Ikhithi yoHlolo lwe-HRM (i-EvaGreen)

Iimbonakalo

■ Isisombululo esiphakamileyo: Yamkela idayi egcwalisiweyo ye-EvaGreen, enesisombululo esiphezulu sokunyibilika kwegophe kwindawo egcwelisiweyo kwaye ekwahlula ukwahluka kwesiseko esinye.
■ Ukucaciswa okuphezulu: Sebenzisa i-hotstart yeDNA polymerase elwa nomzimba ukunciphisa ukukhulisa okungangqalanga kunye nokuphucula ukungangqinelani.
■ uzinzo oluphezulu: Inkqubo ye-Buffer eyenziwe ngononophelo yonyusa uzinzo kwigophe lokunyibilika kunye nokuphucula ukuthembeka kweziphumo.
■ Ukulungiswa kwe-ROX: Idayi ye-ROX ipakishwe yodwa, eguqukayo ngakumbi ukuyisebenzisa kwaye ineziphumo ezichanekileyo.

Ukucaciswa

Uhlobo: I-Antibody eguqulweyo Taq DNA polymerase, EvaGreen.
Izicelo: Ukuchwetheza kwe-SNP eyaziwayo; Uvavanyo lweSNP olungaziwayo; Ukuskena imfuza engaguqukiyo engaziwayo; Uhlalutyo lwe-PCR yeMethylation.

Zonke iimveliso zingenziwa ngokwezifiso kwi-ODM / OEM. Iinkcukacha,nceda ucofe iNkonzo eChongiweyo (i-ODM / i-OEM)

×

I-Genomic DNA yakhutshwa kwi-100 μl yesampulu yegazi lomntu kusetyenziswa i-TIANamp yegazi le-DNA Kit. I-50 ng DNA yayilayishwe ngexesha lokwenyani lokufunyanwa kwe-PCR. Ngokwelayibrari ye-NCBI SNP, i-SNP eyaziwayo ye-FEN1 gene (RS 174538) iyakhethwa kwaye ifunyenwe kusetyenziswa iRoche LightCycle480. Iziphumo zezi zilandelayo:

Iziphumo zibonisa ukuba i-HRM Analysis Kit yeyona ndlela ilungileyo yohlalutyo lwendawo ye-SNP enokuphindeka okuphezulu kokunyibilika kwegophe le-genotype efanayo, ukusonjululwa okucacileyo kwee-genotypes ezahlukeneyo kwaye akukho kugweba gwenxa.

Ulwazi lwe-SNP: …… CGGAAGAACACGTCG [A / G] CAGGAGCAGGCGCCT …… (Alququququency: A = 0.314, G = 0.686)

I-Primer: Yeqhekeza le-bp ye-108 (FEN1_F: CCTCAACGCTCTCACCATTTTG; FEN1_R: GGCACTTCCTTTTCCGGTTGTG)