Ndabere nhazi mbụ (enwere ike idozi nsogbu 99%)

1. Ogologo oge mbụ: Akwụkwọ ọgụgụ chọrọ 15-30bp, na-abụkarị ihe dịka 20bp.Ọnọdụ dị n'ezie ka mma ịbụ 18-24bp iji hụ na nkọwapụta, mana ogologo oge ka mma, ogologo primer ga-ebelata nkọwapụta, ma belata mkpụrụ.

2. Ntugharị ngwa ngwa: 200-500bp kwesịrị ekwesị, na mpempe akwụkwọ nwere ike gbasaa na 10kb n'okpuru ọnọdụ ụfọdụ.

3. Isi mmalite: Ọdịnaya G + C kwesịrị ịbụ 40-60%, obere mmetụta mmụba G + C adịghị mma, oke G + C dị mfe ịpụta ụgbụ na-abụghị nke akọwapụtara.A na-ekesa ATGC kacha mma na-enweghị usoro, na-ezere ụyọkọ karịa 5 purine ma ọ bụ pyrimidine nucleotides.Multi-gc maka njedebe 5 ′ na usoro etiti iji nwekwuo nkwụsi ike, zere GC bara ụba na njedebe 3′, enweghị GC maka ntọala 3 ikpeazụ, ma ọ bụ enweghị GC maka 3 nke ntọala 5 ikpeazụ.

4. Zere usoro nke abụọ na primers, ma zere mmeju n'etiti primers abụọ, karịsịa mmeju na 3 'njedebe, ma ọ bụghị ya, a ga-emepụta primer dimer na-abụghị nke a ga-emepụta bandwit amplified.

5. The bases na 3 'njedebe nke primers, karịsịa ndị ikpeazụ na penultimate bases, kwesịrị ịdị na-akpachapụ anya paired iji zere PCR ọdịda n'ihi unpaired ọnụ bases.

6. primers nwere ma ọ bụ nwere ike ịgbakwunye ya na saịtị nkwụsị kwesịrị ekwesị, na usoro ihe mgbaru ọsọ a na-emewanyewanye kwesịrị inwe ebe nkwụsị kwesịrị ekwesị, nke bara uru nke ukwuu maka nyocha nke nkwụsị ma ọ bụ cloning molecular.

7. Nkọwapụta nke primers: primers ekwesịghị inwe homology doro anya na usoro ndị ọzọ na nchekwa data usoro nucleic acid.

8. Mụta iji sọftụwia: PP5, Oligo6, DNAstar, Vector NTI, primer3 (Nhazi ịntanetị a na-arụ ọrụ kacha mma).

Ọdịnaya dị n'elu nwere ike dozie opekata mpe 99% nke nsogbu imewe nke mbụ.

Jikwaa nkọwa nke nhazi primer

1. Ogologo oge mbụ

General primer ogologo bụ 18 ~ 30 bases.N'ozuzu, ihe kachasị mkpa na-achọpụta okpomọkụ na-ekpo ọkụ nke primer bụ ogologo nke primer.A na-ahọrọ okpomoku na-ekpo ọkụ nke primer (uru Tm -5 ℃), ụfọdụ na-ejikwa uru Tm ozugbo.Enwere ike iji usoro ndị a iji gbakọọ okpomoku na-ekpo ọkụ nke primers.

Mgbe ogologo nke primer na-erughị 20bp: [4(G+C)+2(A+T)]-5℃

Mgbe ogologo nke primer karịrị 20bp: 62.3℃+0.41℃(%GC) -500/ogologo-5℃

Tụkwasị na nke ahụ, a pụkwara iji ọtụtụ ngwanrọ mee ihe iji gbakọọ okpomọkụ na-ekpo ọkụ, ụkpụrụ nchịkọta ga-adị iche, ya mere mgbe ụfọdụ, ọnụ ahịa gbakọrọ nwere ike inwe obere oghere.Iji bulie mmeghachi omume PCR, a na-eji primers kacha dị mkpụmkpụ nke na-ahụ na okpomọkụ nke na-erughị 54 ℃ na-eji maka arụmọrụ na nkọwapụta kacha mma.

N'ozuzu, nkọwa nke primer na-abawanye site na ihe anọ maka nucleotide ọ bụla ọzọ, nke mere na ogologo oge kachasị maka ọtụtụ ngwa bụ 18 nucleotides.Oke elu nke ogologo primer adịghị ezigbo mkpa, ọkachasị metụtara arụmọrụ mmeghachi omume.N'ihi entropy, ka primer na-adị ogologo, na-ebelata ọnụego ọ na-agbanye iji jikọta na DNA ezubere iji mepụta template kwụsiri ike nke nwere eriri abụọ maka DNA polymerase iji kekọta.

