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Fluorescent dUTP-based method for automatically detecting SSR molecular marker

An automatic detection and molecular labeling technology, applied in the field of molecular biology, can solve problems such as affecting the accuracy of spectral band interpretation, weak fluorescence signal, etc., and achieve huge social and economic benefits, large fluorescence signal intensity, and enhanced accuracy.

Inactive Publication Date: 2010-12-01
RES INST OF TROPICAL FORESTRY CHINESE ACAD OF FORESTRY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the fluorescence signal in the above literature is weak, which greatly affects the accuracy of band interpretation.

Method used

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  • Fluorescent dUTP-based method for automatically detecting SSR molecular marker
  • Fluorescent dUTP-based method for automatically detecting SSR molecular marker
  • Fluorescent dUTP-based method for automatically detecting SSR molecular marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] A method for detecting eucalyptus SSR marker Embra189 based on fluorescent dUTP, comprising the following steps:

[0025] (1) Prepare PCR reaction system: take 1.0 μL 10×buffer, dNTP 25 μM, MgCl 2 2.0mM, forward primer (5'-CGTGCTTTTGAGGCTCT-3') 0.5μM, backward primer (5'-GATTGAGGATGAGTGGTC-3') 0.5μM, Taq enzyme 1 unit (Shanghai Shenneng Gaming Company), fluorescent dUTP 10pmol ( Fermentas, Canada) and 5 ng of genomic DNA from an individual Eucalyptus urophylla (E.urophylla) individual, and add ultrapure water to make up to 10 μL;

[0026] (2) Drop-down PCR: 94°C for 4min; 20 cycles: 94°C for 30s, 70 to 60°C for 30s and each cycle lowered by 0.5°C, 72°C for 1min; another 26 cycles: 94°C for 30s, 60°C for 30s, 72 °C for 1 min; finally, 72 °C for 10 min to obtain the PCR product;

[0027] (3) Take 1.0 μL of the PCR product and add 9.34 μL of ultra-pure formamide, 0.16 μL of molecular weight internal standard GeneScan 500LIZ (USA AB Company), 95 °C for 5 minutes, place it...

Embodiment 2

[0029]A fluorescent dUTP-based method for the detection of the Eucalyptus SSR marker Embra147. The specific steps are basically the same as in "Example 1" above, except that the forward primer (5'-GCACGGTACTCGATCATAGA-3') and the backward primer (5'-TGAGATAGGATTGCGCGT-3') are different, and the 70-60°C in the landing PCR is changed to 66-60°C. 56°C, 60°C changed to 56°C in 26 cycles. The test results are attached figure 2 Identified by H. Peak Ⅰ is the molecular weight internal standard, and Ⅱ(a) and Ⅱ(b) are target fragments. The height of the peak represents the strength of the fluorescent signal. Eucalyptus is diploid, and the peaks of II(a) and II(b) indicate that the lengths of the two alleles of this individual on the SSR marker are different, which are 178bp and 184bp respectively.

Embodiment 3

[0031] A fluorescent dUTP-based method for the detection of the medicinal plant-Schisandra chinensis SSR-labeled WWZ-AC25. The specific steps are basically the same as "Example 1" above, except that the forward primer (5'-CATCGGAAAAATCAGAGAAG-3') and the backward primer (5'-CCTATCAAAACTCCCCTCTT-3') are different, and the temperature of 70-60°C in landing PCR is changed to 66-56°C. ℃, 60 ℃ was changed to 56 ℃ in 26 cycles, and Eucalyptus urophylla was changed to Schisandra chinensis. The test results are attached image 3 . I(a) and I(b) peaks are molecular weight internal standards, and II peaks are target fragments. Schisandra is diploid, and peak II indicates that the length of the two alleles of this individual on the SSR marker is the same, which is 201bp.

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Abstract

The invention provides a fluorescent dUTP-based method for automatically detecting an SSR molecular marker. The method comprises the following steps of: (1) preparing a PCR reaction system: mixing 1.0muL of 10*buffer, 25 to 50muM of dNTP, 2.0mM of MgCl2, 0.5muM of forward primer, 0.5muM of backward primer, 1 unit of Taq enzyme, 10pmol of fluorescent dUTP and 5ng of genome DNA, and adding super-pure water into the mixture to supplement the mixture to 10muL; (2) performing touchdown PCR: treating the mixture for 4 minutes at the temperature of 94 DEG C; performing 20 cycles for 30 seconds at the temperature of 94 DEG C, 30 seconds at the temperature of between 70 and 60 DEG C or between 66 and 56 DEG C and 1 minute at the temperature of 72 DEG C, wherein each cycle reduces 0.5 DEG Cm; performing 26 cycles for 30 seconds at the temperature of 94 DEG C, 30 seconds at the temperature of 60 DEG C or 56 DEG C and 1 minute at the temperature of 72 DEG C; and finally, treating the mixture for 1 minute at the temperature of 72 DEG C to obtain a PCR product; and (3) adding 9.34muL of super-pure formamide and 0.16muL of molecular weight internal mark into 1.0muL of PCR product, treating the mixture for 5 minutes at the temperature of 95 DEG C, then quickly cooling the mixture over ice, and performing mark detection on a sequencer.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for automatically detecting SSR molecular markers on a sequencer based on fluorescent dUTP. Background technique [0002] SSR (Simple sequence repeats, also known as microsatellites) is a type of DNA sequence composed of several bases repeated in tandem, and its length is generally short (each unit length is between 1 and 6 bp), widely distributed at different locations in the genome. The variability in the number of repeats in different genetic material leads to the basis for the generation of SSR markers. SSR marker is one of the popular molecular marker technologies today. Its principle is: design a pair of specific primers based on the conserved single-copy sequences at both ends of the SSR, amplify the core SSR sequence in between by PCR technology, and use electrophoresis analysis technology to Get its length polymorphism. In order to meet the needs of high thro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 甘四明李发根翁启杰李梅周长品于晓丽
Owner RES INST OF TROPICAL FORESTRY CHINESE ACAD OF FORESTRY
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