Maziko oyambira (zovuta 99% zitha kuthetsedwa)
1. Kutalika koyambira: Bukuli limafunikira 15-30bp, nthawi zambiri pafupifupi 20bp.Mkhalidwe weniweniwo ndi wabwino kukhala 18-24bp kuti muwonetsetse kuti zenizeni, koma nthawi yayitali, yoyambira yotalika kwambiri idzachepetsanso tsatanetsatane, ndikuchepetsa zokolola.
2. Kutalika kwa kukula kwa Primer: 200-500bp ndi koyenera, ndipo chidutswacho chikhoza kukulitsidwa mpaka 10kb pansi pazikhalidwe zina.
3. Maziko oyambira: Zomwe zili mu G + C ziyenera kukhala 40-60%, zotsatira zochepa kwambiri za G + C sizili bwino, G + C yochuluka ndiyosavuta kuoneka ngati magulu osakhala enieni.ATGC imagawidwa bwino mwachisawawa, kupewa masango opitilira 5 purine kapena pyrimidine nucleotides.Multi-gc kwa 5 'mapeto ndi ndondomeko zapakatikati kuti muwonjezere kukhazikika, pewani GC wolemera pamapeto a 3', palibe GC pazitsulo zomaliza za 3, kapena palibe GC kwa 3 ya maziko otsiriza a 5.
4. Pewani mawonekedwe achiwiri muzoyambira, ndipo pewani kuphatikizika pakati pa zoyambira ziwiri, makamaka kukwanira kumapeto kwa 3, apo ayi primer dimer idzapangidwa ndipo magulu osatchulika adzapangidwa.
5. Zoyambira kumapeto kwa 3 'mapeto a zoyambira, makamaka zomaliza ndi zoyambira, ziyenera kulumikizidwa mosamalitsa kuti PCR isalephereke chifukwa cha zoyambira zosapangana.
6. Ma primers ali ndi kapena akhoza kuwonjezeredwa ndi malo oyenera ophwanyidwa, ndipo ndondomeko yowonjezereka iyenera kukhala ndi malo oyenera ophwanyidwa, omwe ndi opindulitsa kwambiri pakuwunika kwa cleavage kapena molecular cloning.
7. Kudziwika kwa zoyambira: zoyambira siziyenera kukhala ndi homology yodziwikiratu ndi ma sequence ena mu nucleic acid sequence database.
8. Phunzirani kugwiritsa ntchito mapulogalamu: PP5, Oligo6, DNAstar, Vector NTI, primer3 (Mapangidwe apa intaneti amagwira ntchito bwino).
Zomwe zili pamwambazi zitha kuthetsa osachepera 99% yazovuta zamapangidwe oyambira.
Sinthani tsatanetsatane wa mapangidwe oyambira
1. Kutalika koyambira
Kutalika koyambira koyamba ndi 18-30 maziko.Mwambiri, chofunikira kwambiri chomwe chimatsimikizira kutentha kwa annealing kwa primer ndi kutalika kwa primer.Kutentha kwa annealing kwa primer nthawi zambiri kumasankhidwa (Tm value -5 ℃), ndipo ena amagwiritsa ntchito mtengo wa Tm mwachindunji.Njira zotsatirazi zitha kugwiritsidwa ntchito powerengera pafupifupi kutentha kwa annealing kwa zoyambira.
Pamene utali woyambira ndi wochepera 20bp: [4(G+C)+2(A+T)] -5℃
Pamene utali woyambira ndi wamkulu kuposa 20bp: 62.3 ℃+0.41 ℃(%GC) -500/utali-5℃
Kuphatikiza apo, mapulogalamu ambiri angagwiritsidwenso ntchito kuwerengera kutentha kwa annealing, mfundo yowerengera idzakhala yosiyana, kotero nthawi zina mtengo wowerengeka ukhoza kukhala ndi kusiyana kochepa.Kuti muwonjezere machitidwe a PCR, zoyambira zazifupi kwambiri zomwe zimatsimikizira kutentha kosachepera 54 ℃ zimagwiritsidwa ntchito moyenera komanso mwachindunji.
Ponseponse, tsatanetsatane wa zoyambira zimawonjezeka ndi zinayi pa nucleotide iliyonse yowonjezera, kotero kuti utali woyambira wocheperako pamapulogalamu ambiri ndi ma nucleotide 18.Malire apamwamba a kutalika kwa primer sikofunikira kwambiri, makamaka okhudzana ndi magwiridwe antchito.Chifukwa cha entropy, choyambira chikatalikirapo, chimachepetsa kuchuluka komwe chimamangirira ku DNA yomwe mukufuna kuti ipange template yokhazikika yamizere iwiri ya DNA polymerase kuti imangirire.
