Igishushanyo mbonera (99% ibibazo birashobora gukemurwa)
1. Uburebure bwa primer: Igitabo gisaba 15-30bp, mubisanzwe nka 20bp.Imiterere nyayo nibyiza kuba 18-24bp kugirango tumenye neza, ariko igihe kirekire, primer ndende cyane nayo izagabanya umwihariko, kandi igabanye umusaruro.
2. Primer amplification span: 200-500bp irakwiriye, kandi igice gishobora kwagurwa kugera kuri 10kb mubihe byihariye.
3. Ibanze shingiro: Ibiri muri G + C bigomba kuba 40-60%, ingaruka nke za G + C zo kongera imbaraga ntabwo ari nziza, cyane G + C biroroshye kugaragara bande idasanzwe.ATGC ikwirakwizwa neza uko bishakiye, irinda ihuriro rya nucleotide zirenga 5 za purine cyangwa pyrimidine.Multi-gc kuri 5 ′ iherezo hamwe nintera yo hagati kugirango yongere ituze, irinde GC ikize kuri 3 ′ iherezo, nta GC kubirindiro 3 byanyuma, cyangwa nta GC kuri 3 kuri 5 yanyuma.
4. Irinde imiterere ya kabiri muri primers, kandi wirinde kuzuzanya hagati ya primers ebyiri, cyane cyane kuzuzanya kuri 3 'iherezo, bitabaye ibyo primer dimer izashirwaho kandi hashyizweho imirongo idasanzwe idasanzwe.
5. Ibishingwe kuri 3 'iherezo rya primers, cyane cyane ibyanyuma kandi byanyuma, bigomba guhuzwa cyane kugirango birinde PCR kunanirwa kubera ibishingwe bitarangiye.
6. Primers ifite cyangwa irashobora kongerwaho hamwe na site ya clavage ikwiye, kandi urutonde rwongerewe intego rugomba kuba rufite urubuga rukwiye, rufite akamaro kanini kubisesengura rya clavage cyangwa clone ya molekile.
7. Umwihariko wa primers: primers ntigomba kugira homologiya igaragara hamwe nizindi gahunda zikurikirana muri nucleic aside ikurikirana.
8. Wige gukoresha software: PP5, Oligo6, DNAstar, Vector NTI, primer3 (Iki gishushanyo cyo kumurongo gikora neza).
Ibiri hejuru birashobora gukemura byibuze 99% byibibazo byubushakashatsi.
Igenzura ibisobanuro birambuye bya primer
1. Uburebure bwa primer
Uburebure bwa primer rusange ni 18 ~ 30 shingiro.Muri rusange, ikintu cyingenzi kigena ubushyuhe bwa annealing ya primer ni uburebure bwa primer.Ubushyuhe bwa annealing bwa primer bwatoranijwe muri rusange (Tm agaciro -5 ℃), kandi bamwe bakoresha agaciro ka Tm.Inzira zikurikira zirashobora gukoreshwa mukubara hafi ubushyuhe bwa annealing bwa primers.
Iyo uburebure bwa primer buri munsi ya 20bp: [4 (G + C) +2 (A + T)] - 5 ℃
Iyo uburebure bwa primer burenze 20bp: 62.3 ℃ + 0.41 ℃ (% GC) -500 / uburebure-5 ℃
Mubyongeyeho, software nyinshi zirashobora kandi gukoreshwa mukubara ubushyuhe bwa annealing, ihame ryo kubara rizaba ritandukanye, kuburyo rimwe na rimwe agaciro kabaruwe gashobora kugira icyuho gito.Kugirango uhindure imikorere ya PCR, primers ngufi yemeza ko ubushyuhe bwa annealing butari munsi ya 54 ℃ bukoreshwa muburyo bwiza kandi bwihariye.
Muri rusange, primer yihariye yiyongera kubintu bine kuri buri nucleotide yinyongera, kuburyo uburebure bwa primer byibura kuri progaramu nyinshi ni 18 nucleotide.Imipaka yo hejuru yuburebure bwa primer ntabwo ari ingenzi cyane, cyane cyane ijyanye no gukora neza.Kubera entropiya, igihe kirekire primer, niko igipimo kigereranya guhuza na ADN igamije gukora icyitegererezo gihamye cyikubye kabiri kugirango ADN polymerase ihuze.
