Isiseko soyilo sokuqala (iingxaki ezingama-99% zinokusonjululwa)
1. Ubude be-Primer: Incwadi yokufunda ifuna i-15-30bp, ngokuqhelekileyo malunga ne-20bp.Ubume bokwenene bungcono ukuba bube yi-18-24bp ukuze kuqinisekiswe ukuchaneka, kodwa ixesha elide elingcono, i-primer ende kakhulu iya kunciphisa ngokukodwa, kwaye inciphise isivuno.
2. I-Primer amplification span: 200-500bp ifanelekile, kwaye iqhekeza linokwandiswa ukuya kwi-10kb phantsi kweemeko ezithile.
3. Isiseko se-Primer: Umxholo we-G + C kufuneka ube ngu-40-60%, i-G + C encinci kakhulu impembelelo yokukhulisa i-amplification ayilungile, i-G + C eninzi kakhulu kulula ukuvela iibhendi ezingezizo ezodwa.I-ATGC ihanjiswa ngokufanelekileyo ngokungaqhelekanga, ukuphepha amaqoqo angaphezu kwe-5 purine okanye i-pyrimidine nucleotides.I-Multi-gc ye-5 'isiphelo kunye nokulandelelana okuphakathi ukwandisa ukuzinza, ukuphepha i-GC ecebileyo ekupheleni kwe-3, akukho GC yeziseko ezi-3 zokugqibela, okanye akukho GC ye-3 yeziseko ezi-5 zokugqibela.
4. Gwema ukwakhiwa kwesibini kwiiprimers, kwaye ugweme ukuhambelana phakathi kweeprimers ezimbini, ngakumbi ukudibanisa ekupheleni kwe-3, kungenjalo i-primer dimer iya kwenziwa kwaye i-non-specific amplified bands iya kuveliswa.
5. Iziseko eziku-3 'end of primers, ingakumbi iziseko zokugqibela kunye nezokugqibela, mazidityaniswe ngokungqongqo ukuthintela ukusilela kwePCR ngenxa yeziseko zetheminali ezingabhangiswanga.
6. Iiprimers zinako okanye zinokongezwa kunye neendawo ezifanelekileyo zokuqhawula, kwaye ulandelelwano lwethagethi olwandisiweyo kufuneka lube neendawo ezifanelekileyo zokuqhawula, eziluncedo kakhulu kuhlalutyo lokuqhawula okanye i-molecular cloning.
7. Ukuchaneka kwee-primers: ii-primers akufanele zibe ne-homology ecacileyo kunye nolunye ulandelelwano kwi-database yokulandelelana kwe-nucleic acid.
8. Funda ukusebenzisa isoftware: PP5, Oligo6, DNAstar, Vector NTI, primer3 (Olu luyilo lwe-intanethi lusebenza kakuhle).
Umxholo ongentla unokusombulula ubuncinane i-99% yeengxaki zokuyila i-primer.
Lawula iinkcukacha zoyilo lweprimer
1. Ubude be-Primer
Ubude be-primer ngokubanzi yi-18 ~ 30 iziseko.Ngokuqhelekileyo, eyona nto ibalulekileyo ekunqumeni ukushisa kwe-annealing ye-primer ubude be-primer.Ubushushu be-annealing ye-primer bukhethwa ngokubanzi (ixabiso le-Tm -5℃), kwaye abanye basebenzisa ngokuthe ngqo ixabiso le-Tm.Ezi fomyula zilandelayo zingasetyenziselwa ukubala ngokuthe ngqo ubushushu be-annealing ye-primers.
Xa ubude be-primer bungaphantsi kwe-20bp: [4(G+C)+2(A+T)] -5℃
Xa ubude be-primer bungaphezulu kwe-20bp: 62.3℃+0.41℃(%GC) -500/ubude-5℃
Ukongeza, isoftware eninzi ingasetyenziselwa ukubala ubushushu be-annealing, umgaqo wokubala uya kwahluka, ngoko ngamanye amaxesha ixabiso elibaliweyo linokuba nesithuba esincinci.Ukwandisa ukuphendula kwe-PCR, ezona zixhobo zimfutshane ziqinisekisa amaqondo obushushu angekho ngaphantsi kwe-54℃ zisetyenziselwa eyona mpumelelo kunye neenkcukacha.
Ngokubanzi, i-primer specificity inyuka nge-factor of four kwi-nucleotide nganye eyongezelelweyo, ukwenzela ukuba ubude obuncinci be-primer kwizicelo ezininzi buyi-18 nucleotides.Umda ophezulu wobude be-primer awubalulekanga kakhulu, ngokukodwa unxulumene nokusebenza kakuhle kokuphendula.Ngenxa ye-entropy, ixesha elide le-primer, lisezantsi izinga apho lidibanisa khona ukubopha kwi-DNA ekujoliswe kuyo ukwenza itemplate ezinzileyo ephindwe kabini ye-DNA polymerase ukuze ibophe.
Xa usebenzisa isoftware ukuyila iiprimers, ubude beeprimers bunokumiselwa ngexabiso leTM ngokulandelelana, ngakumbi kwiprimers ye-fluorescence quantitative PCR, TM=60℃ okanye ke kufuneka ilawulwe.
