PCR, ngatafatsi PCR, E teng PCR, Phetoho ea PCR, RT-PCR, qPCR (1)-PCR

Re tla hlophisa mehopolo, mehato le lintlha tse fapaneng tsa PCR

Ⅰ. PCR

Polymerase Chain Reaction, e bitsoang PCR, ke theknoloji ea baeloji ea limolek'hule e sebelisetsoang ho holisa likhechana tse itseng tsa DNA.E ka nkoa e le phetiso e khethehileng ea DNA in vitro.DNA polymerase (DNA Polymerase I) e ile ea sibolloa ho tloha ka 1955, 'me Klenow Fragment ea E. Coli, e nang le boleng ba liteko le ho sebetsa, e ile ea sibolloa ke Dr. H. Klenow mathoasong a lilemo tsa bo-1970, empa hobane enzyme ena ha e mamelle mocheso, mocheso o phahameng o ka o senya, kahoo ha o kopane le ketane e phahameng ea mocheso oa polymerase.Li-enzyme tse sebelisoang kajeno (tse bitsoang Taq polymerase), li ne li arohane le Thermus aquaticus, baktheria ea selemo se chesang ka 1976. Tšobotsi ea eona ke hore e khona ho hanyetsa mocheso o phahameng 'me ke enzyme e loketseng, empa e sebelisoa haholo ka mor'a lilemo tsa bo-1980.Khopolo ea pele ea mohlala oa khale oa PCR e tšoana le tokiso ea liphatsa tsa lefutso le ho kopitsa, e hlahisitsoeng ke Dr. KJell Kleppe ka 1971. O ile a hatisa kopi ea pele e bonolo le e khutšoanyane ea liphatsa tsa lefutso (e tšoanang le liphetoho tse peli tsa pele tsa potoloho ea PCR).PCR e ntlafalitsoeng kajeno e entsoe ke Dr. Kary B. Mullis ka 1983. Dr. Mullis o ile a sebeletsa lik'hamphani tsa PE selemong seo, kahoo PE e na le boemo bo khethehileng indastering ea PCR.Dr. Mullis o phatlalalitse ka molao pampiri ea pele e amanang le Saiki le ba bang ka 1985. Ho tloha ka nako eo, tšebeliso ea PCR ke lik'hilomithara tse likete ka letsatsi, 'me boleng ba lipampiri tse amanang le tsona bo ka boleloa hore bo etsa hore mekhoa e meng e mengata ea lipatlisiso e se ke ea latsoa.Kamora moo, theknoloji ea PCR e sebelisoa haholo lipatlisisong tsa mahlale a baeloji le lits'ebetsong tsa bongaka, e fetoha theknoloji ea bohlokoahali ea lipatlisiso tsa baeloji ea limolek'hule.Mullis o boetse a hapa Khau ea Nobel ea 1993 ea Chemistry.

PCRMolao-motheo

Molao-motheo oa motheo oa theknoloji ea PCR o tšoana le mokhoa oa tlhaho oa ho ikatisa oa DNA, 'me ho khetheha ha oona ho itšetlehile ka oligonucleotide primer e tlatsetsang lipheletsong tse peli tsa tatellano ea sepheo.PCR e entsoe ka degeneration-annealing-extensions tse tharo tsa mantlha: ①Degeneration of the template DNA: Kamora hore template ea DNA e futhumale hoo e ka bang 93°C ka nako e itseng, tharollo ea DNA e habeli bakeng sa DNA ea ketane e habeli e entsoeng ke PCR amplification ea thempleite ea template ea DNA Leaving, e le hore e lokise ketane e le 'ngoe hore e kopane.②The annealing (compound) ea thempleite ea DNA le primer: Kamora hore thempleite ea DNA e futhumatsoe le ho senyeha ho ba ketane e le 'ngoe, mocheso o theohela ho hoo e ka bang 55°C.Tatelano e tlatsanang ya primer le thempleite DNA single-chain.③Katoloso ea primer: template ea DNA - tlamahano ea mantlha e ipapisitse le ketso ea TaqDNA polymerase, ka dNTP joalo ka karabelo e tala.Boloka molao-motheo oa ho pheta-pheta, kopanya ketane e ncha ea likopi e bolokiloeng e tlatsanang le ketane ea DNA ea thempleite, 'me u phete cycle degeneration-annealing-extension mekhoa e meraro e ka fumana "semi-reserved copy chain",' me ketane ena e ncha e fumaneha hape E-ba template bakeng sa potoloho e latelang.Ho nka 2-4min ho phethela loop, mofuta oa gene o ka holisoa ka makhetlo a limilione tse 'maloa ka lihora tse 2-3.