Iyo ukoresheje software mugushushanya primers, uburebure bwa primers burashobora kugenwa nagaciro ka TM mugihe kimwe, cyane cyane kuri primers ya fluorescence yuzuye PCR, TM = 60 ℃ cyangwa rero igomba kugenzurwa.
Ibirimo
Mubisanzwe, ibikubiye muri G + C muburyo bwa primer ni 40% ~ 60%, naho GC nibirimo na Tm agaciro kamwe ka primers bigomba guhuzwa.Niba primer ifite icyerekezo gikomeye cya GC cyangwa AT, umubare ukwiye wa A, T cyangwa G na C umurizo urashobora kongerwaho kuri 5 'iherezo rya primer.
3. Ubushyuhe bwa Annealing
Ubushyuhe bwa annealing bugomba kuba munsi ya 5 than munsi yubushyuhe budasanzwe.Niba umubare wibanze wa primer ari muto, ubushyuhe bwa annealing burashobora kwiyongera muburyo bukwiye, bushobora kongera umwihariko wa PCR.Niba umubare wibanze ari munini, ubushyuhe bwa annealing burashobora kugabanuka uko bikwiye.Itandukaniro ry'ubushyuhe bwa annealing hagati ya primers ya 4 ℃ ~ 6 ℃ ntabwo bizagira ingaruka kumusaruro wa PCR, ariko nibyiza ko ubushyuhe bwa annealing bwa primers ni bumwe, bushobora gutandukana hagati ya 55 ℃ ~ 75 ℃.
4. Irinde agace ka kabiri k'imiterere yicyitegererezo
Nibyiza kwirinda urwego rwa kabiri rwakarere rwicyitegererezo mugihe uhitamo igice cyongerewe.Imiterere ihamye ya kabiri yibice byateganijwe irashobora guhanurwa no kugereranywa na software ikora mudasobwa, ifasha muguhitamo inyandikorugero.Ibisubizo byubushakashatsi byerekana ko kwaguka akenshi bitananirwa mugihe ingufu zubusa (△ G) zakarere kagomba kwagurwa zitageze kuri 58.6lkJ / mol.
5. Kudahuza na ADN igamije
Iyo intego ya ADN ikurikiranye ari nini, primer irashobora guhuza ibice byinshi bigize intego ya ADN, bikavamo imirongo myinshi igaragara mubisubizo.Iki gihe birakenewe gukoresha ikizamini cya software ya BLAST, urubuga:http://www.ncbi.nlm.nih.gov/BLAST/.Hitamo Guhuza ibyiciro bibiri (bl2seq).
Kwandika primer ikurikirana kuri zone 1 hamwe na ADN ikurikirana kuri zone 2 irasimburana, kandi BLAST ibara ibyuzuzanya, antisense, nibindi bishoboka, abakoresha rero ntibakeneye kumenya niba iminyururu yombi ari iminyururu yumvikana.Urashobora kandi kwinjiza numero ya GI niba uzi umubare wa GI ukurikirana mububiko, ntugomba rero gushiraho igice kinini cyurukurikirane.Hanyuma, kanda Align kuri 3 kugirango urebe niba primer ifite imbuga nyinshi za homologique muri ADN igenewe.
6. Terminal terminal
3 'iherezo rya primer niho kwaguka gutangirira, ni ngombwa rero gukumira ibidahuye bitangirira aho.Impera ya 3 'ntigomba kurenza G cyangwa C ikurikiranye, kuko ibi bizatera primer kwibeshya mukarere ka G + C gakungahaye.3 'iherezo ntirishobora gukora urwego rwakabiri, usibye muri PCR idasanzwe (AS-PCR), 3 ′ iherezo rya primer ntishobora guhuzwa.Kurugero, niba kodegisi yakuweho, 3 'iherezo rya primer ntigomba guhagarikwa kumwanya wa gatatu wa codon, kubera ko umwanya wa gatatu wa codon ukunda kwangirika, bizagira ingaruka kumikorere no gukora neza.Mugihe ukoresheje annexation primers, reba imbonerahamwe yo gukoresha codon, witondere ibyifuzo byibinyabuzima, ntukoreshe primers ya annexation kuri 3 ′, kandi ukoreshe kwibanda cyane kuri primers (1uM-3uM).