Mgbe ị na-eji software chepụta primers, ogologo nke primers nwere ike ikpebi site TM uru n'aka, karịsịa maka primers nke fluorescence quantitative PCR, TM = 60 ℃ ma ọ bụ otú kwesịrị ịchịkwa.

2.GC ọdịnaya

N'ozuzu, ọdịnaya nke G+C na nhazi usoro bụ 40% ~ 60%, na ọdịnaya GC na uru Tm nke otu ụzọ primers kwesịrị ịhazi.Ọ bụrụ na primer nwere àgwà GC ma ọ bụ AT siri ike, enwere ike ịgbakwunye ọdụ A, T ma ọ bụ G na C kwesịrị ekwesị na njedebe 5 nke primer.

3. Annealing okpomọkụ

Okpomọkụ annealing kwesịrị ịdị 5 ℃ dị ala karịa nke enweghị chain.Ọ bụrụ na ọnụ ọgụgụ nke ntọala primer dị ntakịrị, enwere ike ịbawanye okpomọkụ nke na-ekpo ọkụ nke ọma, nke nwere ike ime ka PCR dịkwuo elu.Ọ bụrụ na ọnụ ọgụgụ nke ntọala buru ibu, enwere ike ibelata okpomọkụ nke annealing nke ọma.The annealing okpomọkụ dị iche n'etiti a ụzọ primers nke 4 ℃ ~ 6 ℃ agaghị emetụta PCR mkpụrụ, ma kacha mma na annealing okpomọkụ nke a ụzọ primers bụ otu, nke nwere ike ịdị iche n'etiti 55 ℃ ~ 75 ℃.

4. Zere ebe nhazi nke abụọ nke template amplification

Ọ kacha mma ka ịzere mpaghara nhazi nke abụọ nke template mgbe ị na-ahọrọ mpekere agbakwunyere.Enwere ike ibu amụma na atụmatụ nhazi nke abụọ kwụsiri ike nke iberibe ebumnuche site na ngwanrọ kọmputa dị mkpa, nke na-enyere aka maka nhọrọ ndebiri.Nsonaazụ nnwale na-egosi na mgbasawanye anaghị enwekarị ihe ịga nke ọma mgbe ike efu (△G) nke mpaghara a ga-agbasa erughị 58.6lkJ/mol.

5. Ekwekọrịtaghị na DNA lekwasịrị anya

Mgbe usoro DNA agbagoro agbagoro buru ibu, primer nwere ike jikọta n'ọtụtụ akụkụ nke DNA ebumnuche, na-ebute ọtụtụ eriri na-apụta na nsonaazụ ya.Oge a ọ dị mkpa iji nyocha ngwanrọ BLAST, webụsaịtị:http://www.ncbi.nlm.nih.gov/BLAST/.Họrọ Hazie usoro abụọ (bl2seq).

Tapawa usoro primer na mpaghara 1 na lekwasịrị anya usoro DNA na mpaghara 2 bụ mgbanwe, na BLAST na-agbakọ mgbakwunye, antisense, na ohere ndị ọzọ, yabụ na ndị ọrụ adịghị mkpa ịchọpụta ma agbụ abụọ a bụ agbụ uche.Ị nwekwara ike tinye nọmba GI ma ọ bụrụ na ị maara nọmba GI nke usoro na nchekwa data, n'ihi ya, ịkwesighi mado nnukwu akụkụ nke usoro ahụ.N'ikpeazụ, pịa Tinyegharịa na 3 ka ịhụ ma primer nwere ọtụtụ saịtị otu na DNA ebumnuche.

6. Primer ọnụ

Ngwugwu 3 nke primer bụ ebe ndọtị na-amalite, n'ihi ya, ọ dị mkpa igbochi mismatches ịmalite ebe ahụ.Ọgwụgwụ nke 3 ekwesịghị ịbụ ihe karịrị 3 n'usoro G ma ọ bụ C, n'ihi na nke a ga-eme ka ihie ụzọ kpalite primer na mpaghara usoro nkwalite G+C.Ọgwụgwụ 3 enweghị ike ịmepụta usoro nke abụọ ọ bụla, belụsọ na mmeghachi omume PCR (AS-PCR) pụrụ iche, njedebe 3′ nke primer enweghị ike ịmekọrịta.Dịka ọmụmaatụ, ọ bụrụ na mpaghara encoding na-abawanye, 3 'njedebe nke primer ekwesịghị ịkwụsị n'ọnọdụ nke atọ nke codon, n'ihi na ọnọdụ nke atọ nke codon na-adịkarị mfe imebi, nke ga-emetụta kpọmkwem na arụmọrụ nke nkwalite.Mgbe ị na-eji primers annexation, rụtụ aka na tebụl ojiji codon, ṅaa ntị na mmasị ndu, ejila ntinye ntinye na njedebe 3′, ma jiri ntinye dị elu nke primers (1uM-3uM).