2.GC umxholo
Ngokuqhelekileyo, umxholo we-G + C ngokulandelelana kwe-primer yi-40% ~ 60%, kunye nomxholo we-GC kunye nexabiso le-Tm leebini zeeprimers kufuneka zilungelelaniswe.Ukuba i-primer ine-GC enzulu okanye i-AT tendency, isixa esifanelekileyo se-A, T okanye i-G kunye nomsila we-C unokongezwa kwi-5 'end of the primer.
3. Ubushushu bokutshisa
Ubushushu be-anneal kufuneka bube 5℃ ngaphantsi kwe-unchain.Ukuba inani leziseko ze-primer lincinci, ukushisa kwe-annealing kunokunyuswa ngokufanelekileyo, okunokunyusa ngokukodwa kwe-PCR.Ukuba inani leziseko likhulu, ukushisa kwe-annealing kungancitshiswa ngokufanelekileyo.Umahluko weqondo lobushushu phakathi kwepere ye-4℃ ~ 6℃ awuyi kuchaphazela isivuno se-PCR, kodwa ngokufanelekileyo iqondo lobushushu le-annealing yeperi yeprimers liyafana, elinokwahluka phakathi kwe-55℃ ~ 75℃.
4. Gwema indawo yesibini yesakhiwo setemplate yokukhulisa
Kungcono ukuphepha ummandla wesakhiwo sesibini se template xa ukhetha iqhekeza elikhulisiwe.Isakhiwo sesibini esizinzileyo seqhekeza ekujoliswe kulo sinokuqikelelwa kwaye siqikelelwe yi-software efanelekileyo yekhompyutheni, eluncedo ekukhethweni kwetemplate.Iziphumo zovavanyo zibonisa ukuba ulwandiso alusoloko luphumelela xa amandla asimahla (△G) ommandla oza kwandiswa engaphantsi kwe-58.6lkJ/mol.
5. Ukungafani ne-DNA ekujoliswe kuyo
Xa ukulandelelana kwe-DNA ekujoliswe kuyo kukhulu, i-primer inokubophelela kwiindawo ezininzi ze-DNA ekujoliswe kuyo, okubangela ukuba kubekho amaqela amaninzi avela kwisiphumo.Ngeli xesha kuyafuneka ukuba usebenzise uvavanyo lwesoftware yeBLAST, iwebhusayithi:http://www.ncbi.nlm.nih.gov/BLAST/.Khetha Lungisa ulandelelwano ezimbini (bl2seq).
Ukuncamathelisa ulandelelwano lwe-primer kwindawo yoku-1 kunye nolandelelwano lwe-DNA ekujoliswe kulo ukuya kwindawo yesi-2 luyatshintsheka, kwaye i-BLAST ibala ukuhambelana, i-antisense, kunye nezinye izinto ezinokwenzeka, ukuze abasebenzisi baqaphele ukuba omabini amatyathanga ngamatyathanga engqiqo.Unako kwakhona ukufaka inombolo ye-GI ukuba uyayazi inani le-GI lolandelelwano kwisiseko sedatha, ukuze ungancamathiseli icandelo elikhulu lolandelelwano.Ekugqibeleni, cofa u-Lungelelanisa ku-3 ukubona ukuba i-primer ineendawo ezininzi ze-homologous kwi-DNA ekujoliswe kuyo.
6. Itheminali yokuqala
I-3 'isiphelo se-primer kulapho ulwandiso luqala khona, ngoko ke kubalulekile ukuthintela ukungahambelani ukusuka apho.Isi-3 'isiphelo akufunekanga sibe ngaphezu kwe-3 elandelelanayo G okanye C, kuba oku kuya kubangela ukuba i-primer iqaliswe ngempazamo kwingingqi ye-G+C yolandelelwano.I-3 'isiphelo ayikwazi ukwenza naluphi na ulwakhiwo lwesibini, ngaphandle kwe-PCR ekhethekileyo (AS-PCR) reactions, i-3′ isiphelo se-primer ayikwazi ukuchasana.Ngokomzekelo, ukuba ummandla we-encoding ukhulisiwe, i-3 'isiphelo se-primer akufanele sipheliswe kwindawo yesithathu ye-codon, kuba indawo yesithathu ye-codon ilungele ukuhla, eya kuchaphazela ukuchaneka kunye nokusebenza kakuhle kokukhulisa.Xa usebenzisa ii-primers ze-annexation, bhekisa kwitheyibhile yokusetyenziswa kwe-codon, qwa lasela ingqalelo kwizinto eziphilayo, ungasebenzisi i-annexation primers ekupheleni kwe-3, kwaye usebenzise i-concentration ephezulu ye-primers (1uM-3uM).
