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Method for developing functional molecular marker related to miRNA

A molecular marker, functional technology, applied in the field of molecular genetics

Inactive Publication Date: 2011-10-19
SOUTHWEST UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The development of miDNA as a new functional marker has not been reported so far

Method used

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  • Method for developing functional molecular marker related to miRNA
  • Method for developing functional molecular marker related to miRNA

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Embodiment Construction

[0026] Taking Brassica napus as an example, developing functional molecular markers related to miRNA.

[0027] (1) Download all miRNA sequences from the miRNA database (http: / / www.mirbase.org / ), and download the partial genome sequence of Brassica napus (not yet sequenced) (http: / / www.ncbi. nlm.nih.gov / ); use the sequence downloaded from miRNA as the seed sequence (query sequence), and then perform BlastN with the published sequence of Brassica napus (http: / / www.ncbi.nlm.nih.gov / BLAST / ) Alignment, miDNA candidate sequences that meet the following parameter conditions:

[0028] The parameters are set as follows:

[0029]Condition 1: There are 25 sequences in the query sequence that are brassica napus miRNA sequences, and the matching sequences tolerate mismatches within 3 bases during alignment; other sequences in the query sequence are tolerant of matching sequences during alignment Mismatches within 5 bases;

[0030] Condition 2: The similarity between the query sequence a...

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Abstract

The invention discloses a method for developing a functional marker related to MicroRNA (miRNA for short). The method comprises the following steps of: performing BlastN comparison on a known miRNA sequence in an miRNA database with a disclosed DNA sequence of a researched species; determining a DNA (miDNA for short) sequence corresponding to the miRNA based on the number and a similarity degree of base mismatch; extending to the left and right for a certain distance until the total length is about 150bp; determining a precursor miDNA site through prediction of a stem loop secondary structure and calculation of the minimum free folding energy coefficient; classifying and comparing all screened precursor miRNA sequences; designing primers according to conserved sequences; electronically comparing the designed primers in the database, and determining as miRNA special sequences; and finally combining according to an annealing temperature of each primer, and developing the functional marker with high efficiency and good polymorphism. The marker system integrates the advantages of the conventional markers such as simple sequence repeat (SSR), amplified fragment length polymorphism (AFLP) and the like, and has good characteristics of higher portability, low cost, polymorphism and stability and the like.

Description

technical field [0001] The invention relates to the technical field of molecular genetics, in particular to a method for developing functional molecular markers related to miRNA. Background technique [0002] PCR-based molecular marker technology plays a very important role in germplasm resource evaluation, genetic diversity analysis, genetic map construction, gene mapping and cloning, and molecular marker-assisted selection breeding. At present, the markers that are widely used are simple sequence repeats (SSR), Amplified fragment length polymorphism (AFLP) and other markers. These marker technologies have many shortcomings, which limit their application. For example, although the SSR marker system has a high stability However, the polymorphism is poor. Generally, the proportion of polymorphisms amplified by primers designed in one species does not exceed 30%, and the number of markers amplified by most SSR primers is only 1-2, less efficient. The polymorphism and efficie...

Claims

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Application Information

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IPC IPC(8): G06F19/16
Inventor 付东辉李加纳沈亮余魏丽娟周庆红
Owner SOUTHWEST UNIVERSITY
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