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Nucleic acid fragment detection system and its application in nucleic acid analysis and related enzyme study

A nucleic acid fragment and detection system technology, applied in the field of gene analysis and related enzyme analysis technology

Inactive Publication Date: 2004-12-22
HUNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention develops a nucleic acid fragment detection system, which is applied in nucleic acid analysis and protein-nucleic acid interaction research to solve the deficiencies of existing gene analysis techniques and to promote the development of related research

Method used

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  • Nucleic acid fragment detection system and its application in nucleic acid analysis and related enzyme study
  • Nucleic acid fragment detection system and its application in nucleic acid analysis and related enzyme study
  • Nucleic acid fragment detection system and its application in nucleic acid analysis and related enzyme study

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Embodiment one (nucleic acid analysis)

[0026] In 120uL ligation system buffer (30mM Tris-HCl (pH8.0), 2.5mM MnCl 2 , 10mM (NH 4 ) 2 SO 4 , 40mM LiCl, 1.2mM EDTA, 0.1mM NAD+, 0.05%BSA), add 400nM molecular beacon 5'-(TAMRA)-CGTTGA TGG TTC CAC TTC TCG TGC GT TCAACG-(DABCYL)-3', 1.0 Unit E. coli DNA ligase, and 300nM a nucleic acid fragment 5'-AGT GGA ACC-3' half-paired with the molecular beacon, incubated at 37°C, while monitoring the fluorescence intensity with a Fluorolog Tau-3 (Jobin Yvon) fluorometer, The excitation wavelength is 555nm and the emission wavelength is 580nm. After the fluorescence is stabilized, add 300nM of another nucleic acid fragment 5'-CGC ACG AGA-3'. At this time, the fluorescence intensity will increase significantly, as shown in Figure 5 (1) shown. Or use three nucleic acid strands instead of two nucleic acid strands: 5'-CG CACG-3', 5'-AG AAG T-3', 5'-GG AAC C-3', as described above, only in the ligation system buffer First add one or t...

Embodiment 2

[0052] Embodiment 2 (real-time monitoring of nucleic acid ligation process)

[0053] Add 400nM molecular beacon 5'-(TAMRA)-CGTTGA TGG TTC CAC TTC TCG TGC GTTCAACG- (DABCYL)-3', 300nM nucleic acid fragment 5'-AGT GGA ACC-3' and 300nM another nucleic acid fragment 5'-CGCACG AGA-3', incubated at 37°C while monitoring the fluorescence intensity (fluorescence detection conditions are the same as the implementation Example 1). After the fluorescence is stable, add a certain amount of T4 DNA ligase, and record the change of fluorescence intensity at the same time, such as Image 6 shown.

[0054] Image 6 (Left) is the real-time monitoring curve of nucleic acid ligation catalyzed by different concentrations of ligase. It can be seen that as the concentration of ligase increases, the initial speed of ligation increases. The initial speed of ligation is plotted against the concentration of ligase. Image 6 (Right), as can be seen from the figure, there is a direct proportional rela...

Embodiment 3

[0055] Embodiment three (real-time monitoring of nucleic acid phosphorylation process)

[0056] Add 400nM molecular beacon 5'-(TAMRA)-CGTTGA TGG TTC CAC TTC TCG TGC GTTCAACG to 120uL phosphorylation system buffer (66mM Tris-HCl (pH8.0), 6.6mM MgCl2, 0.1mM ATP, 10mM DTT) -(DABCYL)-3', 300 nM nucleic acid fragment 5'-AGT GGA ACC-3' and 300 nM another nucleic acid fragment 5'-CGCACG AGA-3' (wherein 5'-AGT GGA ACC-3' has no 5' terminal phosphate group), 1.4 Units of T4 DNA ligase were incubated at 37°C, and the fluorescence intensity was monitored with the same parameters as in Example 1. After the fluorescence was stabilized, different concentrations of T4 polynucleotide kinase were added to obtain the real-time scanning curve of the fluorescence intensity as shown in Figure 7 (left) shown.

[0057] Figure 7 (Left) is the real-time monitoring curve of nucleic acid phosphorylation catalyzed by different concentrations of polynucleotide kinase. It can be seen that with the inc...

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Abstract

A nucleic acid fragment detecting system is composed of molecular beacon's nucleic acid probe, nucleic acid fragment, nucleic acid joining enzyme and buffer solution. Its application in analyzing and researching nucleic acid includes detecting and analyzing nucleic acid fragment, monitoring the phosphorylation procedure of nucleic acid, analyzing polynucleotide kinase activity, monitoring and analyzing nucleic acid joining process, detecting nucleic acid joining enzyme, etc.

Description

technical field [0001] The invention relates to a detection method in the field of molecular biology, in particular to a gene analysis and related enzyme analysis technology. Background technique [0002] In recent years, the research of life science has entered the molecular level such as genes and proteins, and human beings have greatly deepened their understanding of life, which provides a very beneficial opportunity for controlling human diseases and improving the quality of life. For example, the diagnosis, treatment and even prediction of genetic diseases, as well as the research on the occurrence and development mechanism of malignant tumors, have become hot spots in medical and biological research. [0003] Diagnosis and analysis of diseases at the genetic level requires genetic testing techniques with high sensitivity and reliability, especially the analysis of point mutations. Currently, the existing techniques are difficult to provide satisfactory results in both ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 王柯敏唐志文谭蔚泓
Owner HUNAN UNIV
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