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Set of random primers and method for preparing DNA library using same

A DNA library and random primer technology, applied in biochemical equipment and methods, DNA preparation, recombinant DNA technology, etc., can solve the problems of increasing the number and cost of operations, and not reducing the complexity of genomic DNA, and achieve the effect of inhibiting amplification

Active Publication Date: 2020-01-03
TOYOTA JIDOSHA KK
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, it is inferred that the method described in Patent Document 3 requires nucleotide sequence information of the genome to identify the frequency of occurrence of the genome, which will increase the number of operations and cost
Furthermore, according to the method described in Patent Document 3, the entire genome is to be amplified, and the complexity of genomic DNA cannot be reduced, which is disadvantageous

Method used

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  • Set of random primers and method for preparing DNA library using same
  • Set of random primers and method for preparing DNA library using same
  • Set of random primers and method for preparing DNA library using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0233] 1. Flowchart

[0234] In this example, genomic DNA extracted from various types of biological species was used as a template and according to figure 1 DNA libraries were prepared by PCR with various random primer sets shown in the flow chart. Using the prepared DNA library, sequence analysis was also performed using a so-called next-generation sequencer, and based on the read data, the genotype was analyzed.

[0235] 2. Materials

[0236] In this example, genomic DNA was extracted from sugarcane varieties NiF8 and Ni9, 22 hybrid progeny lines thereof, and rice variety Nipponbare using DNeasy Plant Mini Kit (QIAGEN), and the extracted genomic DNA was purified. Purified genomic DNA was used as NiF8-derived genomic DNA, Ni9-derived genomic DNA, 22 hybrid sugarcane progeny-derived genomic DNA, and Nipponbare-derived genomic DNA, respectively. In this example, human genomic DNA was purchased from TakaraBio and used as human-derived genomic DNA.

[0237] 3. Method

[023...

Embodiment 2

[0480] 1. Flowchart

[0481] In this example, using genomic DNA as a template and random primers, according to Figure 107 with Figure 108 In the schematic diagram shown in , a first DNA fragment is prepared by PCR, and then a second DNA fragment is prepared by PCR using the prepared first DNA fragment as a template and primers for a next-generation sequencer. Using the prepared second DNA fragment as a library for a sequencer, sequence analysis is performed using a so-called next-generation sequencer, and based on the obtained read data, genotype is analyzed.

[0482] 2. Materials

[0483] In this example, genomic DNA was extracted from sugarcane variety NiF8 and rice variety Nipponbare using DNeasy Plant Mini Kit (QIAGEN), and the extracted genomic DNA was purified. Purified genomic DNA was used as NiF8-derived genomic DNA and Nipponbare-derived genomic DNA, respectively.

[0484] 3. Method

[0485] 3.1 Inspection of sugarcane variety NiF8

[0486] 3.1.1 Random primer...

Embodiment 3

[0529] 1. Flowchart

[0530] In this example, using genomic DNA as a template and random primers, in the same manner as in Example 2, a first DNA fragment was prepared by PCR, and then the prepared first DNA fragment was used as a template and a primer for a next-generation sequencer , to prepare the second DNA fragment by PCR. Using the prepared second DNA fragment as a library for a sequencer, sequence analysis was performed using a so-called next-generation sequencer, and based on the read data, the genotype was analyzed. In particular, in this example, it was examined whether amplification of a DNA fragment derived from the chloroplast genome could be suppressed depending on the type of random primers used.

[0531] 2. Materials

[0532] In this example, genomic DNA was extracted from rice variety Nipponbare using DNeasy Plant Mini Kit (QIAGEN), and the extracted genomic DNA was purified. Purified genomic DNA was used as rice-derived genomic DNA. The genomic DNAs of co...

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Abstract

When preparing a DNA library via a nucleic acid amplification reaction using a random primer in a convenient and highly reproducible manner, amplification of DNA fragments derived from the chloroplastgenome is reduced to a significant extent. A random primer comprises oligonucleotides selected from oligonucleotides group represented by TAAGAGACAGNN excluding those in which 2 bases at the 3' terminus are TG and oligonucleotides group represented by TAAGAGACAGNNN excluding those in which 3 bases at the 3' terminus are TGC.

Description

technical field [0001] The present invention relates to a random primer set used in a method of preparing a DNA library that can be used for DNA marker analysis and the like and a method of preparing a DNA library using such a random primer set. Background technique [0002] Typically, genome analysis is performed to perform a comprehensive analysis of the genetic information contained in the genome, such as nucleotide sequence information. However, analysis aimed at determining the nucleotide sequence of the whole genome is disadvantageous in terms of the number of processes and cost. Furthermore, in the case of organisms with a large genome size, genome analysis based on nucleotide sequence analysis has limitations due to the complexity of the genome. [0003] Patent Document 1 discloses an amplified fragment length polymorphism (AFLP) marker technology in which a sample-specific marker is incorporated into a restriction enzyme-treated fragment that has been ligated to an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895
CPCC12Q1/6848C12Q1/6853C12Q1/6895C12Q2525/179C12N15/1093C12Q1/6806
Inventor 榎宏征竹内由枝稻森稔
Owner TOYOTA JIDOSHA KK
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