Mukamagwiritsa ntchito mapulogalamu kupanga zoyambira, kutalika kwa zoyambira kumatha kutsimikiziridwa ndi mtengo wa TM, makamaka zoyambira za fluorescence quantitative PCR, TM = 60 ℃ kapena ziyenera kuyendetsedwa.
2.GC zomwe zili
Nthawi zambiri, zomwe zili mu G+C muzotsatira zoyambira ndi 40% ~ 60%, ndipo GC zili ndi mtengo wa Tm wa zoyambira ziwiri ziyenera kulumikizidwa.Ngati choyambira chili ndi GC yayikulu kapena AT chizolowezi, kuchuluka koyenera kwa A, T kapena G ndi C mchira kumatha kuwonjezeredwa ku 5 'kumapeto kwa choyambira.
3. Annealing kutentha
Kutentha kwa annealing kuyenera kukhala 5 ℃ kutsika kuposa kutentha kwa unchain.Ngati chiwerengero cha maziko oyambira ndi ochepa, kutentha kwa annealing kumatha kuonjezedwa moyenera, zomwe zitha kuwonjezera kutsimikizika kwa PCR.Ngati chiwerengero cha mabasi ndi chachikulu, kutentha kwa annealing kumatha kuchepetsedwa moyenera.Kusiyana kwa kutentha kwapakati pakati pa ma primers a 4 ℃ ~ 6 ℃ sikungakhudze kukolola kwa PCR, koma makamaka kutentha kwapawiri koyambira kumakhala kofanana, komwe kumatha kusiyana pakati pa 55 ℃ ~ 75 ℃.
4. Pewani gawo lachiwiri la template yokulitsa
Ndikwabwino kupewa gawo lachiwiri la template posankha gawo lokulitsa.Mapangidwe achiwiri okhazikika a chidutswa chandamale akhoza kuneneratu ndikuyerekeza ndi mapulogalamu apakompyuta oyenerera, omwe ndi othandiza posankha ma template.Zotsatira zoyeserera zikuwonetsa kuti kukulitsa sikumakhala kopambana pamene mphamvu yaulere (△G) ya dera loti ikulitsidwe ili yosakwana 58.6lkJ/mol.
5. Zosagwirizana ndi DNA yomwe mukufuna
Ngati DNA yokulirapo ikhala yayikulu, choyambira chimamanga mbali zingapo za DNA yomwe mukufuna, zomwe zimapangitsa kuti magulu angapo aziwoneka pazotsatira.Nthawi ino ndikofunikira kugwiritsa ntchito kuyesa kwa pulogalamu ya BLAST, tsamba lawebusayiti:http://www.ncbi.nlm.nih.gov/BLAST/.Sankhani Gwirizanitsani magawo awiri (bl2seq).
Kuyika magawo oyambira ku zone 1 ndikutsata ma DNA kugawo 2 kumatha kusinthana, ndipo BLAST imawerengera zowonjezera, antisense, ndi zotheka zina, kotero ogwiritsa ntchito safunika kuzindikira ngati maunyolo onsewa ndi maunyolo.Mutha kuyikanso nambala ya GI ngati mukudziwa nambala ya GI yotsatizana m'dawunilodi, ndiye kuti simuyenera kumata gawo lalikulu lazotsatira.Pomaliza, dinani Align pa 3 kuti muwone ngati choyambiracho chili ndi masamba angapo amtundu wa DNA womwe mukufuna.
6. Choyimira choyambirira
The 3 'mapeto oyambira ndi pamene kutambasuka kumayambira, kotero ndikofunika kupewa mismatch kuyambira pamenepo.Mapeto a 3 'asapitirire 3 motsatizana G kapena C, chifukwa izi zipangitsa kuti choyambiriracho chiyambitsidwe molakwika mu gawo lotsatizana la G+C.Mapeto a 3 'sangathe kupanga dongosolo lililonse lachiwiri, kupatula muzochitika zapadera za PCR (AS-PCR), mapeto a 3' oyambira sangagwirizane.Mwachitsanzo, ngati chigawo cha encoding chikukulitsidwa, 3 'mapeto a primer sayenera kuthetsedwa pa malo achitatu a codon, chifukwa malo achitatu a codon amatha kuwonongeka, zomwe zidzakhudza kutsimikizika ndi mphamvu ya kukulitsa.Mukamagwiritsa ntchito ma annexation primers, onetsani ku tebulo logwiritsira ntchito kodon, samalani zokonda zamoyo, musagwiritse ntchito zoyambira zowonjezera kumapeto kwa 3′, ndipo gwiritsani ntchito zoyambira zambiri (1uM-3uM).
