PCR, shine tsarin sarkar polymerase, wanda ke nufin ƙari na dNTP, Mg2 +, abubuwan haɓakawa da haɓaka abubuwan haɓakawa ga tsarin a ƙarƙashin catalysis na DNA polymerase, ta yin amfani da DNA na iyaye a matsayin samfuri da ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun kayan haɓakawa da haɓaka haɓakawa ga tsarin da ke tattare da DNA polymerase. Ta matakai na denaturation, annealing, tsawo, da dai sauransu, tsarin in vitro replicating 'yar strand DNA daidai da iyaye strand samfuri DNA iya sauri da kuma musamman fadada duk wani manufa DNA a cikin vitro.
1. Hot Start PCR
Lokacin farawa na haɓakawa a cikin PCR na al'ada ba shine sanya injin PCR a cikin injin PCR ba, sannan shirin ya fara haɓakawa.Lokacin da tsarin tsarin ya ƙare, haɓakawa yana farawa, wanda zai iya haifar da haɓakawa mara kyau, kuma PCR mai zafi zai iya magance wannan matsala.
Menene PCR mai zafi?Bayan an shirya tsarin amsawa, ana fitar da gyare-gyaren enzyme a babban zafin jiki (yawanci fiye da 90 ° C) a lokacin farkon yanayin zafi na amsawa ko "farawa mai zafi", don haka an kunna DNA polymerase.Madaidaicin lokacin kunnawa da zafin jiki sun dogara ne akan yanayin DNA polymerase da mai gyara farawa mai zafi.Wannan hanyar galibi tana amfani da masu gyara kamar ƙwayoyin rigakafi, ligands, ko masu gyara sinadarai don hana ayyukan DNA polymerase.Tun lokacin da aka hana aikin DNA polymerase a cikin zafin jiki, fasahar farawa mai zafi tana ba da babban dacewa don shirya tsarin amsawar PCR da yawa a cikin zafin jiki ba tare da sadaukar da takamaiman halayen PCR ba.
2. RT-PCR
RT-PCR (Reverse transcription PCR) dabara ce ta gwaji don juyar da rubutun mRNA zuwa cDNA da amfani da shi azaman samfuri don haɓakawa.Hanyar gwaji ita ce cire jimillar RNA a cikin kyallen takarda ko sel da farko, yi amfani da Oligo (dT) azaman firamare, yi amfani da reverse transcriptase don haɗa cDNA, sannan a yi amfani da cDNA azaman samfuri don haɓaka PCR don samun jigon manufa ko gano maganganun kwayoyin halitta.
3. Fluorescent quantitative PCR
PCR na Fluorescent (PCR mai ƙididdigewa na ainihin lokaci,RT-qPCR) yana nufin hanyar ƙara ƙungiyoyi masu kyalli zuwa tsarin amsawar PCR, ta yin amfani da tarawar siginar kyalli don saka idanu gabaɗayan tsarin PCR a ainihin lokacin, kuma a ƙarshe ta amfani da daidaitaccen lanƙwasa don tantance samfurin ƙima.Hanyoyin qPCR da aka saba amfani da su sun haɗa da SYBR Green I da TaqMan.
4. PCR mai gida
PCR Nsted yana nufin yin amfani da saiti biyu na PCR na farko don zagaye biyu na haɓaka PCR, kuma samfurin ƙarawa na zagaye na biyu shine gutsure kwayoyin halitta.
Idan rashin daidaituwa na nau'i-nau'i na farko (na waje na waje) ya sa samfurin da ba na musamman ba don haɓakawa, yiwuwar yankin da ba na musamman ba da aka gane ta biyu na farko da kuma ci gaba da haɓaka yana da ƙananan ƙananan, don haka haɓakawa ta biyu na farko, an inganta ƙayyadaddun PCR.Ɗaya daga cikin fa'idodin yin zagaye biyu na PCR shine yana taimakawa haɓaka isassun samfuri daga ƙayyadaddun farawa DNA.
5. Tabbataccen PCR
Touchdown PCR hanya ce don inganta ƙayyadaddun halayen PCR ta hanyar daidaita sigogin sake zagayowar PCR.