7. Ubume besibini bee-primers
Ii-primers ngokwazo akufanele zibe nokulandelelana okuhambelanayo, ngaphandle koko ii-primers ngokwazo ziya kugoqa kwizakhiwo ze-hairpin, kwaye esi sakhiwo sesibini siya kuchaphazela ukubotshwa kwee-primers kunye neetemplates ngenxa yokuphazamiseka kwe-steric.Ukuba isigwebo sokwenziwa sisetyenzisiweyo, iziseko ezihambelanayo eziqhubekayo ze-primers ngokwabo akufanele zibe ngaphezu kwe-3bp.Akufuneki kubekho ukuhambelana phakathi kwee-primers ezimbini, ngakumbi ukudibanisa okuhambelanayo kwe-3 'isiphelo kufuneka kuthintelwe ukuthintela ukubunjwa kwe-primer dimers.Ngokubanzi, akufuneki kubekho ngaphezu kwe-4 iziseko ezilandelelanayo ze-homology okanye ukuhambelana phakathi kwepere ye-primers.
8. Yongeza iziphawuli okanye i-loci
Isiphelo sesi-5 sinesiphumo esincinci kwingcaciso yokukhulisa kwaye ke ngoko sinokulungiswa ngaphandle kokuchaphazela ukucaciswa kokukhulisa.Ukuguqulwa kwe-primer 5 'isiphelo kubandakanywa: ukongeza indawo yokuthintela i-enzyme;Ibhalwe nge-biotin, i-fluorescence, i-digoxin, i-Eu3+, njl.Ukwazisa amasayithi okuguqula, ukufaka kunye nokungabikho kweendlela zokuguqula ukuguqulwa kunye nokwazisa ukulandelelana komgqugquzeli, njl.Ukulandelelana okongeziweyo okungekho kulandelelwano olujoliswe kuyo, njengendawo yokuthintela kunye nokulandelelanisa umgqugquzeli, kunokongezwa kwi-5' ekupheleni kwe-primer ngaphandle kokuchaphazela ukuchaneka.Olu landelelwano alufakwanga ekubaleni ixabiso le-primer Tm, kodwa kufuneka livavanywe ukuhambelana kunye nesakhiwo sesibini sangaphakathi.
9. Iisubclones
Amaxesha amaninzi, i-PCR yi-cloning yokuqala kuphela, kwaye ke kufuneka sithobe isiqwenga ekujoliswe kuso kwii-vectors ezahlukeneyo, ngoko ke kufuneka siyile iziseko ezongezelelweyo zomsebenzi olandelayo kwinyathelo le-PCR.
Olunye ulandelelwano oluyilelwe i-subcloning lushwankathelwa ngezantsi.
Indawo yothintelo lwe-endonuclease yongezwa
Ukongeza iisayithi zothintelo lwe-enzyme yeyona ndlela isetyenziswa kakhulu kwi-subcloning yemveliso ye-PCR.Ngokuqhelekileyo, indawo yokuqhawula iziseko ezithandathu, ukongeza kwi-5 'isiphelo sendawo yokuqhawula idinga ukongeza i-2 ~ 3 iziseko zokukhusela.Nangona kunjalo, inani leziseko zokukhusela ezifunwa yi-enzymes ezahlukeneyo zahlukile.Umzekelo, i-SalⅠ ayifuni siseko sokhuselo, i-EcoRⅤ idinga isiseko esi-1 sokukhusela, i-NotⅠ idinga iziseko ezi-2 zokukhusela, kunye ne-Hind Ⅲ idinga iziseko ezi-3 zokukhusela.
I-LIC yongeza umsila
Igama elipheleleyo le-LIC yi-Ligation-Independent cloning, i-cloning method eyaqanjwa ngu-Navogen ngokukodwa inxalenye yayo ye-PET vector.I-peT carrier elungiswe yi-LIC method ine-non-complementary 12-15 yesiseko se-single strand sticky ends, encedisa iziphelo ezincamathelayo ezihambelanayo kwiqhekeza lokufaka ithagethi.Ngeenjongo zokukhulisa, i-primer 5′ ukulandelelana kweqhekeza elifakiweyo kufuneka lincedise i-LIC vector.I-3′→5′ extranect activity ye-T4 DNA polymerase inokwenza isiphelo esincangathi esinye kwisiqwenga esifakiweyo emva kwexesha elifutshane.Ngenxa yokuba imveliso inokuqulunqwa kuphela kwi-annealing edibeneyo yesiqhekeza esilungisiweyo sokufaka kunye ne-vector, le ndlela ikhawuleza kakhulu kwaye iyasebenza, kwaye iqondiswe kwi-cloning.
Iqondiswe TA clone yongeza umsila
I-TA cloning ayikwazanga ukujolisa kwisiqwenga kwi-vector, ngoko ke kamva i-Invitrogen yazisa i-vector enokuthi ijolise kwi-cloning, equlathe iziseko ezine ezibalaseleyo ze-GTGGS kwelinye icala.Ngoko ke, ekuyilweni kwee-primers ze-PCR, ukulandelelana okuhambelanayo kufuneka kufakwe ngokufanelekileyo, ukwenzela ukuba iziqhekeza zibe "zijoliswe".
Ukuba ufutshane ngexesha, ungazama ukudibanisa ngokuthe ngqo, ukudibanisa i-gene kunye ne-vector, into esiyibiza ngokuba yi-ET gene synthesis kwi-musecularists.
