24317 2 × Ezmax® Ultra Universal CloneMix 31201 CRISPR-LFA ssDNA reporter 31202 CRISPR-LFA ssRNA reporter 31203 CRISPR 单系统检测试纸条(FAM/FITC) 32210 T4 DNA Ligase 32217 Recombinant RNase Inhibitor 41101 Tolo SafeRed Nucleic Acid Stain ...
16、优选地,所述ssdna-reporter在5’端fam标记、3’端bhq1标记的单链核苷酸序列或5’端fam标记、3’端biotin标记的单链核苷酸序列。 17、优选地,所述ssdna-reporter为ssdna-reporter-cla或ssdna-reporter-lfa,所述ssdna-reporter-cla的核苷酸序列为5’-fam-ttatt-bhq1-3’; 18、所述ssdna-reporter-lfa的核苷酸...
有ssDNA的反式切割活性(trans-cleavage)。基于Cas12蛋白家族的特异性 切割DNA的这种核酸酶活性,已开发出大量基于CRISPR/Cas12系统的核酸检 测技术,具有很强的推广潜力。 2018年,JenniferADoudna团队开发了DETECTR(DNAendonuclease- targetedCRISPRtransreporter,DNA核酸内切酶靶向CRISPR反式报告分子)平 台,用于快速检测患者...
可选的,所述crispr/cas12a检测体系还包括cas12a蛋白、ssdna报告分子、缓冲液、无核酸酶水;[0020]可选的,ssdna报告分子为5’‑f-ttattatt-q-3’,所述f为荧光素,q为荧光淬灭剂;ssdnalfareporter为5’‑f-ttattatt-b-3’,f为fam,b为生物素。[0021]更可选的,f为6-羧基荧光素,q为荧光淬灭剂bhq1;f为...
25、再或者,用于进行rpa/cas12a荧光检测反应的反应体系包括:sf缓冲液、crrna序列、rna酶抑制剂、ssdna-reporter-cy5或者ssdna-reporter-fam、lbcas12a、rpa扩增产物和depc处理水;具体的是:2μl的sf缓冲液(tris-oac、mgoac、bsa和dtt)、1μl浓度为10μm的crrna序列、1μl酶活力为40 u/μl的rna酶抑制剂、2μ...
(TTTN)序列下游 18-25 nt 的靶 DNA.Casl2a 在 crRNA 的介导下靶向结合 dsDNA, 形成了三元复合物,激活了非特异性 ssDNA 反式裂 解活性[24].基于 Cas12a 这一特殊性质,将 Casl2a 反式切割活性与等温扩增相结合的方法,开发了一 种新 DNA 检测方法—DETECTR(DNA endonuclease targeted CRISPR trans reporter)...
When Cas12’s collateral cleavage activity was triggered by the sensed dsDNA target, an introduced ssDNA quenched fluorescent reporter molecule was cleaved in trans. These two Cas12-based methods used different amplification approaches; in their initial publications, DETECTR employed RPA while HOLMES ...
Compared to E-CRISPR, which uses a conventional linear ssDNA reporter as a sensing interface, hpDNA has an increased accessibility to Cas12a nucleases which dramatically improves electrochemical response change and analytical sensitivity of the method. As low as 30 pM of unamplified target DNA can ...
LbCas12a was preassembled with an HPV16- or HPV18-targeting CRISPR RNA (crRNA) at 37°C for 30 minutes and diluted in 1× binding buffer (20 mM Tris-HCl, pH 7.5, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 5% glycerol, 50 μg/ml heparin) and custom ssDNA-FQ reporter (IDT) (Table 2...
The top peak of the curve shows the freed FAM molecule in cleaved reporter samples, and the bottom peak shows the presence of the uncleaved FAM–biotin reporter. e, Michaelis–Menten plot of LbaCas12a-catalysed ssDNA trans-cleavage upon a representative DNA–crRNA pairing (complementary sequences...