7. Usoro nke abụọ nke primers

Ndị na-emepụta ihe n'onwe ha ekwesịghị inwe usoro mgbakwunye, ma ọ bụghị ya, ndị na-emepụta onwe ha ga-atụgharị n'ime usoro ntutu isi, na usoro nke abụọ a ga-emetụta njikọ nke primers na ndebiri n'ihi nkwụsịtụ siri ike.Ọ bụrụ na a na-eji ikpe mmadụ eme ihe, ntọala nkwado na-aga n'ihu nke primers n'onwe ha ekwesịghị ịkarị 3bp.E kwesịghị inwe complementarity n'etiti abụọ primers, karịsịa mmekọ overlap nke 3 'njedebe kwesịrị izere iji gbochie guzobe primer dimers.N'ozuzu, e kwesịghị inwe ihe karịrị 4 n'usoro bases homology ma ọ bụ nkwado n'etiti otu ụzọ nke primers.

8. Tinye akara ma ọ bụ loci

Ọgwụgwụ 5 ahụ nwere mmetụta dị nta na nkọwapụta mmụba yana yabụ enwere ike gbanwee ya na-emetụtaghị nkọwapụta nkwalite.Ngbanwe nke primer 5 'njedebe gụnyere: na-agbakwunye saịtị mgbochi enzyme;Akpọrọ aha biotin, fluorescence, digoxin, Eu3+, wdg. Webata protein njikọ DNA usoro;Ewebata saịtị mutation, ntinye na-efunahụ usoro ngbanwe na iwebata usoro mgbasa ozi, wdg. Ihe ndabere ndị ọzọ ga-emetụta arụmọrụ nke mmụba ma mee ka ohere nke ịmepụta primer dimer, ma a ghaghị ime ụfọdụ nkwenye maka nzọụkwụ ọzọ.Usoro ndị ọzọ na-adịghị adị n'usoro ebumnuche, dị ka saịtị mmachi na usoro nkwalite, nwere ike ịgbakwunye na njedebe 5′ nke primer na-emetụtaghị nkọwapụta.Agụnyeghị usoro ndị a na ngụkọ nke ụkpụrụ primer Tm, mana ekwesịrị ịnwale maka mmeju na nhazi nke abụọ dị n'ime.

9. Subclones

Ọtụtụ oge, PCR bụ naanị cloning nke mbụ, mgbe ahụ, anyị kwesịrị imechi iberibe ebumnuche ahụ n'ime vectors dị iche iche, yabụ anyị kwesịrị imepụta ntọala ndị ọzọ maka ọrụ ọzọ na nzọụkwụ PCR.

Achịkọtara ụfọdụ usoro emebere maka subcloning n'okpuru.
Agbakwunyere ebe mmachi endonuclease

Ịgbakwunye saịtị mmachi enzyme bụ usoro a na-ejikarị eme ihe maka ibelata ngwaahịa PCR.N'ozuzu, ebe nkwụsịtụ bụ ntọala isii, na mgbakwunye na 5 'njedebe nke ebe nkwụsịtụ kwesịrị ịgbakwunye 2 ~ 3 ntọala nchebe.Otú ọ dị, ọnụ ọgụgụ nke ntọala nchebe chọrọ site na enzymes dị iche iche dị iche iche.Dịka ọmụmaatụ, SalⅠ achọghị ntọala nchebe, EcoRⅤ chọrọ ntọala nchekwa 1, Ọ bụghịⅠ chọrọ ntọala nchekwa 2, na Hind Ⅲ chọrọ ntọala nchekwa 3.

LIC na-agbakwunye ọdụ ahụ

Aha zuru oke nke LIC bụ ligation-independent cloning, usoro cloning nke Navogen chepụtara kpọmkwem maka akụkụ nke vector pET.Onye na-ebu pET nke a kwadebere site na usoro LIC nwere isi 12-15 na-ejikọtaghị ọnụ, nke na-emeju njedebe nnyapade kwekọrọ na ibe ntinye ebumnuche.Maka ebumnuche nkwalite, usoro primer 5′ nke iberibe etinyere kwesịrị ịkwado vector LIC.Ọrụ 3′→5′ extranect nke T4 DNA polymerase nwere ike mepụta otu eriri nnyapade na iberibe etinyere obere oge.N'ihi na ngwaahịa a nwere ike ịmepụta naanị site na nchikota nke ibe ntinye a kwadebere na vector, usoro a dị ngwa ngwa ma dị irè, a na-eduzi ya na cloning.
eduzi TA clone tinye ọdụ
TA cloning enweghị ike idobe iberibe iberibe ahụ n'ime vector, yabụ mgbe e mesịrị, Invitrogen webatara vector nke nwere ike ilekwasị anya na cloning, nke nwere isi GTGGS anọ a ma ama n'otu njedebe.Ya mere, na imewe nke PCR primers, ekwesịrị ịgbakwunye usoro mmekọ ya, nke mere na iberibe nwere ike ịbụ "gbadoro anya".