7. Mapangidwe achiwiri a zoyambira
Zoyambira zokha siziyenera kukhala ndi zotsatizana, apo ayi zoyambira zokha zimapindika m'mapangidwe atsitsi, ndipo kapangidwe kachiwiri kameneka kadzakhudza kumangirira kwa ma primers ndi ma templates chifukwa cha kulepheretsa steric.Ngati chigamulo chochita chikugwiritsidwa ntchito, zoyambira zowonjezera zomwe siziyenera kupitilira 3bp.Sipayenera kukhala kugwirizana pakati pa zoyambira ziwirizi, makamaka kuphatikizika kowonjezera kwa 3 'mapeto kuyenera kupewedwa kuti tipewe kupanga ma primer dimers.Nthawi zambiri, pasakhale zoyambira zinayi zotsatizana za homology kapena kulumikizana pakati pa zoyambira.
8. Onjezani zolembera kapena loci
Mapeto a 5 'amakhudza pang'ono kukulitsa kukhazikika ndipo amatha kusinthidwa popanda kukhudza tsatanetsatane wa matalikidwe.Kusintha kwa primer 5 'mapeto kunaphatikizapo: kuwonjezera malo oletsa ma enzyme;Zolemba za biotin, fluorescence, digoxin, Eu3+, ndi zina zotero. Yambitsani ndondomeko za DNA zomanga mapuloteni;Kuyambitsa malo osintha masinthidwe, kuyika ndi kusowa zotsatizana za masinthidwe ndikuyambitsa zotsatsira zotsatsira, ndi zina zotero. Zoyambira zowonjezera zidzakhudza kwambiri kukulitsa bwino ndikuwonjezera mwayi wopanga ma primer dimer, koma zololeza zina ziyenera kupangidwa pa sitepe yotsatira.Zowonjezera zowonjezera zomwe sizilipo pazotsatira zomwe zatsata, monga malo oletsa ndi zotsatizana zotsatizana, zikhoza kuwonjezeredwa ku 5 'mapeto a chiyambi popanda kukhudza mwachindunji.Zotsatizanazi sizinaphatikizidwe powerengera zamtengo wapatali wa Tm, koma ziyenera kuyesedwa kuti zigwirizane ndi zina zamkati.
9. Ma subclones
Nthawi zambiri, PCR imakhala yongoyamba kumene, ndiyeno tifunika kuyika chidutswa chandamale kukhala ma vector osiyanasiyana, kotero tifunika kupanga maziko owonjezera a opareshoni yotsatira pagawo la PCR.
Njira zina zopangira subcloning zikufotokozedwa mwachidule pansipa.
Malo oletsa endonuclease oletsa adawonjezedwa
Kuwonjezera masamba oletsa ma enzyme ndiyo njira yomwe imagwiritsidwa ntchito kwambiri popangira zinthu za PCR.Nthawi zambiri, cleavage site ndi maziko asanu ndi limodzi, kuwonjezera pa 5 'mapeto a cleavage malo ayenera kuwonjezera 2 ~ 3 maziko zoteteza.Komabe, kuchuluka kwa zoteteza zomwe zimafunikira ma enzymes osiyanasiyana ndizosiyana.Mwachitsanzo, SalⅠ sifunika maziko oteteza, EcoRⅤ imafunika 1 maziko oteteza, NotⅠ imafunika 2 zotetezera, ndipo Hind Ⅲ imafunika 3 zotetezera.
LIC imawonjezera mchira
Dzina lonse la LIC ndi Ligation-Independent cloning, njira yopangira ma cloning yopangidwa ndi Navogen makamaka chifukwa cha gawo lake la vector ya pET.Chonyamulira cha pET chokonzedwa ndi njira ya LIC chimakhala ndi nsonga zomata za 12-15 zoyambira, zomwe zimayenderana ndi nsonga zomata pagawo loyikapo.Pazolinga zokulitsira, zoyambira 5′ zachidutswa chomwe chalowetsedwa ziyenera kugwirizana ndi vekitala ya LIC.3′→5′ extranect activity ya T4 DNA polymerase imatha kupanga chingwe chomata pachidutswacho pakapita nthawi yochepa.Chifukwa chakuti mankhwalawa amatha kupangidwa kuchokera ku mgwirizano wogwirizana wa chidutswa chokonzekera choyikapo ndi vector, njirayi ndi yofulumira komanso yothandiza, ndipo imayendetsedwa ndi cloning.
Directed TA clone kuwonjezera mchira
TA cloning sinathe kuloza chidutswacho kukhala vekitala, kotero pambuyo pake Invitrogen inayambitsa vekitala yomwe ikanalunjika ku cloning, yomwe inali ndi maziko anayi odziwika a GTGGS kumapeto kwake.Chifukwa chake, pamapangidwe a PCR zoyambira, zotsatizana zowonjezera ziyenera kuwonjezeredwa molingana, kuti zidutswa zitha kukhala "zolunjika".
