Primer dhizaini hwaro (99% matambudziko anogona kugadziriswa)
1. Primer urefu: Bhuku rinoda 15-30bp, kazhinji inenge 20bp.Mamiriro chaiwo ari nani kuve 18-24bp kuti ave nechokwadi cheiyo chaiyo, asi iyo yakareba zviri nani, yakarebesa primer zvakare inoderedza iyo chaiyo, uye kuderedza goho.
2. Primer amplification span: 200-500bp yakakodzera, uye chidimbu chinogona kuwedzerwa kusvika ku10kb pasi pemamiriro ezvinhu chaiwo.
3. Primer base: Zvinyorwa zveG + C zvinofanira kuva 40-60%, zvishoma G + C amplification effect haina kunaka, yakawandisa G + C iri nyore kuoneka isiri-chaiyo mabhendi.ATGC inonyanya kugoverwa zvisina tsarukano, kudzivirira masumbu eanopfuura 5 purine kana pyrimidine nucleotides.Multi-gc ye5 ′ yekupedzisira uye yepakati sequences kuti uwedzere kugadzikana, dzivisa mupfumi GC pamagumo e3, hapana GC kune ekupedzisira 3 mabhesi, kana kwete GC ye3 yekupedzisira 5 mabhesi.
4. Dzivisa chimiro chechipiri mumaprimers, uye dzivirira kupindirana pakati pemaprimers maviri, kunyanya kuzadzisa pa 3 'kuguma, kana zvisina kudaro primer dimer ichaumbwa uye mabhendi asina-chaiyo amplified achagadzirwa.
5. Mabhesi pa3 'magumo ekutanga, kunyanya ekupedzisira uye peultimate mabhesi, anofanirwa kunyatsobatanidzwa kudzivirira PCR kutadza nekuda kwekusagadziriswa mabhesi ekupedzisira.
6. Maprimers ane kana anogona kuwedzerwa neakakodzera cleavage nzvimbo, uye amplified target sequence inofanira kunge iine yakakodzera cleavage nzvimbo, iyo inobatsira zvikuru pakuongorora cleavage kana molecular cloning.
7. Mamiriro ezvinhu ekutanga: maprimers haafaniri kuva nehomology yakajeka nemamwe maitiro mu nucleic acid sequence database.
8. Dzidza kushandisa software: PP5, Oligo6, DNAstar, Vector NTI, primer3 (Iyi purogiramu yepaIndaneti inoshanda zvakanakisisa).
Izvo zviri pamusoro apa zvinogona kugadzirisa inokwana 99% yeprimer dhizaini matambudziko.
Dzora zvinyorwa zvekutanga dhizaini
1. Kureba kwekutanga
General primer kureba ndeye 18 ~ 30 mabhesi.Kazhinji, chinhu chinonyanya kukosha chinotaridza tembiricha yeannealing yeprimer kureba kweprimer.Iyo annealing tembiricha ye primer inowanzo sarudzwa (Tm kukosha -5 ℃), uye vamwe vanoshandisa zvakananga Tm kukosha.Aya mafomula anotevera anogona kushandiswa kuverengera annealing tembiricha yeprimers.
Kana kureba kwekutanga kuri pasi pe20bp: [4(G+C)+2(A+T)] -5℃
Kana kureba kweprimer kwakakura kupfuura 20bp: 62.3℃+0.41℃(%GC) -500/kureba-5℃
Mukuwedzera, akawanda software anogonawo kushandiswa kuverenga annealing tembiricha, iyo calculation musimboti ichave yakasiyana, saka dzimwe nguva iyo yakaverengerwa kukosha inogona kuve nediki gap.Kugonesa maitiro ePCR, mapfupi maprimers ayo anovimbisa tembiricha yeannealing isingasviki 54 ℃ anoshandiswa pakuita zvakanaka uye hunyanzvi.
Pakazere, hunyanzvi hwekutanga hunowedzera nechikamu chechina kune imwe neimwe yekuwedzera nucleotide, zvekuti hurefu hwekutanga hwemaapplication mazhinji ndeye 18 nucleotides.Muganho wepamusoro wehurefu hweprimer hauna kunyanya kukosha, kunyanya zvine chekuita nekuita zvakanaka.Nekuda kwe entropy, iyo yakarebesa iyo primer, inodzikira mwero wainosungirira kusungirira kune inonangwa DNA kuti iumbe yakagadzikana ine tambo mbiri template yeDNA polymerase kuti isunge.