StandardPCRReaction System

Lintlha tse hlano tsa karabelo ea PCR

Ho na le haholo mefuta e mehlano ea lintho tse amehang karabelong ea PCR, e leng primer, enzyme, dNTP, template le buffer (Mg2+ ea hlokahala).[Mokhoa oa PCR]

Mokhoa o tloaelehileng oa PCR o arotsoe ka mehato e meraro

1. Ho senyeha ha DNA (90 ° C-96 ° C): Li-templates tsa DNA tse peli-ketane tlas'a ts'ebetso ea mocheso, li-hydrogen bonds li robeha, ho etsa DNA ea ketane e le 'ngoe.

2. Annealing (25℃ -65℃): Thempereichara ea tsamaiso e fokotsehile, primer e kopantsoe le template ea DNA ho etsa ketane ea libaka tse peli.

3. Keketso (70 ℃ -75 ℃): Tlas'a ketso ea Taq enzyme (hoo e ka bang 72 ° C, mosebetsi o motle ka ho fetisisa), dNTP e sebelisoa e le thepa e tala, e atoloha ho tloha qetellong ea 5' ea primer → 3′, synthesis le template li tlatsana le ketane ea DNA.

Potoloho e 'ngoe le e 'ngoe e atolosoa, e atolosoa, 'me e atolosoa, e menahanya litaba tsa DNA habeli.Hona joale, ka lebaka la sebaka se khuts'oane sa amplification, PCR e 'ngoe e ka phetoa ka nako e khutšoanyane haholo le haeba mosebetsi oa enzyme ea Taq o sa nepahale, kahoo o ka fetoloa mehato e' meli, ke hore, annealing le katoloso e ka etsoa ho 60 ° C-65 ° C ka nako e le 'ngoe.E le ho fokotsa mokhoa oa ho phahamisa le ho pholisa le ho ntlafatsa lebelo la karabo.

Likarolo tsa PCR Reaction

● E Nepahetseng Haholo

Lintlha tse ikhethileng tsa karabelo ea PCR ke: ①Motsoako o ikhethileng oa primer le thempleite ea DNA.②Molao-motheo oa ho hokahanya ha motheo.③Botšepehi ba TaqDNA polymerase synthesis reaction.④Tlhahiso le tlhokomelo ea mofuta o lebeletsoeng.

Motsoako o nepahetseng oa li-primers le li-template ke senotlolo.Ho tlama ha primer le template le ho atolosoa ha ketane ea primer ho itšetlehile ka molao-motheo oa ho lumellana ha motheo oa alkaline.Botšepehi ba polymerase synthesis reactions le ho hanyetsa mocheso o phahameng oa Taq DNA polymerase ho etsa tlamahano (motsoako) oa thempleite le primer ka karabelo e ka etsoa ka mocheso o phahameng.Ntho e ikhethang ea motsoako e eketsehile haholo.Sekotwana se ka boloka tekanyo e phahameng ea ho nepahala.Ka ho khetha sebaka se lebeletsoeng sa liphatsa tsa lefutso se nang le tlhokomelo e phahameng le tlhokomelo e phahameng, ho khetheha ha eona ho phahame.

