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Hasashen Fluorescence Quantitative Pcr Principle Techniques And Applications

PCR na ainihi mai kyalli shine hanya don auna jimlar adadin samfur bayan kowane zagayowar sarkar polymerase (PCR) a cikin ƙarar ƙarar DNA ta amfani da fluorophore.Ana amfani da hanyar don ƙididdige takamaiman jerin DNA a cikin samfurin da za a gwada ta hanyoyin tunani na ciki ko na waje.Tun lokacin da aka fara, gwaje-gwajen PCR masu ƙyalƙyali sun ƙara shahara tare da malaman dakin gwaje-gwaje.

Ƙa'idar PCR Fluorescence: Fluorescence PCR, wanda aka fara kiransa TaqManPCR kuma daga baya kuma Real-TimePCR, wata sabuwar dabara ce ta ƙididdigar nucleic acid wadda PE (PerkinElmer) ta haɓaka a cikin Amurka a cikin 1995. Dabarar ta dogara ne akan ƙari na bincike mai suna fluorescently ko daidai. rini mai kyalli zuwa PCR na al'ada don cimma aikin ƙididdigewa.Ka'ida: yayin da aikin PCR ke ci gaba, samfuran amsawar PCR suna taruwa kuma ƙarfin siginar kyalli yana ƙaruwa daidai gwargwado.Tare da kowane zagayowar, ana tattara siginar ƙarfin kyalli ta yadda za mu iya sa ido kan canjin adadin samfur ta canjin ƙarfin haske don haka samun jadawali mai lanƙwasa mai kyalli.

Hasken haske na ainihi3
Hasken-lokaci na ainihi2

Gabaɗaya, za'a iya raba madaidaicin ƙarar haske zuwa matakai uku: lokacin siginar bangon haske, lokacin ƙara ƙarar siginar kyalli da kuma lokacin ƙarawa.Yayin lokacin siginar bango, siginar haɓakar siginar kyalli tana rufe ta da siginar bangon haske kuma ba za a iya tantance canje-canjen adadin samfur ba.A cikin zangon plateau, samfurin ƙarawa ba zai ƙara ƙaruwa ba, babu wata alaƙa ta layi tsakanin adadin ƙarshen samfurin da adadin samfurin farawa, kuma ba za a iya ƙididdige lambar kwafin DNA ta farawa bisa ƙimar samfurin PCR na ƙarshe ba.Sai kawai a cikin lokacin ƙarawa na siginar kyalli akwai dangantaka ta layi tsakanin logarithm na adadin samfurin PCR da adadin samfurin farawa, kuma zamu iya zaɓar ƙididdige wannan a wannan matakin.Don saukaka ƙididdigewa da kwatanta, an gabatar da mahimman ra'ayoyi guda biyu a cikin dabarar PCR mai kyalli na ainihin lokaci: madaidaicin haske da ƙimar CT.

Ƙofar ƙima ce ta wucin gadi da aka saita akan madaidaicin ƙarar haske.Matsakaicin lokaci na haɓakawa na PCR.

Ƙimar Ct: ita ce adadin zagayowar da siginar kyalli a cikin kowane bututun amsawa ya yi don isa wurin da aka saita.

Dangantakar da ke tsakanin ƙimar Ct da samfurin farawa: binciken ya nuna cewa ƙimar Ct na kowane samfuri yana da alaƙa ta layi tare da logarithm na lambar kwafin farawa na wannan samfuri, ƙarin kwafi na lambar kwafin farawa, ƙarami Ct. daraja.Ƙimar Ct suna da ɗan daidaita.Ana iya yin daidaitaccen lanƙwasa ta amfani da ma'auni tare da sanannen lambar kwafin farawa, inda haɗin gwiwar kwance yana wakiltar logarithm na lambar kwafin farawa kuma haɗin kai tsaye yana wakiltar ƙimar Ct kamar yadda aka nuna a hoton da ke ƙasa.