Paunenge uchishandisa software kugadzira maprimers, hurefu hwemaprimers hunogona kutariswa neTM kukosha kwacho, kunyanya kune maprimers efluorescence quantitative PCR, TM = 60 ℃ kana zvakadaro inofanira kudzorwa.
2.GC zvemukati
Kazhinji, zvirimo zveG+C mukutevedzana kwekutanga ndeye 40% ~ 60%, uye GC yemukati uye Tm kukosha kwepeya yekutanga kunofanirwa kurongeka.Kana iyo primer ine A yakakomba GC kana AT maitiro, yakakodzera huwandu hweA, T kana G uye C muswe hunogona kuwedzerwa ku5 'kuguma kwekutanga.
3. Annealing tembiricha
Iyo tembiricha yekudziya inofanira kunge iri 5 ℃ yakaderera pane unchain tembiricha.Kana huwandu hwemabhesi ekutanga ari diki, tembiricha yeannealing inogona kuwedzerwa zvakakodzera, izvo zvinogona kuwedzera kujeka kwePCR.Kana huwandu hwemabhesi hwakakura, tembiricha yeannealing inogona kuderedzwa zvakakodzera.Iwo annealing tembiricha musiyano pakati pemaprimers e4 ℃ ~ 6 ℃ haizokanganise goho rePCR, asi zvine mutsindo tembiricha yekudziya yepeya yeprimers yakafanana, inogona kusiyana pakati pe55℃ ~ 75 ℃.
4. Dzivisa chechipiri chimiro nharaunda yeamplification template
Zvakanakisisa kudzivirira chikamu chechipiri chechimiro che template paunosarudza chidimbu chakasimudzirwa.Iyo yakagadzikana yechipiri chimiro chechidimbu chakanangwa chinogona kufanotaurwa uye kufungidzirwa neakakodzera komputa software, iyo inobatsira pakusarudza template.Migumisiro yekuedza inoratidza kuti kuwedzera hakuwanzobudiriri kana simba remahara (△G) redunhu richawedzerwa riri pasi pe58.6lkJ/mol.
5. Kusapindirana nechinangwa cheDNA
Kana iyo amplified target DNA sequence yakakura, primer inogona kusunga kune dzakawanda zvikamu zveDNA yakanangwa, zvichiita kuti mabhendi akawanda aoneke mumhedzisiro.Panguva ino zvakakosha kushandisa iyo BLAST software yekuyedza, webhusaiti:http://www.ncbi.nlm.nih.gov/BLAST/.Sarudza Gadzirisa mitsetse miviri (bl2seq).
Kumisikidza kutevedzana kwekutanga kune zone 1 uye kunanga DNA kutevedzana kune zone 2 inochinjika, uye BLAST inoverengera inopindirana, antisense, uye zvimwe zvingaitika, saka vashandisi havafanire kuona kana cheni dzese dziri ngetani dzekunzwa.Iwe unogona zvakare kuisa iyo GI nhamba kana iwe uchiziva iyo GI nhamba yekutevedzana mudhatabhesi, saka haufanirwe kunamira chikamu chikuru chekutevedzana.Chekupedzisira, dzvanya Rongedza pa3 kuti uone kana iyo primer ine akawanda homologous saiti mune yakananga DNA.
6. Primer terminal
Iyo 3 'kuguma kweprimer ndiko kunotanga kuwedzera, saka zvakakosha kudzivirira kusawirirana kubva kutanga ipapo.Iyo 3 'kuguma haifanirwe kudarika 3 inoteedzana G kana C, nekuti izvi zvinokonzeresa kuti primer ikonzerese zvisizvo munharaunda yeG+C inoteedzana.Iyo 3 'kuguma haigone kuumba chero chechipiri chimiro, kunze kwekukosha kwePCR (AS-PCR) maitiro, iyo 3' yekupedzisira yeprimer haigone kufananidzwa.Semuenzaniso, kana iyo encoding dunhu yakakwidziridzwa, iyo 3 'kuguma kweprimer haifanirwe kugumiswa panzvimbo yechitatu yekodon, nekuti chinzvimbo chechitatu chekodon chinowanzo derera, izvo zvinokanganisa kujeka uye kugona kweamplification.Paunenge uchishandisa annexation primers, tarisa kune tafura yekushandisa kodon, teerera kune zvipenyu zvinodiwa, usashandise annexation primers pakupera kwe3′, uye shandisa yakakwira yakawanda yeprimers (1uM-3uM).
