Multiple cross isothermal amplification method implemented by aid of AUDG [Antarctic thermal sensitive uracil-DNA (deoxyribonucleic acid)-glycosylase] coupled with self-avoiding molecular recognition systems
A technology of cross constant temperature amplification and constant temperature amplification, applied in biochemical equipment and methods, analytical materials, recombinant DNA technology, etc., can solve the problems of false positive results, cross contamination, etc., to eliminate false positive results, increase specificity performance, ensuring high efficiency
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0062] Embodiment 1.MCDA amplification
[0063] 1. MCDA reaction principle
[0064] The MCDA reaction system includes 10 primers, which recognize 10 regions of the target sequence, including 2 internal cross primers, namely CP1 and CP2 (Cross Primer, CP), 2 displacement primers (Displacement amplification), namely F1 and F2, and 6 Amplification primers, namely D1, C1, R1, D2, C2 and R2. In order to construct a detectable product, select any one of the 10 primers and label the hapten (FITC, fluorescein) at the 5' end, and the newly labeled primers are named F1*, F2*, CP1*, CP2*, C1* , C2*, D1*, D2*, R1* and R2*. In the present invention, C1* is taken as an example to illustrate the principle of the present invention.
[0065] Under the given constant temperature conditions, the double-stranded DNA in the reaction system is in a dynamic equilibrium state of half-dissociation and half-binding. When any primer performs base pairing extension to the complementary part of the dou...
Embodiment 2
[0076] Embodiment 2.SAMRS-MCDA reaction system
[0077] 1. SAMS-MCDA reaction principle
[0078] The reaction principle of SAMS-MCDA is the same as that of ordinary MCDA reaction. Only the ordinary MCDA primers are modified with SAMRS components, which enhances the specificity of the primers, thereby enhancing the specificity of the method, thus eliminating the problems caused by off-target hybridization and primer dimers. False positive results.
[0079] 2. SAMS-MCDA reaction system
[0080] The concentration of the cross primer SAMRS-CP1 was 40pmol, the concentration of the cross primer SAMRS-CP2 was 40pmol, the concentration of the displacement primers SAMRS-F1 and SAMRS-F2 was 10pmol, and the amplification primers SAMRS-C1*, SAMRS-C2, SAMRS-R1, The concentration of SAMRS-R2, SAMRS-D1 and SAMRS-D2 is 20pmol, 2M Betain, 8mM MgSO 4 , 2.5 μL of 10×Bst DNA polymerase buffer, 1.4 mM dATP, 0.7 mM dTTP, 0.7 mM dUTP, 1.38 mM dCTP, 0.02 mM biotin-14-dCTP, 1.4 mM dGTP, 10 U of cha...
Embodiment 3
[0087] The optimal reaction temperature determination of embodiment 3.SAMRS-MCDA technology
[0088] Under the conditions of the SAMRS-MCDA reaction system, the MTC template and the designed corresponding SAMRS-MCDA primers were added, and the template concentration was 2×10 3 copies / microliter (10pg / microliter). The reaction was carried out under constant temperature conditions (59-66°C), and the results were detected by a real-time turbidimeter, and different dynamic curves were obtained at different temperatures, see Figure 5 . 60-62°C is recommended as the optimum reaction temperature for SAMRS-MCDA primers. In the follow-up verification of the present invention, 61° C. was selected as the constant temperature condition for SAMRS-MCDA amplification.
PUM
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com