Ukuxilongwa kweemolekyuli, iteknoloji yePCR esetyenziswa ngokuqhelekileyo kunye nomgaqo

I-PCR, i-polymerase chain reaction, ebhekisela ekongezweni kwe-dNTP, i-Mg2 +, i-elongation factor kunye ne-amplification yokuphucula izinto kwinkqubo phantsi kwe-catalysis ye-DNA polymerase, usebenzisa i-DNA yomzali njenge template kunye ne-primers ethile njengendawo yokuqala yokwandisa, Ngamanyathelo e-denaturation, annealing, extension, njl., inkqubo ye-vitro yokuphindaphinda intombi ye-strand DNA ehambelana nomzali we-strand template DNA inokukhawuleza kwaye ikhulise ngokuthe ngqo nayiphi na i-DNA ekujoliswe kuyo kwi-vitro.

1. Qala i-PCR eshushu

Ixesha lokuqala lokukhulisa kwi-PCR eqhelekileyo ayikuko ukubeka umatshini we-PCR kumatshini we-PCR, emva koko inkqubo iqalise ukukhulisa.Xa uqwalaselo lwenkqubo lugqityiwe, i-amplification iqala, enokubangela ukukhulisa okungangqalanga, kunye ne-PCR eshushu-yokuqala inokusombulula le ngxaki.

Yintoni iPCR yokuqala eshushu?Emva kokuba inkqubo yokusabela ilungisiwe, i-enzyme modifier ikhutshwa kwiqondo lokushisa eliphezulu (ngokuqhelekileyo lingaphezulu kwe-90 ° C) ngexesha lokuqala lokufudumala kwenqanaba lokuphendula okanye "isiqalo esishushu", ukwenzela ukuba i-polymerase ye-DNA isebenze.Ixesha elichanekileyo lokuvula kunye nobushushu buxhomekeke kubume be-polymerase ye-DNA kunye ne-hot-start modifier.Le ndlela isebenzisa ikakhulu iziguquli ezifana ne-antibodies, i-affinity ligands, okanye i-chemical modifiers ukuvimbela umsebenzi we-DNA polymerase.Ekubeni umsebenzi we-DNA polymerase uthintelwe kwiqondo lokushisa, itekhnoloji yokuqala eshushu ibonelela ngokulula kakhulu ukulungiselela iinkqubo ezininzi zokusabela kwe-PCR kwiqondo lobushushu begumbi ngaphandle kokuncama ubume be-PCR.

2. I-RT-PCR

I-RT-PCR (Reverse transcription PCR) bubuchule bokulinga ukukhutshelwa umva ukusuka ku-mRNA ukuya kwi-cDNA kwaye uyisebenzise njengethempleyithi yokukhulisa.Inkqubo yovavanyo kukukhupha i-RNA epheleleyo kwizicubu okanye iiseli kuqala, sebenzisa i-Oligo (dT) njenge-primer, sebenzisa i-reverse transcriptase ukuze udibanise i-cDNA, kwaye emva koko usebenzise i-cDNA njengethemplate yokukhulisa i-PCR ukufumana i-gene ekujoliswe kuyo okanye ukufumana i-gene expression.

3. Fluorescent quantitative PCR

I-Fluorescent quantitative PCR (Ixesha lokwenyani le-PCR yobungakanani,I-RT-qPCR) ibhekisa kwindlela yokongeza amaqela e-fluorescent kwinkqubo yokusabela kwe-PCR, kusetyenziswa ukuqokelelana kwemiqondiso ye-fluorescent ukujonga yonke inkqubo ye-PCR ngexesha langempela, kwaye ekugqibeleni usebenzisa ijika eliqhelekileyo ukuhlalutya ngokobungakanani bethemplethi.Iindlela eziqhele ukusetyenziswa qPCR ziquka SYBR Green I kunye TaqMan.

4. I-PCR esecaleni

I-Nested PCR ibhekisa kusetyenziso lweeseti ezimbini zeeprimer zePCR kwimijikelo emibini yokwandiswa kwe-PCR, kwaye imveliso yokukhulisa umjikelo wesibini liqhekeza lemfuza ekujoliswe kulo.

Ukuba ukungahambelani kwesibini sokuqala (ii-primers zangaphandle) kubangela ukuba imveliso engangqalanga yandiswe, ukuba kunokwenzeka ukuba kwaloo mmandla ungacaciswanga uqatshelwe sisibini sesibini seeprimers kwaye ukuqhubeka nokukhulisa kuncinci kakhulu, ngoko ke ukukhulisa ngesibini sokuqala, ukucaciswa kwe-PCR kuphuculwe.Enye inzuzo yokwenza imijikelo emibini ye-PCR kukuba inceda ukukhulisa imveliso eyaneleyo ukusuka kwi-DNA yokuqala.

