Enzyme reaction method for nucleic acid and composition for separating nucleic acid

An enzymatic reaction and nucleic acid technology, applied in the direction of alkali metal compounds, chemical instruments and methods, biochemical equipment and methods, etc., can solve the problems that cannot be used to remove inhibitors, and achieve the effect of easy preparation

Inactive Publication Date: 2013-05-22
ROCHE DIAGNOSTICS GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to the past method, in order to separate the glass particles from the pollutants, if the particles are separated by centrifugation, if the liquid is filtered through a glass fiber filter. But this is a stuck step, which limits the use of these methods for processing large quantities sample.
[0009] After adding salt and ethanol to produce precipitation, the method of immobilizing nucleic acid with magnetic particles is introduced in Anal.Biochem.201, 166-169 (1992) and PCT GB91 / 00212. In this method, nucleic acid is agglutinated with magnetic particles. By Applying a magnetic field and performing a wash separates the agglutinate from the original solvent. After a wash, the nucleic acid is dissolved in trimethylaminomethane buffer. However, this method has the disadvantage that precipitation is not selective for nucleic acids. Many Solid and dissolved material is also coagulated. As a result, this method cannot be used to remove large quantities of inhibitors of specific enzyme reactions that may be present.

Method used

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  • Enzyme reaction method for nucleic acid and composition for separating nucleic acid
  • Enzyme reaction method for nucleic acid and composition for separating nucleic acid
  • Enzyme reaction method for nucleic acid and composition for separating nucleic acid

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0070] Preparation of magnetic particles of the present invention

[0071] Six sols were used. The sols were prepared as follows:

[0072] Sol 1 (SiO 2 :B 2 0 3 =7:3):

[0073] Synthesis was carried out in a 250ml round flask with constant agitation.

[0074] 86.6ml tetraethylorthosilicate

[0075] +7ml absolute non-denatured ethanol

[0076] +14.1ml0.15M HCl

[0077] A two-phase mixture was produced. Stir at room temperature until single phase. add drop by drop

[0078] +37.8ml trimethyl borate

[0079] The sol was then left at 50°C for 2 hours. join in

[0080] +14.1ml0.15M HCl

[0081] Sol 2 (SiO 2 :B 2 0 3 =4:1):

[0082] Synthesis was carried out in a 250ml round flask with constant agitation.

[0083] 100.5ml tetraethylorthosilicate

[0084] +7ml absolute non-denatured ethanol

[0085] +16.3ml0.15M HCl

[0086] A two-phase mixture was produced. Stir at room temperature until single phase. add drop by drop

[0087] +25.6ml trimethyl borate

[008...

example 2

[0132] Preparation of GMP1, GMP2, GMP3 and GMP4

[0133] GMP1, GMP2, GMP3 and GMP4 are different batches of pigments prepared under the following conditions from sol 1 (example 1) by the process described in example 1:

[0134] parameter GMP1 GMP2 GMP3 GMP4 Sol aging (h) (30℃) 36 36 36 36 Pigment percentage in sol (g / 100ml) 5 15 8 20 Nozzle Airflow (%) 100 100 100 100 air pressure(bar) 6 6 6 3 Nozzle temperature (℃) 135 120 130 143 Densification temperature (°C) 534 534 534 615 Immediate oxygen treatment time (1 hour) (300℃) (300℃) (300℃) (400℃) Pigment yield Low high middle high DNA yield Low high high high

example 3

[0136] Pretreatment of human whole blood PCR samples using magnetic glass particles

[0137] nucleic acid isolation

[0138] 3 parts of glass magnetic particles (GMP2-4), 10 mg per part were put into micro test tubes. The exact sample weights are given in Table 1. Three repeated measurements were carried out.

[0139]Pipette 40 μl proteinase K (20 mg / ml, made by lyophilization) into 200 μl thawed whole blood and mix immediately. Next add 200 μl binding buffer (6M guanidine-HCl, 10 mM trimethylaminomethane -HCl, 10 mM urea, 30% Triton X-100, pH 4.4) and mixed, then incubated at 70°C for 10 minutes. 200 μl of isopropanol was added and the preparation was mixed in a vortex mixer for 10 seconds. The sample was left at room temperature for 20 minutes and then mixed again for 10 seconds. Magnetic separation was performed on a Boehringer Mannheim (ID# 1641794) Magnetic Particle Separator for at least 30 seconds. Remove the supernatant and analyze as described below.

[0140] Mag...

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Abstract

New prods. are (a) magnetic particles with an external glass surface that is nonporous or has pores with a dia. below 10 nm and (b) ferromagnetic particles with a glass surface.

Description

[0001] This application was submitted on June 8, 2006, the filing date is June 6, 1996, the application number is 200610093596.X, and the title of the invention is "the method for performing enzymatic reaction on nucleic acid and the composition for isolating nucleic acid" Divisional application of a patent application. technical field [0002] The present invention relates to magnetic particles having a glass surface, and methods of purifying biological substances, particularly nucleic acids, using glass particles in the presence of chaotropic salts. The invention also relates to methods of isolating these biological substances and concentrating biological substances and Its method of transferring from a high-concentration salt solution to a low-concentration salt solution. Background technique [0003] Many biological substances, especially nucleic acids, present particular difficulties in isolating them from their natural environment. On the one hand, they are often prese...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12P19/34G01N33/48G01N33/552B01J20/28B03CB03C1/01C07HC07H1/08C07H21/00C12NC12N15/09C12N15/10C12N15/11C12QG01N33/553H01F1/00H01F1/11H01F1/36
CPCB03C1/01B82Y25/00C03C3/078C03C3/083C03C3/085C03C3/087C03C3/089C03C3/091C03C3/102C03C3/105C03C3/108C03C3/111C07H21/00C12N15/1013C12Q1/6804C12Q1/6806C12Q1/6834H01F1/0063H01F1/112H01F1/36Y10S428/90Y10S435/814Y10T428/2996
Inventor J·克莱恩T·沃尔特H·哈尔蒂希C·列斯尼亚克M·梅恩伊格M·里德林H·施密特
Owner ROCHE DIAGNOSTICS GMBH
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