● Kutloelo-bohloko e Phahameng

Bophahamo ba tlhahiso ea lihlahisoa tsa PCR bo eketsoa ka index, e ka atolosang template ea ho qala ea Picker (PG = 10-12) ho eketsa boemo ba microcontroller ho isa boemong ba micrograms (μg = -6).Lisele tse shebiloeng li ka bonoa liseleng tse limilione tse 1;ha ho fumanoa likokoana-hloko, kutloisiso ea PCR e ka fihla ho 3 RFUs (libaka tse se nang letho tse entsoeng lihlopha);tekanyo e fokolang ea ho lemoha ho saense ea baktheria ke libaktheria tse 3.

● E bonolo ebile e potlakile

PCR reflection e sebelisa Taq DNA polymerase e phahameng ea mocheso, e eketsang tharollo ea karabelo ka nako e le 'ngoe, ke hore, karabelo ea degeneration-anneal-extension ka tharollo ea amplification ea DNA le pitsa ea ho hlapa metsi.Ka kakaretso, karabelo ea amplification e phetheloa ka lihora tse 2 ho isa ho tse 4.Lihlahisoa tse ekelitsoeng hangata li hlahlojoa ka sabole ea motlakase, 'me ha lia tlameha ho sebelisa li-isotopes, ha ho na tšilafalo ea radioactive, le papatso e bonolo.

● Bohloeki ba mohlala bo tlaase

Ha ho hlokahale ho arola livaerase kapa libaktheria le lisele tsa setso.Lihlahisoa tse tala tsa DNA le RNA li ka sebelisoa e le liamplifier.Ho lemoha ka matlafatso ea DNA ho ka sebelisoa ka kotloloho ho sebelisoa mehlala ea bongaka e kang mali, mokelikeli oa 'mele, metsi a hlatsoang sefuba, moriri, lisele le lisele tse phelang.

PCRmathata a tloaelehileng

● Mashano a fosahetseng, ha a na lihlopha tse atolositsoeng

Mehato ea bohlokoa ea karabelo ea PCR e kenyelletsa: ① ho lokisoa ha li-nucleic acid tsa template, ② boleng le boleng ba li-primers, ③ boleng ba li-enzyme ④ maemo a potoloho ea PCR.Ho fumana lebaka ho boetse ho lokela ho hlahlojoa le ho ithutoa bakeng sa lihokelo tse kaholimo.

Templates: ① Thempleite e na le mefuta e fapaneng ea protheine, ② Thempleite e na le Taq enzyme inhibitor, ③ Protheine e ka har'a template ha e felisoe, haholo-holo protheine ea sehlopha e ho chromosome.⑤ Deminer nucleic acid degeneration ha e na hantle.Ha boleng ba li-enzyme le li-primers li le ntle, ha ho na sehlopha sa amplification, seo mohlomong e leng pheko ea tšilo ea lijo ea mehlala.Ho na le ho hong ho phoso ka ts'ebetso ea ho ntšoa ha template ea nucleic acid, kahoo ho lokisa tharollo e sebetsang le e tsitsitseng ea tšilo ea lijo, ts'ebetso ea eona e lokela ho lokisoa mme e se ke ea fetoloa ka boomo.

Ho se sebetse ha enzyme: enzyme e ncha kapa li-enzyme tse peli tsa khale le tse ncha li lokela ho sebelisoa hammoho ho hlahloba hore na ts'ebetso ea enzyme e lahlehile kapa ha e lekane, e lebisang mefuteng e fosahetseng.Ho lokela ho hlokomeloa hore Taq enzyme kapa ethidium bromide ka linako tse ling e lebaloa.