Ngati muli ndi nthawi yochepa, mutha kuyesa kaphatikizidwe mwachindunji, kuphatikiza jini ndi vector, zomwe timatcha ET gene synthesis mu musecularists.
D. In-Fusion cloning njira
Palibe ligase yofunika, palibe kuyankha kwautali komwe kumafunikira.Malingana ngati kutsatizana kumalekezero onse a vekitala yopangidwa ndi mzere kumayambitsidwa Popanga zoyambira, ndiye kuti mankhwala a PCR ndi makina opangidwa ndi mzere amawonjezedwa mu njira ya in-fusion enzyme yomwe ili ndi BSA ndikuyika kutentha kwa theka la ola, kusintha kungatheke.Njirayi ndiyoyenera makamaka kutembenuka kwa voliyumu yayikulu.
10. Phatikizani zoyambira
Nthawi zina, zidziwitso zochepa zokha zotsatizana zimadziwika pakupanga koyambira.Mwachitsanzo, ngati ma amino acid amatsatizana okha ndi omwe amadziwika, choyambira cholumikizira chikhoza kupangidwa.Choyambira chophatikizira ndi chisakanizo chamayendedwe osiyanasiyana omwe amayimira kuthekera kosiyanasiyana komwe kumayika amino acid imodzi.Kuti muwonjezere kutsimikizika, mutha kulozera ku tebulo logwiritsa ntchito kodon kuti muchepetse kuphatikizika malinga ndi zomwe mumakonda kugwiritsa ntchito zamoyo zosiyanasiyana.Hypoxanthine imatha kuphatikizidwa ndi zoyambira zonse kuti muchepetse kutentha koyambira.Musagwiritse ntchito maziko ophatikizidwa kumapeto kwa 3 'mapeto a chiyambi chifukwa kulumikiza maziko a 3 otsiriza kumapeto kwa 3' ndikokwanira kuyambitsa PCR pamalo olakwika.Zoyambira zapamwamba (1μM mpaka 3μM) zimagwiritsidwa ntchito chifukwa zoyambira muzophatikiza zambiri sizikhala zachindunji.
PCR zopangirakulamulira
1. Kuchuluka koyambira
Kuchuluka kwa chiyambi chilichonse ndi 0.1 ~ 1umol kapena 10 ~ 100pmol.Ndi bwino kutulutsa zotsatira zofunikira ndi kuchuluka kotsika kwambiri kwa primer.Kuchulukirachulukira kwa zoyambira kumayambitsa kusagwirizana komanso kukulitsa kosagwirizana, ndikuwonjezera mwayi wopanga ma dimers pakati pa zoyambira.
2. Kukhazikika koyambira
Kuchuluka kwa zoyambira kumakhudza kutsimikizika.Mulingo woyenera kwambiri woyambira nthawi zambiri umakhala pakati pa 0.1 ndi 0.5μM.Kuchulukirachulukira koyambira kumabweretsa kukulitsa kwazinthu zomwe sizikudziwika.
3. Annealing kutentha kwa primer
Gawo lina lofunikira la zoyambira ndi kutentha kosungunuka (Tm).Uku ndi kutentha pamene 50% ya zoyambira ndi zotsatizana zimayimiridwa ngati mamolekyu a DNA amitundu iwiri.Tm imafunika kukhazikitsa kutentha kwa PCR.Moyenera, kutentha kwa annealing ndi kotsika mokwanira kuti zitsimikizire kuti zoyambira zimatsatiridwa motsatana ndi zomwe mukufuna, koma zimakwera mokwanira kuti zichepetse kumangiriza kosadziwika.Kutentha koyenera kwa annealing kuchokera ku 55 ℃ mpaka 70 ℃.Kutentha kwa Annealing kumayikidwa 5 ℃ kutsika kuposa Tm ya primer.