7. Sekondari chimiro chekutanga
Iwo maprimer pachawo haafanire kuve neanopindirana akateedzana, zvikasadaro maprimer pachawo anopeta kuita hairpin zvimiro, uye ichi chechipiri chimiro chinozokanganisa kusungirirwa kweprimers uye matemplate nekuda kwekuvhiringidza kwesteric.Kana kutonga kwekugadzira kuchishandiswa, zvigadziko zvinopindirana zvemaprimer pachazvo hazvifanirwe kunge zvakakura kupfuura 3bp.Hapafanirwe kuve nekuwirirana pakati pemaprimers maviri, kunyanya kupindirana kweiyo 3 'kuguma kunofanirwa kudzivirirwa kudzivirira kuumbwa kweprimer dimers.Kazhinji, hapafanirwe kuve neanopfuura mana akateedzana mabhesi ehomology kana kuwirirana pakati pemaprimers.
8. Wedzera zviratidzo kana loci
Iyo 5 'kuguma haina mhedzisiro pane yekuwedzera chaiyo uye inogona kugadziridzwa pasina kukanganisa amplification chaiyo.Kugadziriswa kweprimer 5 'kuguma kwaisanganisira: kuwedzera enzyme restriction site;Yakanyorwa biotin, fluorescence, digoxin, Eu3+, etc. Isai mapuroteni anosunga DNA sequences;Kuunza masaiti ekuchinja, kuisa uye kushayikwa kutevedzana kwekuchinja uye kuunza kutevedzana kwevanokurudzira, nezvimwewo. Mabhesi ekuwedzera anozokanganisa zvakanyanya kana kushoma kushanda kweamplification uye kuwedzera mukana wekuumbwa kweprimer dimer, asi zvimwe zvibvumirano zvinofanirwa kuitwa zvedanho rinotevera.Zvimwe zvakatevedzana zvisipo pane zvakatemerwa zvakatevedzana, senge nzvimbo dzekurambidza uye kutevedzana kwekusimudzira, zvinogona kuwedzerwa kune 5 ′ kuguma kweiyo primer pasina kukanganisa chaiyo.Aya akatevedzana haana kuverengerwa mukuverenga kweprimer Tm kukosha, asi inofanirwa kuongororwa kuti inoenderana uye yemukati yechipiri chimiro.
9. Subclones
Kazhinji yenguva, PCR inongori yekutanga cloning, uyezve isu tinoda kuisa pasi chidimbu chakatarwa mumavheji akasiyana, saka tinoda kugadzira mamwe mabhesi ekushanda kunotevera muPCR nhanho.
Mamwe akatevedzana akagadzirirwa subcloning akapfupikiswa pazasi.
Restriction endonuclease restriction site yakawedzerwa
Kuwedzera enzyme yekurambidza masaiti ndiyo inonyanya kushandiswa nzira ye subcloning PCR zvigadzirwa.Kazhinji, iyo cleavage saiti zvigadziko zvitanhatu, kuwedzera kune iyo 5 'kuguma kweiyo cleavage saiti inoda kuwedzera 2 ~ 3 mabhesi ekudzivirira.Nekudaro, huwandu hwemabhesi ekudzivirira anodiwa nema enzymes akasiyana akasiyana.Semuyenzaniso, SalⅠ haidi hwaro hwekudzivirira, EcoRⅤ inoda hwaro hwekudzivirira 1, KweteⅠ inoda mabhesi ekudzivirira maviri, uye Hind Ⅲ inoda mabhesi matatu ekudzivirira.
LIC inowedzera muswe
Iro zita rakazara reLIC iLigation-Yakazvimirira cloning, nzira yekugadzira yakaumbwa naNavogen yakanangana nechikamu chePet vector.Iyo pET inotakura inogadzirwa neLIC nzira ine isingaenderane 12-15 base single strand inonamira magumo, ayo anopindirana anonamira magumo pane chinongedzo chekuisa chidimbu.Nezvinangwa zvekusimudzira, iyo primer 5 ′ kutevedzana kwechimedu chakaiswa chinofanirwa kuenderana neLIC vector.Iyo 3′→5′ extranect activity yeT4 DNA polymerase inogona kuumba tambo imwe inonamira inoperera pachidimbu chakaiswa mushure menguva pfupi.Nokuti chigadzirwa chacho chinogona kuumbwa chete kubva kune pamwe chete kubatanidzwa kwechikamu chakagadzirirwa chekuisa uye vector, iyi nzira inokurumidza uye inoshanda, uye inotungamirirwa cloning.
