Real Time PCR, wanda kuma aka sani da PCR mai ƙididdigewa ko qPCR, hanya ce don saka idanu na ainihin lokaci da nazarin samfuran haɓaka PCR.
Saboda PCR mai ƙididdigewa yana da fa'idodin aiki mai sauƙi, mai sauri da dacewa, babban hankali, maimaitawa mai kyau, da ƙarancin gurɓataccen ƙwayar cuta, ana amfani dashi ko'ina a cikin gwajin likitanci, ƙimar ingancin ƙwayoyi, binciken maganganun kwayoyin halitta, bincike na transgenic, gano kwayar halitta, gano cutar, gano dabbobi da shuka., gwajin abinci da sauran fannoni.
Don haka, ko kun tsunduma cikin bincike na asali a cikin ilimin kimiyyar rayuwa, ko ma'aikatan kamfanonin harhada magunguna, kamfanonin kiwon dabbobi, kamfanonin abinci, ko ma ma'aikatan dubawa-fitowa da ofisoshin keɓewa, sassan kula da muhalli, asibitoci da sauran sassan, za ku kasance ko žasa da fallasa su Ko kuna buƙatar sanin ilimin sanin ƙimar PCR.

Ka'idar Real Time PCR

Real Time PCR wata hanya ce da ake ƙara abubuwa masu kyalli a cikin tsarin amsawar PCR, kuma ana lura da ƙarfin siginar kyalli a cikin aiwatar da amsawar PCR a ainihin lokacin ta hanyar kayan aikin PCR mai ƙididdigewa, kuma a ƙarshe an bincika bayanan gwaji tare da sarrafa su.

【Lanƙwan ƙarawa】shi ne lanƙwan da ke kwatanta tsarin aiki na PCR.Ƙwaƙwalwar haɓakawa na PCR ba ainihin madaidaicin madaidaicin lanƙwasa ba ne, amma lanƙwan sigmoid.

[lokacin dandali na ƙarawa mai lankwasa]Tare da karuwar yawan hawan hawan PCR, rashin kunnawa na DNA polymerase, raguwar dNTPs da primers, da kuma hana haɓakar haɓakawa ta hanyar amsawa ta hanyar samfurin pyrophosphate, da dai sauransu, PCR ba koyaushe yana fadadawa ba., kuma a ƙarshe zai shiga cikin tudu.

[Yankin Girma Mai Girma na Ƙaƙwalwar Ƙarfafawa]Ko da yake lokacin plateau ya bambanta sosai, a cikin wani yanki na yanki mai girma na haɓakar haɓakawa, maimaitawa yana da kyau sosai, wanda yake da mahimmanci ga ƙididdigar ƙididdiga na PCR.

[Ƙimar ƙofa da ƙimar Ct]Mun saita ƙayyadaddun ƙimar gano haske mai haske a matsayi mai dacewa a cikin yanki mai girma na ƙarawa, wato ƙimar kofa (Threshold).Matsakaicin ƙimar kofa da madaidaicin ƙarawa shine ƙimar Ct, wato, ƙimar Ct tana nufin adadin zagayowar (Threshold Cycle) lokacin da ƙimar kofa ta kai.

Jadawalin da ke ƙasa yana nuna a sarari dangantakar tsakanin layin kofa da lanƙwan ƙarawa, bakin kofa da ƙimar Ct.

【Yadda za a ƙididdigewa?】

An tabbatar da ka'idar ilmin lissafi cewa ƙimar Ct tana da alaƙa mai juzu'i tare da logarithm na adadin samfuran farko.Real Time PCR yana sa ido kan samfuran haɓaka PCR a cikin ainihin lokaci kuma yana ƙididdige su yayin lokacin haɓakawa na juzu'i.

Ga kowane zagayowar PCR, DNA ɗin ya ƙaru sosai da sau 2, kuma nan da nan ya isa tudu.