5. Ukuchukumisa iPCR

I-Touchdown PCR yindlela yokuphucula i-spectity ye-PCR reaction ngokulungelelanisa iiparamitha zomjikelezo we-PCR.

Kwi-PCR ye-touchdown, iqondo lobushushu le-annealing lemijikelo embalwa yokuqala lisetwa iidigri ezimbalwa ngaphezu kobushushu obuphezulu be-annealing (Tm) yeeprimers.Ubushushu obuphezulu be-annealing bunokunciphisa ngokufanelekileyo ukukhulisa okungangqalanga, kodwa kwangaxeshanye, ubushushu obuphezulu be-annealing buya kwenza kube mandundu ukwahlulwa kwe-primers kunye nolandelelwano olujoliswe kuyo, okukhokelela ekunciphiseni isivuno sePCR.Ngoko ke, kwimijikelezo embalwa yokuqala, ukushisa kwe-annealing ngokuqhelekileyo kumiselwe ukunciphisa nge-1 ° C ngomjikelezo ngamnye ukunyusa umxholo wejene elijoliswe kuyo kwinkqubo.Xa ubushushu be-annealing buthotywa ukuya kobona bushushu buphezulu, ubushushu be-annealing bugcinwa kwimijikelo eseleyo.

6. I-PCR ethe ngqo

I-PCR ethe ngqo ibhekisela ekwandiseni i-DNA ekujoliswe kuyo ngokuthe ngqo kwisampulu ngaphandle kwesidingo se-nucleic acid yodwa kunye nokuhlanjululwa.

Kukho iindidi ezimbini zePCR ngqo:

indlela ethe ngqo: thatha isampuli encinci kwaye uyifake ngokuthe ngqo kwi-PCR Master Mix yokuchonga i-PCR;

indlela yokuqhekeka: emva kokusampula isampuli, yongeza kwi-lysate, i-lyse ukukhulula i-genome, thatha inani elincinci le-lysed supernatant kwaye uyifake kwi-PCR Master Mix, yenza ukuchongwa kwe-PCR.Le ndlela yenza lula ukuhamba komsebenzi wokulinga, ukunciphisa izandla ngexesha, kwaye igweme ilahleko ye-DNA ngexesha lokucoca.

7. I-SOE PCR

I-Gene splicing ngokwandisa i-PCR (i-SOE PCR) isebenzisa ii-primers ezineziphelo ezihambelanayo ukwenza iimveliso ze-PCR zenze amatyathanga ahlanganayo, ukuze kwimpendulo yokukhulisa i-amplification elandelayo, ngolwandiso lwamatyathanga ahlanganayo, imithombo eyahlukeneyo ye-A apho amaqhekeza akhuselweyo adityaniswa khona. kwaye idibene.Le teknoloji okwangoku inezikhokelo ezimbini eziphambili zesicelo: ukwakhiwa kweejene ze-fusion;gene site-directed uguquko.

8. IPCR

I-PCR eguqukileyo (IPCR) isebenzisa iiprimers ezixhasayo ezibuyela umva ukukhulisa amaqhekeza e-DNA ngaphandle kwee-primers ezimbini, kwaye ikhulise ulandelelwano olungaziwayo kumacala omabini eqhekeza elaziwayo le-DNA.

I-IPCR yayiyilelwe ekuqaleni ukumisela ulandelelwano lwemimandla engaziwayo ekufutshane, kwaye isetyenziswa kakhulu ukufunda ulandelelwano lwabakhuthazi bemfuza;i-oncogenic chromosomal rearrangements, ezifana ne-gene fusion, ukutshintshwa kunye nokutshintshwa;kunye nokudityaniswa kofuzo lwentsholongwane, zikwasetyenziswa ngokuqhelekileyo ngoku Kwi-site-directed mutagenesis, khuphela iplasmid kunye noguquko olufunwayo.

9. dPCR

I-Digital PCR (dPCR) bubuchule bomlinganiselo opheleleyo we-nucleic acid molecules.

Okwangoku, kukho iindlela ezintathu zokulinganisa i-nucleic acid.Ifotoometry isekelwe ekufunxeni kweemolekyuli ze-nucleic acid;I-real-time fluorescent quantitative PCR (i-PCR yeXesha langempela) isekelwe kwixabiso le-Ct, kwaye ixabiso le-Ct libhekisela kwinombolo yomjikelezo ehambelana nexabiso le-fluorescence elinokubonwa;I-PCR yedijithali yeyona teknoloji yamva nje yokuBantu esekwe kwindlela enye ye-PCR yokubala ubungakanani be-nucleic acid yindlela yobungakanani obupheleleyo.