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Method for obtaining gene information and functional genes from species without genome reference sequence

A reference sequence and genome technology, applied in biochemical equipment and methods, microbial measurement/inspection, recombinant DNA technology, etc., can solve problems such as high cost, difficult to eliminate redundant sequences, and impact on animal and plant genome research

Inactive Publication Date: 2011-12-14
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For species that lack genome sequences, the discovery of new genes and functional research have always been the main problems that plague biologists. Traditional gene discovery methods generally use library construction, SAGE technology, MPSS technology, or genome sequencing methods to obtain, however These methods generally have high cost, heavy workload, and obtain a large number of impurity sequences, and it is difficult to eliminate redundant sequences without a reference genome. These problems seriously affect the research on the genome level of animals and plants.

Method used

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  • Method for obtaining gene information and functional genes from species without genome reference sequence
  • Method for obtaining gene information and functional genes from species without genome reference sequence
  • Method for obtaining gene information and functional genes from species without genome reference sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Embodiment 1, obtain DGE-tag

[0100] Taking the DGE-tag analysis of the Asian corn borer as an example, the method steps are as follows, and the concise experimental procedure is shown in figure 1 and figure 2 .

[0101] 1. Extraction of total RNA from Ostrinia officinalis

[0102] Extract by conventional Trizol method, purify by conventional method, and treat with DNase to obtain a Total RNA sample with a concentration ≥ 300ng / ul, a total amount ≥ 6ug, and an OD260 / 280 of 1.8-2.2 (must meet the detection requirements of Agilent 2100).

[0103] 2. Isolation of mRNA and synthesis of cDNA

[0104] The mRNA with polyA was isolated using magnetic beads with oligo-dT, and then the first-strand cDNA was synthesized using random 6-mers and Invitrogen's Superscript II reverse transcriptase kit. cDNA second strand was completed with RNase H (Invitrogen) and DNA polymerase I (New England BioLabs).

[0105] 3. Tag preparation and sequencing

[0106] Utilizing the double-st...

Embodiment 2

[0111] Embodiment 2, DGE-tag analysis

[0112] DGE-tag analysis continues with embodiment 1, and proceeds to the following steps:

[0113] (c) Annotate the Tag and establish the corresponding relationship between the Tag and the gene: since there is no reference gene data for Ostrinia oleifera, the inventors refer to the RNA-seq data of Ostrinia strigae that was completed at the same time, and use software to retrieve the RNA-seq data for Ostrinia striatus For all CATG sites in the database, a reference label database of CATG+17nt bases is generated. Then compare all Clean Tags with the reference tag database, allow up to one base mismatch, perform gene annotation on the tag (Unambiguous Tags) that is uniquely compared to a gene, and count the number of original Clean Tags corresponding to each gene, and then Standardize the original Clean Tag numbers to obtain standardized gene expression levels, so as to measure gene expression levels more accurately and scientifically. Th...

Embodiment 3

[0119] Example 3, RNA-seq analysis

[0120] For sample processing and sequencing procedures, see image 3 . The specific method is as follows:

[0121] 1. Extraction of Total RNA from Ostrinia cerevisiae

[0122] The conventional Trizol method was used to extract, purify, and treat with DNase to obtain a Total RNA sample with a concentration ≥ 300ng / ul, a total amount ≥ 6ug, and an OD260 / 280 of 1.8-2.2 (which must meet the detection requirements of Agilent 2100).

[0123] 2. mRNA isolation and random interruption

[0124] Use magnetic beads with oligo-dT to separate the mRNA with polyA, and then use ultrasonic waves to randomly interrupt and recover fragments of 200-700bp.

[0125] 3. Synthesis of first and second strands of cDNA

[0126] First-strand cDNA synthesis was performed using random 6-mers and Invitrogen's Superscript II reverse transcriptase kit. cDNA second strand was completed with RNase H (Invitrogen) and DNA polymerase I (New England BioLabs).

[0127] 4....

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Abstract

The invention relates to a method for obtaining gene information and functional genes from species without genome reference sequences. The invention also discloses a method for obtaining the gene expression profile of the Asian corn borer. The invention also discloses a method for obtaining transcriptome information and functional genes of the Asian corn borer. The present invention combines DGE-tag technology with RNA-seq technology for the first time to obtain gene expression period, expression level, corresponding metabolic pathway and gene function information from species without genome reference sequence. The method is convenient and fast , accurate and low cost.

Description

technical field [0001] The invention belongs to the field of biotechnology; more specifically, the invention relates to a method for obtaining gene information and functional genes from species without genome reference sequences. Background technique [0002] The rapid and high-throughput discovery of new genes in species lacking genome reference sequences has always been a major problem for biologists. Traditional gene discovery methods generally use library construction, SAGE technology, MPSS technology, or genome sequencing methods However, these methods generally have high cost, heavy workload, and obtain a large number of complex sequences, and it is difficult to eliminate redundant sequences without a reference genome sequence. These problems have seriously affected the research on the genome level of animals and plants. [0003] Digital gene expression tag (DGE-tag) and high-throughput transcriptome analysis (RNA-seq) are based on next-generation sequencing technology...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68
Inventor 苗雪霞李海超王玉冰张浩黄勇平
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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