7. Imiterere ya kabiri ya primers
Intangiriro ubwazo ntizigomba kugira urutonde rwuzuzanya, bitabaye ibyo primers ubwazo zizikubitira mumisatsi yimisatsi, kandi iyi miterere yinyongera izagira ingaruka kubihuza primers na templates kubera inzitizi zikomeye.Niba urubanza rwibihimbano rwakoreshejwe, gukomeza kwuzuzanya gushingiye kuri primers ubwabo ntibigomba kurenza 3bp.Ntabwo hagomba kubaho kuzuzanya hagati ya primers zombi, cyane cyane kuzuzanya kuzuzanya kwa 3 'iherezo bigomba kwirindwa kugirango hirindwe ko habaho primer dimers.Muri rusange, ntihakagombye kubaho ibirenga 4 bikurikiranye homology cyangwa kuzuzanya hagati ya primers.
8. Ongeraho ibimenyetso cyangwa loci
Iherezo rya 5 'ntirigira ingaruka nke muburyo bwo kongera imbaraga kandi birashobora guhinduka rero bitagize ingaruka kumpamvu zihariye.Guhindura primer 5 'iherezo ririmo: kongeramo urubuga rwo kubuza enzyme;Ikimenyetso cya biotine, fluorescence, digoxin, Eu3 +, nibindi.Kumenyekanisha imbuga za mutation, kwinjiza no kubura urutonde rwa mutation no kumenyekanisha gahunda ya porotokoro, nibindi. Ibishingiro byinyongera bizagira ingaruka nyinshi cyangwa nkeya bigira ingaruka kumikorere ya amplification kandi byongere amahirwe yo gushiraho primer dimer, ariko hagomba gukorwa bimwe kugirango intambwe ikurikira.Urutonde rwinyongera rutabaho kurugero rwateganijwe, nkurubuga rwo kubuza hamwe na poroteri ikurikirana, urashobora kongerwaho kuri 5 ′ iherezo rya primer bitagize ingaruka kubintu byihariye.Uru rutonde ntirurimo kubara indangagaciro za primer Tm, ariko zigomba kugeragezwa kubwuzuzanya nuburyo bwimbere.
9. Subclone
Igihe kinini, PCR ni cloni ibanza gusa, hanyuma dukeneye guhinduranya igice cyintego mubice bitandukanye, bityo rero dukeneye gushushanya izindi shingiro kubikorwa bizakurikiraho murwego rwa PCR.
Inzira zimwe zagenewe subcloning zavunaguye hano hepfo.
Kubuza endonuc Please urubuga rwo kubuza rwongeyeho
Ongeraho imbuga zibuza enzyme nuburyo bukoreshwa cyane mugutandukanya ibicuruzwa bya PCR.Mubisanzwe, ikibanza cya clavage ni base esheshatu, hiyongereyeho 5 'iherezo ryikibanza cya clavage gikeneye kongeramo 2 ~ 3 kurinda.Nyamara, umubare wibanze urinda usabwa na enzymes zitandukanye uratandukanye.Kurugero, SalⅠ ntabwo isaba kurinda, EcoRⅤ isaba ibirindiro 1 byo kurinda, NotⅠ isaba ibirindiro 2 birinda, naho Hind Ⅲ ikenera ibirindiro 3 byo kurinda.
LIC yongeraho umurizo
Izina ryuzuye rya LIC ni Ligation-Yigenga ya cloni, uburyo bwa cloni bwahimbwe na Navogen byumwihariko kubice byayo bya pET vector.PET itwara ibintu byateguwe nuburyo bwa LIC ifite ibyuzuzanya 12-15 shingiro imwe yumutwe ufatanye, wuzuza impera zifatika zifatika kumutwe winjizamo igice.Kugirango intego zongerwe imbaraga, primer 5 ′ ikurikiranye ryibice byinjijwe bigomba kuzuza LIC vector.Igikorwa cya 3 ′ → 5 ′ cyo gukuramo T4 ADN polymerase irashobora gukora umurongo umwe wiziritse ku gice cyinjijwe nyuma yigihe gito.Kuberako ibicuruzwa bishobora gukorwa gusa muburyo bwo guhuza ibice byateguwe byinjizwamo hamwe na vector, ubu buryo burihuta cyane kandi neza, kandi buyobora cloni.
Kuyobora TA clone ongeraho umurizo
TA cloni ntiyashoboye kwerekeza igice mubice, nuko nyuma Invitrogen yazanye icyerekezo gishobora gukoronizwa, cyarimo ibice bine byingenzi GTGGS kumpera imwe.Kubwibyo, mugushushanya kwa PCR yibanze, ibyuzuzanya bigomba kongerwaho uko bikwiye, kugirango ibice bishobore "kwerekanwa".
