Isisekelo somklamo wokuqala (izinkinga ezingama-99% zingaxazululwa)

1. Ubude be-Primer: Incwadi yokufunda idinga u-15-30bp, ngokuvamile cishe u-20bp.Isimo sangempela singcono sibe ngu-18-24bp ukuze kuqinisekiswe ukucaciswa, kodwa isikhathi esingcono, i-primer ende kakhulu izonciphisa ukucaciswa, futhi inciphise isivuno.

2. I-Primer amplification span: 200-500bp ifanelekile, futhi ucezu lunganwetshwa lube ngu-10kb ngaphansi kwezimo ezithile.

3. Isisekelo sokuqala: Okuqukethwe kwe-G+C kufanele kube ngu-40-60%, umphumela omncane kakhulu wokukhulisa i-G+C awumuhle, i-G+C eningi kulula ukuvela amabhendi angacacisiwe.I-ATGC isatshalaliswa kangcono ngokungahleliwe, igwema amaqoqo angaphezu kuka-5 we-purine noma i-pyrimidine nucleotides.I-Multi-gc yesiphetho esingu-5′ nokulandelana kwamaphakathi ukuze kwandiswe ukuzinza, gwema i-GC ecebile ekugcineni okungu-3′, ayikho i-GC yezisekelo ezingu-3 zokugcina, noma ayikho i-GC yezisekelo ezingu-3 kwezingu-5 zokugcina.

4. Gwema ukwakheka kwesibili kuma-primer, futhi ugweme ukuhambisana phakathi kwama-primers amabili, ikakhulukazi ukuphelelisa ekugcineni oku-3, ngaphandle kwalokho i-primer dimer izokwakhiwa futhi amabhendi akhuliswe angacacisiwe azokhiqizwa.

5. Izisekelo ekugcineni oku-3 'kokuqala, ikakhulukazi izisekelo zokugcina nezokunqamula, kufanele zimataniswe ngokuqinile ukuze kugwenywe ukwehluleka kwe-PCR ngenxa yezisekelo zamatheminali ezingabhangqiwe.

6. Ama-primer anazo noma anganezelwa ngeziza ezifanele zokuqhekeka, futhi ukulandelana kwethagethi okukhulisiwe kufanele kube neziza ezifanele zokuhlanzwa, okuzuzisa kakhulu ekuhlaziyweni kokuqhekeka noma ukuhlanganisa i-molecular cloning.

7. Ukucaciswa kweziqalo: ama-primers akufanele abe ne-homology esobala nokunye ukulandelana kusizindalwazi sokulandelana kwe-nucleic acid.

8. Funda ukusebenzisa isofthiwe: PP5, Oligo6, DNAstar, Vector NTI, primer3 (Lo mklamo we-inthanethi usebenza kahle kakhulu).

Okuqukethwe okungenhla kungaxazulula okungenani u-99% wezinkinga ze-primer design.

Lawula imininingwane ye-primer design

1. Ubude beprimer

Ubude be-primer obujwayelekile yizisekelo eziyi-18~30.Ngokuvamile, into ebaluleke kakhulu enquma izinga lokushisa lokungena kwe-primer ubude be-primer.Izinga lokushisa le-anneal ye-primer ngokuvamile liyakhethwa (inani le-Tm -5℃), futhi abanye basebenzisa inani le-Tm ngokuqondile.Amafomula alandelayo angasetshenziswa ukuze kubalwe cishe izinga lokushisa le-anneal lama-primers.

Lapho ubude be-primer bungaphansi kuka-20bp: [4(G+C)+2(A+T)]-5℃

Lapho ubude be-primer bukhulu kuno-20bp: 62.3℃+0.41℃(%GC)-500/ubude-5℃

Ngaphezu kwalokho, isofthiwe eminingi ingasetshenziswa futhi ukubala izinga lokushisa le-annealing, isimiso sokubala sizohluka, ngakho ngezinye izikhathi inani elibaliwe lingase libe negebe elincane.Ukuze kuthuthukiswe ukusabela kwe-PCR, ama-primer amafushane kakhulu aqinisekisa amazinga okushisa angaphansi kuka-54℃ asetshenziselwa ukusebenza kahle nokucaciswa okungcono kakhulu.

Sekukonke, ukucaciswa kwe-primer kukhuphuka nge-factor of four ku-nucleotide ngayinye eyengeziwe, ukuze ubude be-primer obuncane bezinhlelo zokusebenza eziningi bube ngama-nucleotide angu-18.Umkhawulo ongaphezulu wobude be-primer awubalulekile kakhulu, ikakhulukazi uhlobene nokusebenza kahle kokusabela.Ngenxa ye-entropy, uma i-primer iba yinde, liyancipha izinga lapho ihlanganisa khona ukuhlanganisa i-DNA eqondiwe ukuze yakhe isifanekiso esinemicu ephindwe kabili ukuze i-DNA polymerase iboshwe.

Uma usebenzisa isofthiwe ukuze udizayine iziqalisi, ubude bezinto zokuqala bunganqunywa inani le-TM ngokushintshana, ikakhulukazi kuma-primers we-fluorescence quantitative PCR, TM=60℃ noma kanjalo kufanele ilawulwe.

