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Establishing method of blue honeysuckle sequence-related amplified polymorphism (SRAP) reaction system

A reaction system and blue fruit technology, applied in the field of molecular biology, can solve problems such as reduction of amplification products, reduction of polymorphic bands, reduction of primer-template binding rate, etc., and achieve the effect of simple operation

Inactive Publication Date: 2019-01-22
NORTHEAST AGRICULTURAL UNIVERSITY
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AI Technical Summary

Problems solved by technology

[0006] The amount of Tag DNA polymerase used in PCR is restricted by factors such as reaction volume, enzyme activity, and heat resistance of the enzyme. The use of high-concentration Tag enzyme not only causes economic waste, but also easily leads to the accumulation of non-specific amplification products. It is diffuse; if the amount of Tag enzyme is too low, the efficiency of new chain synthesis will decrease, resulting in a decrease in the amplification product
[0007] If the primer concentration is low, the binding rate between the primer and the template will be reduced, and the polymorphic bands will be reduced; as the primer concentration increases, the brightness and number of the bands will increase, but the primer concentration should not be too high. The concentration will lead to non-specific amplification and the formation of primer dimers, which will compete with the target sequence for Tag enzyme and dNTPs, affecting the yield of the target sequence

Method used

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  • Establishing method of blue honeysuckle sequence-related amplified polymorphism (SRAP) reaction system

Examples

Experimental program
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Effect test

example 1

[0028] Example 1: (1) The system was optimized by a single factor test. The basic reaction conditions were a total reaction volume of 20 μl, of which 10×PCR buffer 2 μl, MgCl2 2.5 mmol / l, dNTPs 0.15 mmol / l, primer 0.1 μmol / l, template DNA 20ng, Tag DNA polymerase 1.5U, make up the insufficient part with double distilled water, and carry out the reaction.

[0029] (2) On the basis of the basic reaction conditions in step (1), set the concentration of Tag DNA polymerase to 0.5U for reaction. Take 10 μl of the final product of the reaction and add 1 μl of 6× loading buffer to mix, and electrophoresis in 1.8% agarose gel. The electrophoresis buffer was 1×TAE, the electrophoresis electric field strength was 5V / cm, stained with Gofdview, observed, photographed and recorded under the Alphalmager HP gel imaging system. Select the concentration of Tag DNA polymerase with clear and bright bands.

[0030](3) On the basis of the basic reaction conditions in step (1), set the template DN...

example 2

[0035] Example 2: (1) The system was optimized by a single factor test. The basic reaction conditions were a total reaction volume of 20 μl, of which 10×PCR buffer 2 μl, MgCl2 2.5 mmol / l, dNTPs 0.15 mmol / l, primer 0.1 μmol / l, template DNA 20ng, Tag DNA polymerase 1.5U, make up the insufficient part with double distilled water, and carry out the reaction.

[0036] (2) On the basis of the basic reaction conditions in step (1), set the concentration of Tag DNA polymerase to 1.0 U for reaction. Take 10 μl of the final product of the reaction and add 1 μl of 6× loading buffer to mix, and electrophoresis in 1.8% agarose gel. The electrophoresis buffer was 1×TAE, the electrophoresis electric field strength was 5V / cm, stained with Gofdview, observed, photographed and recorded under the Alphalmager HP gel imaging system. Select the concentration of Tag DNA polymerase with clear and bright bands.

[0037] (3) On the basis of the basic reaction conditions in step (1), set the template ...

example 3

[0042] (1) The system was optimized by a single factor test. The basic reaction conditions were a total reaction volume of 20 μl, including 2 μl of 10×PCR buffer, 2.5 mmol / l of MgCl2, 0.15 mmol / l of dNTPs, 0.1 μmol / l of primers, and 20 ng of template DNA , Tag DNA polymerase 1.5U, make up the insufficient part with double distilled water, and carry out the reaction.

[0043] (2) On the basis of the basic reaction conditions in step (1), set the concentration of Tag DNA polymerase to 1.5 U for reaction. Take 10 μl of the final product of the reaction and add 1 μl of 6× loading buffer to mix, and electrophoresis in 1.8% agarose gel. The electrophoresis buffer was 1×TAE, the electrophoresis electric field strength was 5V / cm, stained with Gofdview, observed, photographed and recorded under the Alphalmager HP gel imaging system. Select the concentration of Tag DNA polymerase with clear and bright bands.

[0044] (3) On the basis of the basic reaction conditions in step (1), set t...

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Abstract

The invention belongs to the field of molecular biology, and provides an establishing method of a blue honeysuckle sequence-related amplified polymorphism (SRAP) reaction system. SRAP serves as a novel molecular marker, has the characteristics of being simple in operation, stable and efficient, and is widely applied to researches of map construction, gene mapping and genetic diversity analysis ofdifferent crops; similar to other PCR technologies, the amplified results can be affected by reaction factors such as Tag DNA polymerase, template DNA, dNTPs, Mg<2+> and primers, so that optimizationof the SRAP system is the precondition to obtain a good research result. The blue honeysuckle SRAP reaction system is established, abundant polymorphism DNA can be acquired simply, stably and efficiently, and therefore the establishing method is applied to map construction, gene mapping and genetic diversity analysis and the like of the blue honeysuckle.

Description

Technical field: [0001] The invention belongs to the field of molecular biology, and mainly relates to the establishment of a blue honeysuckle SRAP reaction system. Background technique: [0002] Related sequence amplified polymorphism (sequence-related amplified polymorphism, SRAP), also known as sequence-based amplified polymorphism (sequence-base amplified polymorphism, SBAP), was developed by Dr. Li and Quiros of the Vegetable Crops Department of the University of California in 2001. propose. This labeling technology is also a new labeling system based on simple PCR. It mainly amplifies the open reading frame (ORF) through a unique primer design. The primer consists of a core sequence and 3 selective bases. The core sequence Contains two parts: non-specific stuffer sequence and specific sequence. The SRAP-PCR reaction system uses two primers, a 17-base upstream primer and an 18-base downstream primer. The upstream primers specifically amplify the exons, and the downst...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12Q1/6895
CPCC12Q1/6858C12Q1/6895C12Q2600/156C12Q2531/113C12Q2565/125
Inventor 霍俊伟张妍刘化禹孙丰秦栋
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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