Niba uri mugufi mugihe, urashobora kugerageza synthesis itaziguye, ugahuza gene na vector, aribyo twita ET gene synthesis muri museculariste.
D. Uburyo bwo gukoroniza muri-Fusion
Nta ligase isabwa, nta reaction ndende isabwa.Igihe cyose urukurikirane rwimpande zombi zumurongo rwerekanwe rwerekanwe Mugushushanya kwa primers, hanyuma ibicuruzwa bya PCR hamwe na vector umurongo byongewemo mumuti wa in-fusion enzyme irimo BSA ugashyirwa mubushyuhe bwicyumba mugihe cyigice cyisaha, impinduka irashobora gukorwa.Ubu buryo burakwiriye cyane cyane guhindura amajwi manini.
10. Huza primer
Rimwe na rimwe, gusa amakuru akurikirana arazwi kubijyanye na primer design.Kurugero, niba gusa aminide acide ikurikirana izwi, guhuza primer birashobora gushushanywa.Gukomatanya primer nuruvange rwurutonde rutandukanye rwerekana ibintu byose bitandukanye byashingiweho bigizwe na aside amine imwe.Kugirango wongere umwihariko, urashobora kwifashisha imbonerahamwe ya codon kugirango ugabanye umugereka ukurikije imikoreshereze yibanze y'ibinyabuzima bitandukanye.Hypoxanthine irashobora guhuzwa nifatizo zose kugirango igabanye ubushyuhe bwa annealing ya primer.Ntukoreshe ibishingwe byometse kuri 3 ′ iherezo rya primer kuko guhuza ibice 3 byanyuma kuri 3 ′ birahagije kugirango utangire PCR kurubuga rutari rwo.Kwibanda cyane kwa primer (1μM kugeza 3μM) birakoreshwa kuko primers mumvange myinshi yomugereka ntabwo yihariye icyitegererezo.
Ibikoresho bya PCRkugenzura
1. Umubare wambere
Ubwinshi bwa buri primer ni 0.1 ~ 1umol cyangwa 10 ~ 100pmol.Nibyiza gutanga ibisubizo bisabwa hamwe numubare muto wa primer.Kwibanda cyane kwa primer bizatera kudahuza no kudasanzwe kwa amplification, kandi byongere amahirwe yo gukora ibipimo hagati ya primers.
2. Kwibanda cyane
Kwibanda kwa primers bigira ingaruka kumwihariko.Ibyiza bya primer yibanze muri rusange hagati ya 0.1 na 0.5μM.Kwibanda cyane kwa primer biganisha ku kongera ibicuruzwa bidafite akamaro.
3. Annealing ubushyuhe bwa primer
Ikindi kintu cyingenzi kuri primers ni ubushyuhe bwo gushonga (Tm).Nubushyuhe iyo 50% ya primers hamwe nuruhererekane rwuzuzanya bigaragazwa nka molekile ya ADN ikubye kabiri.Tm irasabwa gushiraho ubushyuhe bwa PCR.Byiza, ubushyuhe bwa annealing buri hasi bihagije kugirango habeho guhuza neza primers hamwe nintego ikurikiranye, ariko hejuru bihagije kugirango ugabanye guhuza bidafite akamaro.Ubushyuhe bufatika buva kuri 55 ℃ kugeza 70 ℃.Ubushyuhe bwa Annealing busanzwe bushyirwaho 5 ℃ munsi ya Tm ya primer.