Pali njira zingapo zokhazikitsira Tm, zomwe zimasiyana kwambiri kutengera fomu yomwe imagwiritsidwa ntchito komanso kutsata kwa zoyambira.Chifukwa mafomula ambiri amapereka mtengo woyerekeza wa Tm, kutentha konseko kumangokhala poyambira.Kufotokozera kungawonjezeke pofufuza zochitika zingapo zomwe zimakweza pang'onopang'ono kutentha kwa anneal.Yambani pansi pa chiyerekezo cha Tm-5 ℃, ndipo pang'onopang'ono muwonjezere kutentha kwa annealing mu increment ya 2 ℃.Kutentha kwakukulu kwa annealing kudzachepetsa mapangidwe a primer dimers ndi zinthu zomwe sizinali zenizeni.Kuti mupeze zotsatira zabwino, zoyambira ziwirizi ziyenera kukhala ndi pafupifupi Tm.Ngati kusiyana kwa Tm kwa ma primer pairs kupitilira 5 ℃, zoyambira zidzawonetsa kuyambika kwabodza pogwiritsa ntchito kutentha kocheperako pozungulira.Ngati zoyambira ziwiri za Tm ndi zosiyana, ikani kutentha kwa annealing ku 5 ℃ kutsika kuposa Tm yotsika kwambiri.Kapenanso, kuti muwonjezere kutsimikizika, mizunguliro isanu imatha kuchitidwa koyamba pa kutentha kwapakati komwe kumapangidwira Tm yokwera, kutsatiridwa ndi mikombero yotsalira pa kutentha kwa annealing komwe kumapangidwira kutsika kwa Tm.Izi zimalola kuti kopi yapang'ono ya template yopita ipezeke pansi pazovuta.
4. Koyamba chiyero ndi bata
Kuyera kokhazikika kwa zoyambira zoyambira ndizokwanira pamapulogalamu ambiri a PCR.Kuchotsa magulu a benzoyl ndi isobutylyl pochotsa mchere ndikochepa choncho sikusokoneza PCR.Ntchito zina zimafuna kuyeretsedwa kuti zichotse zotsatizana zilizonse zomwe sizili zazitali mu kaphatikizidwe.Izi zotsatizanazi zimachitika chifukwa mphamvu ya DNA synthesis chemistry si 100%.Iyi ndi njira yozungulira yomwe imagwiritsa ntchito machitidwe a mankhwala mobwerezabwereza pamene maziko aliwonse amawonjezeredwa kuti apange DNA kuchokera ku 3' mpaka 5'.Mutha kulephera mkombero uliwonse.Zoyambira zazitali, makamaka zoyambira 50, zimakhala ndi magawo ocheperako ndipo zingafunike kuyeretsedwa.
Zokolola za oyambira zimakhudzidwa ndi mphamvu ya chemistry yopanga ndi njira yoyeretsera.Makampani a Biopharmaceutical, monga Cytology ndi Shengong, onse amagwiritsa ntchito gawo lochepa la OD kuti atsimikizire kutulutsa kwathunthu kwa oligonucleoside.Zoyambira zachikhalidwe zimatumizidwa mu mawonekedwe a ufa wowuma.Ndi bwino kusungunulanso zoyambira mu TE kuti ndende yomaliza ikhale 100μM.TE ndi yabwino kuposa madzi a deionized chifukwa pH ya madzi nthawi zambiri imakhala acidic ndipo imayambitsa hydrolysis ya oligonucleosides.
Kukhazikika kwa zoyambira kumatengera kusungirako zinthu.Ufa wouma ndi zoyambira zosungunuka ziyenera kusungidwa pa -20 ℃.Zoyamba zomwe zimasungunuka mu TE pazigawo zazikulu kuposa 10μM zimatha kusungidwa bwino pa -20 ℃ kwa miyezi 6, koma zimatha kusungidwa kutentha (15 ℃ mpaka 30 ℃) kwa sabata yosakwana 1.Zoyambira za ufa wouma zimatha kusungidwa pa -20 C kwa chaka chimodzi komanso kutentha (15 C mpaka 30 C) kwa miyezi iwiri.
5. Ma enzyme ndi kuchuluka kwawo
Pakalipano, Taq DNA polymerase yomwe imagwiritsidwa ntchito ndi jini ya jini yopangidwa ndi mabakiteriya a coliform.Kuchuluka kwa enzyme yofunikira kuti ipangitse machitidwe a PCR ndi pafupifupi 2.5U (amatanthauza kuchuluka kwa 100ul).Ngati ndendeyo ili yokwera kwambiri, imatha kuyambitsa kukulitsa kosaneneka;ngati ndende yotsika kwambiri, kuchuluka kwa mankhwala opangidwa kudzachepetsedwa.
6. Ubwino ndi kuchuluka kwa dNTP
Ubwino wa dNTP umagwirizana kwambiri ndi ndende komanso mphamvu ya kukulitsa kwa PCR.DNTP ufa ndi granular, ndipo kusinthasintha kwake kumataya ntchito yake yachilengedwe ngati sikusungidwa molakwika.Njira ya dNTP ndi acidic, ndipo iyenera kugwiritsidwa ntchito kwambiri, ndi 1M NaOH kapena 1M Tris.HCL yankho la buffer kuti lisinthe PH yake ku 7.0 ~ 7.5, yocheperako yaing'ono, yosungirako mazira pa -20 ℃.Kuundana kangapo kungawononge dNTP.Pochita PCR, dNTP iyenera kukhala 50 ~ 200umol/L.Makamaka, chidwi chiyenera kuperekedwa kwa ndende ya anayi DNTPS ayenera kukhala ofanana (mofanana mole kukonzekera).Ngati kuchuluka kwa aliyense wa iwo ndi kosiyana ndi ena (wapamwamba kapena otsika), kusagwirizana kumayambika.Kutsika kwambiri kumachepetsa zokolola za PCR.dNTP imatha kuphatikiza ndi Mg2+ ndikuchepetsa kuchuluka kwa Mg2+ yaulere.
