Makina a PCR ndi chipangizo cha labotale chomwe chimagwiritsa ntchito njinga yamoto yolondola kuti ithandizire Polymerase Chain Reaction, zomwe zimathandizira kukulitsa mwachangu ma DNA kapena ma RNA kuti aunike. Pogwiritsa ntchito magawo otenthetsera ndi kuziziritsa omwe amafunikira kuti asinthe, annealing, ndi kukulitsa, makina amakono a PCR amapereka kusasinthasintha komanso kuthamanga kofunikira pakugwiritsa ntchito zamankhwala ndi kafukufuku.
Kumvetsetsa ukadaulo wam'makinawa ndikofunikira kwa oyang'anira ma labotale, ofufuza, ndi akatswiri azachipatala omwe akufuna kukhathamiritsa ntchito zawo zowunikira. Kuchokera pazovuta zazinthu zotenthetsera za Peltier kupita kuukadaulo wapamwamba wamakina anthawi yeniyeni, uinjiniya womwe umathandizira kukulitsa kwa DNA ndizovuta komanso zosangalatsa. Nkhaniyi ikupereka kuwunika kwatsatanetsatane kwaukadaulo wa PCR, zida zake zamakina, ndi ntchito zake zosiyanasiyana m'makampani azachipatala padziko lonse lapansi.
Chidule cha Kapangidwe ka Nkhani
| Gawo |
Chidule |
| Momwe Makina a PCR Amagwirira Ntchito |
Kufotokozera za njira zitatu zoyendetsera kutentha kwapang'onopang'ono (kuchepetsa, kukulitsa, ndi kukulitsa) komwe kumalola kukula kwa DNA mokulira. |
| Zigawo zazikulu za PCR Technology |
Kuwonongeka kwaukadaulo kwa midadada yotenthetsera, zinthu za Peltier, ndi masensa omwe amaonetsetsa kuti kutentha kumafanana komanso kulondola. |
| Mitundu Yamakina a PCR ndi Ntchito Zawo |
Kuyerekeza pakati pa ma cyclers wamba, Real-Time (qPCR) machitidwe, ndi matekinoloje a Digital PCR (dPCR). |
| Kufunika kwa Kufanana kwa Thermal |
Kuwunika chifukwa chake kusasinthasintha kwa kutentha pazitsime zonse ndiye chinthu chofunikira kwambiri pazotsatira zodalirika za labotale ya SEO. |
| Mapulogalamu mu Modern Diagnostics |
Momwe ukadaulo wa PCR umagwiritsidwira ntchito pakuyesa matenda opatsirana, oncology, ndi chizindikiritso chazamalamulo. |
| Malangizo Okonzekera ndi Kukhathamiritsa |
Njira zabwino zosinthira ndikusunga zida za PCR kuti zitsimikizire kulondola kwanthawi yayitali komanso magwiridwe antchito. |
Momwe Makina a PCR Amagwirira Ntchito
Kagwiridwe kake ka makina a PCR kumaphatikizapo kupalasa njinga kupyola magawo atatu osiyana a kutentha kuti athandizire kusinthika kwa DNA, kutulutsa koyambira, ndi kukulitsa kwa enzymatic, zomwe zimapangitsa kufananizidwanso kwamtundu womwe mukufuna.
Pakatikati pa ndondomekoyi ndi kuzungulira kwa kutentha. Mu gawo loyamba, denaturation, makina PCR kutentha anachita osakaniza pafupifupi 94 kuti 98 madigiri Celsius. Kutentha kwakukulu kumeneku kumaswa ma hydrogen pakati pa zingwe ziwiri za DNA double helix, zomwe zimapangitsa kuti pakhale ma tempuleti a DNA a chingwe chimodzi. Kulondola panthawiyi n'kofunika, chifukwa kutentha kosakwanira sikungathe kulekanitsa zingwezo, pamene kutentha kwakukulu kungawononge puloteni yokhazikika ya polymerase.
Gawo lachiwiri ndi la annealing, pomwe kutentha kumatsikira pakati pa 50 ndi 65 digiri Celsius. Izi zimathandiza kuti ma DNA afupikitsa otchedwa primers amangirire kuzinthu zawo zowonjezera pazithunzi za DNA zamtundu umodzi. Kutentha kwenikweni kumadalira kutentha kosungunuka kwa zoyambira zomwe zimagwiritsidwa ntchito. Makina apamwamba kwambiri a PCR ayenera kufika ndi kukhazikika pa kutentha kwapadera kumeneku mwamsanga kuti ateteze kumangirira kosakhazikika, zomwe zingayambitse zotsatira zolakwika kapena 'phokoso' mu deta.
