PCR (polymerase chain reaction) nimwe muma tekinoroji ya vitro vitro yo kongera imbaraga, ifite amateka yimyaka irenga 30.

Ikoranabuhanga rya PCR ryatangijwe na Kary Mullis wo muri Cetus, muri Amerika mu 1983. Mullis yasabye ipatanti ya PCR mu 1985 maze asohora impapuro za mbere za PCR ku bumenyi muri uwo mwaka.Mullis yahawe igihembo cyitiriwe Nobel muri chimie mu 1993 kubera akazi yakoze.

Amahame shingiro ya PCR

PCR irashobora kwagura ibice bya ADN inshuro zirenga miliyoni.Ihame riri munsi ya catalizike ya ADN polymerase, ukoresheje ADN umurongo wababyeyi nkicyitegererezo na primer yihariye nkintangiriro yo kwaguka.Yigana muri vitro binyuze mu ntambwe nka denaturation, annealing, no kwaguka.Inzira yumukobwa umurongo ADN yuzuzanya nababyeyi umurongo wicyitegererezo ADN.

Inzira isanzwe ya PCR igabanijwemo intambwe eshatu:

1.Kwiyongera: Koresha ubushyuhe bwo hejuru kugirango utandukanye ADN imirongo ibiri.Ihuza rya hydrogène hagati ya ADN ebyiri zacitse ku bushyuhe bwinshi (93-98 ℃).

2.Gushyira hamwe: ADN imaze gutandukana kabiri, gabanya ubushyuhe kugirango primer ibashe guhuza ADN imwe.

3.Ubwiyongere: ADN polymerase itangira guhuza imirongo yuzuzanya kumurongo wa ADN uhereye kuri primers iboshye mugihe ubushyuhe bwamanutse.Iyo kwagura birangiye, uruziga rurarangira, kandi umubare wa ADN ibice byikubye kabiri

Gusubiramo izi ntambwe eshatu inshuro 25-35, umubare wibice bya ADN uziyongera cyane.

Ubuhanga bwa PCR ni uko primers zitandukanye zishobora gukorerwa genes zitandukanye, kugirango ibice bya gene bishobora kwongerwa mugihe gito.

Kugeza ubu, PCR irashobora kugabanywamo ibyiciro bitatu, aribyo PCR isanzwe, fluorescent quantitative PCR na PCR.

Igisekuru cya mbere cya PCR isanzwe

Koresha igikoresho gisanzwe cya PCR kugirango wongere gene igenewe, hanyuma ukoreshe agarose gel electrophorei kugirango umenye ibicuruzwa, hashobora gukorwa isesengura ryujuje ubuziranenge.

Ingaruka nyamukuru zo mu gisekuru cya mbere PCR:

1.Gukunda amplification idasanzwe hamwe nibisubizo byiza.

2.Kumenya bifata igihe kirekire kandi imikorere iragoye.

3.Ikizamini cyujuje ubuziranenge kirashobora gukorwa

Igisekuru cya kabiri-Igihe-PCR

Real-Time PCR, izwi kandi nka qPCR, ikoresha probe ya fluorescent ishobora kwerekana iterambere rya sisitemu yo kubyitwaramo, ikanagenzura ikusanyirizo ryibicuruzwa byongerewe imbaraga binyuze mu kwegeranya ibimenyetso bya fluorescent, kandi igacira ibisubizo ibisubizo ikoresheje umurongo wa fluorescence.Irashobora kugereranywa hifashishijwe Cq agaciro nigipimo gisanzwe.

Kubera ko tekinoroji ya qPCR ikorerwa muri sisitemu ifunze, amahirwe yo kwandura aragabanuka, kandi ibimenyetso bya fluorescence birashobora gukurikiranwa kugirango hamenyekane umubare, bityo rero niwo ukoreshwa cyane mubikorwa byubuvuzi kandi wabaye ikoranabuhanga ryiganje muri PCR.

Ibintu bya fluorescente bikoreshwa mugihe nyacyo cya fluorescent PCR ishobora kugabanywamo: TaqMan fluorescent probe, beacons ya molekile hamwe n irangi rya fluorescent.

1) Ubushakashatsi bwa TaqMan fluorescent:

Mugihe cyo kongera PCR, iperereza ryihariye rya fluorescent ryongeweho mugihe wongeyeho primer.Iperereza ni oligonucleotide, kandi impande zombi zanditswemo umunyamakuru fluorescent hamwe nitsinda rya fluorescent.

Iyo iperereza ridahwitse, ikimenyetso cya fluorescent cyatanzwe nitsinda ryabanyamakuru cyakirwa nitsinda rizimya;mugihe cyo kongera PCR, ibikorwa bya 5′-3 ′ exonuclease ya Taq enzyme ikora kandi ikanatesha agaciro iperereza, bigatuma umunyamakuru wa fluorescent hamwe na quencher Itsinda rya fluorescent ryatandukanijwe, kugirango sisitemu yo gukurikirana fluorescence ishobora kwakira ibimenyetso bya fluorescence, ni ukuvuga ko burigihe burigihe umurongo wa ADN wongerewe imbaraga, hamwe no gukusanya ibimenyetso bya fluorescence hamwe no gukusanya ibimenyetso bya fluorescence.