A cikin taɓawa PCR, ana saita zafin zafi na ƴan zagayowar farko a ƴan digiri sama da matsakaicin zafin zafin jiki (Tm) na firamare.Mafi girman zafin jiki na annealing zai iya rage haɓakar da ba takamaiman ba, amma a lokaci guda, mafi girman zafin jiki na annealing zai tsananta rarrabuwa na firam da jeri na manufa, yana haifar da rage yawan amfanin PCR.Sabili da haka, a cikin ƴan zagayowar farko, ana saita zafin jiki na annealing don ragewa da 1°C kowace zagaye don ƙara abun ciki na kwayar halittar da aka yi niyya a cikin tsarin.Lokacin da aka saukar da zafin jiki na annealing zuwa mafi kyawun zafin jiki, ana kiyaye zafin jiki na annealing don ragowar hawan keke.
6. PCR kai tsaye
PCR kai tsaye yana nufin haɓaka DNA da aka yi niyya kai tsaye daga samfurin ba tare da buƙatar keɓewar acid nucleic da tsarkakewa ba.
Akwai nau'ikan PCR kai tsaye guda biyu:
Hanyar kai tsaye: ɗauki ɗan ƙaramin samfurin kuma ƙara shi kai tsaye zuwa PCR Master Mix don tantance PCR;
Hanyar cracking: bayan yin samfurin samfurin, ƙara shi a cikin lysate, lyse don saki kwayoyin halitta, Ɗauki ƙaramin adadin lysed supernatant kuma ƙara shi zuwa PCR Master Mix, yi ganewar PCR.Wannan hanya tana sauƙaƙa aikin gwajin gwaji, yana rage hannayen hannu akan lokaci, kuma yana guje wa asarar DNA yayin matakan tsarkakewa.
7. SOE PCR
Gene splicing by overlap extension PCR (SOE PCR) yana amfani da firamare tare da madaidaitan ƙarewa don sanya samfuran PCR su zama sarƙoƙi masu jujjuyawa, ta yadda a cikin haɓakawar haɓakawa ta gaba, ta hanyar haɓaka sarƙoƙi, maɓuɓɓuka daban-daban na A dabarar da ke tattare da ɓangarorin haɓakawa. kuma suka rabu tare.Wannan fasaha a halin yanzu tana da manyan kwatance guda biyu na aikace-aikace: gina kwayoyin halittar fusion;maye gurbi mai jagorancin rukunin kwayoyin halitta.
8. IPCR
Inverse PCR (IPCR) yana amfani da juzu'i na haɗin gwiwa don haɓaka gutsuttsuran DNA ban da madaidaitan abubuwa guda biyu, kuma yana haɓaka jerin da ba a san su ba a ɓangarorin DNA da aka sani.
An tsara IPCR asali don tantance jerin yankuna da ba a san su ba, kuma galibi ana amfani da su don nazarin jerin masu tallata kwayoyin halitta;oncogenic chromosomal sake tsarawa, kamar jinsin fusion, jujjuya da juyi;da hadewar kwayoyin halittar kwayar cuta, kuma ana amfani da su a yanzu Don mutagenesis da ke jagorantar rukunin yanar gizo, kwafi plasmid tare da maye gurbin da ake so.
9. dPCR
Digital PCR (dPCR) wata dabara ce don cikakken ƙididdige ƙwayoyin ƙwayoyin acid nucleic.
A halin yanzu akwai hanyoyi guda uku don ƙididdige ƙwayoyin nucleic acid.Photometry ya dogara ne akan shayar da kwayoyin acid nucleic;PCR na ainihi mai kyalli (Real Time PCR) yana dogara ne akan ƙimar Ct, kuma ƙimar Ct tana nufin lambar sake zagayowar daidai da ƙimar kyalli wanda za'a iya ganowa;PCR dijital ita ce sabuwar fasaha ta ƙididdigewa bisa hanyar PCR mai-kwaya ɗaya don ƙididdige adadin acid nucleic hanya ce ta ƙididdigewa.
Lokacin aikawa: Juni-13-2023