Hariho formulaire nyinshi zo gushiraho Tm, ziratandukanye cyane bitewe na formula yakoreshejwe hamwe nurutonde rwa primers.Kuberako formula nyinshi zitanga agaciro ka Tm, ubushyuhe bwa annealing ni intangiriro gusa.Umwihariko urashobora kunozwa mugusesengura reaction nyinshi zigenda zizamura ubushyuhe bwa annealing.Tangira munsi yikigereranyo cya Tm-5 and, hanyuma wongere buhoro buhoro ubushyuhe bwa annealing mubwiyongere bwa 2 ℃.Ubushyuhe bwo hejuru bwa annealing buzagabanya imiterere ya primer dimers nibicuruzwa bidasanzwe.Kubisubizo byiza, primers ebyiri zigomba kugira agaciro ka Tm.Niba Tm itandukanyirizo ya primer irenze 5 ℃, primers izerekana intangiriro yibinyoma ukoresheje ubushyuhe buke bwa annealing muri cycle.Niba primers ebyiri Tm zitandukanye, shyira ubushyuhe bwa annealing kuri 5 ℃ munsi ya Tm yo hasi.Ubundi, kugirango wongere umwihariko, inzinguzingo eshanu zirashobora gukorwa mbere kubushyuhe bwa annealing bwagenewe Tm yo hejuru, hanyuma hagakurikiraho inzinguzingo zisigaye kuri annealing ubushyuhe bwagenewe Tm yo hepfo.Ibi bituma igice cyicyitegererezo cyicyerekezo kiboneka mugihe gikabije.
4. Isuku yibanze no gutuza
Ubusanzwe ubuziranenge bwibisanzwe birahagije kubikorwa byinshi bya PCR.Gukuraho amatsinda ya benzoyl na isobutylyl mukuyungurura ni make bityo ntibibangamira PCR.Porogaramu zimwe zisaba kwezwa kugirango zikureho icyaricyo cyose kitari cyuzuye murwego rwo guhuza.Uru rutonde rwaciwe rubaho kuko imikorere ya chimie ya ADN ya synthesis ntabwo ari 100%.Ubu ni inzira izenguruka ikoresha imiti igaruka nkuko buri shingiro ryongeweho kugirango ADN kuva 3 ′ kugeza 5 ′.Urashobora kunanirwa muri buri cyiciro.Intangiriro ndende, cyane cyane izirenga 50 shingiro, zifite igice kinini cyurutonde rwaciwe kandi zishobora gusaba kwezwa.
Umusaruro wa primers uhindurwa nubushobozi bwa chimie synthique nuburyo bwo kweza.Ibigo bikoresha imiti, nka Cytology na Shengong, byose bikoresha byibuze OD kugirango harebwe umusaruro wose wa oligonucleoside.Customer primers zoherejwe muburyo bwifu yifu.Nibyiza kongera guhinduranya primers muri TE kugirango kwibanda kwanyuma ni 100μM.TE iruta amazi ya deionised kuko pH yamazi akenshi iba acide kandi bizatera hydrolysis ya oligonucleoside.
Guhagarara kwa primers biterwa nuburyo bwo kubika.Ifu yumye hamwe na primers yashonze bigomba kubikwa kuri -20 ℃.Primers yashonga muri TE yibitekerezo birenze 10μM irashobora kubikwa neza kuri -20 ℃ mumezi 6, ariko irashobora kubikwa gusa mubushyuhe bwicyumba (15 ℃ kugeza 30 ℃) mugihe kitarenze icyumweru.Ifu yumye irashobora kubikwa kuri -20 C byibuze umwaka 1 no mubushyuhe bwicyumba (15 C kugeza 30 C) mugihe cyamezi 2.
5. Enzymes hamwe nibitekerezo byabo
Kugeza ubu, Taq ADN polymerase ikoreshwa ni enzyme ya injeniyeri ya gene ikomatanya na bagiteri ya coliform.Ingano ya enzyme ikenewe kugirango ihagarike PCR isanzwe ni 2.5U (bivuga ubwinshi bwa reaction ya 100ul).Niba kwibandaho ari hejuru cyane, birashobora kuganisha kuri amplification idasanzwe;niba kwibumbira hamwe ari bike cyane, ingano yibicuruzwa bizagabanuka.
6. Ubwiza no kwibanda kuri dNTP
Ubwiza bwa DNTP bufitanye isano rya bugufi no kwibanda hamwe no gukora neza kwa PCR.Ifu ya DNTP ni granular, kandi guhinduka kwayo gutakaza ibikorwa byibinyabuzima niba bibitswe nabi.Igisubizo cya DNTP ni acide, kandi kigomba gukoreshwa muburyo bwinshi, hamwe na 1M NaOH cyangwa 1M Tris. Igisubizo cya buffer cya HCL kugirango gihindure PH kuri 7.0 ~ 7.5, umubare muto wibikoresho bipfunyika, ububiko bwahagaritswe kuri -20 ℃.Gukonjesha inshuro nyinshi bizatesha agaciro DNTP.Mubisubizo bya PCR, dNTP igomba kuba 50 ~ 200umol / L.By'umwihariko, hakwiye kwitabwaho kwibanda kuri bine DNTPS igomba kuba ingana (gutegura mole ingana).Niba kwibumbira hamwe kwimwe murimwe gutandukanye nabandi (hejuru cyangwa munsi), kudahuza bizaterwa.Kwibanda cyane bizagabanya umusaruro wibicuruzwa bya PCR.DNTP irashobora guhuza na Mg2 + ikagabanya kwibumbira hamwe kwa Mg2 + kubuntu.
