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Method for detecting DNA having microsatellite region

A microsatellite and regional technology, applied in the field of hybrids, can solve problems such as high price and achieve the effect of no detection errors and high precision

Inactive Publication Date: 2014-12-10
WAKO PURE CHEMICAL INDUSTRIES +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the case of polymorphism analysis of microsatellite DNA, electrophoresis needs to be performed using a 30 cm capillary or a 40 cm long denaturing polyacrylamide gel, so there are problems such as high cost and the need for specialized skills.

Method used

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  • Method for detecting DNA having microsatellite region
  • Method for detecting DNA having microsatellite region
  • Method for detecting DNA having microsatellite region

Examples

Experimental program
Comparison scheme
Effect test

Synthetic example 1

[0073] Synthesis example 1 has the sample DNA of the sequence of poly-T chain length polymorphism in TOMM40 gene sequence preparation

[0074] (1) Preparation of sample DNA with various poly-T chain length polymorphisms in the rs10524523 marker sequence of the TOMM40 gene

[0075] As poly-T sequences with different chain lengths of the rs10524523 marker sequence of the TOMM40 gene, 12 kinds of synthetic oligonucleotides were designed, their complementary chains were synthesized, and these were used as sample DNAs. The sequence of this synthetic oligonucleotide is shown in Table 1 below.

[0076] The above-mentioned nucleotides were synthesized using the commissioned synthesis service of Sigma-genosys. In addition, in the following synthesis examples and examples of the present invention, oligonucleotide synthesis of primers and probes, labeling of fluorescent dyes, and the like were also performed using the commissioned synthesis service of Sigma-genosys.

[0077] [Table...

Embodiment 1

[0098] Example 1 Recognition and detection of the poly-T chain length polymorphism of the TOMM40 gene based on the method of the present invention

[0099] (1) Preparation of probe for ring hybridization reaction (LH probe)

[0100] LH probes were designed so as not to bind to the microsatellite regions in the DNA listed in Table 2 but to bind to both sides thereof. That is, a 19-base complementary strand from the side of the 5' end of the microsatellite region of the complementary sequence of the single-stranded DNA in Table 2 above is connected to a 71-base complementary strand from the side of the 3' end side, so The resulting sequence was used as an LH probe. Specifically, a probe (cy5delT0) of the following sequence was used.

[0101] (cy5delT0; 90mer, T0 =deleted poly T[serial number 28])

[0102] gacctcaagc tgtcctcttg ccccagccct ccaaagcatt gggattactg gcatgagcca ttgcatctggc( T0 )gagatgggg tctcaccatg

[0103] When the aforementioned LH probe forms a hybrid with th...

Embodiment 2

[0126] Example 2 Taking the long-chain poly-T polymorphism as the detection object and based on the method of the present invention Identification and detection of poly-T chain length polymorphism in TOMM40 gene

[0127] Except using Fx / Q28A, Fx / Q29A, or Fx / Q30A as a sample, the poly-T chain length polymorphism in the TOMM40 gene sequence was determined using the same method as in (2) and (3) of Example 1. Identification detection. show the result in image 3 -a. In the figure, band 28 represents the result of swimming the LH reaction product of Fx / Q28A, band 29 represents the result of swimming the LH reaction product of Fx / Q29A, and band 30 represents the LH reaction of Fx / Q30A The result of swimming the product.

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Abstract

The purpose of the present invention is to provide a method that can accurately detect DNA having a microsatellite region without causing the problem of non-specific reaction products. The present invention relates to a "method for detecting DNA having a microsatellite region by (1) bringing a probe, which does not have a nucleotide sequence complementary to the microsatellite region and which will hybridize with the nucleotide sequences on both sides of the microsatellite region, into contact with DNA having the microsatellite region, and forming a hybrid of the probe and the DNA, said hybrid having a loop structure containing the microsatellite region, (2) separating the obtained hybrid, and (3) detecting the hybrid", and a "hybrid of a probe and DNA, said hybrid having a loop structure containing a microsatellite region and obtained by bringing a probe, which does not have a nucleotide sequence complementary to the microsatellite region and which will hybridize with the nucleotide sequences on both sides of the microsatellite region, into contact with DNA having the microsatellite region".

Description

technical field [0001] The invention relates to a method for detecting DNA with a microsatellite region by using a ring hybridization method and a hybrid obtained by the ring hybridization method. Background technique [0002] A repetitive sequence of 1 to 6 bases repeated as a unit is called a microsatellite region, which is known to exist in large numbers in many mammals such as the human genome sequence, and there are more than 3,000 locations in the human genome. This microsatellite region is known to be useful as a marker for analysis of various genes because it shows polymorphisms in which the number of repetitions (number of repeats) differs. It is also known that when a microsatellite region is located in the protein coding region of a gene, a difference in the number of repeats causes a disease, and various studies have been conducted on the microsatellite region in order to investigate the relationship between genes and diseases. [0003] In a method for analyzing...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12Q1/68
CPCC12Q2600/156C12Q1/6876
Inventor 松隈章一石川友一黑泽龙雄
Owner WAKO PURE CHEMICAL INDUSTRIES