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Primers and method for high-efficiency detection of ACDs genome-wide loci based on NGS sequencing

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

Active Publication Date: 2022-06-07
INST OF CROP SCI CHINESE ACAD OF AGRI SCI +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Primers and method for high-efficiency detection of ACDs genome-wide loci based on NGS sequencing
  • Primers and method for high-efficiency detection of ACDs genome-wide loci based on NGS sequencing
  • Primers and method for high-efficiency detection of ACDs genome-wide loci based on NGS sequencing

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Experimental program
Comparison scheme
Effect test

Embodiment 1A

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

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

Embodiment 2

[0037] Example 2 Enrichment of Ac / Ds loci from the maize genome by a high-throughput NGS sequencing method

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Abstract

The invention relates to a primer and method for efficient detection of AcDs whole genome sites based on NGS sequencing. The primers are the base sequences shown by Primer5 and Primer3. Using these two primers, 60-100 Ac / Ds sites can be enriched from each corn material through the method of mixed library construction PCR and NGS next-generation deep sequencing, the detection rate is close to saturation, and random tags are used The sequence marks each sample, which facilitates the separation of the sequence of each sample from the mixed sequencing sequence, and provides a high-throughput and large-scale solution for Ac / Ds site detection.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to primers and methods 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 species evolution, domestication and the formation of new varieties. At the same time, people make extensive use of transposons to make mutant libraries, create artificial mutations, and create new crops. Variety. AcDs is a kind of transposon first discovered in maize. The characteristics of the transposon are evenly distributed at the whole genome level, and the length varies from 1kb to 4kb. In different maize varieties, Ac / Ds transpositions tend to be inserted in the gene region, thus resulting in a rich and varied phenotype of maize, which is a good tool for creating new materials. The Ac / Ds system has cross-species activity, so it is the most widely used transp...

Claims

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Application Information

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Patent Type & Authority Patents(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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