Ndiyakholelwa ukuba wonke umntu uyakuhlala edibana neengxaki ezinjalo okanye ezinje xa esenza ukusabela kwePCR, kodwa uninzi lwazo lunokuhlelwa ngokweengxaki ezimbini eziphambili:

Ukwandiswa okuncinci kwethemplethi yemfuza (ukukhulisa);
Ukwandiswa kakhulu kofuzo olungajoliyo.
Ukusebenzisa izongezo yenye yeendlela eziqhelekileyo zokusombulula ezi ngxaki.Ngokuqhelekileyo indima yezongezo inemiba emibini:
isakhiwo sesibiniyemfuza (isakhiwo sesibini);
Nciphisa i-priming engangqalanga.
Namhlanje, umhleli uza kukwazisa ngokufutshane izongezo eziqhelekileyo kwiimpendulo zePCR kunye nemisebenzi yazo.
Izongezo ezinciphisa isakhiwo sesibini
sulfoxide(DMSO)
iisampulu zofuzoenomxholo ophezulu weGC .Nangona kunjalo, i-DMSO ikwanciphisa kakhulu umsebenzi we-Taq polymerase.Ke ngoko, wonke umntu kufuneka alinganise ukufikeleleka kwetemplate kunye nomsebenzi wepolymerase.Umhleli ucebisa ukuba ungazama ukugxilwa okwahlukileyo kwe-DSMO, njengokusuka kwi-2% ukuya kwi-10%, ukufumana ugxininiso olulungele uvavanyo lwakho.
Izicoci ezingezizo i-ionic
Ii-detergents ezingenayo i-ionic, ezifana ne-0.1-1% i-Triton X-100, i-Tween 20 okanye i-NP-40, ngokuqhelekileyo inciphisa isakhiwo sesibini se-DNA.Nangona oku kunokonyusa i-amplification ye-template gene, kuya kubangela inkathazo yokukhulisa okungangqalanga.Ke, ezi zongezo zisebenza kakuhle kwimpendulo ye-PCR enesivuno esiphantsi ngaphandle kobutyobo, kodwa ayilunganga kangako kwiimpendulo ze-PRC ezingacocekanga.Enye inzuzo ye-non-ionic detergents kukunciphisa ungcoliseko lwe-SDS.Ngokuqhelekileyo ngexesha lokukhutshwa kwe-DNA, i-SDS iya kuziswa kwisinyathelo se-PCR, esithintela kakhulu umsebenzi wepolymerase.Ngoko ke, ukongeza i-0.5% ye-Tween-20 okanye i-Tween-40 kwi-reaction inokunciphisa imiphumo emibi ye-SDS.
Betaine_
I-Betaine inokuphucula ukukhulisa i-DNA ngokunciphisa ukubunjwa kwesakhiwo sesibini kwaye ngokuqhelekileyo "imfihlelo" yongezwa kwiikiti ze-PCR zorhwebo.Ukuba ufuna ukusebenzisa i-betaine, kufuneka ubeke i-betaine okanye i-betaine mono-hydrate (i-Betaine okanye i-Betaine mono-hydrate), kodwa kungekhona i-betaine hydrochloride (i-Betaine HCl), ulungelelanise kwi-concentration yokugqibela ye-1-1.7M.I-Betaine inokunceda ukuphucula ubuchwephesha kuba isusa ukuxhomekeka kokwakheka kwesibini kwe-DNA yokunyibilika/i-DNA denaturation.
Izongezo zokunciphisa i-priming engangqalanga
Formamide
IFormamide sisidibaniso sePCR esisetyenziswa ngokuqhelekileyo.Inokudibanisa kunye ne-groove enkulu kunye ne-groove encinci kwi-DNA, ngaloo ndlela inciphisa ukuzinza kwe-master DNA i-helix ephindwe kabini kunye nokunciphisa ukushisa okunyibilikayo kwe-DNA.I-concentration ye-formamide esetyenziswe kwiimvavanyo ze-PCR idla ngokuba yi-1% -5%.
I-Tetramethylammonium chloride( TMAC)
I-Tetramethylammonium chloride inokunyusa ukuchasana kwe-hybridization (i-hybridization specificity) kunye nokwandisa ukushisa okunyibilikayo kwe-DNA.Ke, i-TMAC inokususa i-priming engacacanga kwaye inciphise ukudityaniswa kwe-DNA kunye ne-RNA.Ukuba usebenzisaiiprimers eziwohlokayokwi-PCR reaction, khumbula ukongeza i-TMAC, esetyenziswa ngokuqhelekileyo kwi-concentration ye-15-100mM.
Ezinye izongezo eziqhelekileyo
Ukongeza kwiindidi ezimbini zezongezo ezikhankanywe ngasentla, zininzi izongezo eziqhelekileyo kwiimpendulo ze-PCR, nangona zinemisebenzi eyahlukileyo, nazo zibaluleke kakhulu.
Magnesium ion
I-Magnesium ion yicofactor (cofactor) eyimfuneko yepolymerase, oko kukuthi, ngaphandle kwe-magnesium ion, i-polymerase ayisebenzi.Nangona kunjalo, i-ion ye-magnesium eninzi inokuchaphazela ukusebenza kakuhle kwepolymerase.Ukuxinwa kwee-ion ze-magnesium kwi-reaction ye-PCR nganye iya kwahluka.I-Chelating agents (ezifana ne-EDTA okanye i-citrate), i-concentration ye-dNTP kunye neeprotheni zonke zichaphazela ukuxinwa kwee-ion ze-magnesium.Ngoko ke, ukuba uneengxaki ngovavanyo lwakho lwe-PCR, unokuzama ukutshintsha ukugxilwa kwe-ion ye-magnesium eyahlukeneyo, umzekelo, ukusuka kwi-1.0 ukuya kwi-4.0mM, kunye nekhefu le-0.5-1mM phakathi.
Kuyaphawuleka ukuba imijikelo emininzi yokunyibilika komkhenkce ingakhokelela kuxinzelelo lwengqondo lwesisombululo semagnesium chloride.Ke ngoko, kufuneka uyinyibilikise ngokupheleleyo phambi kokusetyenziswa ngakunye, kwaye uyixube kakuhle phambi kokuba uyisebenzise.
I-albumin ye-serum yenkomo(Bovine albumin, BSA)
Kuvavanyo lwekhemikhali yemolekyuli, i-albumin ye-bovine serum sisongezo esixhaphake kakhulu, ngakumbi kuthintelo lokugaya i-enzyme kunye novavanyo lwe-PCR.Kwiimpendulo ze-PCR, i-BSA iluncedo ekunciphiseni ungcoliseko olufana neekhompawundi ze-phenolic.Kwaye kuthiwa kwakhona kunokunciphisa ukubambelela kwee-reactants eludongeni lombhobho wokuvavanya.Kwimpendulo ye-PCR, ngokuqhelekileyo i-concentration ye-BSA eyongeziweyo inokufikelela kwi-0.8 mg / ml.
 
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