PCR, waa fal-celinta silsiladda polymerase, taas oo loola jeedo ku-darka dNTP, Mg2 +, qodobbada kor u qaadista iyo xoojinta qodobbada kor u qaadida nidaamka hoos yimaada catalysis ee DNA-da polymerase, iyadoo la adeegsanayo DNA-da waalidka sida template iyo asal gaar ah oo ah barta bilawga ah ee kordhinta. Iyada oo loo marayo tillaabooyinka denaturation, annealing, fidinta, iwm., habka in vitro dib u soo celinta DNA strand gabadha oo kaabaya template strand template DNA waxay si dhakhso ah oo gaar ah u kordhin kartaa DNA kasta oo la beegsanayo in vitro.
1. Hot Start PCR
Waqtiga bilawga kordhinta PCR-ga caadiga ah maaha in mashiinka PCR la geliyo mashiinka PCR, ka dibna barnaamijku wuxuu bilaabaa inuu kordho.Marka qaabeynta nidaamka la dhammeeyo, cod-weyneyntu waxay bilaabataa, taas oo keeni karta cod-weyneyn aan gaar ahayn, PCR-ga bilawga ah ayaa xallin kara dhibaatadan.
Waa maxay bilowga kulul PCR?Ka dib marka nidaamka falcelinta la diyaariyo, beddelka enzyme ayaa lagu sii daayaa heerkul sare (badanaa ka sarreeya 90 ° C) inta lagu jiro marxaladda kulaylka bilowga ah ee falcelinta ama "bilawga kulul", si markaa DNA polymerase loo hawlgeliyo.Waqtiga saxda ah ee firfircoonida iyo heerkulka waxay ku xiran tahay dabeecadda polymerase DNA-ga iyo wax ka beddelka-bilawga kulul.Habkani wuxuu badiyaa adeegsadaa wax-ka-beddelayaasha sida unugyada difaaca jirka, isku-xidhka isku-xidhka, ama beddelayaasha kiimikaad si loo joojiyo dhaqdhaqaaqa DNA-ga polymerase.Maadaama dhaqdhaqaaqa DNA polymerase lagu xannibay heerkulka qolka, tignoolajiyada bilawga kulul waxay bixisaa ku habboonaanta diyaarinta habab badan oo falcelin PCR ah heerkulka qolka iyada oo aan la hurin gaar ahaan falcelinta PCR.
2. RT-PCR
RT-PCR (Reverse transcription PCR) waa farsamo tijaabo ah oo loogu talagalay ka-noqoshada qoraalka mRNA ee cDNA oo loo isticmaalo qaab-dhismeedka kor u qaadista.Habka tijaabada ah waa in marka hore la soo saaro wadarta RNA ee unugyada ama unugyada, isticmaal Oligo (dT) asal ahaan, isticmaal transcriptase gadaal si aad u abuurto cDNA, ka dibna cDNA u adeegso qaab dhismeedka PCR-ga si loo helo hidda-wadaha bartilmaameedka ah ama loo ogaado muujinta hidda-wadaha.
3. PCR tireed Fluorescent
PCR tirooyinka Fluorescent (Waqtiga-dhabta ah ee PCR,RT-qPCR) waxa loola jeedaa habka loogu darayo kooxaha fluorescent ee nidaamka falcelinta PCR, iyadoo la adeegsanayo ururinta calaamadaha fluorescent si loola socdo dhammaan geeddi-socodka PCR wakhtiga dhabta ah, iyo ugu dambeyntii iyadoo la adeegsanayo qalooca caadiga ah si qiyaas ahaan loo falanqeeyo qaabka.Hababka qPCR ee inta badan la isticmaalo waxaa ka mid ah SYBR Cagaaran I iyo TaqMan.
4. PCR buul leh
PCR Nsted waxaa loola jeedaa isticmaalka laba qaybood oo ah aasaasayaasha PCR ee laba wareeg oo kordhinta PCR, iyo wax soo saarka kordhinta wareegga labaad waa jajabka hiddo-wadaha bartilmaameedka ah.
Haddii is-waafajin la'aanta lammaanaha hore ee asaasiga ah (dib-u-dooriyaha) ay keento badeeco aan gaar ahayn in la kordhiyo, suurtogalnimada in isla gobol aan gaar ahayn lagu aqoonsado lammaanaha labaad ee asaasiga ah oo sii wadida kor u qaadida waa mid aad u yar, sidaas darteed kordhinta labada lamaane ee aasaasiga ah, gaar ahaan PCR waa la hagaajiyay.Mid ka mid ah faa'iidooyinka samaynta laba wareeg ee PCR waa in ay ka caawiso in ay kordhiso badeecad ku filan DNA bilawga xaddidan.
