Ihe ntinye aka na mmeghachi omume PCR

N'oge mmeghachi omume PCR, a na-ezutekarị ihe ụfọdụ na-etinye aka.
N'ihi oke uche nke PCR, a na-ahụta mmetọ dị ka otu n'ime ihe kachasị mkpa na-emetụta nsonaazụ PCR ma nwee ike iwepụta nsonaazụ dị mma.
N'otu aka ahụ dị oke egwu bụ isi mmalite dị iche iche na-eduga na nsonaazụ ụgha na-adịghị mma. Ọ bụrụ na egbochi ma ọ bụ gbochie otu akụkụ ma ọ bụ karịa akụkụ dị mkpa nke ngwakọta PCR ma ọ bụ mmeghachi omume nkwuputa n'onwe ya, enwere ike igbochi nyocha nyocha. Nke a nwere ike iduga mbelata arụmọrụ na ọbụna nsonaazụ ọjọọ ụgha.
Na mgbakwunye na mgbochi, mfu nke iguzosi ike n'ezi ihe nucleic acid nwere ike ime n'ihi mbupu na/ma ọ bụ ọnọdụ nchekwa tupu nkwadebe nlele. Karịsịa, okpomọkụ dị elu ma ọ bụ nchekwa ezughị ezu nwere ike ibute mmebi nke mkpụrụ ndụ na acid nucleic. Ndozi cell na anụ ahụ na ntinye paraffin bụ ihe a maara nke ọma ihe kpatara nkewa DNA na nsogbu na-adịgide adịgide (lee foto 1 na 2). N'okwu ndị a, ọbụnadị oke mma na ịdị ọcha agaghị enyere aka.

Onyonyo 1 | Mmetụta nke immobilization na iguzosi ike n'ezi ihe DNA
Agarose gel electrophoresis gosiri na àgwà DNA dịpụrụ adịpụ na ngalaba paraffin nke autopsies dịgasị iche iche. DNA nke ogologo iberibe iberibe dị iche iche dị na mpụta dabere na usoro nhazi. A na-echekwa DNA naanị mgbe edobere ya n'ụdị ntụ oyi kpọnwụrụ akpọnwụ yana na formalin na-anọpụ iche. Ojiji nke siri ike acidic Bouin fixative ma ọ bụ enweghị nchekwa, formic acid nwere formalin butere nnukwu mfu nke DNA. Iberibe nke fọdụrụ na-ekewa nke ukwuu.
N'aka ekpe, a na-egosipụta ogologo nke iberibe na ụzọ abụọ kilobase (kbp)

Ọgụgụ 2 | Mfu nke iguzosi ike n'ezi ihe nke ebumnuche nucleic acid
(a) Oghere nke 3′-5′ na eriri abụọ ahụ ga-ebute nkwụsịtụ na DNA ebumnuche. njikọ DNA ka ga-eme na obere iberibe. Otú ọ dị, ọ bụrụ na ebe nkwụsị nke primer na-efu na mpempe DNA, ọ bụ naanị mmụba linear na-eme. N'okwu kachasị mma, iberibe iberibe nwere ike megharịa ibe ha, mana mkpụrụ ga-adị ntakịrị na n'okpuru ọkwa nchọpụta.
(b) Ọnwụ nke ntọala, tumadi n'ihi depurination na nhazi nke thymidine dimer, na-eduga n'ịbelata ọnụ ọgụgụ H-bonds na mbelata na Tm. N'oge oge okpomọkụ elongated, primers ga-agbaze na DNA matriks ma ọ gaghị ekpochapụ ọbụna n'okpuru ọnọdụ ndị siri ike.
(c) N'akụkụ thymine bases na-etolite TT dimer.
Nsogbu ọzọ a na-ahụkarị nke na-emekarị na nchọpụta mkpụrụ ndụ bụ ntọhapụ nke acids nucleic lekwasịrị anya na-adịchaghị mma ma e jiri ya tụnyere mmịpụta phenol-chloroform. N'okwu ndị dị oke njọ, nke a nwere ike jikọta ya na ihe ndị na-adịghị mma. Enwere ike ịchekwa oge dị ukwuu site na lysis na-esi esi ma ọ bụ mgbaze enzymatic nke irighiri cell, mana usoro a na-ebutekarị mmetụta PCR dị ala n'ihi enweghị ntọhapụ nucleic acid.