2.GC okuqukethwe

Ngokuvamile, okuqukethwe kwe-G+C ngokulandelana kokuqala kungu-40%~60%, futhi okuqukethwe kwe-GC kanye nenani le-Tm lepheya leziqalo kufanele kuhlanganiswe.Uma i-primer inokuthambekela kwe-GC ebucayi noma kwe-AT, inani elifanele lika-A, T noma G kanye no-C umsila lingangezwa ekupheleni kuka-5 'kwesiqalo.

3. Ukushisa kwe-annealing

Izinga lokushisa le-anneal kufanele libe ngu-5℃ ngaphansi kwezinga lokushisa le-unchain.Uma inani lezisekelo ze-primer lincane, izinga lokushisa le-anneal linganyuswa ngokufanele, okungakhuphula ukucaciswa kwe-PCR.Uma inani lezisekelo likhulu, izinga lokushisa le-anneal lingancishiswa ngokufanele.Umehluko wezinga lokushisa lokudonsa phakathi kwepheya lama-primer angu-4℃ ~ 6℃ ngeke uthinte isivuno se-PCR, kodwa ngokufanelekile izinga lokushisa le-anneal lepheya leziqalo liyefana, elingahluka phakathi kuka-55℃ ~ 75℃.

4. Gwema indawo yesibili yesakhiwo sesifanekiso sokukhulisa umsindo

Kungcono ukugwema isifunda sesakhiwo sesibili sesifanekiso lapho ukhetha ucezu olukhulisiwe.Isakhiwo sesibili esizinzile socezu oluqondiwe singabikezelwa futhi silinganiswe ngesofthiwe efanele yekhompyutha, ewusizo ekukhetheni isifanekiso.Imiphumela yokuhlola ibonisa ukuthi ukunwetshwa ngokuvamile akuphumeleli uma amandla amahhala (△G) esifunda azonwetshwa engaphansi kuka-58.6lkJ/mol.

5. Ukungafani ne-DNA target

Uma ukulandelana kwe-DNA okuqondisiwe okuthuthukisiwe kukukhulu, i-primer ingase ibophezele ezingxenyeni eziningi ze-DNA eqondiwe, okuholela ekuveleni kwamabhande amaningi kumphumela.Kulokhu kuyadingeka ukusebenzisa i-BLAST yokuhlola isofthiwe, iwebhusayithi:http://www.ncbi.nlm.nih.gov/BLAST/.Khetha Qondanisa ukulandelana okubili (bl2seq).

Ukunamathisela ukulandelana kwe-primer kuzoni 1 nokukhomba ukulandelana kwe-DNA endaweni yesi-2 kuyashintsheka, futhi i-BLAST ibala okuhambisanayo, i-antisense, nezinye izinto ezingenzeka, ngakho abasebenzisi akudingeki baqaphele ukuthi womabili amaketango angamaketango emizwa.Ungakwazi futhi ukufaka inombolo ye-GI uma uyayazi inombolo ye-GI yokulandelana kusizindalwazi, ngakho-ke awudingi ukunamathisela ingxenye enkulu yokulandelana.Ekugcineni, chofoza Qondanisa ku-3 ukuze ubone ukuthi i-primer inawo yini amasayithi ama-homologous ku-DNA eqondiwe.

6. Itheminali yokuqala

Ukuphela kwe-3 'kwe-primer yilapho isandiso siqala khona, ngakho-ke kubalulekile ukuvimbela ukungafani ukuthi kuqale lapho.Isiphetho esingu-3 akufanele sibe ngaphezu kuka-3 olandelanayo we-G noma u-C, ngoba lokhu kuzobangela ukuthi i-primer iqaliswe ngephutha endaweni yokulandelanisa ye-G+C.Isiphetho esingu-3 asikwazi ukwakha noma yisiphi isakhiwo sesibili, ngaphandle kokusabela okukhethekile kwe-PCR (AS-PCR), isiphetho esingu-3′ se-primer asikwazi ukungafaniswa.Isibonelo, uma isifunda sombhalo wekhodi sikhulisiwe, isiphetho esingu-3 'se-primer akufanele sinqanyulwe endaweni yesithathu ye-codon, ngoba indawo yesithathu ye-codon ijwayele ukuwohloka, okuzothinta ukucaciswa nokusebenza kahle kokukhulisa.Uma usebenzisa ama-primer annexation, bheka ithebula lokusetshenziswa kwe-codon, naka okuthandwayo kwezinto eziphilayo, ungasebenzisi iziqalo zokunamathisela ekupheleni kuka-3′, futhi sebenzisa ukugxila okuphezulu kweziqalo (1uM-3uM).