Ọ bụrụ na ị dị mkpụmkpụ na oge, ị nwere ike ịnwale njikọ kpọmkwem, ijikọta mkpụrụ ndụ ihe nketa na vector, nke anyị na-akpọ ET gene synthesis na musecularists.

D. In-Fusion cloning usoro

Enweghị ligase chọrọ, ọ dịghị ogologo mmeghachi omume achọrọ.Ọ bụrụhaala na a na-ebute usoro na nsọtụ abụọ nke vector linearized Na nhazi nke primers, mgbe ahụ, a na-agbakwunye ngwaahịa PCR na vector linearized n'ime ngwọta enzyme in-fusion nke nwere BSA ma tinye ya n'ime ụlọ okpomọkụ maka ọkara otu awa, enwere ike ime mgbanwe ahụ.Usoro a dabara adaba maka ntughari olu buru ibu.

10. Jikota primer

Mgbe ụfọdụ, ọ bụ naanị ozi usoro dị ntakịrị ka a maara maka imewe primer.Dịka ọmụmaatụ, ọ bụrụ na a maara naanị usoro amino acid, enwere ike ịmepụta primer merging.Ngwakọta ngwakọ bụ ngwakọta nke usoro dị iche iche na-anọchite anya ohere niile dị iche iche na-etinye akara amino acid.Ka iwelie nkọwapụta, ị nwere ike ịtu aka na tebụl ojiji codon iji belata mgbakwunye dị ka mmasị ojiji nke ihe dị iche iche si dị.Enwere ike ijikọ Hypoxanthine na ntọala niile iji belata okpomọkụ na-ekpo ọkụ nke primer.Ejila ntọala agbakwunyere na njedebe 3′ nke primer n'ihi na nchichi nke ntọala 3 ikpeazụ na njedebe 3 zuru ezu iji malite PCR na saịtị na-ezighi ezi.A na-eji mkpokọta primer dị elu (1μM ruo 3μM) n'ihi na primers n'ọtụtụ ngwakọta mgbakwunye abụghị kpọmkwem maka ndebiri ebumnuche.

PCR akụrụngwanjikwa

1. Ọnụ ọgụgụ mbụ

Ntinye nke primer ọ bụla bụ 0.1 ~ 1umol ma ọ bụ 10 ~ 100pmol.Ọ ka mma ịmepụta nsonaazụ achọrọ na ọnụ ọgụgụ kacha nta nke primer.Ọnụ ọgụgụ dị elu nke primer ga-eme ka ejikọtaghị ọnụ na mmụba na-abụghị nke a kapịrị ọnụ, ma mee ka ohere ịmepụta dimers n'etiti primers.

2. Ntinye uche nke mbụ

Ntinye uche nke primers na-emetụta kpọmkwem.Ọnụ ọgụgụ kachasị mma na-adịkarị n'etiti 0.1 na 0.5μM.Nleta mbụ dị elu na-eduga n'ịkwalite ngwaahịa na-abụghị nke akọwapụtara.

3. Annealing okpomọkụ nke primer

Ihe ọzọ dị mkpa maka primers bụ okpomọkụ na-agbaze (Tm).Nke a bụ ọnọdụ okpomọkụ mgbe 50% nke primers na usoro mmekọ na-anọchi anya dị ka mkpụrụ ndụ DNA nwere eriri abụọ.Achọrọ Tm ka ịtọọ okpomọkụ na-ebelata PCR.Dị ka o kwesịrị, okpomọkụ na-ekpo ọkụ dị ntakịrị iji hụ na anneal nke primers dị irè na usoro ebumnuche, mana ọ dị elu iji belata njide na-enweghị isi.Okpomọkụ na-ekpo ọkụ nwere ezi uche site na 55 ℃ ruo 70 ℃.A na-edobe okpomọkụ na-ekpo ọkụ 5 ℃ dị ala karịa Tm nke primer.

Enwere ọtụtụ usoro maka ịtọ Tm, nke dịgasị iche iche dabere na usoro ejiri na usoro nke primers.N'ihi na ọtụtụ usoro na-enye ọnụ ahịa Tm a na-eme atụmatụ, okpomọkụ niile na-ebelata bụ naanị mmalite.Enwere ike imeziwanye nkọwapụta site n'ịtụle mmeghachi omume dị iche iche na-eji nwayọọ nwayọọ na-ebuli okpomọkụ na-ebelata ahụ.Malite n'okpuru Tm-5℃ a na-eme atụmatụ, ma jiri nwayọọ nwayọọ na-abawanye okpomọkụ na-ekpo ọkụ na mmụba nke 2 ℃.Okpomọkụ dị elu ga-ebelata mmepụta nke primer dimers na ngwaahịa ndị na-abụghị nke a kapịrị ọnụ.Maka nsonaazụ kacha mma, primers abụọ ahụ kwesịrị inwe ụkpụrụ Tm dị nso.Ọ bụrụ na Tm dị iche nke primer ụzọ abụọ karịrị 5 ℃, primers ga-egosi mmalite ụgha dị ịrịba ama site na iji obere annealing okpomọkụ na okirikiri.Ọ bụrụ na primers abụọ Tm dị iche, debe okpomọkụ na-ekpo ọkụ ka ọ bụrụ 5 ℃ ala karịa Tm kacha ala.N'aka nke ọzọ, iji mụbaa nkọwapụta, enwere ike ịme usoro cycles ise na ọnọdụ okpomọkụ emebere maka Tm dị elu, na-esote usoro nke fọdụrụ na okpomọkụ na-ekpo ọkụ emebere maka Tm dị ala.Nke a na-enye ohere ịnweta nnomi akụkụ nke ebe ndebiri n'okpuru ọnọdụ siri ike.

4. Primer ịdị ọcha na nkwụsi ike

Ọkọlọtọ ịdị ọcha nke primers omenala zuru oke maka ọtụtụ ngwa PCR.Mwepụ nke benzoyl na isobutylyl dị iche iche site na ikpochapụ dị ntakịrị ma ya mere anaghị egbochi PCR.Ngwa ụfọdụ chọrọ ịdị ọcha iji wepụ usoro ọ bụla na-enweghị ogologo na usoro njikọ.Usoro ndị a gbajiri agbaji na-eme n'ihi na arụmọrụ nke kemịkalụ DNA abụghị 100%.Nke a bụ usoro okirikiri nke na-eji mmeghachi omume kemịkalụ ugboro ugboro ka etinyere ntọala ọ bụla iji mee DNA site na 3′ ruo 5′.Ị nwere ike ịda n'ime okirikiri ọ bụla.Ihe nrịbama dị ogologo, ọkachasị ndị karịrị ntọala 50, nwere oke nke usoro agbajichara ma nwee ike ịchọ ịdị ọcha.

A na-emetụta mkpụrụ nke primers site na arụmọrụ nke kemịkalụ sịntetik na usoro nchacha.Ụlọ ọrụ biopharmaceutical, dị ka Cytology na Shengong, niile na-eji nkeji OD kacha nta iji hụ na mkpokọta oligonucleoside dị.A na-ebufe primers omenala n'ụdị ntụ ntụ.Ọ kacha mma ịghaghachi primers na TE nke mere na njedebe ikpeazụ bụ 100μM.TE dị mma karịa mmiri deionized n'ihi na pH nke mmiri na-abụkarị acidic na ọ ga-eme ka hydrolysis nke oligonucleosides.

Nkwụsi ike nke primers na-adabere na ọnọdụ nchekwa.Ekwesịrị ịchekwa ntụ ntụ na ntụ ntụ na-agbaze na -20 ℃.A na-echekwa primers gbasasịa na TE na oke karịa 10μM na-echekwa na -20 ℃ maka ọnwa 6, mana enwere ike ịchekwa ya na ụlọ okpomọkụ (15 ℃ ruo 30 ℃) maka ihe na-erughị otu izu.Enwere ike ịchekwa primers ntụ ntụ akọrọ na -20 C ma ọ dịkarịa ala otu afọ yana na ụlọ okpomọkụ (15 C ruo 30 C) ruo ọnwa 2.

5. Enzymes na ntinye ha

Ka ọ dị ugbu a, Taq DNA polymerase ejiri mee ihe bụ n'ụzọ bụ isi enzyme injinia nke nje bacteria coliform mebere.Ọnụ ọgụgụ nke enzyme achọrọ iji kpata mmeghachi omume PCR na-ahụkarị bụ ihe dịka 2.5U (na-ezo aka na mkpokọta mmeghachi omume nke 100ul).Ọ bụrụ na ịta ahụhụ dị oke elu, ọ nwere ike ibute mmụba na-abụghị nke a kapịrị ọnụ;ọ bụrụ na ntinye uche dị oke ala, a ga-ebelata ọnụ ọgụgụ nke ngwaahịa sịntetik.

6. Ogo na itinye uche nke dNTP

Ogo dNTP nwere njikọ chiri anya na ntinye na arụmọrụ nke nkwalite PCR.DNTP ntụ ntụ bụ granular, na mgbanwe ya na-efunahụ ọrụ ndu ya ma ọ bụrụ na echekwara ya nke ọma.dNTP ngwọta bụ acidic, ma ekwesịrị iji ya mee ihe na ntinye uche dị elu, na 1M NaOH ma ọ bụ 1M Tris.HCL ngwọta ngwọta iji dozie PH ya na 7.0 ~ 7.5, obere obere ngwugwu, nchekwa oyi kpọnwụrụ na -20 ℃.Otutu ifriizi-thawing ga-eweda dNTP.Na nzaghachi PCR, dNTP kwesịrị ịbụ 50 ~ 200umol / L.Karịsịa, a ga-akwụ ụgwọ nlebara anya na ntinye nke DNTPS anọ kwesịrị ịdị nhata (nkwadebe mole nha nhata).Ọ bụrụ na ntinye nke nke ọ bụla n'ime ha dị iche na nke ndị ọzọ (nke dị elu ma ọ bụ nke dị ala), a ga-eme ka enweghị nkwekọrịta.Ntinye uche dị ala ga-ebelata mkpụrụ nke ngwaahịa PCR.dNTP nwere ike ijikọ na Mg2+ ma belata mkpokọta Mg2+ efu.

7. Template (content gene) nucleic acid

Ọnụ ego na ogo ịdị ọcha nke template nucleic acid bụ otu n'ime isi njikọ maka ịga nke ọma ma ọ bụ ọdịda nke PCR.Ụzọ nchacha DNA ọdịnala na-ejikarị SDS na protease K iji gbarie ma tụfuo ụdịdị.Ọrụ bụ isi nke SDS bụ: igbari lipids na protein na akpụkpọ ahụ cell, si otú ahụ na-ebibi akpụkpọ ahụ cell site na-agbaze protein protein, na-ekewapụ protein protein na cell, SDS nwekwara ike ikpokọta na protein na precipitate;Protease K nwere ike hydrolyze na mgbari protein, karịsịa histones ejikọtara na DNA, wee jiri organic mgbaze phenol na chloroform wepụ proteins na ndị ọzọ cell components, na-eji ethanol ma ọ bụ isopropyl mmanya na-ebuli nucleic acid.Enwere ike iji acid nucleic ewepụtara dị ka ndebiri maka mmeghachi omume PCR.Maka ihe nleba anya nyocha ụlọ ọgwụ n'ozuzu, enwere ike iji usoro dị ngwa ma dị mfe iji gbazee mkpụrụ ndụ, lysate pathogens, mgbari ma wepụ protein na chromosomes na mkpụrụ ndụ ihe nketa efu, ma jiri ya mee ihe maka nkwalite PCR.Mwepụta template RNA na-ejikarị guanidine isothiocyanate ma ọ bụ usoro protease K iji gbochie RNase imebi RNA.

8.Mg2+ itinye uche

Mg2+ nwere mmetụta dị ukwuu na nkọwapụta na mkpụrụ nke nkwalite PCR.N'ozuzu PCR mmeghachi omume, mgbe ịta nke dị iche iche dNTP bụ 200umol/L, kwesịrị ekwesị ịta nke Mg2+ bụ 1.5 ~ 2.0mmol/L.Mg2 + ntinye uche dị oke elu, nkọwa mmeghachi omume na-ebelata, mmụba na-abụghị nke a na-eme, oke ntinye uche ga-ebelata ọrụ nke Taq DNA polymerase, na-ebute mbelata ngwaahịa mmeghachi omume.

ion Magnesium na-emetụta ọtụtụ akụkụ nke PCR, dị ka ọrụ DNA polymerase, nke na-emetụta mkpụrụ;Ihe atụ ọzọ bụ primer annealing, nke na-emetụta kpọmkwem.dNTP na ndebiri na-ejikọta na magnesium ion, na-ebelata ego nke ion magnesium efu achọrọ maka ọrụ enzyme.Ntụpọ magnesium ion kacha mma dịgasị iche maka ụzọ abụọ na ndebiri dị iche iche, mana PCR na-amalite itinye uche na 200μM dNTP bụ 1.5mM (mara: Maka PCR n'ezie, jiri 3 ruo 5mM magnesium ion solution na nyocha fluorescent).Ọnụ ọgụgụ dị elu nke ion magnesium efu na-abawanye mkpụrụ, mana ọ na-abawanye nkwalite na-enweghị isi ma belata ikwesị ntụkwasị obi.Iji chọpụta mkpokọta kachasị mma, a na-eme titration magnesium ion na mmụba nke 0.5mM site na 1mM ruo 3mM.Iji belata ịdabere na njikarịcha ion magnesium, enwere ike iji Platinum Taq DNA polymerase.Platinum Taq DNA polymerase na-enwe ike ịnọgide na-arụ ọrụ n'ọtụtụ dịgasị iche iche nke magnesium ion mkpokọta karịa Taq DNA polymerase ya mere ọ chọrọ obere njikarịcha.

