隐–荧光DNA扩增  

Amplification of Hidden Fluorescent DNA

作　　者：刘自厚 刘亮伟[2,3] 

机构地区：[1]河南农业大学国际教育学院,河南 郑州 [2]河南农业大学生命科学学院,河南 郑州 [3]农业部农业酶工程重点实验室,河南 郑州

出　　处：《微生物前沿》2023年第1期45-54,共10页Advances in Microbiology

摘　　要：目的:隐–荧光(Hf: hidden fluorescent) DNA通过带荧光素及10 nt之内淬灭基团的Hf引物扩增。方法:本研究设计Hf_Pf引物带有dT12_Fam荧光素-dT16_BHQ1淬灭基团,扩增5851 bp HfDNA pET28a-xylanase,探讨Hf_Pf引物Tm (退火温度)适合计算器、HfDNA扩增条件、T5 DNA酶(T5exo)切割产物检测、及T5exo酶切动力学。结果:Hf_Pf Tm计算器为Oligo、IDT,经非等量引物PCR优化扩增HfDNA,激光共聚焦定性检测HfDNA酶切产物Fam荧光、酶标仪定量检测荧光值19,683 a.u。根据HfDNA浓度–荧光值方程,T5exo酶促动力学参数Km 0.1 nM,弥补解离常数KD不足。结论:本研究扩增隐–荧光HfDNA,提供了DNA酶学研究新材料。Objective: Hidden fluorescent (Hf) DNA substrates can be amplified by Hf primer that has a fluorescein and a quencher within 10 nt. Method: The study designed a 20 nt Hf_Pf having the dT12_Fam and the dT16_BHQ1, amplified a 5851 bp HfDNA pET28a-xylanase, determined suitable analyzers for calculating primer Hf_Pf Tm (melting temperature), optimized PCR to amplify the HfDNA substrates, assayed HfDNA-T5exo digestion product fluorescence. Result: Suitable analyzers were Oligo and IDT, and un-equal PCR was optimized to amplify the HfDNA substrates. Instead of the HfDNA substrates, the HfDNA-T5exo digestion products exhibited dT12_Fam fluorescence in quality under a laser scanning confocal microcopy and a 19,683 a.u fluorescence intensity in quantity under a microreader. According to HfDNA concentration-fluorescence intensity function, T5exo kinetics was determined to have a 0.1 nM Km, fulfilling in-adequate of dissociation constant KD. Conclusion: The study amplified HfDNA substrates, provided a new material for assaying DNase enzyme properties.

关 键 词：隐–荧光DNA 扩增 荧光检测 

分 类 号：R73[医药卫生—肿瘤]