7. Isakhiwo sesibili sama-primers

Ama-primer ngokwawo akufanele abe nokulandelana okuhambisanayo, ngaphandle kwalokho ama-primer ngokwawo azogoqa abe yizakhiwo ze-hairpin, futhi lesi sakhiwo sesibili sizothinta ukuboshwa kwama-primers nezifanekiso ngenxa yesithiyo esiqinile.Uma kusetshenziswa ukwahlulela okwenziwayo, izisekelo eziqhubekayo ezihambisanayo zama-primer ngokwazo akufanele zibe ngaphezu kuka-3bp.Akufanele kube nokuhambisana phakathi kwama-primers amabili, ikakhulukazi ukugqama okuhambisanayo kwesiphetho esingu-3 'kufanele kugwenywe ukuvimbela ukwakheka kwama-primer dimers.Ngokuvamile, akumele kube nezisekelo ezi-4 ezilandelanayo ze-homology noma ukuphelelisana phakathi kwepheya leziqalo.

8. Engeza omaka noma i-loci

Isiphetho esingu-5 sinomthelela omncane ekucacisweni kokukhulisa futhi singashintshwa ngaphandle kokuphazamisa ukucaciswa kokukhulisa.Ukuguqulwa kokuphela kwe-primer 5 kwakuhlanganisa: ukungeza isayithi lokuvinjelwa kwe-enzyme;Ilebuli ye-biotin, i-fluorescence, i-digoxin, i-Eu3+, njll. Yethula ukulandelana kwe-DNA ebopha amaprotheni;Ukwethula amasayithi okuguqula, ukufaka nokushoda ngokulandelana kokuguqula nokwethula ukulandelana komthuthukisi, njll. Izisekelo ezengeziwe zizothinta kancane ukusebenza kahle kokukhulisa futhi kwandise ithuba lokwakheka kwe-primer dimer, kodwa okunye ukuvunyelwa kufanele kwenziwe esinyathelweni esilandelayo.Ukulandelana okwengeziwe okungekho ekulandeleni okuqondiwe, okufana nezingosi zokukhawulela nokulandelana komthuthukisi, kungangezwa ekupheleni kuka-5′ kwesiqalo ngaphandle kokuthikameze ukucaciswa.Lokhu kulandelana akufakiwe ekubalweni kwamanani e-primer Tm, kodwa kufanele kuhlolwe ukuhambisana kanye nesakhiwo sesibili sangaphakathi.

9. Ama-subclones

Isikhathi esiningi, i-PCR iwukwenza kuqala kuphela, bese sidinga ukuhlanganisa ucezu oluqondiwe kuma-vector ahlukahlukene, ngakho-ke sidinga ukuklama izisekelo ezengeziwe zomsebenzi olandelayo esinyathelweni se-PCR.

Okunye ukulandelana okwenzelwe i-subcloning kufinyezwa ngezansi.
Isizinda sokukhawulela i-endonuclease restriction sengeziwe

Ukwengeza amasayithi okuvimbela ama-enzyme kuyindlela evame ukusetshenziswa kakhulu ye-subcloning yemikhiqizo ye-PCR.Ngokuvamile, indawo yokugenca inezisekelo eziyisithupha, ngaphezu kwesi-5 'esiphelweni sendawo yokuqhekeka idinga ukungeza izisekelo eziyisivikelo ezi-2 ~ 3.Kodwa-ke, inani lezisekelo zokuvikela ezidingwa ama-enzyme ahlukene lihlukile.Isibonelo, i-SalⅠ ayidingi isisekelo sokuzivikela, i-EcoRⅤ idinga isisekelo sokuvikela esingu-1, i-NotⅠ idinga izisekelo ezi-2 zokuvikela, kanti i-Hind Ⅲ idinga izisekelo ezi-3 zokuvikela.

I-LIC ingeza umsila

Igama eliphelele le-LIC i-Ligation-Independent cloning, indlela yokwenza i-cloning eyasungulwa ngu-Navogen eqondise ingxenye yayo yevekhtha ye-pet.Inkampani yenethiwekhi ye-peT elungiselelwe indlela ye-LIC ineziphetho ezinamathelayo zesisekelo esingapheleli ezingu-12-15, ezihambisana neziphetho ezinamathelayo kucezu lokufaka okuqondiwe.Ngezinjongo zokukhulisa, ukulandelana kwe-primer 5′ kwesiqephu esifakiwe kufanele kuhambisane nevektha ye-LIC.Umsebenzi we-3′→5′ we-T4 DNA polymerase ungakha isiphetho esinamathelayo somucu owodwa ocezwini olufakiwe ngemva kwesikhathi esifushane.Ngenxa yokuthi umkhiqizo ungakhiwa kuphela kusukela ekuhlanganisweni okuhlangene kwesiqephu sokufaka esilungisiwe kanye ne-vector, le ndlela ishesha kakhulu futhi isebenza kahle, futhi iqondise i-cloning.
I-Directed TA clone engeza umsila
I-TA cloning ayikwazanga ukukhomba ucezu lube ivekhtha, ngakho kamuva i-Invitrogen yethula ivekhtha engaqondisa i-cloning, equkethe i-GTGGS eyisisekelo emine ekugcineni kwesinye.Ngakho-ke, ekwakhiweni kwama-primers e-PCR, ukulandelana okuhambisanayo kufanele kwengezwe ngokufanele, ukuze izingcezu "ziqondiswe".

Uma unesikhathi esifushane, ungazama ukuhlanganisa okuqondile, ukuhlanganisa isakhi sofuzo nevekhtha, okuyilokho esikubiza ngokuthi i-ET gene synthesis kuma-musecularists.

