Tekinoroji yo gusuzuma indwara ya molekulari ikoresha uburyo bwa biyolojiya y’ibinyabuzima kugirango imenye imiterere n’imiterere yimiterere yimiterere yumubiri wumuntu hamwe na virusi zitandukanye, kugirango ugere ku ntego yo guhanura no gusuzuma indwara.

Mu myaka yashize, hamwe no kuzamura no gusubiramo tekinoroji yo gusuzuma ya molekuline, ikoreshwa rya clinique yo gusuzuma indwara ya molekile ryabaye ryinshi kandi ryimbitse, kandi isoko ryo gusuzuma indwara ya molekile ryinjiye mugihe cyiterambere ryihuse.

Umwanditsi yavuze mu ncamake tekinoloji isanzwe isuzumwa ya molekuline ku isoko, kandi igabanyijemo ibice bitatu: igice cya mbere kizana ikoranabuhanga rya PCR, igice cya kabiri gitangiza aside nucleic aside isothermal amplification tekinoroji, naho igice cya kabiri gitangiza ikoranabuhanga rikurikirana.

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Igice cya I: Ikoranabuhanga rya PCR

Ikoranabuhanga rya PCR

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

Ikoranabuhanga rya PCR ryatangijwe mu 1983 na Kary Mullis wo muri Cetus, muri Amerika.Mullis yasabye ipatanti ya PCR mu 1985 asohora impapuro za mbere za PCR kuri Science muri uwo mwaka.Mullis yatsindiye igihembo cyitiriwe Nobel muri Chimie mu 1993.

Amahame shingiro ya PCR

PCR irashobora kwagura ibice bya ADN inshuro zirenga miliyoni.Ihame ni uko munsi ya catalizike ya ADN polymerase, umurongo wa kibyeyi ADN ikoreshwa nkicyitegererezo, naho primer yihariye ikoreshwa 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. Gutandukana: Koresha ubushyuhe bwo hejuru kugirango utandukanye ADN imirongo ibiri.Amazi ya hydrogène hagati ya ADN ebyiri zacitse ku bushyuhe bwinshi (93-98 ° C).

2. Annealing: ADN imaze gutandukana kabiri, ubushyuhe buragabanuka kugirango primer ibashe guhuza ADN imwe.

3. Kwaguka: ADN polymerase itangira guhuza imirongo yuzuzanya kumurongo wa ADN uhereye kuri primers uhambiriye iyo ubushyuhe bugabanutse.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 nuko primers zitandukanye zishobora gukorerwa genes zitandukanye, kugirango ibice bya gen bigenewe bishobora kongerwa 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:

-Gukunda amplification idasanzwe hamwe nibisubizo byiza.

-Kumenya bifata igihe kirekire kandi imikorere iragoye.

-Gupima gusa ubuziranenge birashobora gukorwa.

Igisekuru cya kabiri fluorescence igereranya PCR

Fluorescence yuzuye PCR (Real-Time PCR), izwi kandi nka qPCR, ikoreshwa mugukurikirana ikusanyirizo ryibicuruzwa byongerewe imbaraga binyuze mu kwegeranya ibimenyetso bya fluorescent hongeweho iperereza rya fluorescent rishobora kwerekana iterambere rya sisitemu yo kubyitwaramo, no guca ibisubizo bivuye kumurongo wa fluorescence, kandi Irashobora kubarwa hifashishijwe agaciro ka Cq numurongo usanzwe.

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 igabanijwemo ibice: TaqMan fluorescent probe, beacons ya molekile hamwe n amarangi ya fluorescent.

1) Ubushakashatsi bwa TaqMan fluorescent:

Mugihe cyo kongera PCR, iperereza ryihariye rya fluorescent ryongeweho mugihe wongeyeho primers.Iperereza ni oligonucleotide, kandi impande zombi zanditseho itsinda ryumunyamakuru 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) Amatara ya 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.Gufunga, ntabwo bizatanga fluorescence.

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 agaciro kambere kagira ingaruka, kandi ibisubizo 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 imenyekanisha ryanyuma kandi ntabwo ishingiye ku gaciro ka Ct (inzitizi yikurikiranya), bityo rero 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 bwibisubizo, birashobora kugabanywamo ubwoko butatu: microfluidic, chip na sisitemu.