D. In-Fusion cloning indlela
Akukho ligase efunekayo, akukho mpendulo ende efunekayo.Ngethuba nje ulandelelwano kuzo zombini iziphelo ze-vector yomgca kuqaliswa Ekuqulunqweni kwee-primers, ngoko imveliso ye-PCR kunye ne-vector ye-linearized yongezwa kwisisombululo se-enzyme ye-in-fusion equkethe i-BSA kwaye ibekwe kwindawo yokushisa kwesiqingatha seyure, ukuguqulwa kunokwenziwa.Le ndlela ifanelekile ngokukodwa ukuguqulwa kwevolumu enkulu.
10. Hlanganisa i-primer
Ngamanye amaxesha, ulwazi olulinganiselweyo lolandelelwano lwaziwayo malunga noyilo lwe-primer.Ngokomzekelo, ukuba kuphela ulandelelwano lwe-amino acid luyaziwa, i-primer yokudibanisa inokuqulunqwa.I-primer yokudibanisa ngumxube wolandelelwano olwahlukileyo olumele zonke iziseko ezinokwenzeka ezahlukeneyo ezifaka i-amino acid enye.Ukwandisa ukucaciswa, ungabhekisa kwitheyibhile yokusetyenziswa kwecodon ukunciphisa isihlomelo ngokwesiseko sokusetyenziswa kwezinto eziphilayo ezahlukeneyo.I-Hypoxanthine inokudityaniswa nazo zonke iziseko zokunciphisa ubushushu be-anneal ye-primer.Musa ukusebenzisa iziseko ezihlonyelweyo kwi-3′ ekupheleni kwe-primer kuba ukuhlanjululwa kweziseko ezi-3 zokugqibela kwi-3′ ekupheleni kwanele ukuqalisa i-PCR kwindawo engafanelekanga.Ugxininiso oluphezulu lwe-primer (1μM ukuya kwi-3μM) luyasetyenziswa kuba iiprimers kwimixube yesihlomelo emininzi ayingqalanga kwithemplethi ekujoliswe kuyo.
PCR imathiriyeli ekrwadaulawulo
1. Ubungakanani bokuqala
Ukugxininiswa kwe-primer nganye yi-0.1 ~ 1umol okanye i-10 ~ 100pmol.Kungcono ukuvelisa umphumo ofunekayo kunye nenani eliphantsi le-primer.Ugxininiso oluphezulu lwe-primer luya kubangela ukungafani kunye nokungacaciswanga kwe-amplification, kunye nokwandisa ithuba lokwenza i-dimers phakathi kwe-primers.
2. I-Primer concentration
Ukuxinwa kwee-primers kuchaphazela ngokukodwa.Ugxininiso lwe-primer olulungileyo luphakathi kwe-0.1 kunye ne-0.5μM.Ugxininiso oluphezulu lwe-primer lukhokelela ekwandisweni kweemveliso ezingachazwanga.
3. Ukushisa kwe-anealing ye-primer
Enye iparameter ebalulekileyo ye-primers yiqondo lokushisa elinyibilikayo (Tm).Eli liqondo lokushisa xa i-50% ye-primers kunye nolandelelwano oluncedisayo lumelwe njengeemolekyuli ze-DNA eziphindwe kabini.Tm iyafuneka ukuseta iqondo lobushushu le-PCR.Ngokunqwenelekayo, iqondo lobushushu le-anneal lisezantsi ngokwaneleyo ukuze kuqinisekiswe ukubanjwa okusebenzayo kweeprimers ngolandelelwano olujoliswe kuko, kodwa liphezulu ngokwaneleyo ukunciphisa ukubophelela okungangxengwanga.Iqondo lobushushu elifanelekileyo le-anneal ukusuka kwi-55℃ ukuya kwi-70℃.Ubushushu be-anealing ngokuqhelekileyo busetwa nge-5℃ ngaphantsi kwe-Tm ye-primer.
Kukho iifomyula ezininzi zokuseta i-Tm, eyahluka kakhulu ngokuxhomekeke kwifomula esetyenzisiweyo kunye nokulandelelana kwee-primers.Ngenxa yokuba uninzi lweefomula zibonelela ngexabiso eliqikelelweyo le-Tm, onke amaqondo obushushu okubamba sisiqalo nje.Ukuchaneka kungaphuculwa ngokuhlalutya iimpendulo ezininzi ezithi ngokuthe ngcembe zinyuse amaqondo obushushu be-anneal.Qala ngaphantsi koqikelelo lwe-Tm-5℃, kwaye kancinci kancinci unyuse iqondo lobushushu le-anneal ngokunyuka kwe-2℃.Ukushisa okuphezulu kwe-annealing kuya kunciphisa ukubunjwa kwe-primer dimers kunye neemveliso ezingezizo ezodwa.Ukufumana iziphumo ezingcono, iiprimer ezimbini kufuneka zibe namaxabiso aqikelelwayo e-Tm.Ukuba umahluko we-Tm we-primer pairs ungaphezulu kwe-5 ℃, iiprimers ziya kubonisa isiqalo esingeyonyani esibalulekileyo ngokusebenzisa iqondo lobushushu elisezantsi le-annealing kumjikelo.Ukuba iiprimers ezimbini ze-Tm zahlukile, seta ubushushu be-anneal ukuya kwi-5℃ ngaphantsi kweyona Tm isezantsi.Kungenjalo, ukunyusa okuthe ngqo, imijikelo emihlanu inokwenziwa kuqala kumaqondo obushushu obuyilelwe i-Tm ephezulu, ilandelwe yimijikelo eseleyo kumaqondo obushushu obuyilelwe i-Tm esezantsi.Oku kuvumela ikopi eyinxenye yetemplate yendawo ekuyiwa kuyo ukuba ifunyanwe phantsi kweemeko ezixineneyo.
