Polymerase Chain Reaction, yomwe imadziwika kuti PCR, ikuyimira chimodzi mwazinthu zofunikira kwambiri zaukadaulo m'mbiri ya biology ya mamolekyulu. Kupangidwa mu 1980s, njirayi yasintha kuchoka ku njira yapadera ya labotale kupita ku chida chofunikira chomwe chimagwiritsidwa ntchito pofufuza zachipatala, sayansi yazamalamulo, ndi kafukufuku wa majini. Polola asayansi kutenga chitsanzo chaching'ono cha DNA ndikuchikulitsa kukhala mamiliyoni a makope, PCR yapangitsa kuti zitheke kufufuza majini mwatsatanetsatane, kuzindikira tizilombo toyambitsa matenda molondola kwambiri, ndi kuzindikira zizindikiro za majini zomwe poyamba zinali zosaoneka ndi njira zowunikira.
Njira ya PCR ndi njira ya labotale yomwe imagwiritsidwa ntchito popanga makope angapo a gawo linalake la DNA kudzera mukusintha kwa kutentha, komwe kumakhudza kusinthika, kutulutsa, ndi kukulitsa, motsogozedwa ndi makina apadera a PCR ndi DNA polymerase yokhazikika.
Kumvetsetsa zovuta za njira ya PCR ndikofunikira kwa akatswiri a labotale, ofufuza zamankhwala, ndi opanga mafakitale omwe akukhudzidwa ndi zida zowunikira. Pomwe kufunikira kwa kuyezetsa mwachangu komanso kolondola kwa ma cell kukukulirakulira padziko lonse lapansi, kudalirika kwa Makina a PCR amakhala mwala wapangodya wa zotsatira zopambana za labotale. Nkhaniyi ikupereka chiwongolero chokwanira cha masitepe, kutentha, ndi zofunikira zamakina za njira ya PCR kuti zitsimikizire kukulitsa kwapamwamba kwa DNA ndi zotsatira zoyesa zolimba.
Ndemanga ya Chidule cha Nkhani
| Gawo |
Chidule |
| Kodi PCR ndi chiyani? |
PCR ndi njira ya biology ya mamolekyulu yomwe imagwiritsidwa ntchito kukulitsa momveka bwino matsatidwe a DNA pazinthu zosiyanasiyana zakumunsi. |
| Chofunikira pa PCR? |
PCR yopambana imafuna template ya DNA, zoyambira, ma nucleotides, DNA polymerase yokhazikika, ndi makina opangira matenthedwe apamwamba kwambiri. |
| Kodi masitepe 4 a PCR ndi ati? |
Ndondomekoyi ikutsatira ndondomeko yomveka bwino yoyambira, kutanthauzira, kubwereza, ndi kuwonjezera kuwirikiza kawiri zomwe zili mu DNA kuzungulira kulikonse. |
| Kodi masitepe a makina a PCR ndi chiyani? |
Zipangizozi zimagwiritsa ntchito kusintha kwa kutentha, kuonetsetsa kuti biochemical reaction ikuchitika panthawi yomwe ikufunika. |
| Kodi kutentha komwe kumagwiritsidwa ntchito popanga denature ndi chiyani? |
Kutentha kwakukulu, komwe nthawi zambiri kumakhala pakati pa 94 ° C ndi 98 ° C, amagwiritsidwa ntchito kuthyola ma hydrogen bond ndikulekanitsa DNA yamitundu iwiri. |
| Kodi chimachitika ndi chiyani panthawi ya annealing step? |
Munthawi imeneyi, kutentha kumatsitsidwa kuti ma primers azitha kumangiriza kutsatana kwazomwe akutsata pa DNA ya chingwe chimodzi. |
| Kodi kutentha komwe kumagwiritsidwa ntchito powonjezera ndi kotani? |
Izi nthawi zambiri zimachitika pa 72 ° C, kutentha koyenera kwa Taq polymerase kuti apange chingwe chatsopano cha DNA. |
| Kodi kutentha kwa PCR ndi chiyani? |
Kuthamanga kumaphatikizapo kachitidwe ka njinga kothamanga kwa kutentha kwapamwamba, kotsika, ndi kwapakati komwe kumabwerezedwa mpaka ndende yomwe mukufuna ifike. |
Kodi PCR ndi chiyani?