Pomaliza, gawo lokulitsa limachitika, nthawi zambiri pa madigiri 72 Celsius. Uku ndiye kutentha koyenera kwa DNA polymerase (yomwe nthawi zambiri imatchedwa Taq polymerase) kuti ipange chingwe chatsopano cha DNA powonjezera ma nucleotides ku zoyambira. Pamene mkomberowo ukubwerezedwa, chiwerengero cha makope a DNA chimaŵirikiza kaŵiri nthaŵi iliyonse. Kuti mumvetse bwino zandalama zomwe zimafunikira paukadaulo uwu, mutha kudziwa zambiri za Kodi Makina a PCR ndi Mtengo Wake kuti awunikenso bajeti yofunikira pamagawo ochita bwino kwambiri.
Zimango za Kukula Kwambiri
Mzere 1: 2 makope
Cycle 10: 1,024 makope
Cycle 30: Makope opitilira 1 biliyoni
Zigawo zazikulu za PCR Technology
Ubwino waukadaulo wamakina a PCR umadalira zida zake zamkati, makamaka zinthu zotenthetsera za Peltier, siliva kapena aluminiyamu yotenthetsera block, ndi pulogalamu yowongolera yotsogola yomwe imayang'anira kusintha kwa kutentha mwachangu.
Chigawo chofunikira kwambiri ndi Peltier element, pampu yotentha yotentha yomwe imatulutsa kutentha kuchokera mbali imodzi ya chipangizo kupita ku ina motsutsana ndi kutentha kwa kutentha. Izi zimalola kutenthetsa ndi kuziziritsa kuchokera ku gwero limodzi, kupangitsa makina a PCR kusintha kutentha mumasekondi. Liwiro lomwe masinthidwewa amachitikira-lomwe limatchedwa 'ramp rate' - limatsimikizira mwachindunji momwe malo ogwirira ntchito amagwirira ntchito. Makina apamwamba nthawi zambiri amakhala ndi zitsulo zasiliva zokhala ndi golide kuti apititse patsogolo kutentha kwa kutentha.
Zomverera ndi ma microprocessors amapanga 'ubongo' wa chipangizocho. Zigawozi zimayang'anira kutentha kwa chipika mu nthawi yeniyeni, kusintha mphamvu ku zinthu za Peltier kuti zisawonongeke kutentha kapena kuwombera pansi. Makina amakono amagwiritsa ntchito masensa angapo kudutsa chipikacho kuwonetsetsa kuti chitsime chilichonse chimakhala ndi kutentha komweko. Kwa maofesi akuyang'ana kukweza hardware yawo, kusankha odalirika Wopereka makina a PCR amatsimikizira mwayi wopeza ukadaulo waposachedwa wa sensor komanso uinjiniya wamphamvu.
Chivundikiro chotenthetsera ndi chinthu china chofunikira koma nthawi zambiri chimanyalanyazidwa. Pokhala ndi chivindikiro pa kutentha pang'ono kuposa kutentha kwa chipika (nthawi zambiri pafupifupi madigiri 105 Celsius), makinawo amalepheretsa kutuluka kwa nthunzi ndi condensation ya zomwe zimasakanikirana pazipewa za chubu. Izi zimatsimikizira kuti kuchuluka kwa ma reagents kumakhalabe kosasintha muzaka zonse za 30 mpaka 40, zomwe ndizofunikira kwambiri pakuwongolera kwamankhwala.
Zofunika Kwambiri pa Hardware
Thermal Block: Nthawi zambiri imakhala ndi zitsime 96 kapena 384.
Ramp Rate: Liwiro la kusintha kwa kutentha (kuyezedwa mu °C/s).
Kulondola: Kutentha kwa block kuli pafupi bwanji ndi malo.
Kufanana: Kutentha kosasinthasintha pazitsime zonse.
Mitundu Yamakina a PCR ndi Ntchito Zawo
Biology yamakono ya mamolekyulu imagwiritsa ntchito magulu atatu akuluakulu aukadaulo wa PCR: Gradient PCR pakukhathamiritsa, Real-Time PCR (qPCR) pakuchulukira, ndi Digital PCR yolondola kwambiri pakuzindikira kosowa.