4. Ukucoceka kokuqala kunye nokuzinza
Ukucoceka okusemgangathweni kwee-primers zesiko kwanele kwizicelo ezininzi ze-PCR.Ukususwa kwamaqela e-benzoyl kunye ne-isobutylyl ngokukhupha ityuwa kuncinci kwaye ngoko akuphazamisi i-PCR.Ezinye izicelo zifuna ukuhlanjululwa ukuze kususwe naluphi na ulandelelwano olungenabude obupheleleyo kwinkqubo yokudibanisa.Olu landelelwano lucuthiweyo lwenzeka ngenxa yokuba ukusebenza kakuhle kwe-DNA synthesis chemistry ayikho i-100%.Le yinkqubo yesetyhula esebenzisa ukuphindaphinda kweekhemikhali njengoko isiseko ngasinye songezwa ukwenza i-DNA ukusuka kwi-3 ukuya kwi-5.Ungasilela kuwo nawuphi umjikelo.Iiprimers ezinde, ngakumbi ezo zingaphezulu kweziseko ezingama-50, zinenxalenye enkulu yolandelelwano olucuthiweyo kwaye zinokufuna ukucocwa.
Isivuno se-primers sichaphazeleka ngokusebenza kakuhle kwe-synthetic chemistry kunye nendlela yokucoca.Iinkampani ze-Biopharmaceutical, ezifana ne-Cytology kunye ne-Shengong, zonke zisebenzisa ubuncinci beyunithi ye-OD ukuqinisekisa ukukhutshwa okupheleleyo kwe-oligonucleoside.Iiprimers eziqhelekileyo zithunyelwa kwifom eyomileyo yomgubo.Kungcono ukuphinda uchithe i-primers kwi-TE ukwenzela ukuba ugxininiso lokugqibela lube yi-100μM.I-TE ingcono kunamanzi adibeneyo ngenxa yokuba i-pH yamanzi ihlala i-acidic kwaye iya kubangela i-hydrolysis ye-oligonucleosides.
Ukuzinza kwee-primers kuxhomekeke kwiimeko zokugcina.Umgubo owomileyo kunye neeprimers ezinyibilikisiweyo kufuneka zigcinwe ku -20 ℃.Iiprimers ezinyityilisiweyo kwi-TE kugxininiso olukhulu kune-10μM zinokugcinwa ngokuzinzileyo kwi -20℃ kangangeenyanga ezi-6, kodwa zinokugcinwa kuphela kwiqondo lobushushu legumbi (15℃ ukuya kwi-30℃) ngaphantsi kweveki enye.I-primers ye powder eyomileyo ingagcinwa kwi--20 C ubuncinane ubuncinane be-1 unyaka kunye neqondo lokushisa (15 C ukuya ku-30 C) ukuya kwiinyanga ezi-2.
5. Ii-Enzymes kunye nokugxilwa kwazo
Okwangoku, i-polymerase ye-Taq DNA esetyenziswayo ngokusisiseko yi-enzayim yobunjineli yemfuza eyenziwe yi-coliform bacteria.Ubungakanani be-enzyme efunekayo ukuze i-catalyze reaction ye-PCR eqhelekileyo imalunga ne-2.5U (ibhekisela kumthamo opheleleyo wokusabela we-100ul).Ukuba ugxininiso luphezulu kakhulu, kunokukhokelela ekukhuliseni okungaqhelekanga;ukuba ugxininiso luphantsi kakhulu, inani lemveliso yokwenziwa liya kuncitshiswa.
6. Umgangatho kunye nogxininiso lwe-dNTP
Umgangatho we-dNTP unxulumene ngokusondeleyo nogxininiso kunye nokusebenza kakuhle kwe-PCR yokukhulisa.I-powder ye-dNTP i-granular, kwaye ukuhluka kwayo kulahlekelwa ngumsebenzi wayo we-biological ukuba igcinwe ngendlela engafanelekanga.Isisombululo se-dNTP si-acidic, kwaye kufuneka sisetyenziswe kwi-concentration ephezulu, kunye ne-1M NaOH okanye i-1M Tris.HCL isisombululo se-buffer ukulungelelanisa i-PH yayo kwi-7.0 ~ 7.5, inani elincinci lokupakisha, ukugcinwa kwefriji kwi -20 ℃.Ukunyibilikiswa komkhenkce okuninzi kuya kuthoba umgangatho we-dNTP.Kwimpendulo ye-PCR, i-dNTP kufuneka ibe yi-50 ~ 200umol/L.Ngokukodwa, ingqwalasela kufuneka ihlawulwe kugxininiso lwee-DNTPS ezine kufuneka zilingane (ukulungiswa kwe-mole).Ukuba ukuxinana kwayo nayiphi na enye kuzo kwahlukile kwezinye (eziphezulu okanye ezisezantsi), ukungafani kuya kubangelwa.Ukugxininiswa okuphantsi kakhulu kuya kunciphisa isivuno seemveliso ze-PCR.I-dNTP inokudibanisa ne-Mg2 + kwaye inciphise ukuxinwa kwe-Mg2 + yamahhala.