7. Icyitegererezo (intego ya gene) acide nucleic
Ingano nogusukura urwego rwicyitegererezo nucleic acide nimwe mumurongo wingenzi kugirango intsinzi cyangwa gutsindwa kwa PCR.Uburyo gakondo bwo kweza ADN busanzwe bukoresha SDS na protease K kugirango igogwe kandi itange ingero.Ibikorwa by'ingenzi bya SDS ni: gushonga lipide na proteyine kuri membrane selile, bityo bikangiza ururenda rwa selile mu gushonga poroteyine za membrane, no gutandukanya poroteyine za kirimbuzi mu kagari, SDS irashobora kandi guhuza poroteyine no kugwa;Protease K irashobora hydrolyze no gusya poroteyine, cyane cyane amateka afitanye isano na ADN, hanyuma ugakoresha fenolike ya solol na chloroform kugirango ukuremo poroteyine nibindi bice bigize selile, hanyuma ukoreshe inzoga ya Ethanol cyangwa isopropyl kugirango ugabanye aside nucleic.Acide nucleic yakuweho irashobora gukoreshwa nkicyitegererezo cya PCR.Kubisanzwe muri rusange byerekana ivuriro, uburyo bwihuse kandi bworoshye burashobora gukoreshwa mugushonga ingirabuzimafatizo, lysate pathogens, gusya no kuvana poroteyine muri chromosomes kugeza gen zigenewe ubusa, kandi bigakoreshwa muburyo bwo kongera PCR.Gukuramo inyandikorugero ya RNA mubisanzwe ikoresha guanidine isothiocyanate cyangwa protease K uburyo bwo gukumira RNase gutesha agaciro RNA.
8.Mg2 + kwibanda
Mg2 + igira ingaruka zikomeye kumiterere n'umusaruro wa amplification ya PCR.Muri rusange reaction ya PCR, iyo kwibumbira hamwe kwa DNTP itandukanye ni 200umol / L, kwibanda kwa Mg2 + ni 1.5 ~ 2.0mmol / L.Mg2 + yibanze cyane, reaction yihariye iragabanuka, amplification idasanzwe ibaho, kwibanda cyane bizagabanya ibikorwa bya Taq ADN polymerase, bigatuma igabanuka ryibicuruzwa.
Ioni ya magnesium igira ingaruka mubice byinshi bya PCR, nkibikorwa bya ADN polymerase, bigira ingaruka kumusaruro;Urundi rugero ni primer annealing, bigira ingaruka kumwihariko.DNTP hamwe nicyitegererezo bihuza na magnesium ion, bigabanya ingano ya ion ya magnesium yubusa ikenewe mubikorwa bya enzyme.Ibyiza bya magnesium ion biratandukana kubintu bitandukanye bya primer hamwe na templates, ariko mubisanzwe PCR itangira kwibanda hamwe na 200μM dNTP ni 1.5mM (icyitonderwa: Kubihe nyabyo PCR, koresha 3 kugeza 5mM ya magnesium ion hamwe na probe ya fluorescent).Kwiyongera kwinshi kwa ioni ya magnesium yubusa byongera umusaruro, ariko kandi byongera amplification idafite akamaro kandi bigabanya ubudahemuka.Kugirango umenye neza icyerekezo cyiza, titre ya magnesium ion yakozwe mubwiyongere bwa 0.5mM kuva 1mM kugeza 3mM.Kugira ngo ugabanye gushingira kuri optique ya magnesium ion, Platinum Taq ADN polymerase irashobora gukoreshwa.Platinum Taq ADN polymerase irashobora kugumana imikorere murwego runini rwa magnesium ion kuruta Taq ADN polymerase bityo ikaba idasaba gukora neza.
