Iyo Polymerase Chain Reaction, inozivikanwa zvakanyanya sePCR, inomiririra imwe yeakanyanya kukosha tekinoroji munhoroondo yemamorekuru biology. Yakagadzirwa muma1980s, nzira iyi yakachinja kubva kune yakasarudzika marabhoritari maitiro kuenda kune yakakosha chishandiso chinoshandiswa mukuongororwa kwekurapa, forensic sainzi, uye genetic research. Nekubvumidza masayendisiti kutora sampuli diki yeDNA uye kuikurisa kuita mamirioni emakopi, PCR yaita kuti zvikwanisike kuongorora majini zvakadzama, kuona utachiona hwakanyanya, uye kuona ma genetic marker akange asingaonekwe kune akajairwa nzira dzekuongorora.
Iyo PCR maitiro inzira yemurabhoritari inoshandiswa kugadzira akawanda makopi echimwe chikamu cheDNA kuburikidza nekutenderera kwekushanduka kwetembiricha, inosanganisira denaturation, annealing, uye kuwedzera, inofambiswa nemuchina wakasarudzika wePCR uye inopisa-yakagadzikana DNA polymerase.
Kunzwisisa kuomesesa kwePCR maitiro kwakakosha kune nyanzvi dzemurabhoritari, vaongorori vekurapa, uye vanogadzira maindasitiri vanobatanidzwa mumidziyo yekuongorora. Sezvo kudiwa kwekukurumidza uye kwakaringana kuyedzwa kwema molecular kuri kuramba kuchikura pasi rose, kuvimbika kweiyo PCR muchina unova musimboti wezvakabudirira murabhoritari mhedzisiro. Ichi chinyorwa chinopa gwara rakazara kumatanho, tembiricha, uye zvemuchina zvinodiwa zvePCR process kuti ive nechokwadi chepamusoro-mhando yeDNA kukwidziridzwa uye mhedzisiro yakasimba yekuyedza.
Chinyorwa Summary Tafura
| Chikamu |
Summary |
| Chii chinonzi PCR? |
PCR inzira yemamorekuru biology inoshandiswa kukwidziridza zvakanangana neDNA kutevedzana kweakasiyana ekudzika maapplication. |
| Chii chinodiwa kuPCR? |
Kubudirira kwePCR kunoda template DNA, primers, nucleotides, yakagadzikana DNA polymerase, uye yakakwirira-chaiyo thermal cycler. |
| Ndeapi matanho mana ePCR? |
Maitiro acho anotevera kutevedzana kunonzwisisika kwekutanga, denaturation, annealing, uye kuwedzera kupeta DNA zvirimo kutenderera kwega kwega. |
| Ndeapi matanho ePCR muchina? |
Midziyo inogadzirisa shanduko chaiyo yetembiricha, kuve nechokwadi kuti maitiro ebiochemical anoitika panguva chaiyo inodiwa. |
| Ndeipi tembiricha inoshandiswa padanho re denature? |
Tembiricha yepamusoro, kazhinji iri pakati pe94°C ne98°C, inoshandiswa kutyora mabhondi ehydrogen nekuparadzanisa DNA ine tambo mbiri. |
| Chii chinoitika panguva yekuzvitakura? |
Munguva iyi, tembiricha inodzikiswa kuti zvibvumire maprimers kuti abatanidze kune yavo inopindirana tarisiro yekutevedzana pane imwechete-stranded DNA. |
| Ndeipi tembiricha inoshandiswa padanho rekuwedzera? |
Iyi nhanho inowanzoitika pa72 ° C, iyo yakanyanya tembiricha yeTaq polymerase kuti igadzire tambo itsva yeDNA. |
| Chii chinonzi PCR tembiricha inoyerera? |
Kuyerera kwacho kunosanganisira kukurumidza kuchovha bhasikoro tembiricha yepamusoro, yakaderera, uye yepakati inodzokororwa kusvika iyo inodiwa yasvika. |
Chii chinonzi PCR?
