwekuchengetwa kunogona kukonzera kukuvara kwemasero uye nucleic acids. Kugadzika kwemasero nematishu uye kuiswa kweparafini ndizvo zvinozivikanwa zvinokonzera kupatsanuka kweDNA uye dambudziko rinoramba riripo (ona Mifananidzo 1 ne2). Muzviitiko izvi, kunyangwe kupatsanurwa kwakakwana uye kucheneswa hazvizobatsiri.

Mufananidzo 1 | Mhedzisiro yekusagona kufamba-famba pakuvimbika kweDNA
Agarose gel electrophoresis yakaratidza kuti mhando yeDNA yakabviswa kubva muzvikamu zveparafini zvematumbu yakasiyana zvikuru. DNA yeavhareji yakasiyana yehurefu hwezvidimbu yaivepo muzvidimbu zvichienderana nenzira yekugadzika. DNA yaingochengetedzwa chete kana yagadziriswa muzviyero zvechando zvemuno uye mu buffered neutral formalin. Kushandiswa kweBouin fixative ine acid yakawanda kana isina buffer, formalin ine formalin kwakakonzera kurasikirwa kukuru kweDNA. Chikamu chasara chakapatsanuka zvakanyanya.
Kuruboshwe, kureba kwezvidimbu kunoratidzwa mumapaya e kilobase (kbp)

Mufananidzo 2 | Kurasikirwa kwekuvimbika kwezvinangwa zve nucleic acid
(a) Mukaha we3′-5′ patambo dzese mbiri uchaita kuti DNA iri kutsvairwa ityoke. Kugadzirwa kweDNA kucharamba kuchiitika pachidimbu chidiki. Zvisinei, kana nzvimbo yeprimer annealing isipo pachidimbu cheDNA, kuwedzera kwemutsara chete ndiko kunoitika. Kana zviri nani, zvidimbu zvinogona kuzadzana, asi goho richave diki uye riri pasi pemazinga ekuona.
(b) Kurasikirwa kwezvigadziko, kunyanya nekuda kwekubvisa purination uye kuumbwa kwe thymidine dimer, kunotungamira kudzikira kwehuwandu hweH-bonds uye kudzikira kweTm. Munguva yekudziya kwenguva refu, ma primer anonyunguduka kubva ku matrix DNA uye haazonyungudike kunyangwe mumamiriro ezvinhu akaomarara.
(c) Mabhesi ethymine ari pedyo anoumba TT dimer.
Chimwe chinetso chinowanzoitika mukuongororwa kwemamorekuru ndechekusunungurwa kushoma kwe target nucleic acids zvichienzaniswa nekubviswa kwe phenol-chloroform. Muzviitiko zvakanyanya, izvi zvinogona kusanganiswa ne false negatives. Nguva yakawanda inogona kuchengetedzwa nekubikira lysis kana enzyme digestion yemarara emasero, asi nzira iyi inowanzo konzera kusanzwa kwePCR kwakaderera nekuda kwekusakwana kwe nucleic acid release.

Kudziviswa kwebasa repolymerase panguva yekuwedzera

Kazhinji, kudziviswa kunoshandiswa sepfungwa yemudziyo kutsanangura zvinhu zvese zvinotungamira kumhedzisiro yePCR isinganyatso shanda zvakanaka. Mupfungwa yebiochemical chete, kudziviswa kunogumira pakushanda kweenzyme, kureva kuti, kunoderedza kana kudzivirira kushandurwa kwesubstrate-product kuburikidza nekubatana nenzvimbo inoshanda yeDNA polymerase kana cofactor yayo (semuenzaniso, Mg2+ yeTaq DNA polymerase).
Zvikamu zviri mumuenzaniso kana mabuffer akasiyana-siyana uye zvakatorwa zvine ma reagents zvinogona kudzivirira zvakananga enzyme kana kubata cofactors yayo (semuenzaniso EDTA), nokudaro zvichimisa polymerase uye zvichizotungamira kumhedzisiro yePCR isina kunaka kana yenhema.
Zvisinei, kudyidzana kwakawanda pakati pezvikamu zve reaction uye nucleic acids dzine target kunodanwawo se 'PCR inhibitors'. Kana kusimba kwesero kwakanganiswa nekupatsanurwa uye nucleic acid yaburitswa, kudyidzana pakati pemuenzaniso nemhinduro yayo yakapoteredza uye solid phase kunogona kuitika. Semuenzaniso, 'scavengers' vanogona kusunga DNA imwe chete kana mbiri-tambo kuburikidza nekudyidzana kusiri kwe covalent uye kukanganisa kupatsanurwa nekucheneswa nekuderedza huwandu hwezvinangwa zvinozosvika mumudziyo wePCR reaction.
Kazhinji, zvinodzivirira PCR zvinowanikwa mumvura zhinji dzemuviri uye zvinoshandiswa pakuongorora chirwere (urea muweti, hemoglobin neheparin muropa), zvekudya zvinovaka muviri (organic components, glycogen, mafuta, Ca2+ ions) uye zvinhu zviri munharaunda (phenols, heavy metals)

Mishonga inodzivirira maPCR inowanikwa zvakanyanya inogona kuwanikwa mumabhakitiriya nemasero eukaryotic, DNA isiri iyo inotarirwa, macromolecules anosunga DNA ematissue matrices uye michina yemurabhoritari yakaita semagirovhosi nemapurasitiki. Kucheneswa kwe nucleic acids panguva yekubviswa kana mushure mekubviswa ndiyo nzira inonyanya kufarirwa yekubvisa mishonga inodzivirira maPCR.
Nhasi uno, michina yakasiyana-siyana inoburitsa zvinhu otomatiki inogona kutsiva maprotocol akawanda emaoko, asi kudzoreredza kana kucheneswa kwezvinangwa 100% hakusati kwamboitika. Zvinogona kudzivirira zvinogona kunge zvichiripo mu nucleic acids dzakacheneswa kana kuti zvingave zvatotanga kushanda. Pane nzira dzakasiyana dzekuderedza simba rezvinodzivisa. Kusarudzwa kwepolymerase yakakodzera kunogona kuva nemhedzisiro yakakura pabasa rezvinodzivisa. Dzimwe nzira dzakasimbiswa dzekuderedza PCR inhibition dzinosanganisira kuwedzera huwandu hwepolymerase kana kushandisa zvimwe zvinhu zvakaita seBSA.
Kudziviswa kwePCR reactions kunogona kuratidzwa nekushandisa internal process quality control (IPC).
Zvinofanira kungwarira kubvisa ma reagents ese nedzimwe mhinduro dziri mu extraction kit, dzakadai se ethanol, EDTA, CETAB, LiCl, GuSCN, SDS, isopropanol uye phenol, kubva mu nucleic acid isolate nekugeza zvakakwana. Zvichienderana nehuwandu hwadzo, dzinogona kumutsa kana kudzivirira PCR.