使用改良型分子信标设计对滚环扩增进行实时监测。
Real-time monitoring of rolling-circle amplification using a modified molecular beacon design.
作者信息
Nilsson Mats, Gullberg Mats, Dahl Fredrik, Szuhai Karoly, Raap Anton K
机构信息
Department of Molecular Cell Biology, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands.
出版信息
Nucleic Acids Res. 2002 Jul 15;30(14):e66. doi: 10.1093/nar/gnf065.
Abstract
We describe a method to monitor rolling-circle replication of circular oligonucleotides in dual-color and in real-time using molecular beacons. The method can be used to study the kinetics of the polymerization reaction and to amplify and quantify circularized oligonucleotide probes in a rolling-circle amplification (RCA) reaction. Modified molecular beacons were made of 2'-O-Me-RNA to prevent 3' exonucleolytic degradation by the polymerase used. Moreover, the complement of one of the stem sequences of the molecular beacon was included in the RCA products to avoid fluorescence quenching due to inter-molecular hybridization of neighboring molecular beacons hybridizing to the concatemeric polymerization product. The method allows highly accurate quantification of circularized DNA over a broad concentration range by relating the signal from the test DNA circle to an internal reference DNA circle reporting in a distinct fluorescence color.
摘要
我们描述了一种使用分子信标以双色和实时方式监测环状寡核苷酸滚环复制的方法。该方法可用于研究聚合反应的动力学，并在滚环扩增（RCA）反应中扩增和定量环化的寡核苷酸探针。修饰的分子信标由2'-O-甲基核糖核酸制成，以防止所用聚合酶的3'核酸外切酶降解。此外，RCA产物中包含分子信标一个茎序列的互补序列，以避免相邻分子信标与串联聚合产物杂交导致的分子间杂交引起的荧光猝灭。该方法通过将测试DNA环的信号与以不同荧光颜色报告的内部参考DNA环相关联，能够在很宽的浓度范围内对环化DNA进行高度准确的定量。
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本文引用的文献
1
Rolling Circle DNA Synthesis: Small Circular Oligonucleotides as Efficient Templates for DNA Polymerases.滚环DNA合成：小环状寡核苷酸作为DNA聚合酶的高效模板
J Am Chem Soc. 1996 Feb 21;118(7):1587-1594. doi: 10.1021/ja952786k.
2
Signal amplification by rolling circle amplification on DNA microarrays.基于DNA微阵列的滚环扩增信号放大。
Nucleic Acids Res. 2001 Dec 1;29(23):E118. doi: 10.1093/nar/29.23.e118.
3
Detection of DNA point mutations and mRNA expression levels by rolling circle amplification in individual cells.通过滚环扩增检测单个细胞中的DNA点突变和mRNA表达水平。
Proc Natl Acad Sci U S A. 2001 Dec 4;98(25):14238-43. doi: 10.1073/pnas.251383598. Epub 2001 Nov 27.
4
L-RCA (ligation-rolling circle amplification): a general method for genotyping of single nucleotide polymorphisms (SNPs).L-RCA（连接-滚环扩增）：一种用于单核苷酸多态性（SNP）基因分型的通用方法。
Nucleic Acids Res. 2001 Nov 15;29(22):E116. doi: 10.1093/nar/29.22.e116.
5
High-throughput genotyping of single nucleotide polymorphisms with rolling circle amplification.采用滚环扩增技术对单核苷酸多态性进行高通量基因分型
BMC Genomics. 2001;2:4. doi: 10.1186/1471-2164-2-4. Epub 2001 Aug 1.
6
An invasive cleavage assay for direct quantitation of specific RNAs.一种用于直接定量特定RNA的侵入性切割分析方法。
Nat Biotechnol. 2001 Jul;19(7):673-6. doi: 10.1038/90290.
7
Visualization of oligonucleotide probes and point mutations in interphase nuclei and DNA fibers using rolling circle DNA amplification.利用滚环DNA扩增技术对间期细胞核和DNA纤维中的寡核苷酸探针及点突变进行可视化分析。
Proc Natl Acad Sci U S A. 2001 Mar 27;98(7):3940-5. doi: 10.1073/pnas.061026198.
8
Fluorescent quenching-based quantitative detection of specific DNA/RNA using a BODIPY((R)) FL-labeled probe or primer.使用硼二吡咯（BODIPY）FL标记的探针或引物基于荧光猝灭的特定DNA/RNA定量检测。
Nucleic Acids Res. 2001 Mar 15;29(6):E34. doi: 10.1093/nar/29.6.e34.
9
RNA-templated DNA ligation for transcript analysis.用于转录本分析的RNA模板DNA连接反应。
Nucleic Acids Res. 2001 Jan 15;29(2):578-81. doi: 10.1093/nar/29.2.578.
10
Immunoassays with rolling circle DNA amplification: a versatile platform for ultrasensitive antigen detection.基于滚环DNA扩增的免疫分析：用于超灵敏抗原检测的通用平台。
Proc Natl Acad Sci U S A. 2000 Aug 29;97(18):10113-9. doi: 10.1073/pnas.170237197.
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