• PCR bụ usoro eji eme ka DNA mụbaa site na obere ndebiri DNA.RT-PCR na-eji ntụgharị ntụgharị iji mepụta ndebiri DNA sitere na isi iyi RNA nke enwere ike ịbawanye elu.
• PCR na RT-PCR na-abụkarị mmeghachi omume njedebe, ebe qPCR na RT-qPCR na-eji kinetics nke ọnụego njikọ ngwaahịa n'oge mmeghachi omume PCR iji gụta ọnụọgụ ndebiri dị ugbu a.
• Ụzọ ọhụrụ, dị ka PCR dijitalụ, na-enye ọnụ ọgụgụ zuru oke nke ndebiri DNA mbụ, ebe ụzọ dị ka PCR isothermal na-ebelata mkpa akụrụngwa dị oke ọnụ iji nye nsonaazụ a pụrụ ịdabere na ya.

Mmeghachi omume yinye polymerase (PCR) bụ usoro ihe ọmụmụ ihe omimi dị mfe na nke a na-ejikarị amụba ma chọpụta usoro DNA na RNA.Tụnyere usoro ọdịnala nke DNA cloning na amplification, nke nwere ike were ụbọchị, PCR chọrọ naanị awa ole na ole.PCR na-enwe mmetụta nke ukwuu ma na-achọ obere ndebiri maka nchọpụta na mmụba nke usoro a kapịrị ọnụ.Ụzọ PCR ndị bụ isi esiwo n'ihu na nchọpụta DNA na RNA dị mfe.N'okpuru ebe a, anyị enyela nkọwapụta nke ụzọ PCR dị iche iche na reagents anyị na-enye na Enzo Life Sciences maka mkpa nyocha gị.Anyị bu n'obi inyere ndị ọkà mmụta sayensị aka ịnweta PCR reagents ngwa ngwa iji na ọrụ nyocha ha na-esote!

PCR

Maka PCR ọkọlọtọ, ihe niile ị chọrọ bụ DNA polymerase, magnesium, nucleotides, primers, DNA template ga-ebuwanye ibu, na igwe ọkụ ọkụ.Usoro PCR dị mfe dịka ebumnobi ya: 1) DNA nwere eriri abụọ (dsDNA) na-ekpo ọkụ na-ekpo ọkụ, 2) primers kwekọọ n'otu eriri DNA ahụ, na 3) DNA polymerase na-agbatị primers, na-ebute mbipụta abụọ nke eriri ahụ. eriri DNA mbụ.The denaturation, annealing, na elongation usoro n'elu a usoro nke okpomọkụ na oge a maara dị ka otu okirikiri nke nkwalite (Fig. 1).

Ekwesịrị ịhazi usoro nke ọ bụla nke okirikiri maka ndebiri na ntọala nke ejiri.A na-emegharị okirikiri a ugboro 20-40, ma enwere ike nyochaa ngwaahịa a na-amụbawanye, nke a na-ejikarị agarose gel (Fig 2).

Dị ka PCR bụ usoro nwere mmetụta dị ukwuu ma achọrọ obere mpịakọta maka otu mmeghachi omume, a na-atụ aro nkwadebe nke ngwakọta ukwu maka ọtụtụ mmeghachi omume.Ngwakọta nna ukwu ga-agwakọta nke ọma wee kewaa site na ọnụọgụ mmeghachi omume, na-ahụ na mmeghachi omume ọ bụla ga-enwe otu ego nke enzyme, dNTPs, na primers.Ọtụtụ ndị na-eweta ihe, dị ka Enzo Life Sciences, na-enyekwa ngwakọta PCR nke nwere ihe niile ma e wezụga primers na DNA template.

Mpaghara Guanine/Cytosine-rich (GC-rich) na-anọchite anya ihe ịma aka na usoro PCR ọkọlọtọ.Usoro bara ụba nke GC kwụsiri ike karịa usoro nwere ọdịnaya GC dị ala.Ọzọkwa, usoro bara ụba nke GC na-etolite n'ụdị nke abụọ, dị ka loops ntutu.N'ihi ya, eriri abụọ bara ụba nke GC siri ike ikewa kpam kpam n'oge usoro denaturation.N'ihi ya, DNA polymerase enweghị ike ịmepụta eriri ọhụrụ na-enweghị mgbochi.Okpomọkụ dị elu nke denaturation nwere ike imeziwanye nke a, na mgbanwe n'ebe okpomọkụ dị elu na oge nkwụsịtụ dị mkpụmkpụ nwere ike igbochi njikọ a na-akọwaghị nke GC-ọgaranya primers.Mgbakwunye reagents nwere ike ịkwalite mmụba nke usoro bara ụba nke GC.DMSO, glycerol, na betain na-enyere aka ịkpaghasị usoro nke abụọ nke mmekọrịta GC na-ebute wee si otú ahụ mee ka nkewa nke eriri abụọ ahụ dị mfe.

