PCR, bụ mmeghachi omume nke polymerase, nke na-ezo aka na mgbakwunye nke dNTP, Mg2 +, ihe elongation na nkwalite nkwalite ihe na usoro n'okpuru catalysis nke DNA polymerase, na-eji DNA nne na nna dị ka template na kpọmkwem primers dị ka mmalite nke ndọtị , Site na usoro nke denaturation, annealing, ndọtị, wdg, usoro nke in vitro imegharị nwa nwanyị strand DNA na-akwado nne na nna strand template DNA nwere ike ime ngwa ngwa na kpọmkwem gbasaa DNA ọ bụla e lekwasịrị anya na vitro.
1. Hot Start PCR
Oge mmalite nke nkwalite na PCR nkịtị abụghị itinye PCR igwe n'ime igwe PCR, mgbe ahụ mmemme ahụ ga-amalite ịbawanyewanye.Mgbe emechara nhazi usoro ahụ, mmụba ahụ na-amalite, nke nwere ike ime ka mmụba na-enweghị isi, na PCR na-ekpo ọkụ nwere ike dozie nsogbu a.
Kedu ihe bụ PCR mmalite dị ọkụ?Mgbe emechara usoro mmeghachi omume ahụ, a na-ahapụ onye na-eme mgbanwe enzyme na okpomọkụ dị elu (na-adịkarị elu karịa 90 ° C) n'oge mmalite ikpo ọkụ nke mmeghachi omume ma ọ bụ "mmalite na-ekpo ọkụ", nke mere na DNA polymerase na-arụ ọrụ.Oge ịgbalite kpọmkwem na okpomọkụ na-adabere na ọdịdị nke DNA polymerase na onye na-ekpo ọkụ mmalite.Usoro a na-ejikarị eme mgbanwe dị ka ọgwụ mgbochi, njikọ njikọ, ma ọ bụ ihe mgbanwe kemịkalụ iji gbochie ọrụ DNA polymerase.Ebe ọ bụ na a na-egbochi ọrụ nke DNA polymerase na ụlọ okpomọkụ, nkà na ụzụ mmalite na-ekpo ọkụ na-enye nnukwu mma maka ịkwadebe ọtụtụ usoro mmeghachi omume PCR na ọnụ ụlọ na-enweghị ịchụ àjà kpọmkwem nke mmeghachi omume PCR.
2. RT-PCR
RT-PCR (PCR ntụgharị ntụgharị) bụ usoro nnwale maka ntụgharị ntụgharị site na mRNA banye cDNA na iji ya dị ka ndebiri maka mmụba.Usoro nnwale bụ ibu ụzọ wepụ RNA ngụkọta n'ime anụ ahụ ma ọ bụ sel, jiri Oligo (dT) dị ka primer, jiri reverse transcriptase synthesize cDNA, wee jiri cDNA dị ka ndebiri maka nkwalite PCR iji nweta mkpụrụ ndụ ebumnuche ma ọ bụ chọpụta mkpụrụ ndụ ihe nketa.
3. Fluorescent quantitative PCR
Fluorescent quantitative PCR (PCR nke ezigbo oge,RT-qPCR) na-ezo aka na usoro nke ịgbakwunye otu fluorescent na usoro mmeghachi omume PCR, na-eji nchịkọta nke mgbaàmà fluorescent iji nyochaa usoro PCR dum na ozugbo, na n'ikpeazụ na-eji usoro ọkọlọtọ na-enyocha template.Ụzọ qPCR a na-ejikarị gụnyere SYBR Green I na TaqMan.
4. PCR akwụrụ
PCR Nested na-ezo aka na iji usoro abụọ nke PCR primers maka agba abụọ nke mmụba PCR, na ngwaahịa mmụba nke agba nke abụọ bụ iberibe mkpụrụ ndụ ebumnuche.
Ọ bụrụ na a mismatch nke mbụ ụzọ nke primers (outer primers) na-eme ka ngwaahịa na-abụghị nke a kapịrị ọnụ, ohere nke otu mpaghara na-abụghị nke a na-amata site na nke abụọ nke primers ma na-aga n'ihu na-amụba dị ntakịrị, ya mere, nkwalite site na ụzọ abụọ nke primers , a na-emeziwanye nkọwa nke PCR.Otu uru dị n'ime PCR ugboro abụọ bụ na ọ na-enyere aka ịkwalite ngwaahịa zuru oke site na DNA mmalite mmalite.