7. Template (chandamale jini) nucleic acid
Kuchuluka ndi kuyeretsedwa kwa template nucleic acid ndi imodzi mwamaulalo ofunikira pakuchita bwino kapena kulephera kwa PCR.Njira zoyeretsera DNA nthawi zambiri zimagwiritsa ntchito SDS ndi protease K pogaya ndi kutaya zitsanzo.Ntchito zazikulu za SDS ndi: kusungunula lipids ndi mapuloteni pa khungu la selo, motero kuwononga nembanemba ya selo mwa kusungunula mapuloteni a nembanemba, ndikulekanitsa mapuloteni a nyukiliya mu selo, SDS ikhozanso kuphatikiza ndi mapuloteni ndi mpweya;Protease K imatha hydrolyze ndikugaya mapuloteni, makamaka histones womangidwa ndi DNA, ndiyeno kugwiritsa ntchito organic zosungunulira phenol ndi chloroform kuchotsa mapuloteni ndi zigawo zina za cell, ndi ntchito Mowa kapena isopropyl mowa kuti precipitate nucleic acid.The yotengedwa nucleic acid angagwiritsidwe ntchito ngati template kwa PCR zochita.Kwa zitsanzo zodziwika bwino zachipatala, njira yofulumira komanso yosavuta ingagwiritsidwe ntchito kusungunula maselo, lysate tizilombo toyambitsa matenda, kugaya ndi kuchotsa mapuloteni kuchokera ku ma chromosome kupita ku majini omasuka, ndikugwiritsidwa ntchito mwachindunji kukulitsa kwa PCR.RNA template m'zigawo nthawi zambiri amagwiritsa ntchito guanidine isothiocyanate kapena protease K njira kuteteza RNase ku kunyozetsa RNA.
8.Mg2+ ndende
Mg2+ imakhudza kwambiri tsatanetsatane ndi zokolola za kukulitsa kwa PCR.Ambiri PCR anachita, pamene ndende zosiyanasiyana dNTP ndi 200umol/L, ndende yoyenera ya Mg2+ ndi 1.5 ~ 2.0mmol/L.Kukhazikika kwa Mg2 + ndikokwera kwambiri, momwe zimakhalira zimachepa, kukulitsa kosakhazikika kumachitika, ndende yotsika kwambiri imachepetsa ntchito ya Taq DNA polymerase, zomwe zimapangitsa kuchepa kwa zinthu zomwe zimachitikira.
Magnesium ions amakhudza mbali zingapo za PCR, monga ntchito ya DNA polymerase, yomwe imakhudza zokolola;Chitsanzo china ndi primer annealing, yomwe imakhudza mwachindunji.dNTP ndi template zimamangiriza ku magnesium ion, kuchepetsa kuchuluka kwa magnesium ion yaulere yofunikira pakuchita kwa enzyme.Mulingo woyenera kwambiri wa ma magnesium ion concentration umasiyanasiyana pawiri zoyambira ndi ma templates, koma PCR yoyambira ndende ndi 200μM dNTP ndi 1.5mM (zindikirani: Kwa PCR yochulukira nthawi yeniyeni, gwiritsani ntchito njira ya 3 mpaka 5mM ya magnesium ion yokhala ndi kafukufuku wa fulorosenti).Kuchulukirachulukira kwa ayoni a magnesium aulere kumawonjezera zokolola, komanso kumawonjezera kukulitsa kosafunikira ndikuchepetsa kukhulupirika.Kuti mudziwe ndende yabwino kwambiri, ma magnesium ion titration adachitidwa mu increments ya 0.5mM kuchokera 1mM mpaka 3mM.Kuchepetsa kudalira kukhathamiritsa kwa magnesium ion, Platinum Taq DNA polymerase ingagwiritsidwe ntchito.Platinamu Taq DNA polymerase imatha kugwira ntchito mopitilira muyeso wambiri wa magnesium ion kuposa Taq DNA polymerase motero imafuna kukhathamiritsa pang'ono.
