Nkà na ụzụ nyocha nke mkpụrụ ndụ ihe nketa na-eji usoro ihe ọmụmụ ihe omimi chọpụta okwu na nhazi nke mkpụrụ ndụ ihe nketa nke ahụ mmadụ na ọrịa dị iche iche, iji nweta nzube nke ịkọ na ịchọpụta ọrịa.

N'ime afọ ndị na-adịbeghị anya, site na nkwalite na ntinye nke teknụzụ nyocha nke mkpụrụ ndụ ihe nketa, ngwa ụlọ ọgwụ nke nyocha mkpụrụ ndụ na-abawanye ụba na nke miri emi, na ahịa nyocha mkpụrụ ndụ abanyela n'oge mmepe ngwa ngwa.

Onye edemede ahụ na-achikota teknụzụ nyocha mkpụrụ ndụ nkịtị na ahịa, wee kewaa ụzọ atọ: akụkụ nke mbụ na-ewebata teknụzụ PCR, akụkụ nke abụọ na-ewebata teknụzụ nucleic acid isothermal amplification, akụkụ nke abụọ na-ewebata teknụzụ usoro.

01

Nkebi nke mbụ: Teknụzụ PCR

PCR teknụzụ

PCR (mmeghachi omume yinye polymerase) bụ otu n'ime teknụzụ mmụba DNA in vitro, nwere akụkọ ihe karịrị afọ 30.

Kary Mullis nke Cetus, USA sụrụ ụzọ nkà na ụzụ PCR na 1983.Mullis tinyere akwụkwọ maka patent PCR na 1985 wee bipụta akwụkwọ agụmakwụkwọ PCR mbụ na Sayensị n'otu afọ ahụ.Mullis nwetara Nrite Nobel na Chemistry na 1993.

Ụkpụrụ bụ isi nke PCR

PCR nwere ike ịbawanye iberibe DNA e lekwasịrị anya ihe karịrị otu nde.Ụkpụrụ bụ na n'okpuru catalysis nke DNA polymerase, a na-eji DNA strand nne na nna mee ihe dị ka ndebiri, a na-ejikwa otu primer dị ka mmalite maka mgbatị.A na-emegharị ya na vitro site na usoro dị ka denaturation, annealing, na ndọtị.Usoro nke eriri nwa nwanyị na-akwado DNA strand template DNA.

E kewara usoro PCR ọkọlọtọ n'ime usoro atọ:

1. Denaturation: Jiri elu okpomọkụ kewaa DNA abụọ strands.A na-agbaji njikọ hydrogen n'etiti eriri abụọ DNA na oke okpomọkụ (93-98°C).

2. Annealing: Mgbe e kewasịrị DNA nke nwere eriri abụọ, a na-ebelata okpomọkụ ka primer wee nwee ike jikọta na DNA nwere otu eriri.

3. Mgbatị: DNA polymerase na-amalite ịmepụta eriri ndị agbakwunyere n'akụkụ eriri DNA site na primers na-ejikọta mgbe okpomọkụ na-agbada.Mgbe ndọtị ahụ mechara, a na-emecha okirikiri, ọnụ ọgụgụ nke iberibe DNA na-abawanye okpukpu abụọ.

N'ịkwụghachi usoro atọ ndị a ugboro 25-35, ọnụ ọgụgụ nke iberibe DNA ga-abawanye nke ukwuu.

Amụma nke PCR bụ na enwere ike ịmepụta primers dị iche iche maka mkpụrụ ndụ ihe nketa dị iche iche, nke mere na mkpụrụ ndụ mkpụrụ ndụ ihe nketa nwere ike ịbawanye n'ime obere oge.

Ka ọ dị ugbu a, enwere ike kewaa PCR ụzọ atọ, ya bụ PCR nkịtị, PCR fluorescent quantitative na PCR dijitalụ.

Ọgbọ mbụ nke PCR nkịtị

Jiri ngwa PCR nkịtị na-amụbawanye mkpụrụ ndụ ebumnuche, wee jiri agarose gel electrophoresis chọpụta ngwaahịa ahụ, naanị nyocha nke qualitative nwere ike ime.

Ihe ọghọm dị na PCR nke ọgbọ mbụ:

-Enwere mmasị na nkwalite na-abụghị nke akọwapụtara na nsonaazụ dị mma ụgha.

-Nchọpụta ahụ na-ewe ogologo oge na ọrụ ahụ siri ike.

-Ọ bụ naanị qualitative ule nwere ike ime.

Ọgbọ nke abụọ fluorescence quantitative PCR

A na-eji Fluorescence quantitative PCR (Real-Time PCR), nke a makwaara dị ka qPCR, iji nyochaa nchịkọta nke ngwaahịa ndị a na-amụbawanye site na nchịkọta nke mgbaàmà fluorescent site n'ịgbakwunye fluorescent probes nke nwere ike igosi ọganihu nke usoro mmeghachi omume, na ikpe ikpe site na fluorescence curve, na Ọ nwere ike ịdị na-atụnye ọnụ ahịa na ọnụ ọgụgụ enyemaka.