Primer: boleng ba primer, mahloriso a primer, le hore na mahloriso a li-primers tse peli a lekana.Ke lebaka le tloaelehileng la ho hloleha ha PCR kapa sehlopha se ntseng se eketseha ha se hantle ebile se tloaetse ho hasana.Ho na le mathata ka boleng ba li-primer tsa linomoro tse ling tsa batch.Li-primers tse peli li na le mahloriso a phahameng le mahloriso a tlase, a bakang asymmetric amplification e tlase.Mehato ea ho hanyetsa ke: ① Khetha sebatli se setle ho kopanya likarolo.② Khatello ea primer ha e itšetlehe feela ka boleng ba OD, empa hape e ela hloko mokelikeli oa pele oa primer ho etsa agar sugar gel electrophoresis.Ho tlameha ho ba le sebaka sa "primer strip", 'me khanya ea li-primers tse peli e lokela ho lumellana ka kakaretso.Lebanta, PCR e kanna ea hloleha ka nako ena, 'me e lokela ho rarolloa ka yuniti ea primer synthesis.Haeba primer e phahame, khanya e tlase, 'me mahloriso a eona a tlameha ho leka-lekana ha a hlapolloa.③ The primer e lokela ho lefshoa le ho bolokoa sebakeng se phahameng ho thibela likarolo tse ngata tsa sehatsetsing kapa nako e telele ea sehatsetsing, e leng se tla etsa hore primer e senyehe le ho senyeha.④ Moralo oa primer ha o utloahale, joalo ka ha bolelele ba primer ha bo lekane, mme di cluster e thehoa lipakeng tsa li-primers.

Khokahano ea Mg2 +: Khatiso ea Mg2 + ion e na le tšusumetso e kholo ho matla a ho holisa PCR.Ho tsepamisa mohopolo ho feteletseng ho ka fokotsa bong bo fapaneng ba ho holisa PCR.Haeba mahloriso a le tlase haholo, tlhahiso ea ntlafatso ea PCR e tla etsa hore PCR e hlolehe ntle le sehlopha sa katoloso.

Phetoho ea bophahamo ba karabelo: Bophahamo bo sebelisitsoeng ho ntlafatso ea PCR ke 20ul, 30ul, le 50ul kapa 100uL, palo e kholo ea ts'ebeliso ea ntlafatso ea PCR e behiloe ho latela merero e fapaneng ea lipatlisiso tsa mahlale le tlhahlobo ea bongaka.Ka mor'a ho etsa li-volume tse nyenyane tse kang 20ul, hoa hlokahala ho etsa boemo ba mohala ha u etsa boholo, ho seng joalo bo tla hlōleha.

Mabaka a 'mele: Phetoho e bohlokoa haholo bakeng sa ho holisa PCR.Haeba mocheso oa ho senyeha o le tlase, nako ea ho senyeha e khutšoanyane, e ka 'na ea etsahala ka mefokolo ea bohata;thempereichara e tlase haholo e ka baka matlafatso e sa khetheheng le ho fokotsa katleho e khethehileng ea amplification.E ama haholo motsoako oa li-primers le litempele ho fokotsa katleho ea ho holisa PCR.Ka linako tse ling hoa hlokahala ho sebelisa li-thermometers tse tloaelehileng ho lemoha ho feto-fetoha ha maemo, annealing le mocheso o atolositsoeng ka ho atolosoa kapa moapehi o qhibilihang ka metsi, e leng e 'ngoe ea mabaka a ho hlōleha ha PCR.

Mefuta e fapaneng ea tatelano e reriloeng: Haeba tatellano e reriloeng e etsahala, phetoho kapa ho hlakoloa, motsoako oa prototype le template li kopantsoe, kapa ka lebaka la khaello ea tatellano e reriloeng, primer le template li tla lahleheloa ke tatellano e tlatselletsang, 'me ntlafatso ea PCR e ke ke ea atleha.

● Pono e fosahetseng

Sehlopha sa ntlafatso sa PCR se bonahala se lumellana le sehlopha sa tatellano ea tatellano, 'me ka linako tse ling sehlopha sa sona se makhethe le ho feta.