2) Irangi rya SYBR fluorescent:

Muri sisitemu ya reaction ya PCR, hiyongereyeho irangi rya SYBR fluorescent irangi.Nyuma yo gusiga irangi rya SYBR fluorescent idashyizwe muburyo bwihariye muri ADN ya kabiri, itanga ikimenyetso cya fluorescent.Molekile ya SYBR idasizwe mumurongo ntisohora ibimenyetso bya fluorescente, bityo byemeze ibimenyetso bya fluorescent Kwiyongera kubicuruzwa bya PCR bihujwe rwose no kwiyongera kwibicuruzwa bya PCR.SYBR ihuza gusa na ADN ebyiri-ebyiri, bityo umurongo wo gushonga urashobora gukoreshwa kugirango umenye niba reaction ya PCR yihariye.

3) Itara rya molekulari:

Nibiti bya stem-loop byanditseho oligonucleotide probe ikora imisatsi yimisatsi igera kuri 8 kuri 5 na 3.Urutonde rwa acide nucleic kumpera zombi zuzuzanya, bigatuma itsinda rya fluorescent hamwe nitsinda rizimya gukomera.Hafi, nta fluorescence izakorwa.

Ibicuruzwa bya PCR bimaze kubyara, mugihe cya annealing, igice cyo hagati cyumucyo wa molekuline gihujwe nurutonde rwihariye rwa ADN, kandi gene ya fluorescent itandukanijwe na gene yo kuzimya kugirango itange fluorescence.

Ingaruka nyamukuru zo mu gisekuru cya kabiri PCR:

Sensitivite iracyabura, kandi gutahura ingero nke za kopi ntabwo aribyo.

Hariho ingaruka zinyuma yagaciro, kandi ibisubizo birashobora kwivanga.

Iyo hari PCR inhibitor muri sisitemu yo kubyitwaramo, ibisubizo byo gutahura birashobora kwivanga.

Igisekuru cya gatatu PCR

Digital PCR (DigitalPCR, dPCR, Dig-PCR) ibara numero ya kopi yintego ikurikirana binyuze mumpera yanyuma, kandi irashobora gukora igereranya ryuzuye ryuzuye ridakoresheje igenzura ryimbere hamwe nu murongo usanzwe.

Digital PCR ikoresha amaherezo-yo gutahura kandi ntabwo ishingiye ku gaciro ka Ct (cycle cycle), bityo reaction ya digitale PCR ntabwo ihindurwa cyane nubushobozi bwa amplification, kandi kwihanganira ibiyobora PCR biratera imbere, hamwe nukuri kandi byororoka.

Bitewe nibiranga sensibilité yo hejuru kandi yukuri, ntabwo byoroshye kubangamirwa na PCR inhibitor reaction, kandi irashobora kugera kumubare nyawo rwose udafite ibicuruzwa bisanzwe, byahindutse ubushakashatsi nibishyirwa mubikorwa.

Ukurikije uburyo butandukanye bwibikorwa bya reaction, birashobora kugabanywamo ubwoko butatu bwingenzi: microfluidic, chip na sisitemu.

1) Microfluidic digital PCR, mdPCR:

Ukurikije tekinoroji ya microfluidic, inyandikorugero ya ADN iratandukanye.Tekinoroji ya microfluidic irashobora kumenya icyitegererezo cya nano-kuzamura cyangwa kubyara ibitonyanga bito, ariko ibitonyanga bikenera uburyo bwihariye bwa adsorption hanyuma bigahuzwa na sisitemu ya reaction ya PCR.mdPCR yagiye ikoreshwa buhoro buhoro nubundi buryo bwo gusimbuza.

2) Droplet ishingiye kuri digitale PCR, ddPCR:

Koresha amazi-y-amavuta yamashanyarazi kugirango utunganyirize icyitegererezo mubitonyanga, hanyuma ugabanye sisitemu yogukora irimo molekile ya acide nucleic mo ibihumbi bitonyanga bya nanoscale, buri kimwe muri byo kikaba kitarimo molekile ya acide nucleic igomba gutahurwa, cyangwa ikubiyemo molekile imwe ya nucleic aside igeragezwa.

3) Chip ishingiye kuri digitale PCR, cdPCR:

Koresha uburyo bwa tekinoroji yinzira ya tekinoroji kugirango ushushanye microtubes na microcavities nyinshi kuri wafer ya silicon cyangwa ikirahuri cya quartz, hanyuma ugenzure imigendekere yumuti unyuze mumashanyarazi atandukanye, hanyuma ugabanye amazi yicyitegererezo muri nanometero zingana zingana mumariba ya reaction ya digitale PCR kugirango igere kumubare wuzuye.

Ingaruka nyamukuru zo mu gisekuru cya gatatu PCR:

Ibikoresho na reagent bihenze.

Inyandikorugero yubuziranenge isabwa ni hejuru.Niba ingano yicyitegererezo irenze urugero rwa microsystem, ntibizashoboka kubara, kandi niba ari bito cyane, umubare wuzuye uzagabanuka.

Ibyiza byiza nabyo birashobora kubyara mugihe hari amplification idasanzwe.