Method for testing DNA mononucleotide pleomorphism

A single nucleotide polymorphism and detection method technology, applied in the field of DNA single nucleotide polymorphism detection, can solve the problems of high cost, increased detection time, low sensitivity, etc., and achieve the effect of simple processing and no need for separation

Inactive Publication Date: 2008-07-16
INST OF CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Single nucleotide polymorphism analysis methods often use electrophoresis analysis and solid-phase hybridization methods, such as: DNA sequencing, restriction fragment length polymorphism analysis, single-stranded DNA conformation polymorphism analysis, heteroduplex nucleic acid analysis , allele-specific oligonucleotide hybridization, mismatch analysis, oligonucleotide ligation analysis, etc., require cumbersome separation or washing procedures, increasing the detection time
The above problems can be avoided by using homogeneous detection methods. The current homogeneous detection methods are all based on fluorescence polarization or fluorescence resonance energy transfer between small molecule fluorescent dyes, and the intensity of polarized fluorescence is greatly reduced, resulting in low sensitivity of methods based on fluorescence polarization. High; methods based on fluorescence resonance energy transfer all require double modification of probe molecules, resulting in high cost

Method used

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  • Method for testing DNA mononucleotide pleomorphism
  • Method for testing DNA mononucleotide pleomorphism
  • Method for testing DNA mononucleotide pleomorphism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1. Application of PFP1 to detect single nucleotide polymorphisms at the RS1800469 site in wild-type samples

[0054]

[0055] 1. Synthesis of PFP1

[0056] The structure of PFP1 is shown in formula (II), and the synthesis method is as follows:

[0057] Add 50mL of toluene, 5.2g of neopentyl glycol and 0.5g of p-toluenesulfonic acid to a 100mL single-necked bottle, reflux for 24 hours, evaporate the solvent and separate on a silica gel column (eluent: dichloromethane) to obtain 4.8g of 1,4- Neopentyldiboronate. 1 H NMR (400MHz, CDCl 3 ): δ (ppm) 7.78 (4H, s), 3.77 (8H, s), 1.02 (12H, s).

[0058] Add 5.4mL toluene and 3.6mL 2M potassium carbonate to a 25mL two-necked flask, and after bubbling nitrogen gas for 30 minutes, add 325 mg of 2,7-dibromo-9,9-bis(6-bromohexyl)fluorene, 165 mg 1,4-Neopentyldiborate and 20 mg tetrakis(triphenylphosphine)palladium, stirred and reacted at 95°C under nitrogen for 24 hours, added chloroform and water after cooling, washe...

Embodiment 2

[0079] Example 2. Using PFP1 to detect single nucleotide polymorphisms at the RS1800469 site in mutant samples

[0080] 1. Synthesis of PFP1

[0081] Same as Step 1 of Example 1.

[0082] 2. Extraction of DNA from samples to be tested

[0083] Same as Step 2 of Example 1.

[0084] 3. Amplification of DNA Fragments Containing Target Sites

[0085] Same as Step 3 of Example 1.

[0086] 4. Single base extension reaction

[0087] Same as Step 4 of Example 1.

[0088] 5. Measurement of Fluorescence Spectroscopy

[0089] Same as Step 5 of Example 1.

[0090] Figure 2 shows the fluorescence spectra obtained by single-base extension with primer 1 and primer 2 respectively. When primer 2 is used, there is a significant energy transfer, but when primer 1 is used, only a background signal appears, indicating that the nucleus of the RS1800469 site of the DNA to be tested is The nucleotide is homozygous for the mutation.

[0091] 6. Result Verification

[0092] Same as Step 6 of ...

Embodiment 3

[0094]Example 3. Application of PFP1 to detect single nucleotide polymorphisms at the RS1800469 site in heterozygous samples

[0095] 1. Synthesis of PFP1

[0096] Same as Step 1 of Example 1.

[0097] 2. Extraction of DNA from samples to be tested

[0098] Same as Step 2 of Example 1.

[0099] 3. Amplification of DNA Fragments Containing Target Sites

[0100] Same as Step 3 of Example 1.

[0101] 4. Single base extension reaction

[0102] Same as Step 4 of Example 1.

[0103] 5. Measurement of Fluorescence Spectroscopy

[0104] Same as Step 5 of Example 1.

[0105] Figure 3 shows the fluorescence spectra obtained by single-base extension with primer 1 and primer 2 respectively. Significant energy transfer occurs when primers 1 and 2 are used, indicating that the nucleotide at the RS1800469 site of the DNA to be tested is a hybrid type.

[0106] 6. Result Verification

[0107] Same as Step 6 of Example 1.

[0108] The result proves that there are two kinds of PCR pro...

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Abstract

The invention discloses a detection method of DNA single nucleotide polymorphism. The method which is provided by the invention uses a fluorescent substance for marking the DNA of a sample to be detected, and the nucleotide of the polymorphism site of the target single nucleotide of the DNA to be detected is determined by the fluorescence resonance energy transfer situation of the fluorescent substance and a water-soluble conjugated polymer. The invention has simple and convenient processing, which omits various separation and purification steps; the nucleotide of the polymorphism site of the single nucleotide can be detected by a small amount of DNA, thus the invention has great significance for the development of drugs and the clinical diagnosis of hereditary diseases.

Description

technical field [0001] The invention relates to a method for detecting DNA single nucleotide polymorphism. Background technique [0002] Single nucleotide polymorphism (single nucleotide polymorphism, SNP) mainly refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genome level. [0003] The polymorphism shown by SNP only involves the variation of a single base, which can be caused by the transition or transversion of a single base, or by the insertion or deletion of a base. Theoretically speaking, a SNP may be a two-allelic polymorphism, or a three- or four-allelic polymorphism, but in practice, the latter two are very rare and can almost be ignored. Therefore, the so-called SNPs are biallelic polymorphisms. [0004] Single nucleotide polymorphism is the most common type of heritable variation in humans, accounting for more than 90% of all known polymorphisms. SNPs are widespread in the human genome, with an average of 1 in every ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 王树段新瑞
Owner INST OF CHEM CHINESE ACAD OF SCI
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