Sabili da haka, ta hanyar samun ƙimar Ct na samfurin da ba a san shi ba, ana iya ƙididdige lambar kwafin farawa na wannan samfurin daga daidaitaccen lanƙwasa.

Ƙimar Ct ba ta dawwama kuma ana iya yin tasiri ta hanyar samfurori daban-daban da kayan aiki daban-daban, koda kuwa an maimaita samfurin sau 2 akan kayan aiki iri ɗaya, ƙimar Ct na iya bambanta.

Ƙididdigar ƙididdigewa: Ƙididdiga mai ƙididdigewa za a iya raba ƙididdigan ƙididdiga masu ƙyalli zuwa ɗigon haske da rini mai kyalli dangane da alamomin da aka yi amfani da su.Abubuwan bincike na Fluorescent sun haɗa da fasahar Beacon (fasaha na fitilar kwayoyin halitta, wanda Amurka Tagyi ta wakilta), TaqMan bincike (wanda ABI ke wakilta) da fasaha na FRET (wanda Roche ke wakilta);riniyoyin mai kyalli sun haɗa da ɗimbin rini masu kyalli da kuma rinayen rini masu kyalli, mai wakilci na yau da kullun na rini mai kyalli na SYBRGreen I, wanda aka saba amfani dashi yanzu;cikakken Wakili na yau da kullun na rini mai kyalli maras cikawa shine SYBRGreenⅠ;Cikakken rini mai kyalli sune EvaGreen, LCGreen, da sauransu.

SYBRGreenI shine rini na ɗaurin DNA da aka saba amfani dashi don PCR mai kyalli, wanda ke ɗaure ba musamman ga DNA mai ɗauri biyu ba.A cikin yanayin kyauta, SYBRGreenI yana fitar da haske mai rauni, amma da zarar an ɗaure shi zuwa DNA mai ɗaure biyu, hasken sa yana ƙaruwa sau 1000.Sabili da haka, jimillar siginar kyalli da aka fitar ta hanyar dauki yayi daidai da adadin DNA mai ɗaure biyu da ke akwai kuma yana ƙaruwa yayin da samfurin ƙarawa ya karu.

Amfanin rini mai ɗaurin DNA mai ɗaure biyu: ƙirar gwaji mai sauƙi, kawai 2 na farko da ake buƙata, babu buƙatar ƙira ƙira, babu buƙatar ƙira ƙira da yawa don saurin gwaji na ƙwayoyin halitta da yawa, ikon yin nazarin yanayin narkewa, gwada ƙayyadaddun ƙayyadaddun abubuwan. amsawa ƙarawa, ƙananan farashi na farko, kyakkyawan yanayin gabaɗaya don haka ana amfani da su a cikin bincike a gida da waje.

Hanyar bincike mai walƙiya (Tsarin Taqman): Lokacin da aka yi haɓakawa na PCR, ana ƙara nau'i-nau'i guda biyu tare da takamaiman bincike mai kyalli.Lokacin da binciken ya kasance cikakke, siginar kyalli da ƙungiyar mai ba da rahoto ke fitarwa tana ɗaukar rukunin da aka kashe kuma kayan PCR ba su gano shi ba;a lokacin haɓakawa na PCR (a cikin lokacin haɓakawa), aikin 5'-3' na ɓarna na Taq enzyme yana ƙasƙantar da binciken enzymatically, yana sa ƙungiyar masu haskakawa mai ba da rahoto da ƙungiyar masu haskakawa.

Aikace-aikacen PCR mai ƙididdigewa.

Binciken Halittar Halitta:

1. Quantitative nucleic acid analysis.Ƙididdige ƙididdiga da ƙididdiga na cututtuka masu yaduwa, gano ƙwayoyin cuta ko ƙwayoyin cuta, irin su mura A (H1N1) annoba na kwanan nan, gano lambobin kwafin kwayoyin halitta na tsire-tsire da dabbobi, gano ƙimar RNAi gene inactivation, da dai sauransu.