Hot Start PCR

Nkwalite enweghị nkọwa bụ nsogbu nwere ike ime n'oge PCR.Ọtụtụ DNA polymerases nke a na-eji maka PCR na-arụ ọrụ kacha mma na okpomọkụ gburugburu 68°C ruo 72°C.Otú ọ dị, enzyme ahụ nwekwara ike na-arụ ọrụ na obere okpomọkụ, ọ bụ ezie na ọ dị ntakịrị.N'ebe okpomọkụ dị n'okpuru okpomọkụ nke annealing, primers nwere ike jikọta na-abụghị kpọmkwem ma mee ka mmụba na-abụghị nke a kapịrị ọnụ, ọbụlagodi ma ọ bụrụ na emebere mmeghachi omume na ice.Enwere ike igbochi nke a site na iji polymerase inhibitors nke na-ekewapụ na DNA polymerase naanị otu oge okpomọkụ ruru, ya mere okwu mmalite PCR na-ekpo ọkụ.Onye na-egbochi ya nwere ike ịbụ ihe mgbochi nke na-ejikọta polymerase na denatured na okpomọkụ denaturation mbụ (95°C na-emekarị).

Polymerase Fidelity dị elu

Ọ bụ ezie na polymerases DNA na-amụba nke ọma n'ụzọ ziri ezi na usoro ndebiri mbụ, mmejọ dị na nucleotide dakọtara nwere ike ime.Esemokwu dị na ngwa dị ka cloning nwere ike bute transcript nke mpịaji, yana protein ndị atụgharịghị uche ma ọ bụ na-adịghị arụ ọrụ mgbada.Iji zere ndakọrịta ndị a, polymerases nwere ọrụ "ngụgharị" achọpụtala ma tinye ya n'ọrụ.Achọpụtara polymerase izizi nke mbụ, Pfu, na 1991 na Pyrococcus furiosus.Enzyme Pfu a nwere ọrụ exonuclease 3' ruo 5'.Ka DNA na-abawanye, exonuclease na-ewepụ nucleotides na-adaghị adaba na njedebe 3' nke eriri.A na-edochizi nucleotide ziri ezi, njikọ DNA na-aga n'ihu.Achọpụta usoro nucleotide na-ezighi ezi na-adabere na njikọ njikọ maka triphosphate nucleoside ziri ezi na enzyme, ebe njide na-adịghị mma na-ebelata njikọ ahụ ma na-enye ohere maka ngbanwe ziri ezi.Ọrụ nyocha nke Pfu polymerase na-ebute mperi mperi na usoro ikpeazụ ma e jiri ya tụnyere Taq DNA polymerase.N'ime afọ ndị na-adịbeghị anya, a chọpụtawo enzymes ndị ọzọ na-agụgharị ihe, na mgbanwe nke mbụ Pfu enzyme emewo ka ọ dịkwuo n'ibelata ọnụego njehie n'oge mmụba DNA.

RT-PCR

PCR ntụgharị ntụgharị, ma ọ bụ RT-PCR, na-enye ohere iji RNA dị ka ndebiri.Nzọụkwụ ọzọ na-enye ohere ịchọpụta na mmụba nke RNA.A na-atụgharị RNA ahụ ka ọ bụrụ DNA nkwado (cDNA), na-eji ntụgharị transcriptase.Ogo na ịdị ọcha nke ndebiri RNA dị mkpa maka ịga nke ọma nke RT-PCR.Nzọụkwụ mbụ nke RT-PCR bụ njikọ nke ngwakọ DNA/RNA.Reverse transcriptase nwekwara ọrụ RNase H, nke na-eweda akụkụ RNA nke ngwakọ ahụ.A na-emezi mkpụrụ ndụ DNA nwere otu eriri site na ọrụ DNA polymerase nke dabere na DNA nke ntụgharị transcriptase n'ime cDNA.Ịrụ ọrụ nke mmeghachi omume nke mbụ nwere ike imetụta usoro mmụba.Site ebe a gaa n'ihu, a na-eji usoro PCR ọkọlọtọ eme ka cDNA gbasaa.Enwere ike ịtụgharị RNA na cDNA site na RT-PCR nwere ọtụtụ uru, a na-ejikwa ya maka nyocha okwu mkpụrụ ndụ ihe nketa.RNA nwere otu eriri na enweghị ntụkwasị obi, nke na-eme ka ọ sie ike ịrụ ọrụ na ya.Ọ na-arụkarị ọrụ dị ka nzọụkwụ mbụ na qPCR, nke na-akọwapụta transcript RNA n'ihe nlele nke ndu.

qPCR na RT-qPCR

A na-eji PCR quantitative (qPCR) chọpụta, kọwaa na ọnụọgụ nucleic acid maka ọtụtụ ngwa.Na RT-qPCR, a na-agụtakarị transcript RNA site na ibu ụzọ tụgharịa ya na cDNA, dịka akọwara n'elu, wee mechaa qPCR.Dịka ọ dị na PCR ọkọlọtọ, DNA na-abawanye site na usoro ugboro atọ: denaturation, annealing, na elongation.Agbanyeghị, na qPCR, akara ngosi fluorescent na-enyere aka nnakọta data ka PCR na-aga n'ihu.Usoro a nwere ọtụtụ uru n'ihi usoro dị iche iche na chemistries dị.