7. Isifanekiso (ijene ekujoliswe kuyo) i-nucleic acid
Isixa kunye neqondo lokuhlanjululwa kwe-template nucleic acid yenye yekhonkco eziphambili zokuphumelela okanye ukungaphumeleli kwe-PCR.Iindlela zemveli zokuhlanjululwa kwe-DNA zihlala zisebenzisa i-SDS kunye ne-protease K ukucola nokulahla imizekelo.Imisebenzi ephambili ye-SDS yile: ukunyibilikisa i-lipids kunye neeprotheni kwi-membrane yeseli, ngaloo ndlela utshabalalisa i-membrane yeseli ngokunyibilikisa iiprotheni ze-membrane, kwaye uhlukanise iiproteni zenyukliya kwiseli, i-SDS inokudibanisa neeproteni kunye ne-precipitate;I-Protease K inokwenza i-hydrolyze kwaye igaye iiprotheni, ngakumbi i-histones eboshwe nge-DNA, kwaye emva koko isebenzise i-organic solvent phenol kunye ne-chloroform ukukhupha iiprotheni kunye nezinye iiseli zeseli, kwaye isebenzise i-ethanol okanye i-isopropyl alcohol ukukhupha i-nucleic acid.I-asidi ye-nucleic ekhutshiweyo ingasetyenziswa njenge template ye-PCR reactions.Kwimizekelo yovavanyo lweklinikhi jikelele, indlela ekhawulezayo nelula ingasetyenziselwa ukunyibilikisa iiseli, i-lysate pathogens, ukugaya kunye nokususa iiprotheni kwiichromosomes ukuya kwiijene ezijoliswe kuzo, kwaye zisetyenziswe ngokuthe ngqo kwi-PCR yokukhulisa.I-RNA template extraction idla ngokusebenzisa i-guanidine isothiocyanate okanye indlela ye-protease K ukuthintela i-RNase ekuthobeni i-RNA.
I-8.Mg2 + yoxinaniso
I-Mg2+ inefuthe elibalulekileyo kwi-speciality kunye nesivuno sokwandiswa kwe-PCR.Ngokubanzi ukusabela kwe-PCR, xa uxinaniso lwee-dNTP ezahlukeneyo luyi-200umol/L, i-concentration efanelekileyo ye-Mg2+ yi-1.5 ~ 2.0mmol/L.Ugxininiso lwe-Mg2+ luphezulu kakhulu, ukuchaneka kokusabela kuyancipha, ukukhulisa okungangcaciswanga kwenzeka, ukugxininiswa okuphantsi kakhulu kuya kunciphisa umsebenzi we-Taq DNA polymerase, okukhokelela ekunciphiseni kweemveliso zokusabela.
I-Magnesium ion ichaphazela iinkalo ezininzi ze-PCR, ezifana nomsebenzi we-DNA polymerase, echaphazela isivuno;Omnye umzekelo yi-primer annealing, echaphazela ukucaciswa.I-dNTP kunye ne-template ibophelela kwi-ion ye-magnesium, ukunciphisa inani le-ion ye-magnesium yamahhala efunekayo kumsebenzi we-enzyme.Ugxininiso lwe-ion ye-magnesium elona lufanelekileyo luyahlukahluka kwiiperi ezahlukeneyo ze-primer kunye neetemplates, kodwa i-PCR eqhelekileyo iqala ukugxila kunye ne-200μM dNTP yi-1.5mM (qaphela: Kwi-PCR yexesha langempela, sebenzisa i-3 ukuya kwi-5mM isisombululo se-ion ye-magnesium kunye ne-probe ye-fluorescent).Uxinzelelo oluphezulu lwee-ion ze-magnesium zasimahla zonyusa isivuno, kodwa zikwanyusa ukukhulisa okungacaciswanga kunye nokunciphisa ukuthembeka.Ukumisela ukugxininiswa okuphezulu, i-magnesium ion titrations yenziwa ngokunyuka kwe-0.5mM ukusuka kwi-1mM ukuya kwi-3mM.Ukunciphisa ukuxhomekeka kwi-magnesium ion optimization, iPlatinum Taq DNA polymerase ingasetyenziswa.I-Platinum Taq DNA polymerase iyakwazi ukugcina umsebenzi phezu koluhlu olubanzi lwe-magnesium ion concentrations kune-Taq DNA polymerase kwaye ngoko idinga ukulungiswa okuncinci.