2. Bambance-bambancen maganganun maganganu.Kwatanta bambance-bambancen maganganun kwayoyin halitta tsakanin samfuran da aka kula da su (misali maganin miyagun ƙwayoyi, jiyya ta jiki, jiyya na sinadarai, da dai sauransu), bambance-bambancen maganganu na takamaiman kwayoyin halitta a cikin matakai daban-daban da kuma tabbatar da microarray na cDNA ko sakamako na bambanta.

3. Ganewar SNP.Gano nau'in polymorphisms na nucleotide guda ɗaya yana da mahimmanci don nazarin yiwuwar mutum ga cututtuka daban-daban ko amsawar mutum ga takamaiman kwayoyi, kuma saboda tsarin fasaha na ƙwayoyin ƙwayoyin ƙwayoyin cuta, da zarar an san jerin bayanan SNP, yana da sauƙi kuma daidai. yi amfani da wannan dabarar don gano SNP mai girma.

4. Ganewar methylation.Methylation yana da alaƙa da yawancin cututtukan ɗan adam, musamman ciwon daji, kuma Laird ya ba da rahoton wata dabara mai suna Methylight, wacce ke magance DNA kafin haɓakawa ta yadda cytosine unmethylated ya zama uracil kuma methylated cytosine ba shi da tasiri, ta yin amfani da takamaiman abubuwan da ake buƙata da binciken Taqman don bambance tsakanin DNA methylated da unmethylated DNA. .mafi m.

Binciken likita:

1. Gano ciwon ciki: mutane ba za su iya magance cututtukan da ke haifar da sauye-sauyen kwayoyin halitta ba, kuma ya zuwa yanzu, ba za su iya rage yawan jariran da ke fama da rashin lafiya ba, ta hanyar lura da juna biyu, don hana aukuwar cututtuka daban-daban na gado.Wannan hanya ce da ba ta da hankali wacce mata masu juna biyu ke karba cikin sauki.

2. Ganewar cuta: The fluorescent quantitative PCR assay damar ƙididdige ƙayyadaddun ƙayyadaddun cututtuka irin su gonococcus, Chlamydia trachomatis, Mycoplasma solium, Human papilloma virus, herpes simplex virus, human immunodeficiency virus, hepatitis virus, mura virus, Mycobacterium tarin fuka da cytomevirus, EB.Yana da abũbuwan amfãni na babban hankali, ƙananan samfurin samfurin, sauri da sauƙi idan aka kwatanta da hanyoyin gwaji na gargajiya.

3. Ƙididdigar ingancin ƙwayoyi: ƙididdigar ƙididdiga na ƙwayar cutar hanta (HBV) da cutar hanta ta C (HCV) ya nuna cewa dangantakar dake tsakanin kwayar cutar kwayar cuta da ingancin wasu magunguna.Idan matakin jini na HBV-DNA ya ragu yayin jiyya na lamivudine sannan ya sake karuwa ko ya wuce matakin da ya gabata, yana nuni da maye gurbin kwayar cutar.

4. Gwajin Oncogenetic: Ko da yake tsarin ci gaban ƙwayar cuta bai riga ya bayyana ba, an yarda da shi cewa maye gurbi a cikin kwayoyin halitta masu dacewa shine tushen dalilin canjin oncogenic.Ana iya ganin karuwar magana da maye gurbi na oncogenes a farkon matakan ciwace-ciwacen da yawa.Ƙididdigar ƙididdigewa na ainihin-lokaci PCR ba wai kawai yana da tasiri wajen gano maye gurbi a cikin kwayoyin halitta ba, amma kuma yana iya gano ainihin bayyanar cututtukan oncogenes.An yi amfani da wannan hanya don gano bayyanar cututtuka daban-daban ciki har da telomerase hTERT gene, da kullum granulocytic cutar sankarar bargo WT1 gene, da oncogenic ER gene, prostate ciwon daji PSM gene, da ƙari-dangane da kwayar cutar kwayar cutar.

Fassara da www.DeepL.com/Translator (sigar kyauta)


Lokacin aikawa: Juni-21-2022