Mgbochi nke ọrụ polymerase n'oge mmụba

N'ozuzu, a na-eji mmachi dị ka echiche akpa iji kọwaa ihe niile na-eduga na nsonaazụ PCR dị elu. N'echiche siri ike biochemical, mmachi nwere oke n'ọrụ nke enzyme ahụ, ya bụ, ọ na-ebelata ma ọ bụ gbochie ntụgharị ngwaahịa site na mmekọrịta ya na saịtị nọ n'ọrụ nke DNA polymerase ma ọ bụ onye na-akwado ya (dịka ọmụmaatụ, Mg2+ maka Taq DNA polymerase).
Ihe ndị dị na ihe nlele ma ọ bụ ihe nchekwa dị iche iche na ihe ndị nwere reagents nwere ike igbochi enzyme ma ọ bụ ọnyà ndị na-akwado ya (dịka EDTA), si otú ahụ na-eme ka polymerase kwụsị ma n'aka nke ya na-eduga na mbelata ma ọ bụ ụgha PCR na-adịghị mma.
Agbanyeghị, ọtụtụ mmekọrịta dị n'etiti ihe mmeghachi omume yana acid nucleic nwere ebumnuche ka ahọpụtara ka 'PCR inhibitors'. Ozugbo iguzosi ike n'ezi ihe nke cell mebiri site na mwepu ma wepụta nucleic acid, mmekọrịta dị n'etiti ihe nlele na ngwọta gbara ya gburugburu na oge siri ike nwere ike ime. Dịka ọmụmaatụ, 'ndị na-apụnara mmadụ' nwere ike jikọta DNA nke nwere otu ma ọ bụ okpukpu abụọ site na mmekọrịta ndị na-abụghị nke covalent ma gbochie ikewapụ na ịdị ọcha site n'ibelata ọnụ ọgụgụ ebumnuche ndị na-emecha rute ụgbọ mmiri mmeghachi omume PCR.
N'ozuzu, PCR inhibitors dị na ọtụtụ mmiri mmiri na reagents eji maka nyocha nyocha ụlọ ọgwụ (urea na mmamịrị, hemoglobin na heparin n'ime ọbara), ihe mgbakwunye nri (ihe ndị na-edozi ahụ, glycogen, abụba, Ca2 + ions) na ihe ndị dị na gburugburu ebe obibi (phenols). ọla dị arọ)

Enwere ike ịhụ ihe mgbochi PCR ka ukwuu na nje bacteria na mkpụrụ ndụ eukaryotic, DNA na-abụghị ebumnuche, DNA na-ejikọta macromolecules nke matrices anụ ahụ na akụrụngwa ụlọ nyocha dị ka uwe aka na plastik. Ịdị ọcha nke acid nucleic n'oge ma ọ bụ mgbe mmịpụta gasịrị bụ usoro kachasị mma maka iwepụ PCR inhibitors.
Taa, ngwa dị iche iche na-ewepụta akpaghị aka nwere ike dochie ọtụtụ ụkpụrụ ntuziaka, mana 100% mgbake na/ma ọ bụ sachapụ ebumnuche emebeghị. Ndị na-egbochi ihe nwere ike ka dị na acid nucleic a sachara ma ọ bụ nweelarị mmetụta. Usoro dị iche iche dị iji belata mmetụta nke ndị na-egbochi ya. Nhọrọ nke polymerase kwesịrị ekwesị nwere ike inwe mmetụta dị ukwuu na ọrụ inhibitor. Ụzọ ndị ọzọ egosipụtara iji belata mgbochi PCR na-abawanye mkpokọta polymerase ma ọ bụ itinye ihe mgbakwunye dị ka BSA.
Enwere ike igosipụta mgbochi nke mmeghachi omume PCR site n'iji njikwa ogo nke ime ime (IPC).
Ekwesịrị iji nlezianya wepụ ihe niile reagents na ihe ngwọta ndị ọzọ na ngwa mmịpụta, dị ka ethanol, EDTA, CETAB, LiCl, GuSCN, SDS, isopropanol na phenol, na nucleic acid kewapụrụ site na usoro ịsa ahụ nke ọma. Dabere na ntinye uche ha, ha nwere ike rụọ ọrụ ma ọ bụ gbochie PCR.