Real Time PCR, yomwe imadziwikanso kuti quantitative PCR kapena qPCR, ndi njira yowunikira komanso kusanthula zinthu zakukweza kwa PCR.
Chifukwa kuchuluka kwa PCR kuli ndi ubwino wa ntchito yosavuta, yofulumira komanso yosavuta, yokhudzidwa kwambiri, yobwerezabwereza bwino, komanso yotsika kwambiri yowonongeka, imagwiritsidwa ntchito kwambiri poyezetsa mankhwala, kuyesa mphamvu ya mankhwala, kufufuza kwa jini, kufufuza kwa transgenic, kufufuza majini, kufufuza tizilombo toyambitsa matenda, zinyama ndi zomera., kuyesa zakudya ndi madera ena.
Chifukwa chake, ngakhale mukuchita nawo kafukufuku woyambira mu sayansi ya moyo, kapena ogwira ntchito kumakampani opanga mankhwala, makampani oweta ziweto, makampani azakudya, kapenanso ogwira ntchito yoyang'anira zotuluka ndi malo okhala kwaokha, m'madipatimenti owunika zachilengedwe, zipatala ndi magawo ena, mudzawonetsedwa kapena kuchepera kapena muyenera kudziwa kudziwa zambiri za PCR.

Mfundo ya Real Time PCR

Real Time PCR ndi njira yomwe zinthu za fulorosenti zimawonjezeredwa ku PCR reaction system, ndipo mphamvu ya chizindikiro cha fluorescence muzochitika za PCR imayang'aniridwa mu nthawi yeniyeni ndi chida cha PCR chochuluka, ndipo pamapeto pake deta yoyesera imawunikidwa ndikukonzedwa.

【Amplification curve】ndiye mkhonde wofotokozera machitidwe amphamvu a PCR.Mapiritsi okulitsa a PCR sikuti ndi njira yokhazikika, koma yokhotakhota ya sigmoid.

[Platform phase of amplification curve]Ndi kuchuluka kwa kuchuluka kwa ma PCR, kutsekeka kwa DNA polymerase, kuchepa kwa dNTPs ndi zoyambira, komanso kulepheretsa kaphatikizidwe kazinthu zomwe zimachitika ndi pyrophosphate, ndi zina zambiri, PCR sikuti imakula mokulirapo., ndipo potsirizira pake adzalowa m’phiri.

[Chigawo Chakukulirapo Kwambiri cha Amplification Curve]Ngakhale gawo lamapiri limasiyanasiyana kwambiri, m'dera lina la kukula kwa chigawo cha amplification curve, kubwerezabwereza ndikwabwino kwambiri, zomwe ndizofunikira kwambiri pakuwunika kwa PCR.

[Threshold value ndi Ct value]Timayika malire a kudziwika kwa fluorescence pamalo oyenera mu gawo la kukula kwa curve amplification, ndilo mtengo wapakati (Threshold).Kuphatikizika kwa mtengo wapachiyambi ndi curve yokulitsa ndi mtengo wa Ct, ndiko kuti, mtengo wa Ct umatanthawuza chiwerengero cha mizere (Threshold Cycle) pamene mtengo wapakati wafika.

Grafu yomwe ili pansipa ikuwonetsa bwino mgwirizano pakati pa mzere wapakati ndi ma curve amplification, threshold ndi mtengo wa Ct.

【Momwe mungawerengere?】

Zatsimikiziridwa ndi chiphunzitso cha masamu kuti mtengo wa Ct uli ndi chiyanjano chotsutsana ndi logarithm ya chiwerengero cha ma tempuleti oyambirira.Real Time PCR imayang'anira zinthu zokulitsa za PCR munthawi yeniyeni ndikuziwerengera panthawi yokulitsa.

Pa kuzungulira kulikonse kwa PCR, DNA inakula kwambiri ndi nthawi za 2, ndipo posakhalitsa inafika pamtunda.

