Zinthu zomwe zimapangitsa kuti PCR ichitike sizigwira ntchito bwino

Pa nthawi ya PCR reaction, zinthu zina zosokoneza nthawi zambiri zimakumana nazo.
Chifukwa cha mphamvu ya PCR, kuipitsidwa kumaonedwa kuti ndi chimodzi mwa zinthu zofunika kwambiri zomwe zimakhudza zotsatira za PCR ndipo kungapangitse zotsatira zabodza.
Magwero osiyanasiyana omwe amabweretsa zotsatira zabodza. Ngati gawo limodzi kapena angapo ofunikira a PCR osakaniza kapena amplification reaction yokha alepheretsedwa kapena kusokonezedwa, kuyesa matenda kungalepheretsedwe. Izi zingayambitse kuchepa kwa magwiridwe antchito komanso zotsatira zabodza zabodza.
Kuwonjezera pa kuletsa, kutayika kwa umphumphu wa nucleic acid womwe mukufuna kungachitike chifukwa cha kutumiza ndi/kapena kusunga zinthu zisanakonzedwe. Makamaka, kutentha kwambiri kapena kusasunga bwino kungayambitse kuwonongeka kwa maselo ndi nucleic acid. Kukhazikika kwa maselo ndi minofu ndi kuyika parafini ndi zifukwa zodziwika bwino zomwe zimayambitsa kugawikana kwa DNA komanso vuto losatha (onani Zithunzi 1 ndi 2). Pazochitika izi, ngakhale kudzipatula bwino ndi kuyeretsa sikungathandize.

Chithunzi 1 | Zotsatira za kulephera kuyenda bwino kwa DNA
Agarose gel electrophoresis inasonyeza kuti ubwino wa DNA yochotsedwa ku magawo a parafini a matupi umasiyana kwambiri. DNA ya kutalika kosiyana kwa zidutswa zapakati inalipo m'zochotsazo kutengera njira yokhazikitsira. DNA inasungidwa pokhapokha ikakhazikika mu zitsanzo zozizira zachilengedwe komanso mu buffered neutral formalin. Kugwiritsa ntchito formalin ya Bouin yokhala ndi acidic kwambiri kapena yosasinthika, yokhala ndi formalin kunapangitsa kuti DNA itayike kwambiri. Gawo lotsalalo ndi logawanika kwambiri.
Kumanzere, kutalika kwa zidutswa kumafotokozedwa mu ma kilobase pairs (kbp)

Chithunzi 2 | Kutayika kwa umphumphu wa zolinga za nucleic acid
(a) Mpata wa 3′-5′ pa zingwe zonse ziwiri ungapangitse kuti DNA yomwe ikufunidwa isweke. Kupanga kwa DNA kudzachitikabe pa chidutswa chaching'ono. Komabe, ngati malo oyambira annealing akusowa pa chidutswa cha DNA, kukulitsa kwa mzere kokha kumachitika. Pankhani yabwino kwambiri, zidutswazo zitha kudzazana, koma zokolola zidzakhala zochepa komanso zosakwana mulingo wozindikirika.
(b) Kutayika kwa maziko, makamaka chifukwa cha depurination ndi thymidine dimer kupanga, kumabweretsa kuchepa kwa chiwerengero cha ma H-bond ndi kuchepa kwa Tm. Panthawi yotenthetsera yayitali, ma primers amasungunuka kuchoka ku matrix DNA ndipo sadzasungunuka ngakhale pakakhala zovuta zochepa.
(c) Maziko a thymine omwe ali pafupi amapanga TT dimer.
Vuto lina lofala lomwe limapezeka nthawi zambiri pofufuza za mamolekyulu ndi kutulutsidwa kochepa kwa ma nucleic acids omwe amafunikira poyerekeza ndi kuchotsedwa kwa phenol-chloroform. Nthawi zambiri, izi zitha kugwirizanitsidwa ndi zolakwika zabodza. Nthawi yambiri ingapulumutsidwe pophika lysis kapena enzyme digestion ya zinyalala za maselo, koma njira iyi nthawi zambiri imabweretsa kuchepa kwa mphamvu ya PCR chifukwa cha kutulutsidwa kosakwanira kwa nucleic acid.