D. Indlela ye-In-Fusion cloning

Ayikho i-ligase edingekayo, akukho ukusabela okude okudingekayo.Inqobo nje uma ukulandelana emikhawulweni yomibili yevektha yomugqa kwethulwa Ekwakheni ama-primers, khona-ke umkhiqizo we-PCR kanye nevekhtha ewumugqa wengezwa esixazululweni se-in-fusion enzyme esiqukethe i-BSA futhi ibekwe ekamelweni lokushisa isigamu sehora, ukuguqulwa kungenziwa.Le ndlela ifaneleka ngokukhethekile ukuguqulwa kwevolumu enkulu.

10. Hlanganisa i-primer

Kwesinye isikhathi, ulwazi olukhawulelwe lokulandelana kuphela olwaziwayo mayelana nomklamo wokuqala.Isibonelo, uma kuphela ukulandelana kwe-amino acid kwaziwa, i-primer yokuhlanganisa ingaklanywa.I-primer yokuhlanganisa iyingxube yokulandelana okuhlukene emele wonke amathuba esisekelo ahlukene ahlanganisa i-amino acid eyodwa.Ukwandisa ukucaciswa, ungabhekisela kuthebula lokusetshenziswa kwe-codon ukuze unciphise ukuxhunyaniswa ngokuvumelana nezintandokazi zokusetshenziswa kwezinto eziphilayo ezahlukene.I-Hypoxanthine ingabhangqwa nazo zonke izisekelo ukuze kuncishiswe izinga lokushisa le-anneal le-primer.Ungazisebenzisi izisekelo ezinamathiselwe ekugcineni okungu-3′ kwe-primer ngoba ukufakwa kwezisekelo ezi-3 zokugcina ekugcineni okungu-3′ kwanele ukuqalisa i-PCR endaweni engafanele.Ukugxiliswa kwe-primer ephakeme (1μM kuya ku-3μM) kuyasetshenziswa ngoba ama-primer emixubeni eminingi yokunamathisela awaqondile kusifanekiso esiqondiwe.

Izinto zokusetshenziswa ze-PCRukulawula

1. Inani lokuqala

Ukugxila kwe-primer ngayinye kungu-0.1 ~ 1umol noma 10 ~ 100pmol.Kungcono ukukhiqiza umphumela odingekayo ngenani eliphansi kakhulu le-primer.Ukugxiliswa okuphezulu kwe-primer kuzodala ukungafani kanye nokukhulisa okungaqondile, futhi kwandise ithuba lokwenza ama-dimers phakathi kwama-primer.

2. Ukugxila kokuqala

Ukugxila kwama-primers kuthinta ukucaciswa.I-primer concentration elungile ivamise ukuba phakathi kuka-0.1 no-0.5μM.Ukugxila okuphezulu kwe-primer kuholela ekwandiseni imikhiqizo engacacisiwe.

3. Anealing lokushisa of primer

Enye ipharamitha ebalulekile yama-primers izinga lokushisa elincibilikayo (Tm).Leli izinga lokushisa lapho u-50% weziqalo kanye nokulandelana okuhambisanayo kuvezwa njengama-molecule e-DNA anemicu emibili.I-Tm iyadingeka ukuze usethe izinga lokushisa le-PCR annealing.Ngokufanelekile, izinga lokushisa le-anneal liphansi ngokwanele ukuze kuqinisekiswe ukukhishwa okusebenzayo kwama-primers ngokulandelana okuqondiwe, kodwa liphezulu ngokwanele ukunciphisa ukubopha okungaqondile.Amazinga okushisa anele okukhipha isisu kusuka ku-55℃ kuya ku-70℃.Izinga lokushisa le-annealing ngokuvamile lisethwa ngo-5℃ ngaphansi kune-Tm ye-primer.

Kunamafomula amaningana okusetha i-Tm, ahluka kakhulu kuye ngefomula esetshenzisiwe kanye nokulandelana kweziqalo.Ngenxa yokuthi amafomula amaningi ahlinzeka ngenani elilinganiselwe le-Tm, wonke amazinga okushisa e-anneal ayisiqalo kuphela.Ukucaciswa kungathuthukiswa ngokuhlaziya ukusabela okuningana okukhuphula kancane kancane izinga lokushisa le-anneal.Qala ngaphansi kwesilinganiso se-Tm-5℃, futhi kancane kancane ukhuphule izinga lokushisa lokukhipha isisu ngokunyuka okungu-2℃.Izinga lokushisa eliphakeme le-annealing lizonciphisa ukwakheka kwama-primer dimers kanye nemikhiqizo engaqondile.Ukuze uthole imiphumela engcono kakhulu, iziqalo ezimbili kufanele zibe namanani acishe abe yi-Tm.Uma umehluko we-Tm wamapheya e-primer ungaphezu kuka-5℃, iziqalisi zizobonisa isiqalo esingamanga esibalulekile ngokusebenzisa izinga lokushisa eliphansi le-anneal kumjikelezo.Uma iziqalo ezimbili ze-Tm zihlukile, setha izinga lokushisa le-anneal libe ngu-5℃ ngaphansi kwe-Tm ephansi kakhulu.Okunye, ukuze kwandiswe ukucaciswa, imijikelezo emihlanu ingenziwa kuqala emazingeni okushisa e-anneal adizayinelwe ukuphakama kwe-Tm, kulandele imijikelezo esele emazingeni okushisa okudonsa aklanyelwe u-Tm ophansi.Lokhu kuvumela ikhophi ingxenye yesifanekiso sendawo okuyiwa kuyo ukuthi itholwe ngaphansi kwezimo eziqinile.

