Kupindira kwezvinhu muPCR maitiro

Munguva yePCR reaction, zvimwe zvinokanganisa zvinowanzosangana.
Nekuda kwekunyanya kunzwisiswa kwePCR, kusvibiswa kunoonekwa sechimwe chezvinhu zvakakosha zvinobata PCR mhedzisiro uye inogona kuburitsa mhedzisiro yenhema.
Zvakaenzana kutsoropodza ndiwo akasiyana masosi anotungamira kune nhema-negative mhedzisiro. Kana imwe kana zvimwe zvikamu zvakakosha zvePCR musanganiswa kana iyo amplification reaction pachayo yakavharwa kana kukanganiswa, iyo diagnostic assay inogona kudziviswa. Izvi zvinogona kutungamirira mukuderedza kushanda zvakanaka uye kunyange migumisiro yakaipa yenhema.
Mukuwedzera kune inhibition, kurasikirwa kwechinangwa nucleic acid kutendeseka kunogona kuitika nekuda kwekutumira uye / kana kuchengetedza mamiriro asati agadzirira sampuli. Kunyanya, tembiricha yakakwira kana kusachengetedzwa kwakakwana kunogona kukonzera kukuvara kwemasero uye nucleic acids. Kugadziriswa kwesero nematishu uye kuiswa kweparafini zvinonyatsozivikanwa zvikonzero zvekutsemuka kweDNA uye dambudziko rinoramba riripo (ona Mifananidzo 1 uye 2). Muzviitiko izvi, kunyange kunyatsozviparadzanisa uye kucheneswa hazvizobatsiri.

Mufananidzo 1 | Mhedzisiro yekusafambisa pane DNA kutendeseka
Agarose gel electrophoresis yakaratidza kuti mhando yeDNA yakaparadzaniswa neparafini zvikamu zve autopsies zvakasiyana zvakanyanya. DNA yehurefu hwakasiyana hwepakati chidimbu yaivepo mune zvakaburitswa zvichienderana nekugadzirisa nzira. DNA yaichengetedzwa chete kana yakagadziriswa mumasampuli echando akaomeswa nechando uye mubuffered neutral formalin. Kushandiswa kweiyo yakasimba acidic Bouin inogadzirisa kana isina buffered, formic acid-ine formalin yakakonzera kurasikirwa kukuru kweDNA. Chikamu chasara chakakamurwa zvakanyanya.
Kuruboshwe, kureba kwezvidimbu kunoratidzwa mu kilobase pairs (kbp)

Mufananidzo 2 | Kurasikirwa kwekutendeseka kwezvinangwa zve nucleic acid
(a) A 3'-5′ gap pamatambo ese ari maviri anozoguma nekuputsika muDNA inonangwa. synthesis yeDNA ichiri kuitika pane chidimbu chidiki. Nekudaro, kana iyo primer annealing saiti isipo pane DNA chidimbu, chete mutsara amplification inoitika. Muchiitiko chakanyanya kunaka, zvimedu zvinogona kugadzirisana, asi zvibereko zvichave zviduku uye pasi pemazinga ekuonekwa.
(b) Kurasikirwa kwemabhesi, kunyanya nekuda kwekusvibiswa uye kuumbwa kwethymidine dimer, kunotungamirira kuderera kwenhamba yeH-zvisungo uye kuderera kweTm. Munguva yekudziya kwechikamu chekudziya, maprimers anonyunguduka kubva kune matrix DNA uye haazovharidzi kunyange pasi pemamiriro asina kuomesesa.
(c) Zvigadziko zvethymine zviri pedyo zvinoumba TT dimer.
Rimwe dambudziko rakajairika rinowanzoitika mukuongororwa kwema molecular ndeye kushomeka-kupfuura-kwakakwana kuburitswa kwechinangwa nucleic acids zvichienzaniswa nephenol-chloroform kubviswa. Muzviitiko zvakanyanyisa, izvi zvinogona kusanganiswa nezvakaipa zvenhema. Nguva yakawanda inogona kuponeswa nekubika lysis kana enzymatic digestion yemasero emarara, asi nzira iyi inowanzokonzera kuderera kwePCR senitivity nekuda kwekusakwana nucleic acid kusunungurwa.