Kuletsa ntchito ya polymerase panthawi yokulitsa

Kawirikawiri, kuletsa kumagwiritsidwa ntchito ngati lingaliro la chidebe pofotokoza zinthu zonse zomwe zimapangitsa kuti pakhale zotsatira zabwino kwambiri za PCR. M'lingaliro la biochemical, kuletsa kumangokhala pa ntchito ya enzyme, mwachitsanzo, kumachepetsa kapena kuletsa kusintha kwa substrate-product kudzera mu kuyanjana ndi malo ogwirira ntchito a DNA polymerase kapena cofactor yake (monga, Mg2+ ya Taq DNA polymerase).
Zigawo zomwe zili mu chitsanzo kapena ma buffer osiyanasiyana ndi zotulutsa zomwe zili ndi ma reagents zimatha kuletsa mwachindunji enzyme kapena kugwira ma cofactor ake (monga EDTA), motero zimaletsa polymerase ndipo pamapeto pake zimapangitsa kuti zotsatira za PCR zichepe kapena zabodza.
Komabe, kuyanjana kwakukulu pakati pa zigawo za reaction ndi ma nucleic acid okhala ndi cholinga kumatchedwanso 'PCR inhibitors'. Kukhulupirika kwa selo kukasokonekera chifukwa cha kudzipatula ndipo nucleic acid ikatulutsidwa, kuyanjana pakati pa chitsanzo ndi yankho lake lozungulira ndi gawo lolimba kumatha kuchitika. Mwachitsanzo, 'ochotsa zinthu m'thupi' amatha kumangirira DNA imodzi kapena ziwiri kudzera mu kuyanjana kosalumikizana ndi ma covalent ndikusokoneza kudzipatula ndi kuyeretsa mwa kuchepetsa kuchuluka kwa zolinga zomwe pamapeto pake zimafika ku chotengera cha PCR reaction.
Kawirikawiri, zoletsa za PCR zimapezeka m'madzi ambiri amthupi ndi ma reagents omwe amagwiritsidwa ntchito poyesa matenda (urea mu mkodzo, hemoglobin ndi heparin m'magazi), zowonjezera zakudya (zigawo za organic, glycogen, mafuta, Ca2+ ions) ndi zigawo zomwe zili m'chilengedwe (ma phenols, zitsulo zolemera)

Mankhwala oletsa PCR ofala kwambiri amapezeka m'mabakiteriya ndi maselo a eukaryotic, DNA yosakhala yolunjika, ma macromolecule omangira DNA a matrices a minofu ndi zida za labotale monga magolovesi ndi mapulasitiki. Kuyeretsa ma nucleic acid panthawi yochotsa kapena mutachotsa ndiyo njira yabwino kwambiri yochotsera mankhwala oletsa PCR.
Masiku ano, zida zosiyanasiyana zochotsera zinthu zokha zimatha kusintha ma protocol ambiri opangidwa ndi manja, koma kubwezeretsa ndi/kapena kuyeretsa zolinga 100% sikunakwaniritsidwepo. Zoletsa zomwe zingatheke zitha kukhalapobe mu nucleic acids zoyeretsedwa kapena zitha kukhala zitayamba kale kugwira ntchito. Pali njira zosiyanasiyana zochepetsera mphamvu ya zoletsa. Kusankha polymerase yoyenera kungakhale ndi zotsatirapo zazikulu pa ntchito ya zoletsa. Njira zina zotsimikizika zochepetsera kuletsa kwa PCR ndikuwonjezera kuchuluka kwa polymerase kapena kugwiritsa ntchito zowonjezera monga BSA.
Kuletsa kwa PCR reactions kungawonetsedwe pogwiritsa ntchito internal process quality control (IPC).
Muyenera kusamala kwambiri kuti muchotse zinthu zonse zoyeretsera ndi zina zomwe zili mu chipangizo chochotseramo zinthu, monga ethanol, EDTA, CETAB, LiCl, GuSCN, SDS, isopropanol ndi phenol, kuchokera ku nucleic acid isolate powatsuka bwino. Kutengera ndi kuchuluka kwawo, amatha kuyambitsa kapena kuletsa PCR.