4. Primer ubumsulwa nokuzinza

Ukuhlanzeka okujwayelekile kweziqalo zangokwezifiso kwanele ezinhlelweni eziningi ze-PCR.Ukususwa kwamaqembu e-benzoyl ne-isobutylyl ngokususa usawoti kuncane ngakho-ke akuphazamisi i-PCR.Ezinye izinhlelo zokusebenza zidinga ukuhlanzwa ukuze kususwe noma yikuphi ukulandelana okungagcwele ubude kunqubo yokuhlanganisa.Lokhu kulandelana okuncishisiwe kwenzeka ngoba ukusebenza kahle kwe-DNA synthesis chemistry akuyona i-100%.Lena inqubo eyindilinga esebenzisa ukusabela kwamakhemikhali okuphindaphindiwe njengoba isisekelo ngasinye sengezwa ukwenza i-DNA isuka ku-3′ kuya ku-5′.Ungahluleka kunoma yimuphi umjikelezo.Iziqalo ezinde, ikakhulukazi lezo ezizisekelo ezingaphezu kuka-50, zinengxenye enkulu yokulandelana okuncishisiwe futhi zingadinga ukuhlanzwa.

Isivuno se-primers sithintwa ukusebenza kahle kwe-synthetic chemistry kanye nendlela yokuhlanza.Izinkampani ze-Biopharmaceutical, njenge-Cytology ne-Shengong, zonke zisebenzisa iyunithi ye-OD encane ukuze kuqinisekiswe ukukhishwa okuphelele kwe-oligonucleoside.Ama-primers angokwezifiso athunyelwa ngendlela yempushana eyomile.Kungcono ukuphinda uhlakaze ama-primers ku-TE ukuze ukugxila kokugcina kube ngu-100μM.I-TE ingcono kunamanzi angcolile ngoba i-pH yamanzi ivame ukuba ne-acidic futhi izobangela i-hydrolysis ye-oligonucleosides.

Ukuzinza kwama-primers kuncike ezimeni zokugcina.Impushana eyomile kanye neziqalo ezincibilikisiwe kufanele zigcinwe ku -20 ℃.Ama-Primers ancibilikisiwe ku-TE ekugxilweni okukhulu kuno-10μM angagcinwa ngokuzinzile ku--20℃ izinyanga ezingu-6, kodwa angagcinwa kuphela ekamelweni lokushisa (15℃ kuya ku-30℃) isikhathi esingaphansi kwesonto elingu-1.Iziqalo zempuphu eyomile zingagcinwa ku -20 C okungenani unyaka owodwa kanye nezinga lokushisa elilingana negumbi (15 C kuya ku-30 ​​C) kuze kube yizinyanga ezimbili.

5. Ama-Enzyme nokugxila kwawo

Njengamanje, i-Taq DNA polymerase esetshenziswa ngokuyisisekelo iyi-enzyme yobunjiniyela bezakhi zofuzo ehlanganiswe amagciwane e-coliform.Inani le-enzyme elidingekayo ukuze kuthuthukiswe ukusabela kwe-PCR okujwayelekile licishe libe ngu-2.5U (libhekisela kumthamo wokusabela ophelele we-100ul).Uma ukugxila kuphezulu kakhulu, kungaholela ekukhuliseni okungaqondile;uma ukugxila kuphansi kakhulu, inani lomkhiqizo wokwenziwa lizoncishiswa.

6. Ikhwalithi nokugxila kwe-dNTP

Izinga le-dNTP lihlobene eduze nokugxilisa ingqondo nokusebenza kahle kokukhulisa i-PCR.I-dNTP powder iyi-granular, futhi ukuhlukahluka kwayo kulahlekelwa umsebenzi wayo wezinto eziphilayo uma igcinwe ngendlela engafanele.Isixazululo se-dNTP sine-acidic, futhi kufanele sisetshenziswe ekugxiliseni okuphezulu, nesixazululo sebhafa esingu-1M NaOH noma esingu-1M Tris.HCL ukuze kulungiswe i-PH yakhona ibe ngu-7.0 ~ 7.5, inani elincane lokupakishwa okuncane, isitoreji esiqandisiwe ku -20℃.Ukuncibilika kweqhwa okuningi kuzokwehlisa i-dNTP.Ekuphenduleni kwe-PCR, i-dNTP kufanele ibe ngu-50 ~ 200umol/L.Ikakhulukazi, kufanele kuqashelwe ukugxila kwe-DNTPS ezine kufanele kulingane (ukulungiselela imvukuzane elinganayo).Uma ukugcwala kwanoma iyiphi enye yazo kuhlukile kwezinye (eziphezulu noma eziphansi), ukungafani kuzobangelwa.Ukugxila okuphansi kakhulu kuzonciphisa isivuno semikhiqizo ye-PCR.I-dNTP ingahlangana ne-Mg2+ futhi yehlise ukugcwala kwe-Mg2+ yamahhala.