PCR, kana Polymerase Chain Reaction, inzira inoshandura molecular biology yakagadzirirwa kukurumidza kugadzira mamirioni kusvika kumabhiriyoni emakopi eiyo chaiyo DNA sampuro.
Pakati payo, PCR inoshanda se'biological photocopier.' Isati yagadzirwa, kukudza DNA yaive nzira inononoka uye yakaoma inosanganisira kubatanidzwa kweDNA kuita bhakitiriya. Nekuuya kwe PCR muchina , vaongorori vanogona ikozvino kupatsanura chaiyo jini kana chikamu cheiyo genome uye kuikudza mumaawa mashoma. Kugona uku kwakakosha nekuti ongororo zhinji dzebiochemical dzinoda huwandu hwakakosha hweDNA kuti ibudise chiratidzo chinoyerika, uye masampula echisikigo anowanzo kupa huwandu hwekutevera chete.
Iko kushandiswa kwakasiyana-siyana kweiyi tekinoroji kunoratidzwa mumhando dzakasiyana dzekushandisa mumaindasitiri akasiyana. Munzvimbo dzekiriniki, inoshandiswa kuona huwandu hwehutachiona, senge muCOVID-19 kana kuongororwa HIV. Mune forensics, inobvumira vaongorori kuti vaone vanhu kubva kune microscopic samples yezvinhu zvipenyu. Muchikamu cheindasitiri, PCR inova nechokwadi chekuchena kwezvigadzirwa zvekudya uye nekuonekwa kwezvipenyu zvakagadziridzwa. Kunzwisisa misimboti uye mitengo yePCR tekinoroji yakakosha kumaLab anotarisa kusimudzira kugona kwavo kwekuongorora.
Chii chinodiwa kuPCR?
Kubudirira kwePCR kuita kunoda zvinhu zvishanu zvakakosha: iyo template yeDNA, maprimers chaiwo, deoxynucleotide triphosphates (dNTPs), DNA polymerase inodziya-yakagadzikana (seTaq), uye yakasarudzika buffer mhinduro.
Iyo DNA template inoshanda seyakatanga purani yaunoda kukopa. Maprimers mapfupi, ekugadzira zvidimbu zveDNA izvo zvakagadzirirwa-zvakagadzirwa kuti zvienderane nekutanga uye kupera kwekutevedzana kwechinangwa. Pasina izvi, iyo DNA polymerase yaisazoziva pekutangira kuvaka tambo nyowani. MadNTP (A, T, C, uye G) ndiwo mabhuroko ekuvaka anoshandiswa neenzyme kugadzira cheni itsva yeDNA.
Chinokosha zvakaenzana inharaunda umo mhinduro inoitika. Iyo buffer inopa yakagadzikana makemikari nharaunda, kunyanya yakatarisana nepH uye kuwanda kwema magnesium ion, ayo akakosha cofactors eDNA polymerase enzyme. Pakupedzisira, kuurayiwa kwemuviri kwemaitiro kunoda high-performance thermal cycler, inowanzonzi PCR muchina , iyo inonyatso kudzora kukurumidza kuchinja kwekushisa kunodiwa kukonzera imwe neimwe nhanho yekuita.
Zvikamu zvakakosha zvePCR Mix
Template DNA : Sample ine chinangwa chakatevedzana.
DNA Polymerase : Kazhinji Taq polymerase, iyo inoramba ichishanda pakupisa kwakanyanya.
Primers : Pamberi uye dzosera kumashure tambo dzinotsanangura miganhu yekukudza.
dNTPs : Zvigadziko zvina zve nucleotide zvinoshanda se 'inki' yekukopa.
Buffer uye Ions : Inochengetedza iyo enzymatic kunyatsoita uye kugadzikana.
Ndeapi matanho mana ePCR?