PCR, kapena Polymerase Chain Reaction, ndi njira yosinthira mamolekyulu ya biology yopangidwa kuti ipange mwachangu makope mamiliyoni mpaka mabiliyoni amtundu wina wa DNA.
Pakatikati pake, PCR imagwira ntchito ngati 'biological photocopier.' Isanapangidwe, kukulitsa DNA inali njira yapang'onopang'ono komanso yovuta yomwe imaphatikizapo kupanga DNA kukhala mabakiteriya. Ndi kubwera kwa Makina a PCR , ofufuza tsopano akhoza kusiyanitsa jini kapena gawo linalake la jini ndi kulikulitsa pakangopita maola angapo. Kuthekera kumeneku ndikofunikira chifukwa kusanthula kwamankhwala ambiri kumafunikira DNA yochulukirapo kuti ipereke chizindikiro choyezeka, ndipo zitsanzo zachilengedwe nthawi zambiri zimangopereka zochepa chabe.
Kusinthasintha kwa ukadaulo uwu kumawonekera m'magwiritsidwe ake osiyanasiyana m'mafakitale osiyanasiyana. M'malo azachipatala, amagwiritsidwa ntchito kudziwa kuchuluka kwa ma virus, monga COVID-19 kapena kuyezetsa HIV. Pazambiri zazamalamulo, zimalola ofufuza kuti azindikire anthu kuchokera ku zitsanzo zazing'ono zazinthu zachilengedwe. M'gawo la mafakitale, PCR imatsimikizira chiyero cha zakudya komanso kuzindikira zamoyo zosinthidwa ma genetic. Kumvetsa mfundo ndi ndalama zaukadaulo wa PCR ndizofunikira kwa ma lab omwe akuyang'ana kuti apititse patsogolo luso lawo lozindikira matenda.
Chofunikira pa PCR?
Kuchita bwino kwa PCR kumafuna zigawo zisanu zazikuluzikulu: template ya DNA, zoyambira zenizeni, deoxynucleotide triphosphates (dNTPs), DNA polymerase (monga Taq), ndi njira yapadera yosungiramo kutentha.
DNA template imagwira ntchito ngati pulani yoyambirira yomwe mukufuna kukopera. Zoyambira ndi zazifupi, zopangira za DNA zomwe zimapangidwira kuti zigwirizane ndi chiyambi ndi mapeto a ndondomeko ya chandamale. Popanda izi, DNA polymerase sakanadziwa komwe angayambire kupanga chingwe chatsopanocho. Ma dNTP (A, T, C, ndi G) ndi zomangira zomwe enzyme imagwiritsa ntchito kupanga tcheni cha DNA chatsopano.
Chofunikiranso ndi malo omwe zomwe zimachitikazo zimachitika. Chosungirachi chimapereka malo okhazikika a mankhwala, makamaka kuyang'ana pH ndi kuchuluka kwa ayoni a magnesium, omwe ndi ofunika kwambiri kwa DNA polymerase enzyme. Potsirizira pake, kuphedwa kwa thupi kwa zomwe zimachitika kumafuna woyendetsa kutentha kwapamwamba, nthawi zambiri amatchedwa PCR makina , omwe amawongolera bwino kutentha kwachangu komwe kumafunika kuyambitsa gawo lililonse la zomwe zimachitika.
Zigawo Zofunikira za PCR Mix
Template DNA : Chitsanzo chokhala ndi ndondomeko ya chandamale.
DNA Polymerase : Nthawi zambiri Taq polymerase, yomwe imakhala yogwira ntchito kutentha kwambiri.
Zoyambira : Patsogolo ndi kumbuyo zingwe zomwe zimatanthauzira malire okulitsa.
dNTPs : Maziko anayi a nucleotide omwe amagwira ntchito ngati 'inki' ya makina okopera.
Buffer ndi Ions : Imasunga mphamvu ya enzymatic komanso kukhazikika.
Kodi masitepe 4 a PCR ndi ati?
