Zinthu zosokoneza pamachitidwe a PCR

Panthawi ya PCR, zinthu zina zosokoneza nthawi zambiri zimakumana.
Chifukwa cha kukhudzidwa kwakukulu kwa PCR, kuipitsidwa kumatengedwa kuti ndi chimodzi mwa zinthu zofunika kwambiri zomwe zimakhudza zotsatira za PCR ndipo zimatha kutulutsa zotsatira zabodza.
Zovutanso chimodzimodzi ndi magwero osiyanasiyana omwe amatsogolera ku zotsatira zabodza. Ngati gawo limodzi kapena zingapo zofunika za kusakaniza kwa PCR kapena kukulitsa komweko kumaletsedwa kapena kusokonezedwa, kuyesa kwa matenda kungalephereke. Izi zingayambitse kuchepa kwachangu komanso zotsatira zabodza zoipa.
Kuphatikiza pa kuletsa, kutayika kwa chandamale cha nucleic acid umphumphu kumatha kuchitika chifukwa cha kutumiza ndi / kapena kusungirako zisanachitike kukonzekera kwachitsanzo. Makamaka, kutentha kwambiri kapena kusungidwa kosakwanira kungayambitse kuwonongeka kwa maselo ndi nucleic acid. Kukonza ma cell ndi minofu ndi kuyika parafini ndizodziwika bwino zomwe zimayambitsa kugawika kwa DNA komanso vuto lomwe limapitilira (onani Zithunzi 1 ndi 2). Muzochitika izi, ngakhale kudzipatula koyenera komanso kuyeretsedwa sikungathandize.

Chithunzi 1 | Zotsatira za immobilization pa kukhulupirika kwa DNA
Agarose gel electrophoresis adawonetsa kuti mtundu wa DNA wolekanitsidwa ndi magawo a parafini a autopsies amasiyana kwambiri. DNA ya kutalika kwachidutswa kosiyanasiyana inalipo muzotulutsa kutengera njira yokonzera. DNA imasungidwa pokhapokha atayikidwa mu zitsanzo zachisanu zachibadwidwe komanso m'malo otetezedwa a neutral formalin. Kugwiritsa ntchito mankhwala a Bouin a acidic kwambiri kapena osasunthika, okhala ndi formic acid okhala ndi formalin kunapangitsa kuti DNA iwonongeke kwambiri. Chigawo chotsalacho ndi chogawanika kwambiri.
Kumanzere, kutalika kwa zidutswa kumawonetsedwa mu ma kilobase awiriawiri (kbp)

Chithunzi 2 | Kutayika kwa umphumphu wa zolinga za nucleic acid
(a) Mpata wa 3’-5’ pa nsonga zonsezo udzachititsa kuti DNA imene chandamale ifike. kuphatikizika kwa DNA kudzachitikabe pachidutswa chaching’onocho. Komabe, ngati tsamba loyambira likusowa pachidutswa cha DNA, kukulitsa kwa mzere kokha kumachitika. Pazochitika zabwino kwambiri, zidutswazo zikhoza kuyambiranso, koma zokolola zidzakhala zazing'ono komanso zotsika kwambiri.
(b) Kutayika kwa maziko, makamaka chifukwa cha depurination ndi thymidine dimer mapangidwe, kumabweretsa kuchepa kwa chiwerengero cha H-bond ndi kuchepa kwa Tm. Panthawi yotentha yotalikirapo, zoyambira zimasungunuka kuchoka ku DNA ya matrix ndipo sizidzasungunuka ngakhale pansi pazovuta kwambiri.
(c) Maziko oyandikana ndi thymine amapanga TT dimer.
Vuto linanso lodziwika bwino lomwe nthawi zambiri limapezeka pakuwunika kwa ma cell ndi kutulutsa kocheperako kwa chandamale cha nucleic acid poyerekeza ndi kutulutsa kwa phenol-chloroform. Muzochitika zovuta kwambiri, izi zikhoza kugwirizanitsidwa ndi zolakwika zabodza. Nthawi yochuluka imatha kupulumutsidwa ndi kuwira kwa lysis kapena kugaya kwa enzymatic kwa zinyalala zama cell, koma njira iyi nthawi zambiri imabweretsa kutsika kwa PCR chifukwa cha kusakwanira kwa nucleic acid.

Kuletsa ntchito ya polymerase panthawi yokulitsa

Nthawi zambiri, kuletsa kumagwiritsidwa ntchito ngati lingaliro la chidebe kufotokozera zinthu zonse zomwe zimatsogolera ku zotsatira za PCR. M'lingaliro lokhazikika lazachilengedwe, kuletsa kumangokhala ndi ntchito ya enzyme, mwachitsanzo, kumachepetsa kapena kuletsa kutembenuka kwazinthu zapansi panthaka polumikizana ndi malo a DNA polymerase kapena cofactor yake (mwachitsanzo, Mg2 + ya Taq DNA polymerase).
Zomwe zili mu zitsanzo kapena ma buffers osiyanasiyana ndi zowonjezera zomwe zimakhala ndi ma reagents zimatha kuletsa puloteni kapena kutsekereza ma cofactors ake (mwachitsanzo EDTA), potero amaletsa ma polymerase ndikupangitsa kuti pakhale zotsatira zoyipa kapena zabodza za PCR.
Komabe, kuyanjana kochuluka pakati pa zigawo za reaction ndi ma nucleic acid okhala ndi chandamale kumatchedwanso 'PCR inhibitors'. Umphumphu wa selo ukasokonezedwa ndi kudzipatula ndipo nucleic acid imatulutsidwa, kuyanjana pakati pa chitsanzo ndi yankho lake lozungulira ndi gawo lolimba likhoza kuchitika. Mwachitsanzo, 'scavengers' amatha kumanga DNA imodzi kapena iwiri kudzera muzochita zosagwirizana ndi kusokoneza kudzipatula ndi kuyeretsa pochepetsa chiwerengero cha mipherezero yomwe pamapeto pake imafika ku PCR reaction chotengera.
Nthawi zambiri, PCR inhibitors amapezeka m'madzi ambiri am'thupi ndi ma reagents omwe amagwiritsidwa ntchito poyezetsa matenda (urea mumkodzo, hemoglobin ndi heparin m'magazi), zowonjezera zakudya (zigawo za organic, glycogen, mafuta, Ca2+ ions) ndi zigawo za chilengedwe (phenols). , heavy metal)

Ma PCR inhibitors ochulukirachulukira amapezeka m'mabakiteriya ndi ma cell a eukaryotic, DNA osatsata, ma macromolecules omanga DNA a matrices a minofu ndi zida za labotale monga magolovesi ndi mapulasitiki. Kuyeretsedwa kwa ma nucleic acid mkati kapena pambuyo pochotsa ndi njira yabwino yochotsera PCR inhibitors.
Masiku ano, zida zosiyanasiyana zodzipangira zokha zimatha kusintha ma protocol ambiri amanja, koma kuchira kwa 100% ndi / kapena kuyeretsedwa kwa zolinga sikunakwaniritsidwe. Ma inhibitors omwe angathe kukhalapobe mu ma nucleic acid oyeretsedwa kapena ayamba kale kugwira ntchito. Pali njira zosiyanasiyana zochepetsera zotsatira za inhibitors. Kusankhidwa kwa polymerase yoyenera kumatha kukhudza kwambiri ntchito ya inhibitor. Njira zina zotsimikiziridwa zochepetsera kuletsa kwa PCR ndikuwonjezera ndende ya polymerase kapena kugwiritsa ntchito zowonjezera monga BSA.
Kuletsa machitidwe a PCR kungasonyezedwe pogwiritsa ntchito Internal process Quality Control (IPC).
Chisamaliro chiyenera kutengedwa kuti muchotse ma reagents ndi njira zina muzitsulo zochotsamo, monga ethanol, EDTA, CETAB, LiCl, GuSCN, SDS, isopropanol ndi phenol, kuchokera ku nucleic acid kudzipatula ndi sitepe yosamba bwino. Kutengera ndende yawo, amatha kuyambitsa kapena kuletsa PCR.