Moralo oa mantlha ha oa nepahala: tatellano e khethiloeng ea ho holisa le tatelano e sa eketseng morero e na le homologous, kahoo ha PCR e holisa, lihlahisoa tsa PCR tse atolositsoeng ha li tatellano e seng sepheo.Tatelano ea sepheo e khuts'oane haholo kapa primer e khuts'oane haholo, 'me e na le monyetla oa ho ba le maikutlo a fosahetseng.E hloka ho hlophisoa bocha.

Tšilafalo e fapaneng ea tatellano ea sepheo kapa lihlahisoa tsa ho hōlisa: Ho na le mabaka a mabeli a tšilafalo ena: Ea pele, tšilafalo ea liphatsa tsa lefutso kapa likarolo tse kholo, tse lebisang ho tse fosahetseng.Mofuta ona oa phoso e fosahetseng o ka rarolloa ka mekhoa e latelang: E-ba hlokolosi 'me u be bonolo nakong ea ts'ebetso ho thibela tatellano ea sepheo hore e se ke ea huloa ka sethunya sa sampole kapa ho phatloha ka har'a tube ea centrifugal.Ntle le li-enzyme le lintho tse ke keng tsa mamella mocheso o phahameng, li-reagents tsohle kapa lisebelisoa li lokela ho hlajoa ka disinfected ka khatello e phahameng.Liphaephe tsa centrifugal le lisampole li lokela ho sebelisoa ka nako e le 'ngoe.Ha ho hlokahala, pele o eketsa mehlala, tube ea reaction le reagent e pepesehetse mahlaseli a ultraviolet ho senya nucleic acid e teng.Ea bobeli, likhechana tse nyane moeeng oa tšilafalo.Likaroloana tsena tse nyane li khuts'oane ho feta tatellano e reriloeng, empa li na le homology e itseng.E ka kopanngoa hammoho.Ka mor'a ho tlatsa li-primers, sehlahisoa sa PCR se ka atolosoa, se tla baka tlhahiso e ntle ea bohata.E ka sebelisoa ho fokotsa kapa ho felisa mokhoa oa PCR oa sehlaha.

● Letlapa la amplification le hlahang le sa tobang

Lihlopha tse hlahileng ka mor'a ho phahamisa PCR ha li lumellane le boholo bo lebeletsoeng, kapa tse kholo kapa tse nyenyane, kapa ka nako e le 'ngoe, kapa ka nako e ts'oanang, lihlopha tse khethehileng tsa amplification le lihlopha tse sa tloaelehang tsa amplification.Ho hlaha ha lihlopha tse sa khetheheng ke: Taba ea pele, li-primers ha lia phethahala ho tlatselletsa tatellano ea sepheo, kapa polymerization ea primer ho theha di cluster.Ea bobeli ke hore mahloriso a MG2 + ion a phahame haholo, mocheso oa annealing o tlase haholo, 'me palo ea lipotoloho tsa PCR e amana.Ea bobeli, boleng le bongata ba li-enzyme.Hangata, li-enzyme tsa mehloli e meng li sekamela ho lihlopha tse seng tse khethehileng 'me li-enzyme tsa mohloli o mong ha li etsahale.Ka linako tse ling ho na le ho holisoa ka tsela e sa tobang ha li-enzyme.Mehato ea ho thibela ke: mekhabiso e entsoeng bocha ha ho hlokahala.Fokotsa palo ea enzyme kapa u nke sebaka sa enzyme ea mohloli o mong.Fokotsa palo ea lintho tsa mantlha, eketsa palo ea litempele ka nepo, 'me u fokotse palo ea lipotoloho.Eketsa mocheso ka nepo kapa sebelisa mekhoa e 'meli ea mocheso (93°C degeneration, annealing and extending at about 65°C).

