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Efficient detection primers and method for AcDs whole genome loci based on NGS sequencing

A genome sequence and specific primer technology, applied in the field of molecular biology, can solve the problems of low efficiency, limited development, high cost, etc., and achieve the effect of reducing cost and high efficiency

Active Publication Date: 2021-01-12
INST OF CROP SCI CHINESE ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing detection methods, including Southern, chromosome walking, circular PCR, reverse PCR and resequencing, etc., have low efficiency and high cost, which limits the development of this field. There is an urgent need for an efficient, cheap, High-throughput detection means

Method used

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  • Efficient detection primers and method for AcDs whole genome loci based on NGS sequencing
  • Efficient detection primers and method for AcDs whole genome loci based on NGS sequencing
  • Efficient detection primers and method for AcDs whole genome loci based on NGS sequencing

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Experimental program
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Effect test

Embodiment 1A

[0031] Embodiment 1Ac / Ds specific primer design and inspection

[0032] According to the Ds sequence whose genbank seqID is JF421358.1 in NCBI as a reference, the present invention scans and compares the maize B73 genome to obtain 73 Ac / Ds. According to the consensus sequence characteristics of these Ac / Ds, each 150bp of the conserved sequence at the end was cut out, and the comparison results are shown in Figure 5 . The present invention will finally use NGS sequencing technology to obtain all Ac / Ds sites at the whole genome level. At present, the read length of NGS is generally 150bp at a single end, and the sum of both ends is 150-300bp, which is a short read length. In order to ensure that within this read length range, transposon sequences larger than 50 bp and adjacent genome sequences larger than 50 bp can be obtained, the present invention designs the 5' or 3' end primers on the Ac / Ds to be about 70 bp away from the ends in their respective directions. The sequence...

Embodiment 2

[0037] Example 2 High-throughput NGS sequencing method enriches Ac / Ds loci from the maize genome

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Abstract

The invention relates to efficient detection primers and a method for AcDs whole genome loci based on NGS sequencing. The primers are base sequences as shown in Primer 5 and Primer 3. By using the twoprimers, 60-100 Ac / Ds loci can be enriched from each corn material through a mixed library building PCR and NGS second-generation deep sequencing method, the detection rate is close to saturation, each sample is marked by using a random tag sequence, the sequence of each sample can be conveniently separated from a mixed sequencing sequence, and a high-flux large-scale scheme is provided for Ac / Dslocus detection.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a primer and a method for efficiently detecting AcDs transposon sites at the whole genome level. Background technique [0002] Transposons are widespread in plants and animals, and play an important role in the evolution of species, domestication and the formation of new varieties. At the same time, people use a large number of transposons to make mutant libraries, create artificial mutations, and create new crops Variety. AcDs is the first transposon discovered in maize. The characteristics of the transposon are evenly distributed at the genome level, and the length of the transposon varies from 1kb to 4kb. In different maize varieties, Ac / Ds transposition tends to be inserted in the gene region, thus resulting in rich and varied phenotypes of maize, which is a good tool for creating new materials. The Ac / Ds system has cross-species activity, so it is the most wide...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6806C12N15/11
CPCC12Q1/6895C12Q1/6806C12Q2525/191C12Q2521/501C12Q2531/113C12Q2521/345C12Q2535/122
Inventor 刘君程溪柳王欢吕明杰高英孙莹
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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