N'ihi na a na-eme teknụzụ qPCR na sistemu mechiri emechi, enwere ike ibute ọrịa na-ebelata, enwere ike nyochaa akara ngosi fluorescence maka nchọpụta ọnụọgụ, yabụ na ọ bụ nke a na-ejikarị eme ihe na ụlọ ọgwụ wee bụrụ teknụzụ kachasị na PCR.

Enwere ike kewaa ihe fluorescent a na-eji na ezigbo oge fluorescent quantitative PCR n'ime: TaqMan fluorescent probes, mgbama molikula na ihe eji esiji fluorescent.

1) Nyocha fluorescent TaqMan:

N'oge nkwalite PCR, a na-agbakwunye otu nyocha fluorescent ka ọ na-agbakwunye otu ụzọ primers.Nyocha ahụ bụ oligonucleotide, na njedebe abụọ ka a na-akpọ otu onye na-ede akụkọ fluorescent otu na otu onye na-ekpo ọkụ ọkụ.

Mgbe nyocha ahụ adịghị emebi emebi, mgbaàmà fluorescent nke ndị otu nta akụkọ na-ewepụta na-etinye uche na otu ndị na-emenyụ ọkụ;n'oge nkwalite PCR, ọrụ 5'-3' exonuclease nke Taq enzyme na-agbaji ma na-eweda nyocha ahụ, na-eme ka onye nta akụkọ fluorescent otu na quencher The fluorescent otu kewapụrụ, nke mere na fluorescence nlekota oru usoro nwere ike na-enweta fluorescence mgbaàmà, ya bụ, mgbe ọ bụla eriri DNA na-amụbawanye, a fluorescent na-emepụta na fluorescent molekul. na nguzobe nke PCR ngwaahịa.

2) SYBR fluorescent e ji esiji ákwà:

N'ime usoro mmeghachi omume PCR, a na-agbakwunye oke nke SYBR fluorescent agba.Mgbe agbakọchara SYBR fluorescent na-abụghị nke etinyere n'ime eriri abụọ nke DNA, ọ na-ewepụta mgbama fluorescent.Mkpụrụ ndụ SYBR dye nke na-etinyeghị n'ime agbụ agaghị ewepụta akara ọ bụla nke fluorescent, si otú ahụ na-ahụ na mgbama fluorescent Mmụba nke ngwaahịa PCR na-ejikọta ya na mmụba nke ngwaahịa PCR.SYBR na-ejikọ naanị na DNA nwere eriri abụọ, yabụ enwere ike iji mgbaze agbaze iji chọpụta ma mmeghachi omume PCR ọ̀ bụ kpọmkwem.

3) mgbama molecular

Ọ bụ ihe nyocha oligonucleotide nwere aha-oligonucleotide nke na-etolite nhazi ntutu nke ihe dịka ntọala 8 na 5 na 3 nsọtụ.Usoro nucleic acid dị na nsọtụ abụọ ahụ na-ejikọta ọnụ, na-eme ka otu fluorescent na otu quenching na-esi ike.Na nso, ọ gaghị emepụta fluorescence.

Mgbe emepụtara ngwaahịa PCR, n'oge usoro mgbakasị ahụ, akụkụ etiti nke mgbaama molekụla na-ejikọta ya na usoro DNA akọwapụtara, na mkpụrụ ndụ fluorescent na-ekewapụ na gene quencher iji mepụta fluorescence.

Ihe ọghọm dị na PCR ọgbọ nke abụọ:

Mmetụta ka na-adịkwaghị, na nchọta nke obere ntule ezighi ezi.

Enwere mmetụta uru ndabere, yana nsonaazụ ya nwere ike ịdaba na nnyonye anya.

Ọgbọ nke atọ dijitalụ PCR

PCR dijitalụ (DigitalPCR, dPCR, Dig-PCR) na-agbakọ nọmba nnomi nke usoro ebumnuche site na nchọpụta njedebe, ma nwee ike ịrụ nchọpụta ọnụọgụ zuru oke na-ejighi njikwa ime na usoro ọkọlọtọ.

Digital PCR na-eji nchọpụta njedebe na-adabereghị na uru Ct (ọnụ ụzọ okirikiri), ya mere mmeghachi omume PCR dijitalụ na-adịchaghị emetụta site na arụmọrụ nkwalite, na nnabata nke PCR mmeghachi omume mmechi na-emeziwanye, na oke ziri ezi na reproducibility.

N'ihi njiri mara nke uche dị elu na izi ezi dị elu, ndị na-egbochi mmeghachi omume PCR anaghị egbochi ya ngwa ngwa, yana ọ nwere ike nweta ọnụọgụ zuru oke na-enweghị ngwaahịa ọkọlọtọ, nke ghọrọ ebe nyocha na ngwa ngwa.

Dị ka ụdị mmeghachi omume dị iche iche si dị, enwere ike kewaa ya na ụdị atọ: microfluidic, mgbawa na usoro droplet.