Kungoganiza kuti kuchuluka kwa DNA koyambira ndi A₀ , pambuyo pa n kuzungulira, kuchuluka kwa chiphunzitso cha DNA kungafotokozedwe motere:

A _n =A ₀ ×2 paⁿ

Kenako, kuchuluka kwa DNA koyambirira kwa A 0 ndi, m'pamenenso kuchuluka kwa mankhwalawa kumafika pamtengo wodziwikiratu An , ndipo kuchuluka kwa mizere ikafika ku An ndi mtengo wa Ct.Ndiye kuti, kuchuluka koyambirira kwa DNA A 0 kumakhala koyambirira, m'pamenenso nsonga yokulirapo imakwera, ndipo momwemonso kuchuluka kofunikira kwa mikombero n kumakhala kochepa.

Timapanga kuchepetsedwa kwa gradient ya muyezo wodziwika bwino ndikuugwiritsa ntchito ngati template ya Real Time PCR, ndipo ma curve angapo okulitsa adzapezedwa molingana ndi dongosolo loyambira kuchuluka kwa DNA kuchokera kuchulukira mpaka kuchepera.Malinga ndi mgwirizano wa mzere pakati pa mtengo wa Ct ndi logarithm ya chiwerengero cha ma tempuleti oyambira, a[mzere wopindika] ukhoza kupangidwa .

Mwa kulowetsa mtengo wa Ct wa chitsanzo ndi ndende yosadziwika muzitsulo zokhazikika, chiwerengero choyambirira cha template cha chitsanzo chosadziwika bwino chikhoza kupezedwa, chomwe ndi chiwerengero cha Real Time PCR.

Njira yodziwira Real Time PCR

Real Time PCR imazindikira zinthu zokulitsa za PCR pozindikira mphamvu ya fluorescence mumayendedwe.

Mfundo ya Njira Yoyikira utoto wa Fluorescent】

Mitundu ya fluorescent, monga TB Green ®, akhoza kumangiriza mosadziwika ku DNA yamitundu iwiri mu machitidwe a PCR ndi fluoresce pomanga.

Kuchuluka kwa fluorescence mumayendedwe kachitidwe kakuwonjezeka kwambiri ndi kuchuluka kwa ma PCR.Pozindikira mphamvu ya fluorescence, kuchuluka kwa kukulitsa kwa DNA mumayendedwe kachitidwe kumatha kuyang'aniridwa mu nthawi yeniyeni, ndiyeno kuchuluka kwa template yoyambira pachitsanzo kumatha kuwerengedwa mosinthika.

【Mfundo ya fulorosenti probe njira】

fulorosenti kafukufukundi gulu la nucleic acid ndi gulu la fulorosenti kumapeto kwa 5 ndi gulu lozimitsa kumapeto kwa 3, lomwe lingathe kumangirira template.Pamene kafukufukuyo ali bwino, fluorescence yotulutsidwa ndi fluorophore imazimitsidwa ndi gulu lozimitsa ndipo silingathe kutulutsa fluoresce.Pamene kafukufukuyo wavunda, chinthu cha fulorosenti chidzalekanitsa ndi kutulutsa fulorosisi.

Pulogalamu ya fulorosenti imawonjezeredwa ku yankho la PCR reaction.Panthawi ya annealing, kufufuza kwa fulorosenti kumangiriza ku malo enieni a template.Pakukulitsa, 5′→3′ exonuclease ntchito ya PCR enzyme imatha kuwola kafukufuku wa fulorosenti wosakanizidwa ndi template, ndipo chinthu cha fulorosenti chimasiyanitsidwa kuti chitulutse fulorosenti.Pozindikira mphamvu ya fulorosenti ya kafukufukuyo mumayendedwe kachitidwe, cholinga chowunika kuchuluka kwazinthu za PCR zitha kukwaniritsidwa.

【Kusankha Njira Yodziwira Fluorescence】

Ngati ikugwiritsidwa ntchito kusiyanitsa zotsatizana ndi ma homology apamwamba ndikuzindikira kuchuluka kwa PCR monga kusanthula kwa SNP, njira yofufuzira fulorosenti ndi yosasinthika.
Kwa kuyesa kwina kwa Real Time PCR, njira yosavuta, yosavuta komanso yotsika mtengo ya fluorescent chimera ingagwiritsidwe ntchito.

probesKukhazikika kwamphamvu, komwe kumatha kuchulukitsa PCR

Kuperewera

Zofunikira zapadera pakukulitsa;

Multiplex PCR sichingachitikeKufunika kupanga zofufuza zenizeni, zokwera mtengo;

nthawi zina kupanga kafukufuku kumakhala kovuta

Zogwirizana nazo: