SI23245A - High performance isolation of nucleic acids - Google Patents

Abstract

High performance isolation of nucleic acids from various types of samples is applicable in molecular biology, microbiological, biotechnological or pharmaceutical laboratories dealing with a great number of samples, where samples can be pure archaebacterial and eubacterial cultures on solid or liquid culture media, cell cultures of eucaryotic cells, tissue cultures, viruses, biological samples from the environment, swabs, tissues and other samples from which NA are extracted, and where samples can be fresh, frozen, fixed or preprepared in some other manner. The isolation is characteristic in that the reagent mixture contains cation exchangers in a concentration from 1 to 20% for chelation of bivalent cations and that other substances, required for disintegration of cells, can be added to the reagent mixture. The reagent mixture is preprepared and packed in two or several microtubes or in a microtiter plate.

Description

HIGH PERFORMANCE NUCLEIC ACID INSULATION

The subject of the invention is high performance nucleic acid isolation from different types of samples, useful in molecular biological, microbiological, biotechnological or pharmaceutical laboratories with a large number of samples. The essential advantages of the invention are the suitability for automation, speed and economy of insulation and non-toxicity for users. The isolation process according to the invention is based on the thermal or other degradation of cell membranes in a suitable cation exchanger solution and may also include other substances, e.g. buffer, a stabilizer that prevents the degradation of isolated nucleic acids.

The present invention relates to the new use of rapid nucleic acid isolation methods, hereinafter referred to as NK, from various types of samples, which enables automation and thus high performance isolation in routine, development and research laboratories, where rapid NK isolation from a large number of samples is crucial. Examples of such laboratories are molecular biological, microbiological, sanitary, biotechnological, pharmaceutical, and others, and samples may be pure bacterial cultures, that is, archaeal and eubacterial cultures on solid or liquid media, cell cultures of eukaryotic cells, tissue cultures, viruses, biological samples from the environment, e.g. soil, air, water, sediment, surfaces, swabs, tissues and other samples from which NK is isolated.

Methods of isolating NK from samples are known and known in the art. The standard procedure is chemical and / or physical cell disintegration followed by NK purification, precipitation and stabilization. Most processes are time consuming because they use several separate procedures for individual isolation stages, which are user-demanding, because it requires a series of devices and different reagent mixes to be successful in isolation and consequently unsuitable for automation. While high-performance processes exist and are described in the prior art, they use the condensed or simplified standard procedures described above, usually using membrane columns, which maintains high cost and long lasting insulation.

NK isolation is carried out using physical and chemical processes to break down the cell wall and cell membrane, followed by purification stabilization of isolated NK. Physical procedures are simpler and may be based on mechanical disintegration, with heating or / and cooling, and using elevated pressures. Chemical methods are based on the use of various enzymes to break down the cell wall, detergents to dissolve the cell membrane, the use of combinations of salts for the precipitation and purification of nucleic acids, and their stabilization and to prevent enzymatic degradation.

Upon breaking down the cell wall and membrane, NK is released into the medium together with specific and non-specific

-2 Nucleases that begin to break down nucleic acids immediately, thereby reducing insulin leakage. Solutions are in use of processes for the removal of all proteins or inactivation of nucleases by boiling or chelation of the divalent cations that the nucleases require for action (Marmus J. (1961) J. Mol. Biol. 3: 208-18). For chelation of divalent cations, cation exchangers may be used, for example: styrene divinylbenzene with bound iminodiacetate ions, zeolite, EDTA, 8-hydroxyquinoline, 2,3 dihydroxypyridine, 4,6 dihydroxypyridine, 2-pteridinol, 2,4, 2,3, quinolindinol dihydroxyquinoxaline, 2,4 pteridindiol. The whole process involving cation exchangers may, however, comprise the disintegration of cells in the abovementioned physical and / or chemical means and the stabilization of NK in buffers or reagents for stabilizing ribonucleic acids. All described procedures are carried out in individual tubes, usually (micro) centrifuges.

High performance isolation methods are available in microcolumns in microtiter format: Mo Bio UltraClean®-htp 96 Well Microbial DNA Kit, Zymoresearch ZR-96 Fungal / Bacterial DNA Kit ™, Qiagen Generation Capture Plate Kit, which are extremely complex because they contain multistage insulation in membrane microcolumns, insulation lasts for several hours and / or uses toxic chemicals.