Inhibition ye polymerase chiitiko panguva yekusimudza

Kazhinji, inhibition inoshandiswa sechinhu chemudziyo kutsanangura zvinhu zvese zvinotungamira kune suboptimal PCR mhedzisiro. Muchidimbu chebiochemical pfungwa, inhibition inogumira kune basa re enzyme, kureva, inoderedza kana kudzivirira substrate-chigadzirwa kutendeuka kuburikidza nekubatana nenzvimbo inoshanda yeDNA polymerase kana cofactor yayo (semuenzaniso, Mg2 + yeTaq DNA polymerase).
Zvikamu zviri mumuenzaniso kana zvakasiyana-siyana mabhafa uye zvakatorwa zvine reagents zvinogona kuvharidzira zvakananga enzyme kana kuteya macofactors ayo (semuenzaniso EDTA), zvichizoregedza iyo polymerase uye zvichizotungamira kune yakadzikira kana yenhema isina kunaka PCR mhedzisiro.
Nekudaro, kupindirana kwakawanda pakati pezvinoita zvinhu uye chinangwa-ine nucleic acids zvakare chinodanwa se 'PCR inhibitors'. Kamwe kuvimbika kwesero kuchikanganiswa nekuzviparadzanisa uye nucleic acid inosunungurwa, kuwirirana pakati pemuenzaniso uye mhinduro yaro yakakomberedza uye chikamu chakasimba chinogona kuitika. Semuenzaniso, 'scavengers' vanogona kusunga imwe- kana kaviri-yakasungwa DNA kuburikidza nekusabatana kwekubatana uye kukanganisa kuparadzaniswa nekucheneswa nekudzikisa huwandu hwezvinangwa zvinozosvika paPCR reaction mudziyo.
Kazhinji, PCR inhibitors inowanikwa mumvura zhinji yemuviri uye reagents anoshandiswa pakiriniki yekuongorora bvunzo (urea muweti, hemoglobin uye heparin muropa), zvekudya zvinowedzera (organic components, glycogen, mafuta, Ca2+ ions) uye zvikamu zviri munharaunda (phenols. , heavy metal)

PCR inhibitors yakawanda inowanikwa mumabhakitiriya uye masero eukaryotic, DNA-inosunga macromolecules ematishu matrices uye midziyo yerabhoritari yakaita segurovhosi nemapurasitiki. Kucheneswa kwe nucleic acids panguva kana mushure mekubviswa ndiyo nzira inosarudzwa yekubvisa PCR inhibitors.
Nhasi, akasiyana-siyana otomatiki ekudhonza midziyo anogona kutsiva akawanda mapuroteni, asi 100% kudzoreredza uye / kana kucheneswa kwezvinangwa hakuna kumboitika. Potential inhibitors inogona kunge ichiripo mune yakacheneswa nucleic acids kana inogona kunge yatotanga kushanda. Pane nzira dzakasiyana dzekudzikisa kukanganiswa kwemainhibitors. Sarudzo yeakakodzera polymerase inogona kuve nemhedzisiro yakakosha pane inhibitor chiitiko. Dzimwe nzira dzakapupurirwa dzekudzikisa PCR inhibition dziri kuwedzera polymerase concentration kana kushandisa zvinowedzerwa zvakaita seBSA.
Inhibition yePCR reactions inogona kuratidzwa nekushandiswa kwemukati maitiro emhando yekudzora (IPC).
Kuchengetedza kunofanira kutorwa kubvisa zvose reagents uye zvimwe zvinogadziriswa mukiti yekuchera, zvakadai se ethanol, EDTA, CETAB, LiCl, GuSCN, SDS, isopropanol uye phenol, kubva kune nucleic acid inoparadzaniswa nekunyatsogeza danho. Zvichienderana nekufunga kwavo, vanogona kumisa kana kumisa PCR.