Njira ya PCR ili ndi magawo anayi oyambira: Kuyambitsa, Kuchotsa, Kuwonjezera, ndi Kukulitsa (komwe kumadziwikanso kuti Elongation).
Gawo loyamba, Initialization, ndi chochitika cha nthawi imodzi pomwe chipinda chochitiramo chimatenthedwa mpaka kutentha kwambiri kuti zitsimikizire kuti DNA polymerase yatsegulidwa mokwanira ndipo zonyansa zilizonse sizimachotsedwa. Pambuyo pa izi, kuzungulira kwa Denaturation kumayamba, pomwe DNA yamitundu iwiri imalekanitsidwa. Izi zikutsatiridwa ndi Annealing, kumene oyambira amapeza zolinga zawo, ndipo potsiriza Kuwonjezera, kumene DNA yatsopano imapangidwira. Kuzungulira kwa magawo atatu awa (Denaturation, Annealing, Extension) kumabwerezedwa 25 mpaka 40.
Chifukwa kuchuluka kwa DNA kumawirikiza kawiri ndi kayendedwe kalikonse kopambana, kukula kwake kumakulirakulira. Mwachitsanzo, pambuyo pa mizungu 30, molekyu imodzi ya DNA imatha kusinthidwa kukhala makope oposa biliyoni imodzi. Mwachangu izi ndi zimene zimapanga ma labotale matenthedwe ma cyclers amakono ofunikira kwambiri pa sayansi yamakono. Popanda zitsulo zotentha kwambiri komanso zoziziritsa zomwe zimapezeka pamakina apamwamba kwambiri a PCR , njirayi ingakhale yochedwa kwambiri kuti igwiritsidwe ntchito m'madera ozindikira matenda apamwamba kwambiri.
Kodi masitepe a makina a PCR ndi chiyani?
Mayendedwe amakina a PCR amaphatikiza kuyendetsa pawokha kutentha kwa kutentha kudzera pamagetsi olondola amagetsi otchinga, kuwongolera liwiro, nthawi yogwira, komanso kuziziritsa komaliza.
Makina a PCR amagwira ntchito pogwiritsa ntchito zinthu za Peltier kuti azitenthetsa komanso kuziziritsa chipika chachitsulo chomwe chimasunga machubu. 'Masitepe' kuchokera pamawonekedwe a makinawa akuphatikizapo 'Ramp,' yomwe ndi liwiro la kusintha pakati pa kutentha, ndi 'Hold,' yomwe ndi nthawi yomwe makina amasunga kutentha kwake. Makina apamwamba amapangidwa kuti azikhala ndi mitengo yothamanga kwambiri kuti achepetse nthawi yomwe amasintha, zomwe zimachepetsa chiopsezo chomangirira kapena kuwonongeka kwa enzyme.
Mapulogalamu mkati mwa makina amalola ogwiritsa ntchito kupanga ma protocol ovuta. Izi zimaphatikizapo kutentha koyambirira, malupu obwerezabwereza a magawo atatu akuluakulu, ndi sitepe yomaliza yogwira pa kutentha kozizira (kawirikawiri 4 ° C) kusunga zitsanzo mpaka katswiri atazitenga. Mawonekedwe amakono a digito pamakina a PCR amalolanso kuyang'anira zenizeni zomwe zikuchitika, kuwonetsetsa kuti mbiri yamafuta ikutsatiridwa ndendende momwe idakonzedwera kuti ipangikenso.
Kodi kutentha komwe kumagwiritsidwa ntchito popanga denature ndi chiyani?
Njira ya denaturation imagwiritsa ntchito kutentha kwapakati pa 94 ° C ndi 98 ° C kuti athe kuthyoka kwa ma hydrogen bond pakati pa zingwe za DNA.
Pa kutentha kwakukulu kumeneku, kapangidwe ka DNA kaŵirikaŵiri kaŵirikaŵiri kumakhala kosakhazikika. Ma hydrogen omwe amamangira ma awiri awiri a adenine-thymine ndi cytosine-guanine amasungunuka, zomwe zimapangitsa kuti pakhale timizere tiwiri tokha ta DNA. Ichi ndi chofunikira kwambiri pamasitepe otsatirawa, popeza zoyambira ndi DNA polymerase enzyme zimatha kulumikizana ndi ma tempuleti amodzi. Ngati kutentha kuli kotsika kwambiri, DNA sidzadzilekanitsa mokwanira, zomwe zimapangitsa kuti kulephera kukulitsa kapena kusakwanira.