Ma Standard Thermal Cyclers ndi omwe amagwiritsidwa ntchito kwambiri pofufuza ndi maphunziro. Makinawa adapangidwira 'mapeto-malo' PCR, kutanthauza kuti zotsatira zake zimawunikidwa pambuyo pomaliza, nthawi zambiri kudzera pa gel electrophoresis. Kusiyanasiyana kwa izi ndi Gradient PCR makina , omwe amalola wogwiritsa ntchito kukhazikitsa kutentha kosiyana kudutsa chipika panthawi imodzi. Izi ndizothandiza kwambiri pozindikira kutentha koyenera kwa zoyambira zatsopano, kupulumutsa maola oyesera ndi zolakwika.
Makina a Real-Time PCR (qPCR) akuyimira kulumpha kwakukulu kwaukadaulo. Zipangizozi zimaphatikizapo makina ophatikizira ophatikizika (ma lasers kapena ma LED ndi zowunikira) kuti aziyang'anira ma fluorescence a zitsanzo za DNA panthawi yakukulitsa. Izi zimalola kusanthula kwa 'kuchulukira', komwe ofufuza amatha kudziwa ndendende kuchuluka kwa ma DNA omwe adatsatana ndi zomwe zidalipo pachitsanzo choyambirira. Tekinoloje iyi ndi mulingo wagolide woyesa kuchuluka kwa ma virus komanso maphunziro a gene expression. Kumvetsa mtengo ndi mafotokozedwe a mayunitsi a PCR ndizofunikira posankha pakati pa woyendetsa wamba ndi makina apamwamba a qPCR.
Digital PCR (dPCR) ndiye chisinthiko chaposachedwa, chopereka kuchulukira kokwanira popanda kufunikira kokhotakhota kokhazikika. Zitsanzozo zimagawidwa m'madontho ang'onoang'ono zikwizikwi, ndipo machitidwe a PCR amachitika pamtundu uliwonse. Powerengera kuchuluka kwa madontho a 'positive' ndi 'negative', makinawa amapereka chiwerengero cholondola kwambiri cha mamolekyu a DNA. Izi ndizothandiza makamaka pakuzindikira masinthidwe osowa m'ma biopsies amadzimadzi kapena zitsanzo zachilengedwe pomwe DNA yomwe mukufuna imapezeka m'malo otsika kwambiri.
Kuyerekeza kwa PCR Technologies
| Mbali |
PCR yokhazikika |
Real-Time PCR (qPCR) |
Digital PCR (dPCR) |
| Kuzindikira |
Mapeto-malo |
Pompopompo |
Mapeto (Absolute) |
| Quantification |
Mkhalidwe |
Wachibale/Kuchuluka |
Mtheradi |
| Kumverera |
Wapakati |
Wapamwamba |
Kwambiri Kwambiri |
| Kuvuta |
Zochepa |
Wapakati |
Wapamwamba |
Kufunika kwa Kufanana kwa Thermal
Kufanana kwa kutentha kumatanthawuza kusasinthasintha kwa kutentha pazitsime zonse za chipika cha PCR, chinthu chofunika kwambiri chomwe chimatsimikizira kuti chitsanzo chilichonse mu mbale ya 96-chitsime chimakulitsidwa pamlingo womwewo ndipo chimapereka zotsatira zobwerezabwereza.
Ngati ngodya imodzi ya chotchinga chotenthetsera imakhala yozizirirapo ngakhale madigiri 0.5 Celsius kuposa pakatikati, DNA yomwe ili m'zitsimezo siingathe kusinthika, kapena zoyambira zimatha kumangika mosagwirizana. Izi zimatsogolera ku 'kusinthika kwabwino,' komwe kungathe kuwononga kuyesa konse kapena, choyipa, kupangitsa kuti pakhale vuto lolakwika pazachipatala. Opanga makina apamwamba kwambiri a PCR amaika ndalama zambiri pakupanga makina olondola komanso kutentha kwamitundu ingapo kuti achepetse zoopsazi ndikupatsanso kufanana.
Mapulogalamu apamwamba a mapulogalamu amagwiritsidwa ntchito kubwezera 'zotsatira za m'mphepete,' kumene zitsime zomwe zili pamphepete mwa chipika zimataya kutentha mofulumira kusiyana ndi zomwe zili pakati. Poyang'anira madera osiyanasiyana a Peltier pawokha, makinawo amatha kupopera kutentha kwambiri mpaka m'mphepete kuti asunge mawonekedwe otenthetsera. Pofufuza zida, ndizopindulitsa kuyang'ana a makina apamwamba kwambiri a PCR kuti afananize momwe mitundu yosiyanasiyana imagwirira ntchito kasamalidwe ka matenthedwe ndi kuwongolera kwa block.
Reproducibility ndiye maziko a sayansi. Kaya kuyesa ku London kapena ku Shanghai, makina a PCR amayenera kupereka magwiridwe antchito ofanana. Ichi ndichifukwa chake miyezo yapadziko lonse lapansi komanso kusanja pafupipafupi ndikofunikira pama labu azachipatala. Makina osafanana bwino adzatulutsa 'Ct' (kuzungulira malire) mikhalidwe yosagwirizana mu qPCR, zomwe zimapangitsa kuti zikhale zosatheka kuwerengera molondola kuchuluka kwa ma virus kapena zolembera ma genetic pamayendedwe osiyanasiyana kapena ma laboratories.
Zinthu Zomwe Zimakhudza Kufanana
Zida Zotchinga: Siliva ndiyopambana kuposa aluminiyumu yogawa kutentha.
Ubwino wa Peltier: Ma Peltier apamwamba amapereka moyo wautali komanso kutentha kosasintha.
Kuyika kwa Sensor: Masensa angapo amapereka mayankho abwino pa loop yowongolera.
Mapulogalamu mu Modern Diagnostics
Kugwiritsiridwa ntchito kwaukadaulo wa PCR kumadutsa pakuzindikiritsa matenda opatsirana, oncology pakuzindikiritsa chizindikiro cha khansa, ndi sayansi yazamalamulo, komwe amagwiritsidwa ntchito popanga mbiri ya DNA kuchokera ku zitsanzo zazing'ono zamoyo.
M'malo a matenda opatsirana, makina a PCR akhala chida chofunikira kwambiri. Zimalola kuzindikira tizilombo toyambitsa matenda monga HIV, Hepatitis, ndi mavairasi opuma omwe ali ndi chidwi kwambiri komanso mofulumira kuposa njira zachikhalidwe. Chifukwa makinawa amatha kuzindikira kukhalapo kwa ma virus mwachindunji, amatha kuzindikira matenda nthawi yayitali wodwalayo asanakhale ndi chitetezo chamthupi kapena zizindikiro, zomwe zimalola kulowererapo koyambirira ndikuwongolera bwino thanzi la anthu.
Oncology ndi gawo lina lomwe limapindula kwambiri ndiukadaulo uwu. PCR imagwiritsidwa ntchito pozindikira masinthidwe enieni okhudzana ndi mitundu ina ya khansa. Izi zimalola 'mankhwala apadera,' komwe madokotala angasankhe chithandizo chothandiza kwambiri potengera chibadwa cha chotupacho. Kwa iwo omwe ali ndi chidwi ndi ntchito yaukadaulo pazaumoyo, kuwerenga za mtengo ndi zofunikira za machitidwe a PCR zimapereka chidziwitso cha momwe zipatala zimagwirizanirana ndiukadaulo wapamwamba kwambiri ndi bajeti zogwirira ntchito.
Sayansi yazamalamulo imadalira PCR kukulitsa DNA kuchokera ku zochitika zaupandu, monga tsitsi limodzi kapena dontho la magazi. Poyang'ana madera ena amtundu wa anthu omwe amasiyana pakati pa anthu (STRs), asayansi azamalamulo amatha kupanga chala chapadera cha DNA. Ukadaulowu wasintha kwambiri machitidwe azamalamulo, ndikupereka umboni wamphamvu kwa onse omwe adapezeka kuti ndi olakwa komanso kumasulidwa kwa omwe akuimbidwa mlandu.
Magawo Akuluakulu Ogwiritsa Ntchito
Kuzindikira Zachipatala: Kuyesa kwa tizilombo toyambitsa matenda ndi kuyezetsa asanabadwe.
Chitetezo Chakudya: Kuzindikira ma GMO kapena tizilombo toyambitsa matenda monga Salmonella.
Sayansi Yachilengedwe: Kuyang'anira kuchuluka kwa tizilombo tating'onoting'ono m'nthaka kapena m'madzi.
Research: Cloning, sequencing, and gene expression analysis.
