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 teknụzụ PCR na 1983. Mullis tinyere akwụkwọ maka patent PCR na 1985 wee bipụta akwụkwọ agụmakwụkwọ PCR mbụ na Sayensị n'otu afọ ahụ.E nyere Mullis ihe nrite Nobel na kemistri na 1993 maka ọrụ ya.

Ụkpụrụ bụ isi nke PCR

PCR nwere ike ịbawanye iberibe DNA e lekwasịrị anya ihe karịrị otu nde.Ụkpụrụ ahụ dị n'okpuru catalysis nke DNA polymerase, na-eji DNA strand nne na nna dị ka ndebiri na kpọmkwem primer dị ka mmalite maka ndọtị.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.Akwụsịla njikọ hydrogen n'etiti eriri abụọ DNA na oke okpomọkụ (93-98 ℃).

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

3.Extension: DNA polymerase na-amalite ịmepụta eriri ndị na-akwado ya n'akụkụ DNA strands 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.

Nkà ihe ọmụma nke PCR bụ na enwere ike ịmepụta primers dị iche iche maka mkpụrụ ndụ ihe nketa dị iche iche, nke mere na 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:

1.Prone na-abụghị kpọmkwem mmụba na ụgha nti utịp.

2.Nchọpụta ahụ na-ewe ogologo oge na ọrụ ahụ dị njọ.

3.Only qualitative ule nwere ike ime

PCR Real-Time ọgbọ nke abụọ

Real-Time PCR, nke a makwaara dị ka qPCR, na-eji nyocha fluorescent nke nwere ike igosi ọganihu nke usoro mmeghachi omume, ma na-enyocha nchịkọta nke ngwaahịa ndị a na-amụbawanye site na nchịkọta nke mgbaàmà fluorescent, na-ekpekwa nsonaazụ site na ntụgharị ọkụ.Enwere ike ịgụta ya site n'enyemaka nke uru Cq na usoro mgbanaka ọkọlọtọ.

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 PCR ezigbo oge fluorescent quantitative: TaqMan nyocha fluorescent, mgbama molekụla na ọcha fluorescent.

1) TaqMan nyocha fluorescent:

N'oge nkwalite PCR, a na-agbakwunye otu nyocha fluorescent ka ọ na-agbakwunye otu ụzọ primer.Nyocha ahụ bụ oligonucleotide, na njedebe abụọ ka ejiri otu ndị nta akụkọ fluorescent na otu ndị na-ekpo ọkụ na-ekpo 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 agba:

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, a 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 nke uru n'azụ, na nsonaazụ ya nwere ike ịdaba na nnyonye anya.

Mgbe enwere PCR inhibitors na sistemu mmeghachi omume, nsonaazụ nchoputa na-enwe ike ibute 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 na 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 dị iche iche nke mmeghachi omume unit si kwuo, enwere ike kewaa ya n'ime isi atọ: microfluidic, mgbawa na usoro droplet.

1) Microfluidic dijitalụ PCR, mdPCR:

Dabere na teknụzụ microfluidic, ụdị DNA kewapụrụ.Teknụzụ microfluidic nwere ike ịghọta ihe nlele nano-upgrading ma ọ bụ ọgbọ nke obere ụmụ irighiri mmiri, mana ụmụ irighiri mmiri ahụ chọrọ usoro mgbasa ozi pụrụ iche wee jikọta ya na sistemụ mmeghachi omume PCR.mdPCR ejirila ụzọ ndị ọzọ dochie anya nke nta nke nta.

2) PCR dijitalụ dabere na Droplet, ddPCR:

Jiri teknụzụ ọgbọ nke mmiri n'ime mmanụ hazie ihe nlele ahụ n'ime ụmụ irighiri mmiri, wee kewaa sistemu mmeghachi omume nke nwere ụmụ irighiri ihe nucleic acid n'ime puku kwuru puku nanoscale ụmụ irighiri mmiri, nke ọ bụla n'ime ha enweghị mkpụrụ ndụ nucleic acid lekwasịrị anya nke a ga-achọpụta, ma ọ bụ nwee otu n'ime ọtụtụ mkpụrụ ndụ nucleic acid a ga-anwale.

3) PCR dijitalụ dabere na mgbawa, cdPCR:

Jiri teknụzụ ụzọ mmiri jikọtara ọnụ iji kanye ọtụtụ microtubes na microcavities na silicon wafers ma ọ bụ iko quartz, ma na-achịkwa usoro nke ngwọta site na valvụ njikwa dị iche iche, wee kewaa mmiri mmiri n'ime nanometers nke otu nha n'ime olulu mmiri mmeghachi omume maka mmeghachi omume PCR dijitalụ iji nweta ọnụọgụ zuru oke.

Ihe ọghọm dị na PCR nke atọ:

Akụrụngwa na reagents dị oke ọnụ.

Ihe achọrọ àgwà template dị elu.Ọ bụrụ na ọnụọgụ ndebiri karịrị ọnụọgụ microsystem, ọ gaghị ekwe omume ịkọwapụta, ma ọ bụrụ na ọ dị ntakịrị, a ga-ebelata nha nha.

Enwere ike iwepụta ihe dị mma mgbe enwere nkwalite na-abụghị nke akọwapụtara.