Yakatungamirwa TA clone wedzera muswe
TA cloning haina kukwanisa kunanga chimedu muveta, saka gare gare Invitrogen yakaunza vheji yaigona kunanga cloning, iyo yaive neina yakakurumbira base GTGGS kune imwe magumo.Nokudaro, mukugadzirwa kwePCR primers, kuenzanisa kunopindirana kunofanira kuwedzerwa maererano, kuitira kuti zvidimbu zvinogona "kutungamirirwa".
Kana iwe uri mupfupi nenguva, unogona kuedza yakananga synthesis, kusanganisa jena nevector, inova yatinodaidza kuti ET gene synthesis mune musecularists.
D. In-Fusion cloning nzira
Hapana ligase inodiwa, hapana kuita kwenguva refu kunodiwa.Chero bedzi kutevedzana kumagumo maviri eiyo linearized vector inotangwa Mukugadzirwa kweprimers, ipapo PCR chigadzirwa uye linearized vector inowedzerwa mu-fusion enzyme mhinduro ine BSA uye inoiswa pakamuri tembiricha kwehafu yeawa, shanduko inogona kuitwa.Iyi nzira inonyanya kukodzera kushandurwa kwevhoriyamu huru.
10. Batanidza primer
Dzimwe nguva, ruzivo rushoma rwekutevedzana rwunozivikanwa nezve primer dhizaini.Semuenzaniso, kana chete amino acid sequence ichizivikanwa, iyo yekutanga yekubatanidza inogona kugadzirwa.Mubatanidzwa wekutanga musanganiswa wekutevedzana kwakasiyana unomiririra ese akasiyana base angave anokodha imwe amino acid.Kuti uwedzere kujeka, unogona kureva tafura yekushandisa kodon kuti uderedze kuwedzeredza zvinoenderana nekushandiswa kwepasi zvido zvezvipenyu zvakasiyana.Hypoxanthine inogona kupetwa nemabhesi ese ekudzikisa tembiricha yekudziya yeprimer.Usashandise mabhesi akabatanidzwa pa 3 ′ kumagumo ekutanga nekuti annealing ekupedzisira 3 mabhesi kumagumo e3 anokwana kuti atange PCR panzvimbo isiriyo.Yepamusoro primer concentrations (1μM kusvika 3μM) inoshandiswa nekuti maprimers mumisanganiswa yakawanda yeannexation haina kujeka kune yakananga template.
PCR mbishi zvinhucontrol
1. Primer uwandu
Iko kusungirirwa kwekutanga kwega kwega ndeye 0.1 ~ 1umol kana 10 ~ 100pmol.Zviri nani kuburitsa mhedzisiro inodiwa nehuwandu hwakaderera hweprimer.Kukwirisa kwepamusoro kweprimer kunokonzeresa kusawirirana uye kusiri-chaiyo amplification, uye kuwedzera mukana wekugadzira dimers pakati pekutanga.
2. Primer concentration
Iko kusungirirwa kwemaprimers kunokanganisa hunhu.Iyo yakakwana primer concentration inowanzo iri pakati pe0.1 uye 0.5μM.Yepamusoro primer concentrations inotungamira mukukwidziridzwa kwezvinhu zvisina kujeka.
3. Annealing tembiricha ye primer
Imwe paramende yakakosha yeprimers tembiricha yekunyungudika (Tm).Iyi ndiyo tembiricha apo 50% yemaprimers uye anowirirana akateedzana anomiririrwa seakapetwa kaviri-tambo dzeDNA mamorekuru.Tm inodiwa kuseta PCR annealing tembiricha.Sezvineiwo, tembiricha yekudzikisira yakadzikira zvakakwana kuti ive nechokwadi chekumisikidzwa kwemaprimers nekutevedzana kwakanangana, asi yakakwirira zvakakwana kudzikisa kusungirirwa kusingatarisike.Zvinonzwisisika annealing tembiricha kubva 55 ℃ kusvika 70 ℃.Annealing tembiricha inowanzosetwa 5 ℃ yakaderera pane Tm yeprimer.
