Polymerase Chain Reaction, dav hu ua PCR, sawv cev rau ib qho ntawm cov txheej txheem tseem ceeb tshaj plaws hauv keeb kwm ntawm molecular biology. Tsim los ntawm xyoo 1980s, cov txheej txheem no tau hloov pauv los ntawm cov txheej txheem kuaj tshwj xeeb mus rau cov cuab yeej siv hauv kev kuaj mob, kev tshawb fawb forensic, thiab kev tshawb nrhiav caj ces. Los ntawm kev tso cai rau cov kws tshawb fawb los coj cov qauv me me ntawm DNA thiab nthuav dav rau ntau lab luam, PCR tau ua kom nws tuaj yeem kawm cov noob hauv kev nthuav dav, tshawb xyuas cov kab mob nrog qhov tseeb, thiab txheeb xyuas cov cim caj ces uas yav dhau los pom tsis tau rau cov qauv kev tshuaj ntsuam xyuas.
Cov txheej txheem PCR yog cov txheej txheem kuaj siv los ua ntau daim ntawv theej ntawm ib ntu DNA tshwj xeeb los ntawm lub voj voog ntawm kev hloov pauv kub, suav nrog denaturation, annealing, thiab txuas ntxiv, txhawb nqa los ntawm lub tshuab PCR tshwj xeeb thiab lub tshav kub-stable DNA polymerase.
Kev nkag siab txog qhov tsis sib xws ntawm cov txheej txheem PCR yog qhov tseem ceeb rau cov kws tshaj lij, cov kws tshawb fawb kho mob, thiab cov tuam txhab lag luam koom nrog hauv cov cuab yeej kuaj mob. Raws li qhov kev thov rau kev sim tshuaj molecular sai thiab raug txuas ntxiv mus thoob ntiaj teb, kev ntseeg siab ntawm cov Lub tshuab PCR dhau los ua lub hauv paus ntawm kev ua tiav kev sim ua tiav. Kab lus no muab cov lus qhia dav dav rau cov kauj ruam, qhov kub thiab txias, thiab cov kev xav tau ntawm cov txheej txheem PCR kom ntseeg tau tias qhov zoo ntawm DNA amplification thiab cov txiaj ntsig kev sim muaj zog.
Tshooj lus Summary Table
| Tshooj |
Cov ntsiab lus |
| PCR yog dab tsi? |
PCR yog cov txheej txheem molecular biology siv los nthuav qhia cov DNA ua ntu zus rau ntau yam kev siv hauv qab. |
| Dab tsi yog qhov yuav tsum tau rau PCR? |
Kev ua tiav PCR yuav tsum muaj cov qauv DNA, primers, nucleotides, ruaj khov DNA polymerase, thiab cov khoom siv hluav taws xob siab. |
| 4 kauj ruam ntawm PCR yog dab tsi? |
Cov txheej txheem ua raws li qhov kev xav tau ntawm qhov pib, denaturation, annealing, thiab txuas ntxiv rau ob npaug ntawm DNA cov ntsiab lus txhua lub voj voog. |
| PCR tshuab cov kauj ruam yog dab tsi? |
Cov cuab yeej automates cov kev hloov pauv ntawm qhov kub thiab txias, kom ntseeg tau tias cov tshuaj tiv thaiv biochemical tshwm sim ntawm lub sijhawm xav tau. |
| Dab tsi yog qhov kub thiab txias siv rau denature kauj ruam? |
Qhov kub thiab txias, feem ntau ntawm 94 ° C thiab 98 ° C, yog siv los ua txhaum hydrogen bonds thiab cais ob-stranded DNA. |
| Yuav ua li cas thaum lub sij hawm annealing kauj ruam? |
Thaum lub sijhawm no, qhov kub thiab txias tau qis dua kom tso cai rau cov primers los khi tshwj xeeb rau lawv cov hom phiaj sib txuas ntawm ib leeg-stranded DNA. |
| Dab tsi yog qhov ntsuas kub siv rau cov kauj ruam txuas ntxiv? |
Cov kauj ruam no feem ntau tshwm sim ntawm 72 ° C, qhov zoo tshaj plaws kub rau Taq polymerase los ua ke cov DNA tshiab strand. |
| PCR kub khiav yog dab tsi? |
Cov dej ntws muaj xws li kev caij tsheb kauj vab nrawm ntawm qhov siab, qis, thiab nruab nrab kub uas rov ua dua kom txog rau thaum qhov xav tau concentration. |
PCR yog dab tsi?