Komabe, kusunga kutentha kumeneku kumafuna DNA polymerase yolimba kwambiri. Ichi ndichifukwa chake kutulukira kwa Taq polymerase, wolekanitsidwa ndi bakiteriya wokonda kutentha Thermus aquaticus , kunali kosintha kwambiri. Ma enzymes okhazikika amatha kuwonongeka pa 95 ° C, koma Taq imagwirabe ntchito. Ma Laboratories akuyenera kuwonetsetsa kuti makina awo a PCR akupereka kutentha kofananira pazitsime zonse kuti ateteze 'malo ozizira' pomwe mawonekedwe amatha kulephera, chomwe ndi gawo lofunikira kwambiri zida zapamwamba za biology ya maselo.
Kodi chimachitika ndi chiyani panthawi ya PCR?
Pa sitepe ya annealing, kutentha kumatsikira pakati pa 50 ° C ndi 65 ° C, zomwe zimapangitsa kuti DNA primers igwirizane ndi ndondomeko zawo zowonjezera pa ma tempuleti a DNA a chingwe chimodzi.
Gawo ili ndilofunika kwambiri pazochitika za PCR. Kutentha kwapadera komwe kumagwiritsidwa ntchito kumadalira kutentha kosungunuka (Tm) kwa zoyambira zomwe zimagwiritsidwa ntchito. Ngati kutentha kuli kokwera kwambiri, zoyambira sizingagwirizane ndi template. Ngati ndizotsika kwambiri, zoyambira zitha kumangika kumayendedwe omwe ali 'pang'ono' ofanana, zomwe zimatsogolera kukulitsa kosagwirizana ndi zotsatira zake. Makina a PCR ayenera kugunda kutentha kwa chandamalechi ndi digiri yapamwamba yolondola (nthawi zambiri mkati mwa 0.1 ° C).
Kutalika kwa gawo la annealing nthawi zambiri kumakhala masekondi 20 mpaka 40. Pazenera lachidule ili, zoyambira zimayendetsa kusakanikirana kwa mamolekyu ndikulowera pamalo omwe mukufuna. Zoyambira zikayamba, zimapereka poyambira kuti DNA polymerase iyambe kuwonjezera ma nucleotides. Kulumikizana bwino kumeneku ndi komwe kumapangitsa kuti munthu azindikire kusintha kwa ma genetic kapena tizilombo toyambitsa matenda muzotengera zovuta zachilengedwe, zomwe zimapangitsa ndalama zamakina ozindikira matenda ndizofunikira kwambiri pamakalabu azachipatala.
Kodi kutentha komwe kumagwiritsidwa ntchito powonjezera ndi kotani?
Njira yowonjezera nthawi zambiri imachitika pa 72 ° C, komwe ndi kutentha koyenera kwa DNA polymerase yosasunthika kuti ipange chingwe chatsopano cha DNA.
Pa 72 ° C, enzyme ya DNA polymerase imagwira ntchito bwino kwambiri. Imayambira patsamba loyambira ndikuyamba kuwonjezera ma dNTP kumapeto kwa 3' koyambira, ndikusunthira pamzere wa template. Enzayimu 'imawerenga' template ndikuyika maziko owonjezera mu chingwe chatsopano. Mwachitsanzo, ngati template ili ndi adenine, polymerase imawonjezera Thymine. Liwiro la izi ndi lochititsa chidwi; Taq polymerase imatha kuwonjezera pafupifupi 1,000 base pa mphindi imodzi.