Zaton cewa adadin fara DNA shine A₀ , bayan n cycles, ana iya bayyana adadin ka'idar samfurin DNA kamar:

A _n =A ₀ ×2ⁿ

Sa'an nan, yawan adadin DNA na farko A 0, da jimawa adadin haɓakar samfurin ya kai darajar ganowa An, kuma adadin zagayowar lokacin isa An shine ƙimar Ct.Wato, yawan adadin adadin DNA na farko A 0, da farko madaidaicin lanƙwan ƙarawa, kuma daidai da adadin da ake buƙata na hawan keke n ya ƙaru.

Muna aiwatar da dilution gradient na daidaitaccen taro da aka sani kuma muna amfani da shi azaman samfuri don PCR na Real Time, kuma za a sami jerin muryoyin ƙarawa a daidai daidaitattun tazara a cikin tsari na fara adadin DNA daga ƙari zuwa ƙasa.Dangane da alaƙar linzamin kwamfuta tsakanin ƙimar Ct da logarithm na adadin samfuran farawa, aAna iya ƙirƙirar [misali mai lankwasa].

Ta hanyar maye gurbin ƙimar Ct na samfurin tare da ƙaddamarwar da ba a sani ba a cikin daidaitaccen ma'auni, ana iya samun adadin samfurin farko na samfurin tare da ƙaddamarwar da ba a sani ba, wanda shine ƙa'idar ƙididdiga ta Real Time PCR.

Hanyar Ganewa na Real Time PCR

Real Time PCR yana gano samfuran haɓaka PCR ta hanyar gano ƙarfin haske a cikin tsarin amsawa.

Ƙa'idar Hanyar Haɗa Rini mai Fluorescent】

Rini mai haske, irin su TB Green ® , na iya haɗawa da DNA mai ma'ana biyu a cikin tsarin PCR da fluoresce akan ɗaure.

Ƙarfin haske a cikin tsarin amsawa ya karu da yawa tare da haɓakar hawan PCR.Ta hanyar gano ƙarfin haske, adadin ƙarar DNA a cikin tsarin amsawa za a iya saka idanu a ainihin lokacin, sa'an nan kuma za a iya ƙididdige adadin samfurin farawa a cikin samfurin.

【Ƙa'idar hanyar bincike mai walƙiya】

bincike mai kyallishi ne jerin nucleic acid tare da ƙungiyar mai kyalli a ƙarshen 5′ da ƙungiyar kashewa a ƙarshen 3′, wanda zai iya ɗaure musamman ga samfurin.Lokacin da binciken ya kasance cikakke, hasken da ke fitar da fluorophore yana kashe ta ƙungiyar masu kashewa kuma ba za ta iya yin haske ba.Lokacin da binciken ya bazu, abin da ke da kyalli zai rabu kuma ya fitar da haske.

Ana ƙara bincike mai kyalli zuwa maganin amsawar PCR.A lokacin aikin cirewa, binciken mai kyalli zai ɗaure zuwa takamaiman matsayi na samfuri.A lokacin tsarin tsawaitawa, aikin 5′→3′ exonuclease na enzyme na PCR zai iya lalata binciken da aka haɗa tare da samfuri, kuma abu mai kyalli ya rabu don fitar da haske.Ta hanyar gano tsananin haske na bincike a cikin tsarin amsawa, ana iya cimma manufar sa ido kan adadin ƙarar samfurin PCR.

【Zaɓin Hanyar Gane Fluorescence】

Idan aka yi amfani da shi don bambance jeri tare da babban homology da aiwatar da ganowar PCR mai yawa kamar nazarin buga rubutu na SNP, hanyar bincike mai kyalli ba za ta iya maye gurbinsa ba.
Don sauran gwaje-gwajen PCR na Real Time, ana iya amfani da hanyar chimera mai sauƙi, mai sauƙi da arha mai sauƙi.

bincika ƙayyadaddun ƙaƙƙarfan ƙaƙƙarfan ƙaƙƙarfan ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun bayanai, mai ikon yin multix PCR

Nakasa

Babban ƙayyadaddun buƙatun don haɓakawa;

Multix PCR ba za a iya yin Bukatar tsara takamaiman bincike, tsada mai tsada;

wani lokaci zanen bincike yana da wahala

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