9. Ihe mgbakwunye PCr na-akwalite

Ịkwalite ọnọdụ okpomọkụ na-ekpo ọkụ, nhazi primer, na ntinye uche nke magnesium ion zuru ezu maka mmụba nke ukwuu nke ọtụtụ ndebiri;agbanyeghị, ụfọdụ ndebiri, gụnyere ndị nwere ọdịnaya GC dị elu, chọrọ usoro ndị ọzọ.Ihe mgbakwunye na-emetụta okpomọkụ na-agbaze nke DNA na-enye ụzọ ọzọ iji melite nkọwa ngwaahịa na mkpụrụ.Achọrọ denaturation zuru ezu nke ndebiri maka nsonaazụ kacha mma.

Na mgbakwunye, usoro nke abụọ na-egbochi njikọ nke primer na ndọtị enzyme.

Ihe mgbakwunye PCR, gụnyere foramide, DMSO, glycerin, betaine, na PCRx Enhancer Solution, na-akwalite nkwalite.Usoro ha nwere ike ime bụ ibelata okpomọkụ na-agbaze, si otú ahụ na-enyere aka nkwụsị nke primers na inyere DNA polymerase ndọtị site na mpaghara nhazi nke abụọ.Ngwọta PCRx nwere uru ndị ọzọ.A na-achọ njikarịcha magnesium ion kacha nta mgbe ejiri Platinum Taq DNA polymerase na Platinum Pfx DNA polymerase.Ya mere, a na-ejikọta usoro Platinum na ihe mgbakwunye iji mee ka nkọwapụta dịkwuo elu ma na-ebelata ndabere nke ụzọ nke atọ, njikarịcha ion magnesium.Maka nsonaazụ kacha mma, a ga-emeziwanye ntinye nke mgbakwunye, ọkachasị DMSO, formamide, na glycerol, nke na-egbochi Taq DNA polymerase.

 Ihe omuma nke DNA Polymerase

10. Mmalite na-ekpo ọkụ

PCR na-ekpo ọkụ bụ otu n'ime ụzọ kachasị mkpa iji melite kpọmkwem PCR na mgbakwunye na nhazi nke ọma.Ọ bụ ezie na okpomọkụ elongation kacha mma nke Taq DNA polymerase bụ 72 ℃, polymerase na-anọgide na-arụ ọrụ na okpomọkụ ụlọ.Ya mere, a na-emepụta ngwaahịa ndị na-abụghị nke a na-emepụta mgbe njide okpomọkụ dị ala karịa okpomọkụ na-ekpo ọkụ n'oge nkwadebe nke mmeghachi omume PCR na na mmalite nke usoro okpomọkụ.Ozugbo emebere ya, ngwaahịa ndị a enweghị nkọwa na-abawanye nke ọma.PCR na-ekpo ọkụ na-arụ ọrụ nke ọma mgbe saịtị ndị a na-eji maka imepụta primer na-ejedebe site na ọnọdụ nke mkpụrụ ndụ ihe nketa, dị ka mmụgharị na-eduzi saịtị, okwu cloning, ma ọ bụ iwu na nhazi nke mkpụrụ ndụ ihe nketa ejiri maka injinịa DNA.

Usoro a na-ahụkarị iji gbochie ọrụ Taq DNA polymerase bụ ịkwadebe ngwọta nzaghachi PCR na ice ma tinye ya na ngwa PCR ekpo ọkụ.Usoro a dị mfe ma dị ọnụ ala, mana ọ naghị emecha ọrụ nke enzyme ahụ, ya mere ọ dịghị ewepụ kpamkpam nkwalite nke ngwaahịa ndị na-abụghị nke a kapịrị ọnụ.

Thermal priming na-egbu oge njikọ DNA site na igbochi ihe dị mkpa ruo mgbe ngwa PCR ruru okpomọkụ denaturation.Imirikiti ụzọ mmalite ọkụ ọkụ akwụkwọ ntuziaka, gụnyere mgbakwunye na-egbu oge nke Taq DNA polymerase, dị egwu, ọkachasị maka ngwa ewepụtara dị elu.Ụzọ priming ndị ọzọ na-ekpo ọkụ na-eji ọta wax kpuchido akụkụ dị mkpa, gụnyere ion magnesium ma ọ bụ enzymes, ma ọ bụ kewapụ ihe ndị na-emeghachi omume, dị ka ndebiri na ihe nchekwa.N'oge okirikiri okpomọkụ, a na-ahapụ ihe dị iche iche ma jikọta ọnụ ka wax na-agbaze.Dị ka usoro mmalite ọkụ ọkụ nke akwụkwọ ntuziaka, usoro ọta wax na-esi ike ma na-enwekarị mmetọ na ọ dịghị adabara maka ngwa mmepụta dị elu.