9. Izongezo zokukhuthaza iPCr
Ukuphucula ubushushu bokufudumala, uyilo lwe-primer, kunye noxinzelelo lwe-ion ye-magnesium kwanele ukukhulisa ngokuthe ngqo uninzi lweetemplates;nangona kunjalo, ezinye iitemplates, kubandakanywa nezo zinomxholo ophezulu weGC, zifuna imilinganiselo eyongezelelweyo.Izongezo ezichaphazela ukushisa okunyibilikayo kwe-DNA zinika enye indlela yokuphucula ukucaciswa kwemveliso kunye nesivuno.Ukuchazwa ngokupheleleyo kwethemplethi kuyafuneka ngeziphumo ezigqwesileyo.
Ukongezelela, isakhiwo sesibini sithintela ukubopha i-primer kunye nokwandiswa kwe-enzyme.
Izongezo ze-PCR, ezibandakanya i-formamide, i-DMSO, i-glycerin, i-betaine, kunye ne-PCRx Enhancer Solution, iphucula ukukhulisa.Indlela yabo enokwenzeka kukunciphisa ubushushu obunyibilikayo, ngaloo ndlela inceda ukufakwa kweeprimers kunye nokuncedisa ukwandiswa kwe-DNA polymerase ngommandla wesakhiwo sesibini.Isisombululo sePCRx sinezinye iingenelo.Ukulungiswa kwe-ion ye-magnesium encinci iyafuneka xa isetyenziswe kunye nePlatinum Taq DNA polymerase kunye nePlatinum Pfx DNA polymerase.Ke, ubuchule bePlatinam budityaniswe kunye nesongezo sokwandisa ukucaciswa ngelixa kunciphisa ukuxhomekeka kwendlela yesithathu, ukulungiswa kwe-ion ye-magnesium.Ukufumana iziphumo ezilungileyo, ukuxinwa kwezongezo kufuneka kuphuculwe, ngakumbi i-DMSO, i-formamide, kunye ne-glycerol, evimbela i-Taq DNA polymerase.
10. Ukuqala okushushu
Isiqalo esishushu iPCR yenye yezona ndlela zibalulekileyo zokuphucula ukucaciswa kwePCR ukongeza kuyilo olulungileyo lweprimer.Nangona elona qondo lobushushu lobude be-Taq DNA polymerase yi-72℃, i-polymerase ihlala isebenza kwiqondo lobushushu begumbi.Ngaloo ndlela, iimveliso ezingezizo ezodwa ziveliswa xa ubushushu bokubamba buphantsi kunobushushu be-annealing ngexesha lokulungiswa kwe-PCR reaction kwaye ekuqaleni komjikelezo we-thermal.Nje ukuba zenziwe, ezi mveliso zingachazwanga ziye zandiswa ngokufanelekileyo.I-PCR yokuqala eshushu isebenza ngokukodwa xa iisayithi ezisetyenziselwa ukuyilwa kweprimer zithintelwe yindawo yezakhi zemfuza, ezinje ngotshintsho oluqondiswe kwindawo, i-expression cloning, okanye ukwakhiwa kunye nokuguqulwa kwezinto zemfuzo ezisetyenziselwa ubunjineli be-DNA.
Indlela eqhelekileyo yokunciphisa umsebenzi we-Taq DNA polymerase kukulungisa isisombululo se-PCR yokusabela kumkhenkce kwaye usibeke kwi-PCR eshushu ngaphambili.Le ndlela ilula kwaye ayibizi, kodwa ayiwugqibi umsebenzi we-enzyme kwaye ngoko ayikuphelisi ngokupheleleyo ukukhulisa iimveliso ezingezizo ezodwa.
Ukulibaziseka kwe-Thermal priming kubambezeleka kwi-DNA synthesis ngokunqanda icandelo elibalulekileyo de isixhobo se-PCR sifikelele kwiqondo lobushushu.Uninzi lweendlela zokuqaliswa kwe-thermal manual, ezibandakanya ukulibaziseka kokongeza kwe-Taq DNA polymerase, zinzima, ngakumbi kwizicelo eziphezulu.Ezinye iindlela ze-thermal priming zisebenzisa i-wax shield ukuvala icandelo elibalulekileyo, kubandakanya i-magnesium ion okanye i-enzymes, okanye ukwahlula ngokwasemzimbeni amacandelo asebenzayo, afana neetemplates kunye ne-buffers.Ngethuba lomjikelezo we-thermal, amacandelo ahlukeneyo akhululwa kwaye axutywe kunye njengoko i-wax inyibilika.Njengendlela yendlela yokuqalisa eshushu, indlela yekhaka lewax inzima kwaye ithanda ukungcoliseka kwaye ayifanelekanga kwizicelo eziphezulu zokuphuma.