7. Isifanekiso (i-target gene) i-nucleic acid

Inani kanye neziqu zokuhlanzwa kwesifanekiso se-nucleic acid kungenye yezixhumanisi ezibalulekile zempumelelo noma ukwehluleka kwe-PCR.Izindlela zokuhlanza i-DNA ngokuvamile zisebenzisa i-SDS ne-protease K ukuze zigaye futhi zilahle izifanekiso.Imisebenzi eyinhloko ye-SDS yilezi: ncibilikisa i-lipids namaprotheni kulwelwesi lweseli, ngaleyo ndlela ucekele phansi ulwelwesi lwamangqamuzana ngokuhlakaza amaprotheni e-membrane, futhi uhlukanise amaprotheni enuzi esitokisini, i-SDS ingakwazi futhi ukuhlangana namaprotheni kanye ne-precipitate;I-Protease K ingakwazi ukwenza i-hydrolyze futhi igaye amaprotheni, ikakhulukazi ama-histones ahlanganiswe ne-DNA, bese isebenzisa i-organic solvent phenol ne-chloroform ukuze ikhiphe amaprotheni nezinye izingxenye zamangqamuzana, futhi isebenzise i-ethanol noma i-isopropyl alcohol ukuze iqhubekisele i-nucleic acid.I-nucleic acid ekhishiwe ingasetshenziswa njengesifanekiso sokusabela kwe-PCR.Ukuze uthole izibonelo ezijwayelekile zokutholwa komtholampilo, indlela esheshayo nelula ingasetshenziswa ukuncibilikisa amaseli, i-lysate pathogens, ukugaya kanye nokukhipha amaprotheni kuma-chromosome ukuya ezakhini zofuzo eziqondiwe mahhala, futhi zisetshenziselwe ngokuqondile ukukhulisa i-PCR.Ukukhishwa kwesifanekiso se-RNA kuvame ukusebenzisa indlela ye-guanidine isothiocyanate noma ye-protease K ukuvimbela i-RNase ekwehliseni isithunzi se-RNA.

8.Mg2+ ukugxila

I-Mg2+ inomthelela obalulekile ekucaciseni nasekukhiqizeni i-PCR amplification.Ngokujwayelekile ukusabela kwe-PCR, lapho ukuhlangana kwe-dNTP ehlukahlukene kungu-200umol/L, ukuhlushwa okufanele kwe-Mg2+ ngu-1.5 ~ 2.0mmol/L.Ukugxiliswa kwe-Mg2+ kuphezulu kakhulu, ukucaciswa kokusabela kuyehla, ukukhulisa okungaqondile okwenzekayo, ukugxilisa ingqondo okuphansi kakhulu kuzonciphisa umsebenzi we-Taq DNA polymerase, okuholela ekuncipheni kwemikhiqizo yokusabela.

I-Magnesium ions ithinta izici eziningana ze-PCR, njenge-DNA polymerase umsebenzi, othinta isivuno;Esinye isibonelo i-primer annealing, ethinta ukucaciswa.I-dNTP nesifanekiso kubophezela ku-ion ye-magnesium, kunciphisa inani le-ion ye-magnesium yamahhala edingekayo emsebenzini we-enzyme.Ukugxiliswa kwe-ion ye-magnesium efanelekile kuyahlukahluka kumapheya ahlukene nezifanekiso, kodwa i-PCR evamile eqala ukugxilisa ingqondo ngo-200μM dNTP ingu-1.5mM (inothi: Ukuze uthole i-PCR yobuningi besikhathi sangempela, sebenzisa isixazululo se-ion ye-magnesium engu-3 kuya ku-5mM nge-fluorescent probe).Ukugxila okuphezulu kwama-ion amahhala e-magnesium kukhulisa isivuno, kodwa futhi kukhulisa ukukhuliswa okungaqondile futhi kunciphise ukwethembeka.Ukuze kunqunywe ukugxiliswa okuphelele, ukulinganisa kwe-magnesium ion kwenziwa ngokunyuka okungu-0.5mM kusuka ku-1mM kuya ku-3mM.Ukunciphisa ukuncika ekusebenziseni i-magnesium ion, iPlatinum Taq DNA polymerase ingasetshenziswa.I-Platinum Taq DNA polymerase iyakwazi ukugcina ukusebenza phezu kohlu olubanzi lokugxila kwe-magnesium ion kune-Taq DNA polymerase ngakho-ke idinga ukwenziwa kahle okuncane.

9. Izithasiselo ezikhuthaza i-Pcr

Ukwenziwa ngcono kwezinga lokushisa le-annealing, i-primer design, kanye nokugxiliswa kwe-magnesium ion kwanele ekukhuliseni okucacile kwezifanekiso eziningi;Nokho, ezinye izifanekiso, kuhlanganise nalezo ezinokuqukethwe okuphezulu kwe-GC, zidinga izinyathelo ezengeziwe.Izithasiselo ezithinta izinga lokushisa elincibilikayo le-DNA zinikeza enye indlela yokuthuthukisa ukucaciswa komkhiqizo kanye nesivuno.Ukuchazwa okugcwele kwesifanekiso kuyadingeka ukuze kube nemiphumela engcono kakhulu.