Platinum DNA polymerase dabara adaba ma rụọ ọrụ nke ọma maka PCR na-ekpo ọkụ akpaka.Platinum Taq DNA polymerase nwere recombinant Taq DNA polymerase jikọtara ya na mgbochi monoclonal megide Taq DNA polymerase.PCR na-emepụta ọgwụ mgbochi ọrịa iji gbochie ọrụ enzyme n'oge njide okpomọkụ dị ogologo.A tọhapụrụ Taq DNA polymerase na mmeghachi omume n'oge mkpuchi 94 ℃ nke denaturation, na-eweghachi ọrụ polymerase zuru oke.N'adịghị ka Taq DNA polymerase gbanwere kemịkalụ maka mmalite thermal, Platinum enzyme anaghị achọ mkpuchi ogologo oge na 94 ℃ (nkeji 10 ruo 15) iji mee ka polymerase rụọ ọrụ.Site na PlatinumTaq DNA polymerase, 90% nke ọrụ Taq DNA polymerase eweghachiri mgbe nkeji 2 gachara na 94 ℃.

Ihe atụ nke HS Taq DNA Polymerase

11. Nest-PCR

Mgbatị nkwalite na-aga n'ihu site na iji primers akwu nwere ike melite nkọwapụta na nghọta.Agba nke mbụ bụ nkwalite ọkọlọtọ nke okirikiri 15 ruo 20.A gbajiri obere akụkụ nke ngwaahịa nkwalite izizi ugboro 100 ruo 1000 wee tinye ya na agba nke abụọ nke nkwalite maka okirikiri 15 ruo 20.N'aka nke ọzọ, ngwaahịa mbụ agbagoro agbagoro nwere ike nha site na nchacha gel.A na-eji primer akwụ ụgwọ na agba nke abụọ nke mmụba, nke nwere ike jikọta na usoro ebumnuche n'ime primer nke mbụ.Iji PCR akwụghị ụgwọ na-ebelata ohere nke mmụba nke ọtụtụ saịtị ebumnuche n'ihi na enwere usoro ebumnuche ole na ole na-akwado usoro abụọ nke primers.Otu ngụkọta ọnụọgụ okirikiri (30 ruo 40) nwere otu primers mere ka saịtị ndị ahụ akọwapụtaghị ya.PCR akwụghị ụgwọ na-abawanye nghọta nke usoro ebumnuche nwere oke (dịka ọmụmaatụ, mrnas na-adịghị ahụkebe) ma na-akwalite nkọwapụta nke PCRS siri ike (dịka 5′ RACE).

12. PCR na-agbada

Ịrịdata PCR na-eme ka nkọwapụta dị mma site n'iji ọnọdụ mgbakasị ahụ siri ike maka nkeji ole na ole mbụ nke PCR.Oge okirikiri na-amalite na okpomọkụ na-ekpo ọkụ dị ihe dị ka 5 ℃ dị elu karịa Tm a na-eme atụmatụ, mgbe ahụ, usoro nke ọ bụla na-ebelata site na 1℃ ruo 2 ℃ ruo mgbe okpomọkụ na-ekpo ọkụ dị n'okpuru Tm 5℃.Naanị ndebiri ebe a na-aga nwere homology kacha elu ka a ga-abawanye.Ngwaahịa ndị a na-aga n'ihu na-agbasawanye n'okirikiri ndị na-esote, na-ekpochapụ ngwaahịa ndị na-abụghị nke a kapịrị ọnụ.Ịrịdata PCR bara uru maka ụzọ ebe amaghị ogo homology n'etiti primer na ndebiri ebumnuche, dị ka akara mkpịsị aka DNA AFLP.

Ngwa PCR emetụtara

 PCR Easyᵀᴹ (Ya na Dye)

2 × PCR dike^TMSistemụ ngwakọta nwere nnabata dị elu nye ndị na-egbochi PCR karịa sistemu PCR Mix nkịtị, ma nwee ike ịnagide nkwalite PCR dị iche iche dị mgbagwoju anya.Usoro mmeghachi omume pụrụ iche na Taq Hero dị elu na-eme ka mmeghachi omume PCR nwee arụmọrụ nkwalite dị elu, nkọwapụta na uche.

 PCR Hero (Ya na Dye)

Ịrụ ọrụ nkwalite dị elu

Ọ nwere 5'→3' DNA polymerase ọrụ na 5'→ 3' exonuclease ọrụ, na-enweghị 3'→5' exonuclease ọrụ.

Ezigbo oge PCR Easyᵀᴹ-SYBR Green I Kit

Ihe nchekwa akọwapụtara nke ọma na mmalite Taq enzyme nwere ike igbochi nkwalite na-abụghị nke akọwapụtara yana nguzobe primer dimer.

Mmetụta dị elu-nwere ike ịchọpụta obere ndebiri nke ndebiri

RT-PCR Easyᵀᴹ I (Otu Nzọụkwụ)

Ngwa ahụ na-eji ihe ntụgharị ntụgharị pụrụ iche nke Foregene na Foregene HotStar Taq DNA Polymerase jikọtara ya na sistemu mmeghachi omume pụrụ iche iji melite arụmọrụ nkwalite na nkọwapụta mmeghachi omume ahụ nke ọma.