Maitiro ePCR ane matanho mana ekutanga anoshanda: Kutanga, Denaturation, Annealing, uye Kuwedzera (inozivikanwawo seElongation).
Danho rekutanga, Initialization, chiitiko chenguva imwe chete apo kamuri yekupindura inodziya kusvika kune tembiricha yakakwira kuti ive nechokwadi chekuti DNA polymerase yakaitwa zvizere uye chero zvinosvibisa hazvibatike. Kutevera izvi, kutenderera kweDenaturation kunotanga, uko DNA ine tambo mbiri inoparadzaniswa. Izvi zvinoteverwa neAnnealing, uko maprimers anowana zvinangwa zvavo, uye pakupedzisira Kuwedzera, uko DNA nyowani inogadzirwa. Iyi nhanho-nhanho nhatu (Denaturation, Annealing, Extension) inodzokororwa 25 kusvika ku40 nguva.
Nekuti huwandu hweDNA hunopetwa nechero denderedzwa rakabudirira, kukura kunowedzera. Somuenzaniso, pashure pemapeji 30, morekuru rimwe reDNA rinogona kuchinjwa kuva makopi anopfuura bhiriyoni. Kushanda uku ndiko kunogadzira emazuva ano murabhoritari inopisa mabhasikoro akakosha kune sainzi yemazuva ano. Pasina kupisa kwepamusoro-soro uye zvivharo zvekutonhodza zvinowanikwa mumuchina wepamusoro-soro wePCR , maitiro acho angave akanonoka zvikuru kuti ashandiswe munzvimbo dzakakwirira dzekuongorora.
Ndeapi matanho ePCR muchina?
Matanho emuchina wePCR anosanganisira otomatiki bhasikoro retembiricha kuburikidza nemagetsi chaiwo ekutonga kwechivharo chinopisa, kutonga chiyero cherampu, nguva yekubata, uye kutonhora kwekupedzisira.
Muchina wePCR unoshanda nekushandisa Peltier zvinhu kukurumidza kupisa nekutonhodza simbi inobata machubhu anoita. 'matanho' kubva pamaonero emuchina anosanganisira 'Ramp,' inova kasipidhi yekuchinja pakati petembiricha, uye 'Bata,' inova nguva iyo muchina unochengetedza tembiricha yakati. Michina yepamusoro-yekupedzisira yakagadzirirwa kuve nekumhanya-mhanya mazinga ekudzikisa nguva inopedzerwa mukushanduka, izvo zvinoderedza njodzi yekusanyatso sunga kana kuparara kwe enzyme.
Iyo software mukati memuchina inobvumira vashandisi kuronga maprotocol akaoma. Izvi zvinosanganisira kupisa kwekutanga, zvishwe zvinodzokorora zvematanho matatu makuru, uye danho rekupedzisira rekubata pane inotonhora tembiricha (kazhinji 4 ° C) kuchengetedza samples kusvikira technician akwanisa kuzvitora. Mazuva ano madhijitari anopindirana pamushini wePCR anobvumirawo kutarisisa-chaiyo-nguva yemaitiro, kuve nechokwadi chekuti iyo inopisa mbiri iri kuteverwa chaizvo sekurongedzerwa kwepamusoro kuberekana.
Ndeipi tembiricha inoshandiswa padanho re denature?
Danho rekuita denaturation rinowanzo shandisa tembiricha dziri pakati pe94°C kusvika 98°C kufambisa kudambuka kwehydrogen bond pakati pematambo eDNA.
Pakupisa uku kwakanyanya, iyo double-helix chimiro cheDNA inova isina kugadzikana. Ma hydrogen bond ane adenine-thymine ne cytosine-guanine peya pamwe chete anonyunguduka, zvoita kuti pave netutambo tuviri twakazvimirira eDNA. Ichi chinhu chakakosha chinodiwa pamatanho anotevera, sezvo maprimers uye DNA polymerase enzyme inogona kungopindirana nematemplate ane tambo imwe chete. Kana tembiricha yakadzikira, DNA haizoparadzanise zvizere, zvichitungamira mukutadza kana kusasimba kwekusimudzira.