Ngaphezu kwalokho, isakhiwo sesibili sivimbela ukubopha kwe-primer kanye nokwandiswa kwe-enzyme.

Izithasiselo ze-PCR, okuhlanganisa i-formamide, i-DMSO, i-glycerin, i-betaine, ne-PCRx Enhancer Solution, zithuthukisa ukukhuliswa.Indlela yabo engase ibe khona ukunciphisa izinga lokushisa elincibilikayo, ngaleyo ndlela kusize ukukhishwa kwama-primers futhi kusize ukunwetshwa kwe-DNA polymerase ngokusebenzisa indawo yesibili yesakhiwo.I-PCRx Solution inezinye izinzuzo.Ukulungiswa kwe-ion ye-magnesium encane kuyadingeka uma kusetshenziswa ne-Platinum Taq DNA polymerase ne-Platinum Pfx DNA polymerase.Ngakho-ke, inqubo yePlatinum ihlanganiswe nesithasiselo sokwandisa ukucaciswa ngenkathi kunciphisa ukuncika kwendlela yesithathu, ukulungiswa kwe-magnesium ion.Ukuze uthole imiphumela engcono kakhulu, ukugcwala kwezithasiselo kufanele kuthuthukiswe, ikakhulukazi i-DMSO, i-formamide, ne-glycerol, evimbela i-Taq DNA polymerase.

 I-Foreasy Taq DNA Polymerase

10. Isiqalo esishisayo

I-PCR yokuqala eshisayo ingenye yezindlela ezibaluleke kakhulu zokuthuthukisa ukucaciswa kwe-PCR ngaphezu kokuklama okuhle kwe-primer.Nakuba izinga lokushisa elifanele le-elongation le-Taq DNA polymerase lingu-72℃, i-polymerase ihlala isebenza ekamelweni lokushisa.Ngakho-ke, imikhiqizo engaqondile ikhiqizwa lapho izinga lokushisa lokubamba liphansi kunokushisa kwe-annealing ngesikhathi sokulungiswa kokusabela kwe-PCR nasekuqaleni komjikelezo oshisayo.Uma yakhiwe, le mikhiqizo engacacisiwe ikhuliswa ngempumelelo.I-Hot-start PCR isebenza kahle kakhulu lapho amasayithi asetshenziselwa ukwakhiwa kokuqala ekhawulwa indawo yezakhi zofuzo, njengokuguquguquka okuqondiswe kusayithi, i-expression cloning, noma ukwakhiwa kanye nokukhohlisa kwezakhi zofuzo ezisetshenziselwa ubunjiniyela be-DNA.

Indlela evamile yokukhawulela umsebenzi we-Taq DNA polymerase ukulungisa isisombululo sokusabela kwe-PCR eqhweni bese usibeka emshinini we-PCR oshiswe ngaphambilini.Le ndlela ilula futhi ayibizi, kepha ayiqedi umsebenzi we-enzyme ngakho-ke ayikuqedi ngokuphelele ukukhuliswa kwemikhiqizo engaqondile.

I-Thermal Priming ibambezela ukuhlanganiswa kwe-DNA ngokuvimbela ingxenye ebalulekile kuze kube yilapho umshini we-PCR ufinyelela izinga lokushisa le-denaturation.Izindlela eziningi zokuqalisa ezishisayo ezenziwa ngesandla, okuhlanganisa ukulibaziseka kokwengezwa kwe-Taq DNA polymerase, zinzima, ikakhulukazi ezinhlelweni zokusebenza eziphuma phambili.Ezinye izindlela zokushisa ezishisayo zisebenzisa isivikelo se-wax ukuvala ingxenye ebalulekile, okuhlanganisa ama-ion e-magnesium noma ama-enzyme, noma ukuhlukanisa ngokoqobo izingxenye ezisebenzayo, njengezifanekiso namabhafa.Phakathi nomjikelezo wokushisa, izingxenye ezihlukahlukene zikhishwa futhi zixutshwe ndawonye njengoba i-wax incibilika.Njengendlela yokuqalisa ukushisa okwenziwa ngesandla, indlela yesivikelo se-wax inzima futhi ijwayele ukungcoliswa futhi ayifanele ukusetshenziswa kokuphuma okuphezulu.