Therefore, there is a need for a high-performance NK isolation method from different samples, which is suitable for automation and is quick and easy, and economical for insulation and non-toxicity for users are important.

The object and object of the invention is a process and a kit for faster and cheaper high-performance NK insulation than currently known. According to the invention, the task is solved by the preparation of reagents, their quota or its m into microtubes or microtiter plate, and a user process for NK isolation according to claims.

By preparing the reagents and the method according to the invention, it is possible to isolate from the same sample quantity a comparable amount of NK significantly simpler and faster from the same amount of sample. The difference is that, according to the invention, methods of rapid cell disintegration, cation exchanger are used to inhibit NK degradation and preparation of solutions in small volumes, which enables high performance isolation and process automation. It is very important to combine the individual but separate physical and chemical stages of isolation with the aim of simplifying the process.

-3Description of the invention

The invention will be presented by means of pictures showing:

Figure 1. Microtube strips with prepared cation exchanger solution, Figure 2. Microtiter plate with prepared cation exchanger solution.

High performance isolation reagents according to the invention are prepared in a way that combines several stages of NK isolation and may, but not necessarily, contain chemical agents for cell disintegration such as lysozyme, anion exchanger, cation exchanger, neutral exchanger, detergent, proteinase K, proteinase K in combination with heating and cooling, magnets, nanoparticles for oxidative lysis, and disintegration can also be physical by heating and / or cooling.

Purification of the leachate may, but not necessarily, be carried out using magnets, glass particles, guanidine thiocyanate, guanidine hydrochloride, Sephadex or Sepharose.

An essential and always necessary component of the reaction mixture are, for the inactivation of specific and nonspecific nuclease nuclease, cation exchanger such as styrene divinylbenzene with bound iminodiacetate ions, zeolite, EDTA, 8-hydroxyquinoline, 2,3 dihydroxypyridine, 4,6 dihydroxypyridine, 4,6 dihydroxypyridine, 4,6 dihydroxypyridine 2,4, quinolindiol, 2,3 dihydroxyquinoxaline, 2,4 pteridindiol, or others, which chelate divalent cations in a concentration range of 1 to 20%, depending on the type of sample. According to the invention, from 1% to 20%, preferably from 3% to 7%, is a cation exchanger solution for isolation of NK from pure bacterial cultures. As inactivators of all enzymes, phenol / chloroform, DTT, DEPC, heparin, iodoacetate, polyvinyl sulfate, cationic surfactant, bentonite, clay macaloids, 3% peroxide, sodium hydroxide, SDS, cesium chloride may, but not necessarily, be in the reaction mixture. ammonium sulphate, and may also have an increased ionic strength.

To stabilize NK, Tris EDTA, TE buffer, COUNT or sucrose may, but not necessarily, be present in the reaction mixture.

In order to achieve the high NK insulation performance of the invention, the reagents must be prepared in either microtubes in strips of at least two or in microtiter plates with at least two wells.

The NK insulation process is performed according to the type of sample. Pre-preparation may, but not necessarily, be required • · · • · ·

-4 of the sample, ie the addition of boiling point salt, the addition of alcohols, the addition of huminic acid-binding polyvinylpyrrolidone, the addition of lysozyme, lysostafin, streptolysin or any other enzymatic agent for cell wall degradation, isolation or concentrating the cells from the matrix or for the preparation of protoplasts, followed by the transfer of samples into microtubes or microtiter plates, by hand using a multi-channel pipette, a robotic system or the like.

An important part of the invention is the fact that NK isolation is henceforth automatic, since all the reagents are prepared in individual microtubes or tubes. the well as well as the NK extraction itself takes place in the same well. This enables high-performance NK insulation and thus automation of the insulation process.

The final part of the NK isolation process may, but may not be necessary, is concentrating the isolates with NaCl, LiCI, magnets or N-butanol and transferring them to other NK treatment processes manually with a multi-channel pipette or with a robotic system. For storage of isolated NK, it can be used as a TE stabilizer buffer or ethanol, or it can be stored at + 4 ° C, at -20 ° C, or -80 ° C. All these final parts of the process depend on the individual post-isolation procedures and are part of the standard techniques in the laboratories mentioned above.