5. PCR taabashada
PCR taabashada waa hab lagu wanaajiyo gaar ahaaneed ee falcelinta PCR iyadoo la hagaajinayo cabirrada wareegga PCR.
Marka la taabto PCR, heerkulka annealing ee dhowrka wareeg ee ugu horreeya waxaa lagu dejiyaa dhowr darajo oo ka sarreeya heerkulka ugu sarreeya (Tm) ee asaasiga ah.Heerkulka sare-u-qaadista ayaa si wax ku ool ah u yareeyn karta kordhinta aan gaarka ahayn, laakiin isla mar ahaantaana, heerkulka kor u kaca ayaa ka sii dari doona kala soocida asaasiga ah iyo taxanaha bartilmaameedka, taas oo keeneysa hoos u dhaca wax-soo-saarka PCR.Sidaa darteed, dhowrka wareeg ee ugu horreeya, heerkulka annealing ayaa sida caadiga ah lagu wadaa inuu hoos u 1 ° C halkii wareeg si loo kordhiyo nuxurka hiddasidaha bartilmaameedka ee nidaamka.Marka heerkulbeegga annealing hoos loo dhigo heerkulka ugu wanaagsan, heerkulka annealing waxaa lagu hayaa wareegyada hadhay.
6. PCR toos ah
PCR tooska ah waxa ay tilmaamaysaa kordhinta DNA-da la beegsanayo si toos ah muunadda iyada oo aan loo baahnayn go'doomin iyo nadiifin aashitada nucleic ah.
Waxaa jira laba nooc oo PCR toos ah:
Habka tooska ah: qaado qadar yar oo muunad ah oo si toos ah ugu dar PCR Master Mix si loo aqoonsado PCR;
Habka dildilaaca: ka dib marka muunada la qaado, ku dar lysate-ka, lyse si loo sii daayo genome-ga, qaado xaddi yar oo lysed supernatant ah oo ku dar PCR Master Mix, samee aqoonsiga PCR.Habkani wuxuu fududeeyaa socodka shaqada ee tijaabada ah, wuxuu yareeyaa gacmaha waqtiga, wuxuuna ka fogaanayaa luminta DNA-da inta lagu jiro tillaabooyinka nadiifinta.
7. SOE PCR
Kala-soocidda hidda-wadaha PCR (SOE PCR) waxay isticmaashaa aasaasayaal leh darafyo dhammaystiran si ay badeecadaha PCR u sameeyaan silsilado is-dul-saaran, si markaa fal-celinta cod-weyneynta ee xigta, iyada oo la kordhinayo silsiladaha is-dul-saaran, ilo kala duwan oo Farsamo ah oo jajabyo la xoojiyay ay is-dul-saaran yihiin. oo la isku daray.Tignoolajiyadani hadda waxay leedahay laba tilmaamood oo codsi ah: dhismaha hidda-wadaha isku-dhafka ah;isu beddelka goobta hidde-sidaha.
8. IPCR
PCR-ga rogan (IPCR) waxay isticmaashaa sooyaalka dhamaystirka gadaale si ay u xoojiso jajabyada DNA-da oo aan ahayn labada asaasiga ah, waxayna kordhisaa taxanaha aan la garanayn ee labada dhinac ee jajabka DNA-da ee la yaqaan.
IPCR waxaa markii hore loogu talagalay in lagu go'aamiyo isku xigxiga gobollada ku dhow ee aan la garanayn, waxaana inta badan loo isticmaalaa in lagu barto taxanaha dhiirrigeliyaha hidda-wadaha;dib-u-habaynta koromosoomyada oncogenic, sida isku-dhafka hidda-wadaha, beddelidda iyo beddelidda;iyo is dhexgalka hidda-wadaha fayraska, ayaa sidoo kale hadda loo isticmaalaa mutagenesis-ku-hagaha, koobiye plasmid oo leh isbeddelka la rabo.
9. dPCR
PCR dhijitaalka ah (dPCR) waa farsamo lagu qiyaaso buuxda ee molecules acid nucleic.
Hadda waxa jira saddex hab oo lagu qiyaaso unugyada nucleic acid.Photometry waxay ku salaysan tahay nuugista molecules acid nucleic;waqtiga dhabta ah ee fluorescent quantitative PCR (Real Time PCR) waxay ku salaysan tahay qiimaha Ct, iyo qiimaha Ct waxa loola jeedaa lambarka meertada ee u dhiganta qiimaha fluorescence ee la ogaan karo;PCR dhijitaalka ah waa tignoolajiyada ugu dambeeyay ee tirada ee ku salaysan habka PCR-ka-molecule ee tirinta qiyaasaha aashitada nucleic waa hab qiyaaseed buuxa.
Waqtiga boostada: Juun-13-2023