Kune akati wandei mafomula ekuseta Tm, ayo anosiyana zvakanyanya zvichienderana neformula inoshandiswa uye kutevedzana kwekutanga.Nekuti akawanda mafomula anopa hunofungidzirwa Tm kukosha, ese annealing tembiricha ingori pekutanga.Kunyatsojeka kunogona kuvandudzwa nekuongorora maitiro akati wandei ayo anosimudza zvishoma nezvishoma tembiricha ye annealing.Tanga pazasi peinofungidzirwa Tm-5 ℃, uye zvishoma nezvishoma wowedzera tembiricha yekuisa mukuwedzera kwe2 ℃.Yepamusoro annealing tembiricha inoderedza kuumbwa kweprimer dimers uye zvisiri-zvakananga zvigadzirwa.Kuti uwane mhedzisiro yakanakisa, iwo maviri ekutanga anofanirwa kuve neakaenzana Tm kukosha.Kana mutsauko weTm wemaprimer pairs uchipfuura 5 ℃, maprimers acharatidza kutanga kwenhema nekushandisa tembiricha yakaderera mukutenderera.Kana maprimers maviri Tm akasiyana, isa tembiricha yekumisa kusvika 5℃ yakaderera pane yakaderera Tm.Neimwe nzira, kuti uwedzere kujeka, mitsetse mishanu inogona kuitwa kutanga pakudziya tembiricha dzakagadzirirwa kukwirira Tm, ichiteverwa neasara matenderedzwa pakudziya tembiricha akagadzirirwa yakaderera Tm.Izvi zvinobvumira chikamu chekopi yenzvimbo yekuenda template kuti iwanikwe pasi pemamiriro akaoma.
4. Primer kuchena uye kugadzikana
Iyo yakajairwa kuchena kweyakajairwa primers inokwana kune akawanda PCR maapplication.Kubviswa kwemapoka ebenzoyl neisobutylyl nekuita desalting kushoma saka hazvikanganise PCR.Mamwe maapplication anoda kucheneswa kuti abvise chero isiri-yakazara-kureba kutevedzana mune synthesis maitiro.Aya madiki akateedzana anoitika nekuti kugona kweDNA synthesis chemistry haisi 100%.Iyi idenderedzwa inoshandisa maitikiro emakemikari anodzokororwa sezvo hwaro hwega hwega hunowedzerwa kuita DNA kubva pa3′ kusvika pa5′.Unogona kukundikana mune chero kutenderera.Mapuranga marefu, kunyanya ayo anopfuura mabhesi makumi mashanu, ane chikamu chikuru chemitsetse yakatemwa uye inogona kuda kucheneswa.
Goho remaprimers rinokanganiswa nekubudirira kwesynthetic chemistry uye nzira yekuchenesa.Makambani eBiopharmaceutical, akadai seCytology neShengong, ese anoshandisa diki OD unit kuti ave nechokwadi chekubuda kweoligonucleoside.Tsika dzekutanga dzinotumirwa dzakaoma poda fomu.Zvakanakisa kunyungudusa zvakare maprimers muTE kuitira kuti iyo yekupedzisira yekumisikidza iri 100μM.TE iri nani pane mvura yakasvibiswa nokuti pH yemvura inowanzoita acidic uye inokonzera hydrolysis ye oligonucleosides.
Kugadzikana kweprimers kunoenderana nekuchengetedza mamiriro.Dry poda uye yakanyungudika primers inofanira kuchengetwa pa -20 ℃.MaPrimers akanyungudika muTE pamitsetse yakakura kupfuura 10μM anogona kuchengetwa zvakatsiga pa -20 ℃ kwemwedzi mitanhatu, asi anogona kuchengetwa pakamuri tembiricha (15 ℃ kusvika 30 ℃) isingasviki vhiki imwe.Dry poda primers inogona kuchengetwa pa -20 C kweinenge gore 1 uye pamhepo tembiricha (15 C kusvika 30 C) kusvika kumwedzi miviri.
5. Enzymes uye kunyanya kwavo
Parizvino, iyo Taq DNA polymerase inoshandiswa ndiyo gene engineering enzyme inogadzirwa necoliform bacteria.Huwandu hwe enzyme inodiwa kukonzeresa yakajairika PCR maitiro ingangoita 2.5U (inoreva huwandu hwekuita vhoriyamu ye100ul).Kana iyo yekuisa yakanyanya kuwanda, inogona kutungamirira kune isiri-yakananga amplification;kana iyo yakanyanyisa kuderera, huwandu hwechigadzirwa chekugadzira huchaderedzwa.