High-performance nucleic acid isolation therefore takes place in two phases:

Preparation of reagents: Prepare an aqueous solution of the cation exchanger and add the additives described above if necessary. This solution is autoclaved. otherwise sterilized. The solution is then transferred by shaking with a multichannel pipette into a sterile microtiter plate or sterile microtube strips, which are then sealed with self-adhesive foil or caps. This phase is preferably done industrially, it may also be robotic, and the reagent with optional additives may be packed and enclosed in a microtiter plate or sterile microtube strips.

Isolation process: The user uses a pre-prepared reagent packed and enclosed in a microtiter plate or sterile microtube strips and inserts samples into individual wells on prepared e.g. microtiter plate. In the wells, the NK extraction then proceeds automatically, and after the extraction is complete, the plate is centrifuged and the supernatant transferred from each well to a new microtiter plate. The NK isolates thus prepared are directly applicable for subsequent molecular biological analyzes. Depending on the preparation, the procedures can be performed robotically.

An implementation example

High performance isolation of nucleic acids from bacteria growing on solid substrates

-5 Reagent Preparation Procedure: A 5% aqueous solution of the cation exchanger of styrene divinylbenzene with bound iminodiacetate ions is pre-prepared. This is autoclaved for 20 minutes at 121 ° C and a pressure of 1.1 bar. The suspension is cooled to room temperature and transferred to a sterile microtiter plate or sterile microtube strips during shaking, which are then sealed with self-adhesive foil or caps.

Isolation process: The user, with an ezo or sterile toothpick, enters the bacterial cells from the colonies on solid medium into individual wells on prepared e.g. microtiter plate. The microtiter plate was then heated at 99 ° C for 15 minutes, then cooled and then centrifuged at 3000 rpm at room temperature for 10 minutes. After centrifugation, it transfers 70 pl of the supernatant from each well into a new microtiter plate. The NK isolates thus prepared are directly applicable for subsequent molecular biological analyzes.

High performance nucleic acid isolation from different sample types is useful in molecular biological, microbiological, biotechnology or pharmaceutical laboratories with a large number of samples, the samples being pure arche- and eubacterial cultures on solid or liquid media, eukaryotic cell cultures, tissue cultures, viruses, biological samples from the environment, swabs, tissues and other samples from which NK is isolated and may be fresh, frozen, fixed or otherwise prepared. The invention is characterized in that the reagent mixture contains cation exchangers in a concentration of from 1 to 20%, preferably from 3% to 7%, which chelate the divalent cations, to which other substances necessary for cell disintegration may be added to the reagent mixture and is reagent mixture prepared and packed in two or more microtubes or microtiter plate. The cation exchanger is styrene divinylbenzene with bound iminodiacetate ions, zeolite, EDTA, 8-hydroxyquinoline,

2,3 dihydroxypyridine, 4,6 dihydroxypyridine, 2-pteridinol, 2,4, quinolindiol, 2,3 dihydroxyquinoxaline, 2,4 pteridindiol or the other, wherein the reaction mixture may be one type of cation exchanger or a mixture of two or more. Substances necessary for cell disintegration, enzyme inactivation, purification of leachate and NK stabilization may be added to the reagent mixture.

Claims (3)

Patent claims 1. High-performance nucleic acid isolation from various sample types, usable in molecular biological, microbiological, biotechnology or pharmaceutical laboratories with a large number of samples, the samples being pure arche- and eubacterial cultures on solid or liquid media, cell cultures of eukaryotic cells , tissue cultures, viruses, biological samples from the environment, swabs, tissues and other samples from which NK is isolated and the samples may be fresh, frozen, fixed or otherwise prepared, characterized in that the reagent mixture contains cation exchanger at a concentration of 1% to 20%, preferably 3% to 7%, chelating the divalent cations, wherein other substances necessary for cell disintegration may be added to the reagent mixture and the reagent mixture is prepared and packaged in two or more microtubes or microtiter plates. Isolation according to claim 1, characterized in that the cation exchanger is styrene divinylbenzene with bound iminodiacetate ions, zeolite, EDTA, 8-hydroxyquinoline, 2,3 dihydroxypyridine, 4,6 dihydroxypyridine, 2pteridinol, 2,4, quinolindiol 3 dihydroxyquinoxaline, 2,4 pteridindiol or the other, wherein in the reaction mixture may be one type of cation exchanger or a mixture of two or more. Isolation according to claim 1, characterized in that other substances are added to the reagent mixture of substances required for cell disintegration, inactivation of enzymes, purification of the leachate and stabilization of NK.