Kutalika kwa nthawi ya sitepeyi kumadalira kutalika kwa gawo la DNA lomwe likukopera. Ngati ndondomeko yomwe mukufuna kutsata ndi 1,000 base pairs kutalika, sitepe yowonjezera ikhoza kukhazikitsidwa kwa mphindi imodzi. Ngati chandamale ndi lalifupi, nthawi akhoza kuchepetsedwa kupulumutsa wonse processing nthawi. Kuonetsetsa kuti makina a PCR akukhalabe ndi 72 ° C mosasunthika panthawi yonseyi n'kofunika kwambiri kuti zingwe za DNA zithe.
Kodi kutentha kwa PCR ndi chiyani?
Kuthamanga kwa kutentha kwa PCR kumatsatira kubwereza mobwerezabwereza kwa kutentha kwapamwamba, kutsekemera kwa kutentha pang'ono, ndi kutentha kwapakati-kutentha, kumapanga 'sawtooth' mbiri ya kutentha.
Kuthamanga uku kudapangidwa kuti kukulitsa kukula kwa geometric kwa kuchuluka kwa DNA. Mwachizoloŵezi, makinawo amayamba pa 95 ° C kwa mphindi 2 (Denaturation Yoyamba), kenako amalowetsa kuzungulira: 95 ° C kwa masekondi 30, 55 ° C kwa masekondi 30, ndi 72 ° C kwa masekondi 60. Lupu ili likubwereza ka 30. Pomaliza, pali 'Final Extension' pa 72 ° C kwa mphindi 5-10 kuwonetsetsa kuti DNA yonse ya chingwe chimodzi imangiriridwa kawiri makina asanazizira mpaka 4 ° C kuti asungidwe.
Kulondola kwa kutentha kumeneku kumakhudza mwachindunji zokolola ndi chiyero cha mankhwala a PCR. Ngati kutuluka kwake sikukugwirizana, enzyme imatha kutaya ntchito kapena zoyambira zimatha kupanga 'primer dimers,' zomwe sizimathandiza kwenikweni. Chifukwa cha izi, ma calibration ndi kufanana kwa kutentha kwa makina a PCR ndizofunikira kwambiri pa labotale iliyonse yomwe imachita kafukufuku wa maselo kapena kafukufuku.
Chidule cha Kutentha kwa Flow
| Gawo |
Kutentha Kwambiri |
Cholinga |
| Kuyambitsa |
94°C – 96°C |
Imayendetsa enzyme, denatures DNA yovuta. |
| Denaturation |
94°C – 98°C |
Amalekanitsa DNA ya zingwe ziwiri kukhala chingwe chimodzi. |
| Annealing |
50°C – 65°C |
Imalola zoyambira kuti zigwirizane ndi zomwe mukufuna. |
| Kuwonjezera |
72°C |
DNA polymerase imapanga zingwe zatsopano za DNA. |
| Final Hold |
4°C – 10°C |
Kusungirako kwakanthawi kochepa kwa mankhwala okulitsa. |
Mapeto
Polymerase Chain Reaction ndi chida chokongola komanso champhamvu chomwe chasintha mawonekedwe a sayansi yachilengedwe. Potsatira mosamalitsa masitepe a denaturation, annealing, and extension, asayansi amatha kuvumbulutsa zinsinsi zomwe zili mkati mwa DNA, ndikupereka mayankho ku mafunso ovuta azachipatala ndi azamalamulo. Kupambana kwa njirayi kumadalira kwambiri ubwino wa ma reagents ndi kulondola kwa makina a PCR omwe amagwiritsidwa ntchito popanga matenthedwe.
Kwa labotale iliyonse yomwe ikuyang'ana kuti ikwaniritse zotsatira zokhazikika komanso zodalirika, kumvetsetsa zowongolera kutentha ndi kayendetsedwe ka kayendetsedwe kake ndikofunikira. Kaya mukuchita kafukufuku wofunikira kapena zowunikira zambiri zachipatala, kusankha zida ndikutsatira ndondomeko zokongoletsedwa zidzatanthawuza kulondola kwa ntchito yanu. Kwa iwo omwe ali ndi chidwi ndi gawo lokonzekera kukhazikitsa ma labu a molekyulu, kufufuza mtengo ndi ukadaulo wa machitidwe amakono a PCR ndiye gawo lotsatira lomveka pakupititsa patsogolo luso lanu lozindikira matenda.