6. Hunhu uye kusungirirwa kwe dNTP
Hunhu hwe dNTP hune hukama hwepedyo nekudzika uye nekubudirira kwePCR amplification.Iyo dNTP powder ine granular, uye kusiyana kwayo kunorasikirwa nehupenyu hwehupenyu kana yakachengetwa zvisina kunaka.dNTP mhinduro ine acidic, uye inofanira kushandiswa muhutano hwepamusoro, ne 1M NaOH kana 1M Tris.HCL buffer solution yekugadzirisa PH yayo kusvika 7.0 ~ 7.5, shoma shoma yekuputira, kuchengetedzwa kwechando pa -20 ℃.Kuwanda kwechando-kunyunguduka kuchasvibisa dNTP.Mukuita kwePCR, dNTP inofanirwa kunge iri 50 ~ 200umol/L.Kunyanya, kutarisisa kunofanirwa kubhadharwa kune kusungwa kweiyo ina DNTPS inofanirwa kuenzana (yakaenzana mole kugadzirira).Kana kusanganiswa kwechimwe chazvo kwakasiyana kubva kune vamwe (yepamusoro kana pasi), kusawirirana kuchakonzerwa.Kunyanya kuderera kunoderedza goho rePCR zvigadzirwa.dNTP inogona kusanganisa neMg2+ uye kuderedza kuwanda kwemahara Mg2+.
7. Template (target gene) nucleic acid
Huwandu uye dhigirii rekucheneswa kwetemplate nucleic acid ndeimwe yeakakosha malink ekubudirira kana kutadza kwePCR.Nzira dzechinyakare dzekuchenesa DNA dzinowanzoshandisa SDS neprotease K kugaya nekurasa zvienzaniso.Mabasa makuru eSDS ndeaya: kunyungudutsa lipids uye mapuroteni pane membrane yesero, nokudaro kuparadza sero membrane nekunyungudutsa membrane mapuroteni, uye kuparadzanisa mapuroteni enyukireya muchitokisi, SDS inogonawo kusangana nemapuroteni uye precipitate;Protease K inogona hydrolyze uye kugaya mapuroteni, kunyanya histones akasungwa neDNA, uyezve kushandisa organic solvent phenol uye chloroform kuburitsa mapuroteni nezvimwe zvikamu zvesero, uye kushandisa ethanol kana isopropyl doro kuti riite nucleic acid.Iyo yakabviswa nucleic acid inogona kushandiswa se template yePCR maitiro.Kune zvakajairwa zvekuonekwa kwekiriniki, nzira inokurumidza uye iri nyore inogona kushandiswa kunyungudutsa maseru, lysate utachiona, kugaya uye kubvisa mapuroteni kubva kumakromosomes kuenda kune emahara chinangwa genes, uye yakananga kushandiswa kwePCR amplification.RNA template extraction inowanzoshandisa guanidine isothiocyanate kana protease K nzira kudzivirira RNase kubva pakusvibisa RNA.
8.Mg2+ concentration
Mg2 + ine mhedzisiro yakakosha pane chaiyo uye goho rePCR amplification.Muzhinji PCR maitiro, kana kusangana kweakasiyana dNTP kuri 200umol/L, iko kwakaringana kweMg2+ i1.5 ~ 2.0mmol/L.Mg2 + kusungirirwa kwakanyanyisa, maitiro ekuita anodzikira, kusiri-chaiyo amplification inoitika, yakanyanya kuderera inoderedza chiitiko cheTaq DNA polymerase, zvichikonzera kudzikiswa kwemaitiro ezvigadzirwa.
Magnesium ions inokanganisa maitiro akawanda ePCR, akadai seDNA polymerase basa, inokanganisa goho;Mumwe muenzaniso ndeye primer annealing, iyo inokanganisa chaiyo.dNTP uye template inosunga kune magnesium ion, ichidzikisa huwandu hwemahara magnesium ion inodiwa kuita enzyme.Iyo yakakwana magnesium ion concentration inosiyana kune akasiyana primer pairs uye templates, asi yakajairika PCR kutanga kukanda ne 200μM dNTP i1.5mM (chiziviso: Kune chaiyo-nguva quantitative PCR, shandisa 3 kusvika 5mM magnesium ion solution ine fluorescent probe).Yakakwira yakakwira yemahara magnesium ion inowedzera goho, asi zvakare inowedzera nonspecific amplification uye kuderedza kutendeka.Kuti uone kuwanda kwakanyanya, magnesium ion titrations yakaitwa mukuwedzera kwe0.5mM kubva 1mM kusvika 3mM.Kudzikisa kutsamira pane magnesium ion optimization, Platinum Taq DNA polymerase inogona kushandiswa.Platinum Taq DNA polymerase inokwanisa kuchengetedza basa pamusoro pehupamhi hwehuwandu hwemagesium ion concentrations kupfuura Taq DNA polymerase uye nekudaro inoda kushoma optimization.