5. PCR na-emetụ aka
PCR mmetụ aka bụ usoro iji kwalite nkọwapụta mmeghachi omume PCR site na ịhazigharị okirikiri PCR.
Na PCR mmetụ aka, a na-edobe okpomoku na-ekpo ọkụ maka obere okirikiri nke mbụ n'ogo ole na ole n'elu okpomoku na-ekpo ọkụ (Tm) nke primers.Okpomọkụ dị elu nwere ike belata mmụba na-abụghị nke a kapịrị ọnụ, mana n'otu oge ahụ, okpomọkụ dị elu ga-eme ka nkewa nke primers na usoro ebumnuche ka njọ, na-ebute mbelata PCR.Ya mere, n'ime nkeji ole na ole mbụ, a na-edobe okpomọkụ na-ekpo ọkụ ka ọ na-ebelata site na 1 Celsius C kwa okirikiri iji mee ka ọdịnaya nke mkpụrụ ndụ ihe nketa dị na sistemụ.Mgbe a na-eweda ọnọdụ okpomọkụ na-ekpo ọkụ na okpomọkụ kachasị mma, a na-edobe okpomọkụ na-ekpo ọkụ maka okirikiri ndị fọdụrụ.
6. PCR kpọmkwem
PCR Direct na-ezo aka na mmụba nke DNA lekwasịrị anya ozugbo site na nlele ahụ na-enweghị mkpa maka ikewapụ acid nucleic na ịdị ọcha.
Enwere ụdị PCR abụọ:
usoro ozugbo: were obere nlele ma tinye ya ozugbo na PCR Master Mix maka njirimara PCR;
usoro mgbawa: mgbe ịlele ihe nlele ahụ, gbakwunye ya na lysate, lyse iji hapụ genome, were obere lysed supernatant ma tinye ya na PCR Master Mix, mee nchọpụta PCR.Usoro a na-eme ka usoro nnwale ahụ dị mfe, na-ebelata aka na oge, ma na-ezere mfu DNA n'oge usoro nchacha.
7. SOE PCR
Gene splicing site overlap extension PCR (SOE PCR) na-eji primers na njedebe mmekọ na-eme ka ngwaahịa PCR na-etolite n'agbụ, nke mere na n'ime mmeghachi omume mmụba na-esote, site na ndọtị nke agbụ ndị na-ekpuchi elu, ụzọ dị iche iche nke usoro nke a na-ejikọta iberi ndị na-emepụta ihe. ma gbakọta ọnụ.Nke a na nkà na ụzụ ugbu a nwere ụzọ abụọ bụ isi ngwa ntụziaka: iwu nke fusion genes;ngbanwe nke saịtị mkpụrụ ndụ ihe nketa.
8. IPCR
PCR Inverse (IPCR) na-eji reverse complementary primers na-amụba iberibe DNA ndị na-abụghị primers abụọ ahụ, ma na-amụba usoro amaghị ama n'akụkụ abụọ nke iberibe DNA amaara.
Emebere IPCR na mbụ iji chọpụta usoro nke mpaghara amaghị ama, ma a na-ejikarị amụ usoro ndị na-akwalite mkpụrụ ndụ ihe nketa;nhazigharị chromosomal oncogenic, dị ka njikọ mkpụrụ ndụ ihe nketa, ntụgharị na ntụgharị;na njikọ nje virus, ka a na-ejikarị ugbu a Maka mutagenesis na-eduzi saịtị, detuo plasmid nwere ngbanwe achọrọ.
9. dPCR
PCR dijitalụ (dPCR) bụ usoro maka nleba anya zuru oke nke molekul acid nucleic.
Enwere ụzọ atọ ugbu a maka ọnụọgụ nke mkpụrụ ndụ nucleic acid.Photometry dabere na nnabata nke mkpụrụ ndụ nucleic acid;ezigbo oge fluorescent quantitative PCR (Real Time PCR) dabere na uru Ct, yana uru Ct na-ezo aka na ọnụọgụ okirikiri kwekọrọ na uru fluorescence nke enwere ike ịchọpụta;PCR dijitalụ bụ teknụzụ ọnụọgụ kacha ọhụrụ dabere na usoro PCR otu-molecule maka ịgụta ọnụọgụ nucleic acid bụ usoro ọnụọgụ zuru oke.
Oge nzipu: Jun-13-2023