9. Pcr-kulimbikitsa zowonjezera
Kukhathamiritsa kwa kutentha kwa annealing, mapangidwe a primer, ndi ndende ya magnesium ion ndizokwanira kukulitsa ma tempuleti ambiri;komabe, ma templates ena, kuphatikizapo omwe ali ndi GC yapamwamba, amafunikira njira zowonjezera.Zowonjezera zomwe zimakhudza kutentha kwa kusungunuka kwa DNA zimapereka njira ina yopititsira patsogolo katchulidwe kazinthu ndi zokolola.Kufotokozera kwathunthu kwa template kumafunikira kuti mupeze zotsatira zabwino.
Kuphatikiza apo, mawonekedwe achiwiri amalepheretsa kumangirira koyambirira komanso kukulitsa ma enzyme.
Zowonjezera za PCR, kuphatikiza formamide, DMSO, glycerin, betaine, ndi PCRx Enhancer Solution, zimakulitsa kukulitsa.Njira yawo yomwe ingatheke ndikuchepetsa kutentha kosungunuka, motero kumathandizira kuti ma primers azitha komanso kuthandizira kufalikira kwa DNA polymerase kudzera m'chigawo chachiwiri.PCRx Solution ili ndi zabwino zina.Kukhathamiritsa pang'ono kwa magnesium ion kumafunika mukagwiritsidwa ntchito ndi Platinum Taq DNA polymerase ndi Platinum Pfx DNA polymerase.Choncho, njira ya Platinamu imaphatikizidwa ndi chowonjezera kuti chiwonjezeke mwachindunji pamene kuchepetsa kudalira njira yachitatu, kukhathamiritsa kwa magnesium ion.Kuti mupeze zotsatira zabwino, kuchuluka kwa zowonjezera ziyenera kukonzedwa, makamaka DMSO, formamide, ndi glycerol, zomwe zimalepheretsa Taq DNA polymerase.
10. Kutentha koyambira
Hot start PCR ndi imodzi mwa njira zofunika kwambiri zosinthira mawonekedwe a PCR kuphatikiza pakupanga koyambira bwino.Ngakhale kutentha kwabwino kwa Taq DNA polymerase ndi 72 ℃, polymerase imakhalabe yogwira kutentha.Choncho, mankhwala osakhala achindunji amapangidwa pamene kutentha kumakhala kochepa kusiyana ndi kutentha kwa annealing panthawi yokonzekera PCR komanso kumayambiriro kwa kutentha.Akapangidwa, zinthu zosafunikira izi zimakulitsidwa bwino.Hot-start PCR imakhala yothandiza makamaka ngati masamba omwe amagwiritsidwa ntchito popanga zoyambira amakhala ochepa potengera momwe ma genetic alili, monga masinthidwe otsogozedwa ndi tsamba, kulinganiza mawu, kapena kupanga ndikusintha ma genetic omwe amagwiritsidwa ntchito popanga DNA.
Njira yodziwika bwino yochepetsera ntchito ya Taq DNA polymerase ndikukonzekeretsa njira ya PCR pa ayezi ndikuyiyika mu chipangizo cha PCR chotenthedwa.Njirayi ndiyosavuta komanso yotsika mtengo, koma siyimaliza ntchito ya enzyme ndipo chifukwa chake sichimathetsa kukulitsa kwazinthu zomwe sizinali zenizeni.
Kutentha kotentha kumachedwetsa kaphatikizidwe ka DNA poletsa chinthu chofunikira mpaka zida za PCR zifika kutentha kwa denaturation.Njira zambiri zoyambira kutentha kwapamanja, kuphatikiza kuchedwa kuwonjezera kwa Taq DNA polymerase, ndizovuta, makamaka pazogwiritsa ntchito kwambiri.Njira zina zopangira matenthedwe zimagwiritsa ntchito chishango cha sera kutsekereza chinthu chofunikira, kuphatikiza ma ayoni a magnesium kapena ma enzymes, kapena kudzipatula mwakuthupi zigawo zogwira ntchito, monga ma tempulo ndi ma buffer.Panthawi yotentha, zigawo zosiyanasiyana zimatulutsidwa ndikusakanikirana pamodzi pamene sera imasungunuka.Mofanana ndi njira yoyambira yotentha yamanja, njira yotchingira sera ndi yovuta komanso imakonda kuipitsidwa ndipo siyoyenera kugwiritsa ntchito zida zambiri.