Nekudaro, kuchengetedza tembiricha iyi kunoda yakasimba kwazvo DNA polymerase. Ichi ndicho chikonzero kuwanikwa kweTaq polymerase, yakaparadzaniswa nebhakitiriya rinoda kupisa Thermus aquaticus , kwakachinja zvakanyanya. Maenzayimu akajairwa aizoparadzwa pa95°C, asi Taq inoramba ichishanda. Marabhoritari anofanirwa kuona kuti muchina wavo wePCR unopa kupisa kwakafanana mumatsime ese kudzivirira 'nzvimbo dzinotonhora' uko denaturation ingatadza, chinova chinhu chakakosha midziyo yemhando yepamusoro yemamolecular biology.
Chii chinoitika panguva yekutora nhanho yePCR?
Munguva yenhanho yekumisa, tembiricha inodzikiswa kusvika pakati pe50°C ne65°C, zvichibvumira maprimers eDNA kuti abatanidze kutevedzana kwawo kunoenderana nematemplate eDNA ane tambo imwe chete.
Iyi nhanho ine nharo ndiyo inonyanya kuomesesa chikamu chePCR maitiro. Iyo chaiyo tembiricha inoshandiswa zvinoenderana nekunyunguduka tembiricha (Tm) yemaprimers ari kushandiswa. Kana tembiricha yakawandisa, maprimers haazosungiri kune template. Kana yakanyanya kuderera, maprimers anogona kusungirira kune akatevedzana anongori 'chikamu' akafanana, zvichitungamira kune isiri-chaiyo amplification uye messy mhedzisiro. Muchina wePCR unofanirwa kukwanisa kurova tembiricha iyi yakanangana nehupamhi hwechokwadi (kazhinji mukati me0.1 ° C).
Nguva yenhanho yeannealing inowanzoita 20 kusvika 40 seconds. Munguva pfupi iyi hwindo, maprimers anofambisa musanganiswa wekuita kuburikidza nekufamba kwema molecular uye anosvetukira panzvimbo inotarirwa. Kana maprimers apinda, anopa pekutangira kuti DNA polymerase itange kuwedzera nucleotides. Uku kurongeka chaiko ndiko kunobvumira kuwonekwa kwechaiyo genetic mutations kana hutachiona mune yakaoma biological sample, zvichiita mari mumichina yekuongorora yehunyanzvi chinhu chakakosha kumakiriniki labs.
Ndeipi tembiricha inoshandiswa padanho rekuwedzera?
Danho rekuwedzera rinowanzoitwa pa72 ° C, inova ndiyo yakanyanya tembiricha inoshanda kune inopisa-yakagadzika DNA polymerase kugadzira iyo itsva DNA strand.
Pa 72°C, DNA polymerase enzyme iri padanho rayo repamusoro. Inotanga panzvimbo yekutanga uye inotanga kuwedzera dNTPs kumagumo e3' ekutanga, ichifamba pamwe chete ne template strand. Iyo enzyme 'inoverenga' template uye inoisa hwaro hwekuwedzera mutambo itsva. Semuenzaniso, kana template ine adenine, iyo polymerase inowedzera Thymine. Kumhanya kwekuita uku kunoshamisa; Taq polymerase inogona kuwedzera zviuru zvishanu paminiti.
Urefu hwenguva yedanho iri hunoenderana nehurefu hwechikamu cheDNA chiri kukopwa. Kana kutevedzana kwechinangwa kuri 1,000 base pairs kureba, danho rekuwedzera rinogona kusetwa kweminiti imwe. Kana chinangwa chiri chipfupi, nguva inogona kuderedzwa kuchengetedza yakazara nguva yekugadzirisa. Kuona kuti muchina wePCR unoramba wakatsiga 72°C mukati mechikamu ichi kwakakosha pakupedzwa kwetambo dzeDNA dzakazara.
