%0 Journal Article
%T 隐–荧光DNA扩增<br>Amplification of Hidden Fluorescent DNA
%A 刘自厚
%A 刘亮伟
%J Advances in Microbiology
%P 45-54
%@ 2327-0829
%D 2023
%I Hans Publishing
%R 10.12677/AMB.2023.121006
%X 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。<br />
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.
%K 隐–荧光DNA，扩增，荧光检测<br>Hidden Fluorescent DNA
%K Amplification
%K Fluorescence Assay
%U http://www.hanspub.org/journal/PaperInformation.aspx?PaperID=63439