9. Pcr-kukurudzira additives
Kukwidziridza tembiricha yekudziya, dhizaini yekutanga, uye magnesium ion concentration inokwana kune yakanyanya kukwidziridzwa yematemplate mazhinji;zvisinei, mamwe matemplate, kusanganisira ayo ane yakakwirira GC zvinyorwa, zvinoda mamwe matanho.Zviwedzere zvinokanganisa kupisa kwekunyunguduka kweDNA zvinopa imwe nzira yekuvandudza hunhu hwechigadzirwa uye goho.Denaturation yakazara yetemplate inodiwa kuti uwane mhedzisiro yakanaka.
Mukuwedzera, chimiro chechipiri chinodzivirira primer kusunga uye kuwedzera enzyme.
PCR additives, zvinosanganisira formamide, DMSO, glycerin, betaine, uye PCRx Enhancer Solution, inosimudzira kusimudzira.Nzira yavo inogoneka ndeyekudzikisa tembiricha yekunyunguduka, nekudaro kubatsira kukwenenzverwa kwemaprimers uye kubatsira DNA polymerase kuwedzera kuburikidza neyechipiri chimiro dunhu.PCRx Solution ine zvimwe zvakanakira.Minimal magnesium ion optimization inodiwa kana ichishandiswa nePlatinum Taq DNA polymerase uye Platinum Pfx DNA polymerase.Nekudaro, iyo Platinum tekinoroji inosanganiswa neyekuwedzera kuti iwedzere hunhu uku ichidzikisa kutsamira kwenzira yechitatu, magnesium ion optimization.Kuti uwane mhedzisiro yakanakisa, kuwanda kwezvinowedzera kunofanirwa kuvandudzwa, kunyanya DMSO, formamide, uye glycerol, iyo inhibit Taq DNA polymerase.
10. Kupisa kutanga
Kupisa kutanga PCR ndiyo imwe yedzakanyanya kukosha nzira dzekuvandudza PCR chaiyo mukuwedzera kune yakanaka primer dhizaini.Kunyangwe iyo yakakwana tembiricha yekureba yeTaq DNA polymerase iri 72 ℃, iyo polymerase inoramba ichishanda pakudziya kwekamuri.Saka, zvigadzirwa zvisiri-chaiyo zvinogadzirwa kana tembiricha yekubata yakadzikira pane tembiricha yeannealing panguva yekugadzirira kwePCR reaction uye pakutanga kwekudziya kutenderera.Kana zvangogadzirwa, zvigadzirwa zvisina tsarukano izvi zvinowedzerwa zvinobudirira.Kupisa-kutanga PCR inonyanya kushanda kana masaiti anoshandiswa pakugadzira dhizaini akaganhurirwa nenzvimbo yemajini zvinhu, senge saiti-inonangidzirwa shanduko, kutaura cloning, kana kuvaka uye kunyengera kwemajini zvinhu zvinoshandiswa kuDNA engineering.
Nzira yakajairika yekudzikamisa chiitiko cheTaq DNA polymerase kugadzirira PCR reaction solution paaizi uye kuiisa mune preheated PCR midziyo.Iyi nzira iri nyore uye isingadhuri, asi haipedzi basa re enzyme uye saka haibvisi zvachose kukwidziridzwa kwezvinhu zvisiri izvo.
Thermal priming inonotsa DNA synthesis nekuvharira chinhu chakakosha kusvika PCR apparatus yasvika denaturation tembiricha.Mazhinji manyorerwo ekupisa kwekutanga nzira, kusanganisira kunonoka kuwedzera kweTaq DNA polymerase, inonetsa, kunyanya kune yakakwira-kuburikidza maapplication.Dzimwe nzira dzekupisa dzinoshandisa nhovo yewakisi kuvharira chinhu chakakosha, kusanganisira magnesium ion kana maenzayimu, kana kuparadzanisa nemuviri zvinhu zvinobata basa, senge matemplate uye mabuffer.Munguva yekupisa, zvikamu zvakasiyana-siyana zvinosunungurwa uye zvinosanganiswa pamwe chete apo wakisi inonyunguduka.Semanyorero ekutanga kupisa nzira, nzira yewakisi shield inonetsa uye inotapukira uye haina kukodzera kushandiswa kwepamusoro.