Na qPCR dabere na agba (nke a na-acha akwụkwọ ndụ akwụkwọ ndụ), akara ngosi fluorescent na-enye ohere nleba anya nke mkpụrụ ndụ DNA agbagoro site n'iji iji dsDNA ejide agbaji.N'oge okirikiri nke ọ bụla, a na-atụ fluorescence.Mgbama fluorescence na-abawanye nha anya na ọnụọgụ DNA emegharịgharịrị.N'ihi ya, a na-atụle DNA na "ezigbo oge" (Fig 3).Ọdịmma dị na qPCR dabere na agba bụ na naanị otu ebumnuche ka enwere ike nyochaa n'otu oge yana na agba ga-ejikọta na ds-DNA ọ bụla dị na nlele.

Na qPCR dabere na nyocha, enwere ike ịchọpụta ọtụtụ ebumnuche n'otu oge na nlele ọ bụla, mana nke a chọrọ njikarịcha na imepụta nyocha (s) akọwapụtara nke ejiri na mgbakwunye na primers.Ọtụtụ ụdị nyocha nyocha dị, mana ụdị a na-ahụkarị bụ nyocha hydrolysis, nke gụnyere fluorophore na quencher.Fluorescence resonance energy transfer (FRET) na-egbochi mpụta nke fluorophore site na quencher mgbe nyocha adịghị.Otú ọ dị, n'oge mmeghachi omume PCR, a na-eme nyocha ahụ n'oge mgbatị primer na mmụba nke usoro a kapịrị ọnụ ọ ga-ejikọta.Nkwụsị nke nyocha ahụ na-ekewa fluorophore site na onye na-eme ka ọ pụta ìhè na-eme ka mmụba dabere na fluorescence (Fig 4).Ya mere, mgbaàmà fluorescence sitere na mmeghachi omume qPCR dabere na nyocha dabara na ọnụọgụ nke usoro ebumnuche nyocha dị na nlele.N'ihi na qPCR dabere na nyocha akọwapụtara karịa qPCR dabere na agba, ọ na-abụkarị teknụzụ ejiri na nyocha nyocha dabere qPCR.

Isothermal Amplification

Usoro PCR nke a kpọtụrụ aha n'elu chọrọ akụrụngwa temometa dị oke ọnụ iji gbalite na mgbada okpomọkụ nke ọnụ ụlọ maka usoro nkwụsị, nkwụsịtụ na mgbatị.E mepụtala ọtụtụ usoro ndị na-adịghị mkpa ngwaọrụ ndị dị otú ahụ ziri ezi ma nwee ike ịme ya na mmiri mmiri dị mfe ma ọ bụ ọbụna n'ime sel nke mmasị.A na-akpọ usoro ndị a mkpokọta isothermal amplification na ọrụ dabere na exponential, linear, ma ọ bụ cascade amplification.

Ụdị nkwalite isothermal kacha mara amara bụ mgbako isothermal, ma ọ bụ LAMP.LAMP na-eji mmụba exponential na 65⁰C iji kwalite ndebiri DNA ma ọ bụ RNA.Mgbe a na-eme LAMP, a na-eji DNA polymerase na-emepụta ihe mbụ anọ ma ọ bụ isii dabara na mpaghara DNA ezubere iche.Abụọ n'ime primers ndị a nwere usoro nnabata nke na-amata usoro dị na primers ndị ọzọ wee kechie ha, na-enye ohere ka usoro "loop" guzobe n'ime DNA arụpụtara ọhụrụ nke na-enye aka na-eme ka primer annealing na-esote nke nkwalite.Enwere ike ịhụ oriọna site n'ọtụtụ ụzọ, gụnyere fluorescence, agarose gel electrophoresis, ma ọ bụ colorimetry.Ọ dị mfe ilele anya na ịchọpụta ọnụnọ ma ọ bụ enweghị ngwaahịa site na colorimetry na enweghị akụrụngwa dị oke ọnụ achọrọ mere LAMP ka ọ bụrụ nhọrọ dabara adaba maka nnwale SARS-CoV-2 na mpaghara ebe nnwale ụlọ ọgwụ adịghị adị ngwa ngwa, ma ọ bụ nchekwa na ibufe ihe nlele. agaghị ekwe omume, ma ọ bụ n'ụlọ nyocha nke na-enwebu PCR akụrụngwa thermocycling.