Platinamu DNA polymerase ndiyosavuta komanso yothandiza poyambira kutentha kwa PCR.Platin Taq DNA polymerase imakhala ndi recombinant Taq DNA polymerase kuphatikiza ndi monoclonal antibody motsutsana ndi Taq DNA polymerase.Ma antibodies amapangidwa ndi PCR kuti aletse ntchito ya ma enzyme panthawi yogwira kutentha kwa nthawi yayitali.Taq DNA polymerase idatulutsidwa momwemo panthawi ya 94 ℃ insulation of the denaturation step, kubwezeretsa ntchito zonse za polymerase.Mosiyana ndi Taq DNA polymerase yosinthidwa ndi mankhwala kuti ayambitse matenthedwe, puloteni ya Platinamu safuna kutsekereza kwanthawi yayitali pa 94 ℃ (10 mpaka 15 mphindi) kuti ayambitse polymerase.Ndi PlatinumTaq DNA polymerase, 90% ya ntchito ya Taq DNA polymerase idabwezeretsedwa patatha mphindi ziwiri pa 94 ℃.
11. Nest-PCR
Kuzungulira motsatizana kwa kukulitsa pogwiritsa ntchito ma primers okhala ndi zisa kumatha kupititsa patsogolo kukhazikika komanso chidwi.Kuzungulira koyamba ndikukulitsa kozungulira kwa 15 mpaka 20.Gawo laling'ono lazinthu zokulitsa zoyambira zidachepetsedwa 100 mpaka 1000 ndikuwonjezeredwa kugawo lachiwiri la kukulitsa kwa mizere 15 mpaka 20.Kapenanso, chinthu choyambirira chokulitsa chimatha kukula ndi kuyeretsedwa kwa gel.Choyambira chokhazikika chimagwiritsidwa ntchito mugawo lachiwiri lakukulitsa, lomwe limatha kumangirira kutsata zomwe mukufuna mkati mwa choyambirira.Kugwiritsiridwa ntchito kwa PCR yokhala ndi zisa kumachepetsa kuthekera kwa kukulitsa kwa malo angapo omwe akuwunikidwa chifukwa pali mindandanda yocheperako yomwe ikugwirizana ndi ma seti onse awiri.Chiwerengero chofanana cha mizunguliro (30 mpaka 40) chokhala ndi zoyambira zomwezo zidakulitsa masamba osatchulika.Nested PCR imawonjezera kukhudzika kwa mindandanda yocheperako (mwachitsanzo, ma mrna osowa) ndikuwongolera kutsimikizika kwa PCRS yovuta (monga 5′ RACE).
12. Kutsika kwa PCR
Kutsika kwa PCR kumapangitsa kuti pakhale tsatanetsatane mwa kugwiritsa ntchito mikhalidwe yolimba pamagawo angapo oyamba a PCR.Kuzungulira kumayambira pa kutentha kwa annealing pafupifupi 5 ℃ kuposa momwe amaganizira Tm, kenako kuzungulira kulikonse kumachepetsedwa ndi 1 ℃ mpaka 2 ℃ mpaka kutentha kwa annealing kumakhala pansi pa Tm 5 ℃.Ndi template yopita yokha yomwe ili ndi homology yapamwamba kwambiri ndiyo idzakulitsidwa.Zogulitsazi zikupitilira kukula m'mizere yotsatila, kutsekereza zinthu zomwe sizinatchulidwe.Kutsika kwa PCR ndikothandiza panjira zomwe kuchuluka kwa homology pakati pa choyambirira ndi template yomwe mukufuna sikudziwika, monga kusindikiza zala za AFLP DNA.
Zogwirizana ndi PCR Kits
2 × PCR HeroTMMix system ili ndi kulolerana kwakukulu kwa PCR inhibitors kuposa njira wamba ya PCR Mix, ndipo imatha kuthana ndi kukulitsa kwa PCR kwama tempulo osiyanasiyana ovuta.Dongosolo lapadera la machitidwe a Taq Hero komanso kuchita bwino kwambiri kwa Taq Hero kumapangitsa kuti machitidwe a PCR akhale ndi magwiridwe antchito apamwamba, kutsimikizika komanso chidwi.
Kuchita bwino kwambiri kwa amplification
Ili ndi 5'→ 3' DNA polymerase ntchito ndi 5'→ 3' exonuclease ntchito, popanda 3'→ 5' ntchito exonuclease.
Real Time PCR Easyᵀᴹ-SYBR Green I Kit
Specific-optimized buffer ndi hot-start Taq enzyme imatha kuteteza kukulitsa kosakhazikika komanso mapangidwe a dimer.
Kumverera kwakukulu - kumatha kuzindikira makope otsika a template
RT-PCR Easyᵀᴹ I (Chigawo Chimodzi)
Zidazi zimagwiritsa ntchito makina apadera a Foregene reverse transcript reagent ndi Foregene HotStar Taq DNA Polymerase ophatikizidwa ndi njira yapadera yamachitidwe kuti apititse patsogolo kukulitsa bwino komanso momwe zimachitikira.
Nthawi yotumiza: May-09-2023