IPlatinam DNA polymerase ikulungele kwaye iyasebenza kwiPCR yokuqala eshushu ngokuzenzekelayo.I-Platinum Taq DNA polymerase iqukethe i-recombinant Taq DNA polymerase idityaniswe ne-monoclonal antibody ngokuchasene ne-Taq DNA polymerase.Ii-antibodies zenziwa yi-PCR ukuthintela umsebenzi we-enzyme ngexesha lokubamba ubushushu ixesha elide.I-Taq DNA polymerase yakhutshelwa kwimpendulo ngexesha le-94 ℃ ukugquma kwenyathelo le-denaturation, ukubuyisela umsebenzi opheleleyo wepolymerase.Ngokwahlukileyo kwi-chemically modified Taq DNA polymerase yokuqaliswa kwe-thermal, i-enzyme ye-Platinum ayifuni ukugquma ixesha elide kwi-94 ℃ (imizuzu eyi-10 ukuya kwe-15) ukuze isebenze i-polymerase.NgePlatinumTaq DNA polymerase, i-90% yomsebenzi we-Taq DNA polymerase ibuyiselwe emva kwemizuzu emi-2 kwi-94 ℃.
11. I-Nest-PCR
Imijikelo elandelelanayo yokukhulisa usebenzisa iiprimers ezifakwe kwindlwane inokuphucula ubuchwephesha kunye nobuntununtunu.Umjikelo wokuqala kukwandiswa okusemgangathweni kwemijikelo eyi-15 ukuya kwengama-20.Iqhekeza elincinci lemveliso yokuqala yokukhulisa yahlanjululwa ngamaxesha angama-100 ukuya kuma-1000 kwaye yongezwa kumjikelo wesibini wokukhulisa i-15 ukuya kwi-20 imijikelezo.Ngaphandle koko, imveliso yokuqala eyandisiweyo inokulinganiswa ngokucoca ijeli.I-primer ene-nested isetyenziswe kwinqanaba lesibini lokukhulisa, elinokuthi libophe ukulandelelana okujoliswe kuyo ngaphakathi kwe-primer yokuqala.Ukusetyenziswa kwe-PCR efakwe kwindlwane kunciphisa ukuba nokwenzeka kokwandiswa kweesayithi ezininzi ekujoliswe kuzo ngenxa yokuba kukho ulandelelwano lweethagethi ezimbalwa ezihambelanayo kuzo zombini iiseti zeeprimers.Eli nani lilonke lemijikelo (30 ukuya ku-40) eneeprimers ezifanayo landisa iindawo ezingacacanga.I-Nested PCR yonyusa ubuntununtunu bolandelelwano lweethagethi ezilinganiselweyo (umz., iimrnas ezinqabileyo) kwaye iphucula ukuchana kwe-PCRS enzima (umz. 5′ RACE).
12. Ukwehla kwePCR
Ukwehla kwe-PCR kuphucula ubuchwephesha ngokusebenzisa iimeko zokuvala eziqinileyo kwimijikelo embalwa yokuqala ye-PCR.Umjikelo uqala kwiqondo lobushushu elimalunga ne-5℃ ngaphezulu kwe-Tm eqikelelweyo, emva koko umjikelo ngamnye ucuthwe nge-1℃ ukuya kwi-2℃ de iqondo lobushushu le-anneal libe ngaphantsi kwe-Tm 5℃.Kuphela yithempleyithi yendawo ekuyiwa kuyo eneyona homology iphezulu eya kwandiswa.Ezi mveliso ziyaqhubeka nokukhula kwimijikelo elandelayo, zixinzelela ngaphandle iimveliso ezingezizo ezongeziweyo.Ukuhla kwe-PCR kuluncedo kwiindlela apho iqondo le-homology phakathi kwe-primer kunye ne-target template ayaziwa, njenge-AFLP DNA fingerprinting.
Iikhithi zePCR ezinxulumeneyo
I-2 × PCR HeroTMInkqubo yokuxuba inokunyamezela okuphezulu kwi-PCR inhibitors kunenkqubo eqhelekileyo ye-PCR Mix, kwaye inokumelana ngokulula nokwandiswa kwe-PCR yeetemplates ezahlukeneyo ezinzima.Inkqubo yokusabela ekhethekileyo kunye ne-Taq Hero ephezulu yenza ukuba i-PCR iphendule ibe nempumelelo ephezulu yokukhulisa, ukuchaneka kunye novakalelo.
Ukusebenza kokwandisa okuphezulu
Ino-5'→3' DNA polymerase umsebenzi kunye 5'→3' exonuclease umsebenzi, ngaphandle 3'→5' umsebenzi exonuclease.
Ixesha lokwenyani PCR Easyᵀᴹ-SYBR Green I Kit
I-Specific-optimized buffer kunye ne-Hot-start Taq enzyme inokuthintela ukukhulisa okungangqalanga kunye nokwakheka kwe-primer dimer.
Uvakalelo oluphezulu-luyakwazi ukubona iikopi eziphantsi zetemplate
I-RT-PCR Easyᵀᴹ I(Inyathelo elinye)
Ikhithi isebenzisa i-reagent ekhethekileyo ye-Foregene reverse transcription reagent kunye ne-Foregene HotStar Taq DNA Polymerase edityaniswe nenkqubo yokusabela ekhethekileyo ukuze kuphuculwe ngokufanelekileyo ukusebenza kakuhle kokukhulisa kunye neenkcukacha zokuphendula.
Ixesha lokuposa: May-09-2023