PCR, los yog Polymerase Chain Reaction, yog ib txoj kev hloov pauv molecular biology tsim los sai sai tsim ntau lab mus rau billions ntawm cov ntawv luam ntawm cov qauv DNA tshwj xeeb.
Ntawm nws cov tub ntxhais, PCR ua raws li 'biological photocopier.' Ua ntej nws tsim, amplifying DNA yog ib tug qeeb thiab cumbersome txheej txheem cuam tshuam nrog lub cloning ntawm DNA rau hauv cov kab mob. Nrog lub advent ntawm lub PCR tshuab , cov kws tshawb fawb tam sim no tuaj yeem cais cov noob tshwj xeeb lossis ntu ntawm cov genome thiab nthuav dav nws hauv ib teev. Qhov kev muaj peev xwm no tseem ceeb heev vim tias feem ntau cov kev tshuaj ntsuam biochemical xav tau ntau npaum li cas ntawm DNA los ua cov teeb liab ntsuas, thiab cov qauv ntuj feem ntau muab cov lej xwb.
Lub versatility ntawm no technology yog reflected nyob rau hauv nws ntau yam kev siv nyob rau hauv txawv industries. Hauv chaw kho mob, nws yog siv los kuaj xyuas cov kab mob kis, xws li hauv COVID-19 lossis kuaj HIV. Nyob rau hauv forensics, nws tso cai rau cov neeg tshawb xyuas los txheeb xyuas cov tib neeg los ntawm cov qauv microscopic ntawm cov khoom siv roj ntsha. Hauv kev lag luam kev lag luam, PCR ua kom huv si ntawm cov khoom noj khoom haus thiab kev tshawb nrhiav cov kab mob hloov caj ces. To taub cov cov hauv paus ntsiab lus thiab cov nqi ntawm PCR thev naus laus zis yog qhov tseem ceeb rau cov chaw sim nrhiav kev txhim kho lawv cov peev xwm kuaj mob.
Dab tsi yog qhov yuav tsum tau rau PCR?
Kev ua tiav PCR cov tshuaj tiv thaiv yuav tsum muaj tsib yam tseem ceeb: cov qauv DNA, cov txheej txheem tshwj xeeb, deoxynucleotide triphosphates (dNTPs), lub tshav kub-stable DNA polymerase (xws li Taq), thiab cov tshuaj tsis haum tshwj xeeb.
Cov qauv DNA ua haujlwm raws li cov qauv qauv qub uas koj xav luam tawm. Primers yog luv luv, cov khoom hluavtaws ntawm DNA uas yog tsim los ua kom haum rau qhov pib thiab qhov kawg ntawm lub hom phiaj sib lawv liag. Yog tsis muaj cov no, DNA polymerase yuav tsis paub qhov twg los pib tsim txoj hlua tshiab. Cov dNTPs (A, T, C, thiab G) yog cov khoom siv raw uas cov enzyme siv los tsim cov saw tshiab DNA.
Qhov tseem ceeb sib npaug yog ib puag ncig uas cov tshuaj tiv thaiv tshwm sim. Cov tsis muaj tshuaj lom neeg ruaj khov, tshwj xeeb tshaj yog tsom rau pH thiab cov concentration ntawm magnesium ions, uas yog cov cofactors tseem ceeb rau DNA polymerase enzyme. Thaum kawg, kev ua tiav lub cev ntawm cov tshuaj tiv thaiv yuav tsum muaj kev ua haujlwm siab thermal cycler, feem ntau hu ua PCR tshuab , uas tswj cov kev hloov kub ceev uas xav tau los ua rau txhua theem ntawm cov tshuaj tiv thaiv.
Cov Cheebtsam tseem ceeb ntawm PCR Mix
Template DNA : Cov qauv uas muaj lub hom phiaj sib lawv liag.
DNA Polymerase : Feem ntau Taq polymerase, uas tseem ua haujlwm ntawm qhov kub thiab txias.
Primers : Forward thiab thim rov qab strands uas txhais tau tias amplification ciam teb.
dNTPs : Plaub lub hauv paus nucleotide uas ua haujlwm raws li 'ink' rau lub tshuab luam ntawv.
Buffer thiab Ions : Tswj cov enzymatic efficiency thiab stability.
4 kauj ruam ntawm PCR yog dab tsi?
Cov txheej txheem PCR muaj plaub theem pib ua haujlwm: Initialization, Denaturation, Annealing, thiab Extension (tseem hu ua Elongation).
Thawj theem, Initialization, yog ib qho kev tshwm sim ib zaug uas cov tshuaj tiv thaiv chamber tau rhuab mus rau qhov kub thiab txias kom ntseeg tau tias DNA polymerase tau ua tiav tag nrho thiab txhua yam kab mob tsis zoo. Ua raws li qhov no, lub voj voog ntawm Denaturation pib, qhov twg DNA ob npaug sib cais. Qhov no yog ua raws li Annealing, qhov twg cov primers pom lawv lub hom phiaj, thiab thaum kawg Extension, qhov twg DNA tshiab yog synthesized. Lub voj voog peb-kauj ruam no (Denaturation, Annealing, Extension) rov ua dua 25 txog 40 zaug.
Vim tias tus nqi ntawm DNA ob npaug nrog txhua lub voj voog ua tiav, kev loj hlob yog exponential. Piv txwv li, tom qab 30 lub voj voog, ib qho molecule ntawm DNA tuaj yeem hloov mus rau ntau tshaj li ib lab luam. Qhov no efficiency yog dab tsi ua niaj hnub kuaj thermal cyclers thiaj li tseem ceeb rau niaj hnub science. Yog tias tsis muaj cov cua kub ceev thiab cua txias uas muaj nyob rau hauv lub tshuab PCR zoo , cov txheej txheem yuav qeeb dhau rau kev siv tswv yim nyob rau hauv high-throughput diagnostic ib puag ncig.
PCR tshuab cov kauj ruam yog dab tsi?
Lub tshuab PCR cov kauj ruam suav nrog kev siv tsheb kauj vab ntawm qhov kub thiab txias los ntawm kev tswj hluav taws xob zoo ntawm lub thermal thaiv, tswj tus nqi ramp, tuav lub sijhawm, thiab txias kawg.
Lub tshuab PCR ua haujlwm los ntawm kev siv Peltier cov ntsiab lus kom sov sai thiab txias cov hlau thaiv uas tuav cov hlab ntsha. Cov 'cov kauj ruam' los ntawm lub tshuab xam pom suav nrog 'Ramp,' uas yog qhov hloov pauv ceev ntawm qhov kub thiab txias, thiab 'Tuav,' uas yog lub sijhawm uas lub tshuab tswj tau qhov kub thiab txias. Cov tshuab high-end yog tsim los kom muaj qhov ceev ceev ceev kom txo tau lub sij hawm siv nyob rau hauv kev hloov pauv, uas txo qhov kev pheej hmoo ntawm kev sib khi tsis tshwj xeeb lossis enzyme degradation.
Lub software nyob rau hauv lub tshuab tso cai rau cov neeg siv los tsim cov txheej txheem complex. Qhov no suav nrog kev pib ua kom sov, rov ua dua ntawm peb theem tseem ceeb, thiab qhov kawg tuav cov kauj ruam ntawm qhov txias txias (feem ntau yog 4 ° C) kom khaws cov qauv kom txog thaum tus kws tshaj lij tuaj yeem khaws lawv. Niaj hnub nimno digital interfaces ntawm lub tshuab PCR kuj tso cai rau kev saib xyuas lub sijhawm tiag tiag ntawm cov tshuaj tiv thaiv, kom ntseeg tau tias cov thermal profile tau ua raws nraim li qhov programmed rau qhov siab tshaj plaws kev tsim tawm.
Dab tsi yog qhov kub thiab txias siv rau denature kauj ruam?
Cov kauj ruam denaturation feem ntau siv qhov kub ntawm 94 ° C thiab 98 ° C los pab txhawb kev tawg ntawm hydrogen bonds ntawm DNA strands.
Thaum tshav kub kub no, cov qauv ob-helix ntawm DNA ua tsis ruaj khov. Cov ntawv cog lus hydrogen uas tuav cov adenine-thymine thiab cytosine-guanine ua ke ua ke ploj mus, ua rau ob txoj kev ywj pheej ntawm DNA. Qhov no yog qhov tseem ceeb ua ntej rau cov kauj ruam tom ntej, vim tias cov primers thiab DNA polymerase enzyme tsuas tuaj yeem cuam tshuam nrog cov qauv ib leeg. Yog tias qhov ntsuas kub qis dhau lawm, DNA yuav tsis sib cais tag nrho, ua rau muaj kev ua tsis tiav lossis tsis muaj txiaj ntsig.
Txawm li cas los xij, kev tswj xyuas qhov ntsuas kub no yuav tsum muaj DNA polymerase muaj zog heev. Qhov no yog vim li cas qhov kev tshawb pom ntawm Taq polymerase, cais tawm los ntawm cov kab mob kub-hlub Thermus aquaticus , yog li hloov pauv. Tus qauv enzymes yuav raug rhuav tshem ntawm 95 ° C, tab sis Taq tseem ua haujlwm. Cov chaw soj nstuam yuav tsum xyuas kom lawv lub tshuab PCR muab cov cua sov sib xws thoob plaws txhua qhov dej kom tiv thaiv 'qhov chaw txias' qhov twg denaturation yuav ua tsis tau, uas yog qhov tseem ceeb ntawm zoo molecular biology khoom.
Yuav ua li cas thaum lub sij hawm annealing ntawm PCR?
Thaum lub sij hawm annealing kauj ruam, qhov kub thiab txias yog txo mus rau nruab nrab ntawm 50 ° C thiab 65 ° C, tso cai rau cov DNA primers khi rau lawv complementary sequences ntawm ib leeg-stranded DNA templates.
Cov kauj ruam no yog arguably qhov tseem ceeb tshaj plaws ntawm cov txheej txheem PCR. Qhov tshwj xeeb kub siv yog nyob ntawm qhov kub melting (Tm) ntawm cov primers siv. Yog tias qhov kub thiab txias heev, cov primers yuav tsis khi rau lub template. Yog tias nws qis dhau, cov primers yuav khi rau cov kab ke uas tsuas yog 'ib nrab' zoo sib xws, ua rau tsis muaj kev nthuav dav thiab cov txiaj ntsig tsis zoo. Lub tshuab PCR yuav tsum muaj peev xwm ntaus lub hom phiaj kub nrog qhov siab ntawm qhov tseeb (feem ntau nyob rau hauv 0.1 ° C).
Lub sijhawm ntawm cov kauj ruam annealing feem ntau yog 20 mus rau 40 vib nas this. Thaum lub sij hawm luv luv no, cov primers navigate cov tshuaj tiv thaiv sib tov los ntawm molecular motion thiab snap mus rau lub hom phiaj site. Thaum cov primers tau annealed, lawv muab qhov pib rau DNA polymerase pib ntxiv nucleotides. Qhov kev sib koom ua ke meej no yog qhov tso cai rau kev tshawb nrhiav cov kev hloov pauv caj ces lossis kab mob hauv cov qauv lom neeg, ua rau kev nqis peev hauv cov tshuab kuaj mob tshaj lij yog qhov tseem ceeb rau cov chaw kuaj mob.
Dab tsi yog qhov ntsuas kub siv rau cov kauj ruam txuas ntxiv?
Cov kauj ruam txuas ntxiv yog feem ntau ua tiav ntawm 72 ° C, uas yog qhov zoo tshaj plaws ua haujlwm kub rau lub tshav kub-stable DNA polymerase los ua ke cov DNA tshiab strand.
Ntawm 72 ° C, DNA polymerase enzyme yog nyob ntawm nws qhov ua tau zoo tshaj plaws. Nws pib ntawm qhov chaw primer thiab pib ntxiv dNTPs rau 3' kawg ntawm cov primer, txav raws cov qauv strand. Lub enzyme 'nyeem' tus qauv thiab tso lub hauv paus ntxiv rau hauv txoj hlua tshiab. Piv txwv li, yog tias tus qauv muaj Adenine, cov polymerase ntxiv Thymine. Qhov ceev ntawm cov tshuaj tiv thaiv no yog impressive; Taq polymerase tuaj yeem ntxiv txog 1,000 puag khub ib feeb.
Lub sijhawm ntev ntawm cov kauj ruam no nyob ntawm qhov ntev ntawm DNA ntu uas tau theej. Yog tias lub hom phiaj ua ntu zus yog 1,000 lub hauv paus khub ntev, cov kauj ruam txuas ntxiv yuav raug teem rau ib feeb. Yog tias lub hom phiaj luv dua, lub sijhawm yuav raug txo kom txuag tau tag nrho cov sijhawm ua haujlwm. Ua kom lub tshuab PCR tuav ruaj khov 72 ° C thoob plaws hauv theem no yog qhov tseem ceeb rau kev ua tiav ntawm tag nrho cov DNA strands.