● E shebahala e le boreleli kapa teipi ea smear

PCR amplification ka linako tse ling e bonahala e sebelisoa kapa e entsoe ka shelled kapa lebanta le kang k'hapete.Ka lebaka leo, ka lebaka la bongata bo feteletseng ba li-enzyme kapa boleng bo bobe ba enzyme, khatello ea dNTP e phahame haholo, khatello ea Mg2 + e phahame haholo, mocheso oa anneal o tlase haholo, 'me palo ea lipotoloho e ngata haholo.Mehato ea ho hanyetsa ke: ① Fokotsa bongata ba li-enzyme, kapa fetola enzyme ea mohloli o mong.② Fokotsa khatello ea dNTP ③ fokotsa khatello ea Mg2+ ka nepo.④Eketsa bongata ba litempele le ho fokotsa palo ea lipotoloho.

Lihlahisoa tse Amanang

PCR Heroᵀᴹ (Ka dae)

◮ Botšepehi bo phahameng: makhetlo a 6 ho feta a enzyme e tloaelehileng ea Taq;

◮ Lebelo la ho holisa kapele

◮ Ho ikamahanya le maemo ho feta ha template

◮ Kakaretso e phahameng ea matlafatso

◮ Mamello ea tikoloho e matla: e behiloe ho 37 ° C ka beke, ho boloka ts'ebetso e fetang 90%;

◮ E na le ts'ebetso ea 5'→3' DNA polymerase le 5'→3' exonuclease, ntle le ts'ebetso ea 3'→5' ea exonuclease.

PCR Easyᵀᴹ (Ka dae)

Sistimi e ikhethang ea karabelo le ts'ebetso e phahameng ea Taq DNA Polymerase li etsa hore karabelo ea PCR e be le katleho e phahameng ea ho holisa, boits'oaro le kutlo.

RT-qPCR Easyᵀᴹ (Mohato o le Mong) -SYBR Green I

◮ Kit ea mohato o le mong e etsa hore ho fetoleloe ka morao le qPCR ka makhetlo a mabeli ka har'a tube e le 'ngoe, e hloka feela ho eketsa template ea RNA, li-primer tsa PCR le RNase-Free ddH.₂O.

◮ The Kit e ka hlahloba RNA ea vaerase kapele le ka mokhoa o atlehileng kapa ea sala morao RNA.

◮ Kit e sebelisa mochini o ikhethileng oa Foregene reverse transcription reagent le Foregene HotStar Taq DNA Polymerase e kopantsoeng le sistimi e ikhethang ea karabelo ho ntlafatsa ka nepo matla a ho holisa le ho nepahala ha karabelo.

◮ Sistimi e ntlafalitsoeng ea karabelo e etsa hore karabelo e be le kutlo e phahameng ea ho lemoha, botsitso bo matla ba mocheso, le mamello e betere.

◮ RT-qPCR e bonolo^TM(Mohato o le mong) -SYBR Green I kit e tla le dae ea ka hare ea ROX, e ka sebelisetsoang ho tlosa semelo sa matšoao le liphoso tsa matšoao lipakeng tsa liliba, tse loketseng bareki ho li sebelisa mefuteng e fapaneng ea lisebelisoa tsa PCR tse ngata.

RT Bonolo^TMII (Master Premix bakeng sa pele-strand cDNA synthesis bakeng saPCR ea Nako ea 'Nete)

-Bokhoni bo botle ba ho tlosa gDNA, e ka tlosa gDNA ka template ka hare ho metsotso ea 2.

-Sistimi e sebetsang hantle ea ho fetolela, ho nka metsotso e 15 feela ho phethela motsoako oa cDNA ea pele.

Li-templates tse rarahaneng: litempele tse nang le litaba tse phahameng tsa GC le sebopeho sa bobeli se rarahaneng le tsona li ka khutlisoa ka katleho e phahameng.

-High-sensitivity reverse transcription system, litempele tsa pg-level le tsona li ka fumana cDNA ea boleng bo holimo.

-The reverse transcript system e na le botsitso bo phahameng ba mocheso, mocheso o nepahetseng oa ho arabela ke 42℃, 'me e ntse e na le ts'ebetso e ntle ea ho fetolela ho 50℃.