9. Inyongera ya Pcr
Gukwirakwiza ubushyuhe bwa annealing, igishushanyo mbonera, hamwe na magnesium ion yibanze birahagije kugirango byongerwe cyane inyandikorugero nyinshi;icyakora, inyandikorugero zimwe, zirimo izifite GC nyinshi, zisaba ingamba zinyongera.Inyongeramusaruro zigira ingaruka ku bushyuhe bwa ADN zitanga ubundi buryo bwo kunoza ibicuruzwa n'umusaruro.Gutandukanya byuzuye byicyitegererezo birakenewe kubisubizo byiza.
Mubyongeyeho, imiterere ya kabiri irinda primer guhuza no kwagura enzyme.
Inyongera za PCR, zirimo formamide, DMSO, glycerine, betaine, na PCRx Enhancer Solution, byongera amplification.Uburyo bwabo bushoboka ni ukugabanya ubushyuhe bwo gushonga, bityo bigafasha guhuza primers no gufasha kwagura ADN polymerase binyuze mukarere ka kabiri.PCRx Igisubizo ifite izindi nyungu.Hafi ya magnesium ion ikenewe cyane iyo ikoreshejwe na Platinum Taq ADN polymerase na Platinum Pfx ADN polymerase.Rero, tekinike ya Platinum ihujwe ninyongeramusaruro kugirango yongere umwihariko mugihe igabanya gushingira kuburyo bwa gatatu, optique ya magnesium.Kubisubizo byiza, kwibumbira hamwe byongeweho bigomba kuba byiza, cyane cyane DMSO, formamide, na glycerol, bibuza Taq ADN polymerase.
10. Gutangira bishyushye
Gutangira bishyushye PCR nimwe muburyo bwingenzi bwo kunoza PCR yihariye hiyongereyeho igishushanyo mbonera cyiza.Nubwo ubushyuhe bwiza bwo kuramba bwa Taq ADN polymerase ari 72 ℃, polymerase ikomeza gukora mubushyuhe bwicyumba.Kubwibyo, ibicuruzwa bidasanzwe byakozwe mugihe ubushyuhe bwo gufata buri munsi yubushyuhe bwa annealing mugihe cyo gutegura reaction ya PCR no mugitangira cyizuba.Iyo bimaze gushingwa, ibyo bicuruzwa bidafite akamaro byongerewe neza.Hot-itangira PCR ikora cyane cyane mugihe imbuga zikoreshwa mugushushanya primer zigarukira kumwanya wibintu bya genetike, nka mutation yerekanwe kurubuga, guhinduranya imvugo, cyangwa kubaka no gukoresha ibintu bya genetike bikoreshwa mubuhanga bwa ADN.
Uburyo busanzwe bwo kugabanya ibikorwa bya Taq ADN polymerase nugutegura igisubizo cya PCR kubibarafu hanyuma ukabishyira mubikoresho byashushe PCR.Ubu buryo buroroshye kandi buhendutse, ariko ntabwo burangiza ibikorwa bya enzyme bityo ntibikuraho burundu amplification yibicuruzwa bidasanzwe.
Ubushuhe bwa primaque butinda synthesis ya ADN muguhagarika igice cyingenzi kugeza ibikoresho bya PCR bigeze ku bushyuhe bwa denatration.Uburyo bwinshi bwo gutangiza amashyuza, harimo gutinda kongeramo Taq ADN polymerase, biragoye, cyane cyane kubisohoka cyane.Ubundi buryo bwa primaire yubushyuhe bukoresha ibishashara kugirango ushiremo ikintu cyingenzi, harimo ioni ya magnesium cyangwa enzymes, cyangwa gutandukanya umubiri ibintu bitagaragara, nka templates na buffers.Mugihe cyizuba cyumuriro, ibice bitandukanye birekurwa kandi bikavangwa hamwe uko ibishashara bishonga.Kimwe nintoki zishyushye zo gutangira, uburyo bwo gukingira ibishashara biragoye kandi bikunda kwanduzwa kandi ntibikwiriye gukoreshwa cyane.
Platinum ADN polymerase iroroshye kandi ikora neza kugirango itangire PCR ishyushye.Platinum Taq ADN polymerase igizwe na recombinant Taq ADN polymerase ihujwe na antibody ya monoclonal irwanya Taq ADN polymerase.Antibodies zakozwe na PCR kugirango zibuze ibikorwa bya enzyme mugihe kirekire.Taq ADN polymerase yarekuwe mubyakozwe mugihe cya 94 ℃ izitera intambwe yo gutandukana, igarura ibikorwa bya polymerase byuzuye.Bitandukanye na chimique ya Taq ADN polymerase kugirango itangire ubushyuhe, enzyme ya platine ntisaba gukingirwa igihe kirekire kuri 94 ℃ (iminota 10 kugeza 15) kugirango ikore polymerase.Hamwe na PlatinumTaq ADN polymerase, 90% yibikorwa bya Taq DNA polymerase byagaruwe nyuma yiminota 2 kuri 94 ℃.