PCR kub khiav yog dab tsi?
Lub PCR kub ntws ua raws li lub voj voog rov ua dua ntawm cov kub kub denaturation, qis cua sov annealing, thiab nruab nrab-kub extension, tsim ib 'sawtooth' thermal profile.
Qhov kev khiav dej num no yog tsim los ua kom qhov loj tshaj qhov kev nce qib ntawm cov DNA ntau. Nyob rau hauv ib txwm khiav, lub tshuab pib ntawm 95 ° C rau 2 feeb (Initial Denaturation), ces nkag mus rau ib lub voj: 95 ° C rau 30 vib nas this, 55 ° C rau 30 vib nas this, thiab 72 ° C rau 60 vib nas this. Lub voj no rov ua dua 30 zaug. Thaum kawg, muaj 'Final Extension' ntawm 72 ° C rau 5-10 feeb los xyuas kom meej tag nrho ib leeg-stranded DNA yog tag nrho ob-stranded ua ntej lub tshuab txias rau 4 ° C rau cia.
Qhov tseeb ntawm qhov ntsuas kub no cuam tshuam ncaj qha rau cov txiaj ntsig thiab kev coj dawb huv ntawm cov khoom PCR. Yog tias qhov ntws tsis sib haum, cov enzyme yuav poob haujlwm lossis cov primers yuav tsim 'primer dimers,' uas yog qhov tseem ceeb ntawm cov khoom siv tsis zoo ntawm cov tshuaj tiv thaiv. Vim li no, calibration thiab thermal uniformity ntawm lub tshuab PCR yog qhov tseem ceeb tshaj plaws rau txhua qhov chaw kuaj xyuas kev kuaj mob lossis kev tshawb fawb.
Summary Table of Temperature Flow
| Theem |
Qhov kub thiab txias |
Lub hom phiaj |
| Pib pib |
94 ° C - 96 ° C |
Activates enzyme, denatures complex DNA. |
| Denaturation |
94 ° C - 98 ° C |
Sib cais ob-stranded DNA rau hauv ib leeg. |
| Annealing |
50 ° C - 65 ° C |
Tso cai rau cov primers khi rau lub hom phiaj ua ntu zus. |
| Txuas ntxiv |
72 ° C |
DNA polymerase synthesizes DNA tshiab strands. |
| Zaum Kawg |
4 ° C - 10 ° C |
Lub sij hawm luv luv ntawm cov khoom lag luam amplified. |
Xaus
Polymerase Chain Reaction yog cov cuab yeej zoo nkauj thiab muaj zog uas tau hloov kho thaj chaw ntawm kev tshawb fawb lom neeg. Los ntawm kev ua raws li cov kauj ruam zoo ntawm denaturation, annealing, thiab txuas ntxiv, cov kws tshawb fawb tuaj yeem qhib qhov zais cia hauv DNA, muab cov lus teb rau cov lus nug nyuaj thiab kev tshawb fawb. Qhov kev vam meej ntawm cov txheej txheem no yog nyob ntawm qhov zoo ntawm cov reagents thiab qhov tseeb ntawm lub tshuab PCR siv los ua kom tiav cov txheej txheem thermal.
Rau txhua qhov chaw kuaj xyuas kom ua tiav cov txiaj ntsig zoo ib yam thiab txhim khu kev qha, kev nkag siab txog nuances ntawm kev tswj qhov kub thiab txias thiab kev tswj lub voj voog yog qhov tseem ceeb. Txawm hais tias koj tab tom ua cov kev tshawb fawb yooj yim lossis cov kev kuaj mob siab, kev xaiv cov cuab yeej thiab kev ua raws li cov txheej txheem ua kom zoo yuav txhais tau qhov tseeb ntawm koj txoj haujlwm. Rau cov neeg txaus siab nyob rau sab logistical ntawm kev teeb tsa lub chaw kuaj molecular, tshawb txog qhov cov nqi thiab cov kev qhia tshwj xeeb ntawm cov tshuab PCR niaj hnub no yog cov kauj ruam tom ntej hauv kev txhim kho koj lub peev xwm kuaj mob.