Platinum DNA polymerase iri nyore uye inoshanda kune otomatiki inopisa kutanga PCR.Platinum Taq DNA polymerase ine recombinant Taq DNA polymerase yakasanganiswa ne monoclonal antibody inopesana neTaq DNA polymerase.Masoja ekudzivirira chirwere anogadzirwa nePCR kuvharidzira kuita kwe enzyme panguva yekubata tembiricha kwenguva refu.Taq DNA polymerase yakaburitswa mukuita panguva ye94 ℃ insulation ye denaturation nhanho, kudzoreredza yakazara polymerase chiitiko.Kusiyana nemakemikari akagadziridzwa Taq DNA polymerase yekutanga kupisa, Platinum enzyme haidi kuvharirwa kwenguva refu pa94 ℃ (10 kusvika 15 maminetsi) kuti ishandise iyo polymerase.NePlatinumTaq DNA polymerase, 90% yeTaq DNA polymerase chiitiko yakadzoserwa mushure memaminitsi maviri pa94 ℃.
11. Nest-PCR
Kutevedzana kutenderera kwekusimudzira uchishandisa nested primers kunogona kuvandudza hunhu uye kunzwa.Dunhu rekutanga nderekukwidziridzwa kwegumi nemashanu kusvika makumi maviri.Chikamu chidiki chechigadzirwa chekutanga chekusimudzira chakanatswa ka100 kusvika ku1000 uye chakawedzerwa kuchikamu chechipiri chekusimudzira kwegumi nemashanu kusvika makumi maviri.Neimwe nzira, iyo yekutanga yakakwidziridzwa chigadzirwa inogona kuyerwa nekucheneswa kwegel.A nested primer inoshandiswa muchikamu chechipiri chekusimudzira, chinogona kusunga kune chinangwa chakatevedzana mukati mekutanga kwekutanga.Iko kushandiswa kwePCR nested kunoderedza mukana wekukwidziridzwa kwemasaiti akawanda anotariswa nekuti kune mashoma anoteedzana anoteedzana anopindirana kune ese maviri seti ekutanga.Huwandu hwakafanana hwematenderedzwa (30 kusvika 40) ane maprimers akafanana akawedzera nzvimbo dzisina kujeka.Nested PCR inowedzera kunzwisiswa kwezvishoma zvinoteedzana zvinoteedzana (semuenzaniso, zvisingawanzo mrnas) uye inovandudza iyo chaiyo yePCRS yakaoma (semuenzaniso 5' RACE).
12. Kudzika PCR
Kudzika PCR kunonatsiridza hunhu nekushandisa yakasimba annealing mamiriro ekutanga mashoma ePCR.Kutenderera kunotanga pane tembiricha inodzika ingangoita 5 ℃ yakakwira kupfuura iyo inofungidzirwa Tm, ipapo kutenderera kwega kwega kunoderedzwa ne1 ℃ kusvika 2 ℃ kusvika tembiricha yekuvharira iri pasi peTm 5 ℃.Chete template yekuenda ine yakanyanya homology ndiyo inokwidziridzwa.Zvigadzirwa izvi zvinoramba zvichikura mumatenderedzwa anotevera, zvichivharira kunze zvakasimudzirwa zvisiri izvo.Kudzika PCR kunobatsira kune nzira uko dhigirii rehomology pakati peprimer uye tarisiro template isingazivikanwe, seAFLP DNA zvigunwe.
Inoenderana PCR Kits
Iyo 2 × PCR GambaTMMix system ine kushivirira kwepamusoro kune PCR inhibitors pane yakajairwa PCR Mix system, uye inogona kutarisana zviri nyore nePCR kukwidziridzwa kwematemplate akasiyana akaomarara.Iyo yakasarudzika maitiro sisitimu uye yakakwirira-inoshanda Taq Gamba inoita kuti PCR iite ive nepamusoro amplification kunyatsoita, kujeka uye kunzwa.
Kukwirisa kwepamusoro kushanda zvakanaka
Iine 5'→ 3' DNA polymerase chiitiko uye 5'→ 3' exonuclease chiitiko, isina 3'→ 5' exonuclease chiitiko.
Real Time PCR Easyᵀᴹ-SYBR Green I Kit
Yakananga-yakagadziridzwa buffer uye inopisa-kutanga Taq enzyme inogona kudzivirira isina-chaiyo amplification uye primer dimer kuumbwa.
Kunzwa kwakanyanya-inogona kuona yakaderera makopi etemplate
Iyo kit inoshandisa yakasarudzika yeForegene reverse transcription reagent uye Foregene HotStar Taq DNA Polymerase yakasanganiswa neyakasarudzika maitiro sisitimu kuti inyatso kunatsiridza kugona kwekusimudzira uye kujeka kwemaitiro.
Nguva yekutumira: Chivabvu-09-2023