I-Platinamu DNA polymerase ilula futhi isebenza kahle ekuqaliseni okuzenzakalelayo kwe-PCR.I-Platinum Taq DNA polymerase iqukethe i-recombinant Taq DNA polymerase ehlanganiswe ne-monoclonal antibody ngokumelene ne-Taq DNA polymerase.Amasosha omzimba akhiwa yi-PCR ukuze avimbele umsebenzi we-enzyme ngesikhathi sokubamba izinga lokushisa isikhathi eside.I-Taq DNA polymerase ikhishelwe ekuphenduleni ngesikhathi se-94 ℃ insulation of the step denaturation, ukubuyisela umsebenzi ogcwele we-polymerase.Ngokuphambene ne-Taq DNA polymerase eguquliwe ngamakhemikhali ukuze kuqaliswe ukushisa, i-enzyme ye-Platinum ayidingi ukwahlukanisa isikhathi eside ku-94℃ (imizuzu eyi-10 ukuya kweyi-15) ukuze iqalise i-polymerase.Nge-PlatinumTaq DNA polymerase, u-90% womsebenzi we-Taq DNA polymerase ubuyiselwe ngemva kwemizuzu emi-2 ku-94 ℃.

I-Foreasy HS Taq DNA Polymerase

11. I-Nest-PCR

Imijikelezo elandelanayo yokukhulisa i-amplification kusetshenziswa iziqalo ezifakwe esidlekeni ingathuthukisa ukucaciswa nokuzwela.Umjikelezo wokuqala uwukukhuliswa okujwayelekile kwemijikelezo eyi-15 kuye kwengama-20.Ingxenye encane yomkhiqizo wokuqala wokukhulisa i-amplification yahlanjululwa izikhathi eziyi-100 kuya kweziyi-1000 futhi yengezwa emzuliswaneni wesibili wokukhulisa imijikelezo eyi-15 kuye kwengama-20.Ngaphandle kwalokho, umkhiqizo wokuqala okhulisiwe ungalinganiswa ngokuhlanzwa kwejeli.I-primer efakwe isidleke isetshenziswa emzuliswaneni wesibili wokukhulisa, ongahlanganisa ukulandelana okuqondiwe ngaphakathi kwe-primer yokuqala.Ukusetshenziswa kwe-PCR efakwe esidlekeni kunciphisa amathuba okukhulisa amasayithi amaningi okuqondiwe ngoba kukhona ukulandelana okuqondiwe okumbalwa okuhambisana nawo womabili amasethi weziqalisi.Inani elifanayo lengqikithi yemijikelezo (30 kuya ku-40) eneziqalo ezifanayo likhulise amasayithi angaqondile.I-Nested PCR inyusa ukuzwela kokulandelana kwethagethi elinganiselwe (isb, ama-mrnas ayivelakancane) futhi ithuthukisa ukucaciswa kwe-PCRS enzima (isb. 5′ RACE).

12. Ukwehla kwe-PCR

Ukwehlisa i-PCR kuthuthukisa ukucaciswa ngokusebenzisa izimo zokubopha eziqinile emijikelezweni embalwa yokuqala ye-PCR.Umjikelezo uqala ngezinga lokushisa lokuphefumula elicishe libe ngu-5℃ ngaphezu kwe-Tm elinganiselwe, bese umjikelezo ngamunye wehliswa ngo-1℃ ukuya ku-2℃ kuze kube yilapho izinga lokushisa lokukhipha isisu libe ngaphansi kuka-Tm 5℃.Isifanekiso sendawo kuphela esine-homology ephezulu kakhulu esizothuthukiswa.Le mikhiqizo iyaqhubeka nokukhula emijikelezweni elandelayo, icindezela ngaphandle kwemikhiqizo engacacisiwe ekhulisiwe.Ukwehlisa i-PCR kuyasiza ezindleleni lapho izinga le-homology phakathi kwe-primer nesifanekiso esiqondiwe lingaziwa, njengokugxivizwa kweminwe kwe-AFLP DNA.

Ahlobene PCR Kits

 I-PCR Easyᵀᴹ (Ngedayi)

Iqhawe le-2 × PCR^TMIsistimu ye-Mix inokubekezelela okuphezulu kuma-PCR inhibitors kunesistimu evamile ye-PCR Mix, futhi ingabhekana kalula nokukhulisa i-PCR yezifanekiso eziyinkimbinkimbi ezihlukahlukene.Isistimu yokusabela eyingqayizivele kanye nokusebenza kahle okuphezulu kwe-Taq Hero kwenza ukusabela kwe-PCR kube nokusebenza kahle kokukhulisa ukuphakama, ukucacisa nokuzwela.

 I-PCR Heroᵀᴹ (With Dye)

Ukusebenza kahle kwe-amplification ephezulu

Inomsebenzi we-DNA polymerase ongu-5'→3' kanye nomsebenzi we-exonuclease ongu-5'→3', ngaphandle komsebenzi we-exonuclease ongu-3'→5'.

Isikhathi Sangempela PCR Easyᵀᴹ-SYBR Green I Kit

I-Specific-optimized buffer kanye ne-hot-start Taq enzyme ingavimbela ukukhulisa okungaqondile kanye nokwakheka kwe-primer dimer

Ukuzwela okuphezulu-kungathola amakhophi aphansi esifanekiso

I-RT-PCR Easyᵀᴹ I(Isinyathelo esisodwa)

Ikhithi isebenzisa i-Foregene reverse transcription reagent eyingqayizivele kanye ne-Foregene HotStar Taq DNA Polymerase ehlanganiswe nesistimu yokusabela ehlukile ukuze kuthuthukiswe ngempumelelo ukusebenza kahle kokukhulisa nokucaciswa kokusabela.