Malangizo Okonzekera ndi Kukhathamiritsa
Kuti makina a PCR azitha kukhala ndi moyo wautali komanso olondola, ma labotale amayenera kukhazikitsa njira zosamalira bwino, kuphatikiza kuyeretsa nthawi zonse, kuyika chivindikiro, komanso kutsimikizira kwaukadaulo kwamafuta.
Kuipitsidwa ndi mdani wamkulu wa PCR. Ngakhale molekyu imodzi ya DNA kuchokera kuthamangitsidwa koyambirira imatha kukulitsidwa, zomwe zimapangitsa kuti pakhale zotsatira zabodza. Choncho, chipika chotentha cha makina a PCR chiyenera kutsukidwa nthawi zonse ndi njira zapadera zochotsera DNA kapena 10% bleach wotsatiridwa ndi ethanol. Ndikofunikiranso kuwonetsetsa kuti machubu amasindikizidwa bwino kuti asatayike m'zitsime, zomwe zimatha kuwononga malo otchinga ndikusokoneza kusamutsa kwamafuta.
Kuwongolera pafupipafupi ndikofunikira. M'kupita kwa nthawi, zinthu za Peltier zimatha kuwonongeka, zomwe zimapangitsa kuti pakhale mayendedwe apang'onopang'ono kapena kuchepa kwa kutentha. Akatswiri oyesa ma calibration amagwiritsa ntchito 'multiprobe' poyezera kutentha kwa zitsime zingapo nthawi imodzi panthawi yachipongwe. Izi zimatsimikizira kuti makinawo akugwirabe ntchito mkati mwa kulolerana kwapadera kwa wopanga. Kwa ma lab omwe amafunafuna zida zolimba zokhala ndi mbiri yosavuta yokonza, funsani akatswiri apadera Wothandizira zida za PCR atha kuthandizira posankha mitundu yopangidwira ntchito zolemetsa.
Zosintha zamapulogalamu siziyenera kunyalanyazidwa. Opanga nthawi zambiri amatulutsa zosintha za firmware zomwe zimasintha ma aligorivimu owongolera kutentha kapena kuwonjezera zatsopano pazogwiritsa ntchito. Kusunga mapulogalamu amakina amakono kumatsimikizira kuti mukupindula ndi kukhathamiritsa kwaposachedwa pakuwongolera kutentha. Kuonjezera apo, nthawi zonse muzigwiritsa ntchito zipangizo zamakono za PCR (machubu ndi mbale), monga mapulasitiki okhala ndi mipanda yopyapyala amapangidwira kuti athetse kutentha kwachangu kuchokera ku chipika kupita kumalo osakaniza.
Mndandanda wa Kusamalira
Tsiku ndi Tsiku: Pukutani pansi ndikuyang'ana chivindikiro chotenthedwa.
Mlungu uliwonse: Yang'anani zitsime za zinyalala kapena zowonongeka; yeretsani chipikacho.
Mwezi ndi mwezi: Yesetsani kudziyesa nokha ngati zilipo mu pulogalamu.
Chaka ndi chaka: Kutsimikizira kwaukadaulo kwaukadaulo ndi kusanja.
Mapeto
Ukadaulo wamakina a PCR ndiwopambana muukadaulo wolondola komanso chemistry yama cell. Popereka malo olamulidwa kuti achulukitse DNA, zidazi zatsegula zinsinsi za ma genetic code ndikupereka zida zofunikira polimbana ndi miliri yapadziko lonse lapansi, kuchiza khansa, komanso kuthetsa milandu. Pamene tikulowera ku machitidwe ophatikizika komanso a digito, mfundo zazikuluzikulu za njinga zamatenthedwe zimakhalabe zofunikira monga kale.
Kwa ma laboratories ndi zipatala, kusankha ukadaulo wolondola wa PCR ndi liwiro, kulondola, komanso kutsika mtengo. Kaya ndi makina opangira matenthedwe opangira maphunziro kapena njira yeniyeni yeniyeni yodziwira matenda ovuta, kumvetsetsa makina omwe ali kumbuyo kwa makinawo ndi sitepe yoyamba yopezera zotsatira zodalirika komanso zogwira mtima. Tsogolo laukadaulo wa PCR limalonjeza zotsatira zofulumira, kusuntha kwakukulu, komanso kukhudzika kwakukulu, kupitiliza udindo wake monga mwala wapangodya wa kupita patsogolo kwa sayansi.