11. Icyari-PCR
Uruzinduko rukurikiranye rwa amplification ukoresheje primer nested irashobora kunoza umwihariko no kumva.Icyiciro cya mbere ni amplification isanzwe ya 15 kugeza 20.Agace gato k'ibicuruzwa byambere byongerewe imbaraga byongewemo inshuro 100 kugeza ku 1000 hanyuma byongerwaho icyiciro cya kabiri cyo kongera imbaraga kuri 15 kugeza 20.Ubundi, ibicuruzwa byambere byongerewe imbaraga birashobora kuba binini mugusukura gel.Icyerekezo cyateguwe gikoreshwa mugice cya kabiri cya amplification, gishobora guhuza intego ikurikiranye imbere ya primer yambere.Ikoreshwa rya PCR ryagabanijwe rigabanya amahirwe yo kwagura imbuga nyinshi zintego kuko hariho intego nke zikurikirana zuzuzanya zombi za primers.Umubare umwe wizunguruka (30 kugeza 40) hamwe na primers imwe yongereye imbuga zidafite akamaro.Nested PCR yongerera ibyiyumvo byintego ntarengwa (urugero, mrnasi idasanzwe) kandi itezimbere umwihariko wa PCRS igoye (urugero 5 ′ AMOKO).
12. Kumanuka PCR
Kumanuka PCR itezimbere umwihariko ukoresheje uburyo bukomeye bwa annealing kubintu bike byambere bya PCR.Umuzenguruko utangirira ku bushyuhe bwa annealing hafi 5 ℃ hejuru ya Tm yagereranijwe, hanyuma buri cyiciro kigabanukaho 1 ℃ kugeza kuri 2 ℃ kugeza ubushyuhe bwa annealing buri munsi ya Tm 5 ℃.Gusa icyerekezo cyerekanwe hamwe na homologiya yo hejuru izongerwaho.Ibicuruzwa bikomeje kwaguka mubyiciro byakurikiyeho, byuzuyemo ibicuruzwa byongerewe agaciro bidafite akamaro.Kumanuka PCR ni ingirakamaro muburyo aho urwego rwa homology hagati ya primer nicyitegererezo ntiruzwi, nko gutunga urutoki rwa ADLP ADN.
Bifitanye isano nibikoresho bya PCR
PCR Byoroshyeᵀᴹ (Hamwe n'irangi)
2 × Intwari ya PCRTMSisitemu yo kuvanga ifite kwihanganira cyane inhibitori za PCR kuruta sisitemu isanzwe ya PCR ivanze, kandi irashobora guhangana byoroshye na PCR ikwirakwizwa rya templates zitandukanye.Sisitemu idasanzwe ya reaction hamwe nubushobozi buhanitse Taq Intwari ituma reaction ya PCR igira ubushobozi bwo kongera amplification, umwihariko hamwe na sensitivite.
Intwari ya PCR (Hamwe n'irangi)
Gukora neza cyane
Ifite ibikorwa bya 5 '→ 3' ADN polymerase na 5 '→ 3' ibikorwa bya exonuclease, nta gikorwa cya 3 '→ 5'.
Igihe nyacyo PCR Byoroshyeᵀᴹ-SYBR Icyatsi I Kit
Byihariye-byateguwe neza na hot-itangira Taq enzyme irashobora gukumira amplification idasanzwe hamwe na primer dimer
Ubukangurambaga bukabije - bushobora kumenya kopi ntoya yicyitegererezo
RT-PCR Byoroshyeᵀᴹ I (Intambwe imwe)
Igikoresho gikoresha uburyo bwihariye bwa Foregene reverse transcription reagent hamwe na Foregene HotStar Taq ADN Polymerase ihujwe na sisitemu idasanzwe yogukora kugirango tunoze neza imikorere ya amplification hamwe nibisobanuro bya reaction.
Igihe cyo kohereza: Gicurasi-09-2023