Chii chinonzi PCR tembiricha inoyerera?
Kuyerera kwePCR tembiricha kunotevera kudzokorora kutenderera kweyakanyanya-kupisa denaturation, yakaderera-kupisa annealing, uye ine mwero-kupisa kuwedzera, kugadzira 'sawtooth' yekupisa mbiri.
Kuyerera uku kwakagadzirirwa kuwedzera kufambira mberi kwejometri yehuwandu hweDNA. Mukumhanya kwakajairika, muchina unotanga pa95 ° C kwemaminetsi maviri (Yekutanga Denaturation), wozopinda muchiuno: 95 ° C kwemasekonzi makumi matatu, 55 ° C kwemasekonzi makumi matatu, uye 72 ° C kwemasekonzi makumi matanhatu. Loop iyi inodzokorora ka30. Chekupedzisira, pane 'Final Extension' pa72°C kwemaminetsi mashanu kusvika gumi kuitira kuti DNA ine tambo imwe chete yanyatsokombwa kaviri muchina usati watonhora kusvika 4°C kuti uchengetedzwe.
Iyo chaiyo yekuyerera kwekushisa uku inobata zvakananga goho uye kuchena kwePCR chigadzirwa. Kana kuyerera kusingawirirani, iyo enzyme inogona kurasikirwa nekuita kana maprimers anogona kuumba 'primer dimers,' izvo zvisina basa zvigadziriso zvemaitiro. Nekuda kweizvi, iyo calibration uye kufananidzwa kwekupisa kwePCR muchina ndizvo zvakanyanya kukosha kune chero marabhoritari inoita mamolecular diagnostics kana tsvagiridzo.
Summary Table yeTemperature Flow
| Phase |
Typical Temperature |
Chinangwa |
| Initialization |
94°C – 96°C |
Inomutsa enzyme, denatures yakaoma DNA. |
| Denaturation |
94°C – 98°C |
Inoparadzanisa DNA ine tambo mbiri kuita tambo imwe chete. |
| Annealing |
50°C – 65°C |
Inobvumira maprimers kusungirira kune chinangwa chakatevedzana. |
| Extension |
72°C |
DNA polymerase inogadzira tambo itsva dzeDNA. |
| Final Hold |
4°C – 10°C |
Kuchengetwa kwenguva pfupi kwechigadzirwa chakawedzerwa. |
Mhedziso
Iyo Polymerase Chain Reaction chishandiso chinoyevedza uye chine simba chakashandura mamiriro esainzi yebiology. Nekutevera nhanho dzehungwaru dze denaturation, annealing, uye kuwedzera, masayendisiti anogona kuvhura zvakavanzika zvakachengetwa mukati meDNA, achipa mhinduro kumibvunzo yakaoma yekurapa neye forensic. Kubudirira kweichi chiitiko kunoenderana zvakanyanya nemhando yemareagents uye iko kurongeka kwePCR muchina unoshandiswa kuita kutenderera kwekupisa.
Kune chero marabhoritari inotarisa kuwana inowirirana uye yakavimbika mhedzisiro, kunzwisisa nuances ekudzora tembiricha uye kutenderera manejimendi kwakakosha. Kunyangwe iwe uri kuita tsvakiridzo yekutanga kana yakakwira-vhoriyamu yekiriniki diagnostics, kusarudzwa kwemidziyo uye kuomerera kune akakwenenzverwa maprotocol anotsanangura chokwadi chebasa rako. Kune avo vanofarira kudivi rekugadzirisa rekumisikidza molecular lab, kuongorora iyo mitengo uye tekinoroji maratidziro emazuva ano PCR masisitimu ndiyo inotevera ine musoro nhanho mukusimudzira kugona kwako kwekuongorora.
