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Method of increasing specificity of nucleic acid hybridization using zwitterionic compound

A nucleic acid hybridization and zwitterion technology, which is applied in the field of improving nucleic acid hybridization specificity with zwitterionic compounds, and can solve the problem of low hybridization specificity

Inactive Publication Date: 2010-11-17
SAMSUNG ELECTRONICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, if the hybridization specificity is low during annealing, in addition to many target bands, many bands will appear on the electrophoresis gel, causing problems in the analysis of multiplex PCR results

Method used

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  • Method of increasing specificity of nucleic acid hybridization using zwitterionic compound
  • Method of increasing specificity of nucleic acid hybridization using zwitterionic compound
  • Method of increasing specificity of nucleic acid hybridization using zwitterionic compound

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Influence of CAPS on Nucleic Acid Hybridization Specificity in Multiplex PCR

[0052] Genomic DNA (gDNA) was isolated from Haemophilus influenza (from Samsung Medical Center), a major pathogen of respiratory infection, and as a template for multiplex PCR, the isolated gDNA was used in an amount of 0.2 ng or 1 ng. 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), which is a zwitterionic compound according to an embodiment of the present invention, was used at a concentration of 0.5% or 1% to perform multiplex PCR. With GeneAmp PCR system 9700 (ABI), PCR mixture (template gDNA 0.2ng or 1ng, CAPS 0.5% or 1%, 1×Taq Pol buffer solution, dNTP mixture each 200 μ M, PCR primer each 400 nM (SEQ ID NO: 1 to SEQ ID NO: 10), and Taq Pol 5 units), at 95°C for 1 minute to completely denature the DNA, and then carry out 25 PCR cycles on the PCR mixture (95°C for 5 seconds, 62°C for 13 seconds, 72 °C for 15 seconds), and a final extension at 72°C for 1 minute, thus perform...

Embodiment 2

[0057] Example 2: Influence of CAPSO and CHES on nucleic acid hybridization specificity in multiplex PCR

[0058] To study the nucleic acid hybridization specificity of different types of zwitterionic compounds in multiplex PCR, CAPSO and CHES were also used. The same experiment was performed in the same manner as in Example 1, except that CAPSO and CHES were added to the multiplex PCR at concentrations of 0.2%, 0.4%, 0.8% and 1.6%, respectively, and 1 ng of Haemophilus influenzae gDNA was used template.

[0059] image 3 A photograph showing the results of electrophoretic analysis of PCR products obtained when CAPSO was added to the multiplex PCR, showing a decrease in the yield of non-specific hybridization products. see image 3 , Multiplex PCR without CAPSO addition was performed on the control. Depend on image 3 It can be seen that the intensity of non-specific hybridization product bands was significantly reduced with the addition of CAPSO compared to the case with...

Embodiment 3

[0060] Example 3: Effect of Types of Zwitterionic Compounds on Nucleic Acid Hybridization Specificity in Multiplex PCR

[0061] Study of nucleic acid hybridization specificity of different classes of zwitterionic compounds in multiplex PCR using betaine. The same test was carried out in the same manner as in Example 1, except that 2.5 μl (0.25×), 5 μl (0.5×), 10 μl (1 ×), and 15 μl (1.5×) of betaine were added to the multiplex PCR, and the Haemophilus influenzae gDNA template was used in amounts of 1 ng, 0.1 ng and 0.01 ng, respectively.

[0062] Figure 5 A photograph showing the results of electrophoretic analysis of PCR products obtained when betaine was added to multiplex PCR, showing a decrease in the yield of non-specific hybridization products. see Figure 5 , the control implements multiplex PCR without adding betaine, and the numbers "1", "2" and "3" refer to the amount of template gDNA used for PCR, for example, 1ng, 0.1ng and 0.01ng, respectively. Depend on Fi...

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Abstract

A method of increasing the specificity of nucleic acid hybridization, comprising hybridizing nucleic acid in a solution containing a zwitterionic compound selected from the group consisting of 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), 3-(cyclohexylamino)-2-hydroxy-1-propane sulfonate (CAPSO), and 2-(cyclohexylamino)ethane sulfonate (CHES) is provided. The method allows a reduction in the yield of non-specific hybridization products in multiplex PCRs while maintaining the yield of target hybridization products, thereby increasing the specificity of nucleic acid hybridization. Thus, the method can be effectively used for identification of specific pathogens, diagnosis of genetic diseases, analysis of gene sequences, and so on.

Description

[0001] This application is a divisional application of Chinese application 200610168642.8. The filing date of the parent application is December 20, 2006. This divisional application adopts the same invention title as the parent application. technical field [0002] The present invention relates to methods for increasing the specificity of nucleic acid hybridization using specific zwitterionic compounds. Background technique [0003] Hybridization specificity (also referred to as hybridization stringency) refers to the degree to which complementary nucleic acids form specific hybrids with each other when two complementary and non-complementary nucleic acids are hybridized. In general, attempts have been made to obtain higher hybridization specificity by adjusting the salt concentration in the buffer and the reaction temperature, or by adding additives such as Denhart's solution, or adding non-specific DNA or RNA to induce hybridization competition. [0004] Polymerase chain ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12P19/34
CPCC12Q1/6832C12Q1/6848C12Q2527/125C12Q2537/143
Inventor 李贞男白象铉
Owner SAMSUNG ELECTRONICS CO LTD
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