CS263173B1 - Determination of homologic sections of nucleic acids - Google Patents

Abstract

The solution concerns presence determination nucleic acid homologous regions, most often the gene on the chromosome. determination the presence of nucleic acid homologous regions Acids are obtained by being single-stranded the nucleic acid acts probe and the presence of complement the labeled monoclonal double-stranded nucleic acid antibody acid. Possibility of using them in medicine human and veterinary, in determining the presence of the gene on the chromosome, in biotechnology procedures, microbiological diagnosis, looking for nucleic acid homologous regions in the evolutionary context of organs.

Description

The invention relates to a method for determining the presence of homologous regions of nucleic acids, most often a gene on a chromosome.

So far, the presence of a gene on a chromosome has been most commonly screened by radiolabeled probes by cutting the deoxyribonucleic acid to be examined into small regions using restriction endonucleases. The fragments are separated by electrophoresis and transferred to filter paper. The sample thus obtained is transferred to a radiolabeled probe environment. In the hybridization process, the probes bind to the complementary region of deoxyribonucleic acid and record the radioactive region on the photosensitive layer. Another known gene mapping method is based on chromosomes obtained after cultivation by a basic procedure.

They are treated with ribonuclease, followed by the process of probe hybridization, silver treatment and re-recording on the photosensitive layer. Both chromosomes and loci with the gene present are displayed. The disadvantage is several-day processing, financial costs, laborious demands, handling of radioactive! substances, their short half-life.

These disadvantages are eliminated by the method of determining the presence of homologous nucleic acid regions, most often a gene on the chromosome, which consists in that a reaction system comprising a carrier, for example nitrocellulose, a nylon-fixed, examined single-stranded nucleic acid and the probe itself, optionally incorporated into a double-stranded vector nucleic acid The specific DNA-antibody is added and evaluated, for example by adding a substrate to form a color reaction.

The advantages of the proposed method of the invention are that it allows to replace the work with radioactive substances with enzymatic labeling, reduces the laboriousness of the procedure, enables gene examination in heterozygotes in medicine, microbiological diagnostics and biotechnological procedures. Reduces financial costs.

The accompanying drawings show examples of the procedure for detecting homologous stretches of nucleic acids. In FIG. 1 and 2 show an example using a single-fiber probe. In FIG. 3 shows a method using a single stranded nucleic acid probe complex and a double stranded nucleic acid. The probe is either added to the fuxed nucleic acid under investigation in FIG. 1, 2, 3, or the probe is fixed and the dinaturated nucleic acid of FIG. 4th

Example 1

The unknown nucleic acid 2 ^is fixed ^{in the} form of a single strand on carrier 2 and probe 3 is added. At the complementary region, hybridization occurs and a double strand is formed.

4. The sample is treated with a specific double-stranded nucleic acid antibody 5, the antibody may be labeled with, for example, the radionuclides of FIG. no. 1, or the unlabeled antibody of FIG. no. 2. Binding of unlabeled antibody 2 ^{to the} double-stranded nucleic acid 4 is confirmed by adding labeled antibody 2 against specific antibody 5.

Example 2

The unknown nucleic acid is fixed in the form of a single strand to the carrier 2, probe 2 is added, a double strand 4 is formed at the complementary region. Substrate 8 is added. The result of reaction 7 is read.

Example 3

The sodium 2 ^is fixed to the support 2, is added, the unknown nucleic acid · 2 ^within the homologous region of the nucleic acid forming a double strand 4, the sample is treated with enzyme-labeled antibody was added · 2, the substrate 2 ^{and the} result of the reaction is read 7th

Example:

The denatured single-stranded deoxyribonucleic acid 1 is transferred to a 5 to 30 µl nitrocellulose filter 2, which is left under vacuum at 80 degrees Celsius for 2 hours. Then enters the predetermined hybridization probe 2 ⁱⁿ an amount of 1-1000 ng / ml for 3 to 20 hours in hybridization buffer in the presence of 45 to 55% formamide at 42 ° C. The probe used is a single stranded nucleic acid region. It has the ability to hybridize with an unknown acid at a site that has a complementary base / Maniatis arrangement, Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor,

NY, 1982]. A single-stranded or complexed probe may be used, wherein the single-strand forms a specific region and the other portion may be a double strand. An example is the single-stranded portion of the vector (Hu over Messing, Gene, 17, 271-277, 1982). A vector is a small portion of a dphoxyribonucleic acid that serves as a carrier for a diverse DNA, such as phages, plasmids, and others. After hybridization, a double stranded 4 nucleic acid is formed at the site of the homologous regions. The sample is overlapped to allow monoclonal antibody 3 against double-stranded nucleic acid 4, its ability to bind only to double-stranded structures forms a complex. Binding can be demonstrated by labeling the antibody. Preferably, enzymatic 3 or radioisotopes 6 are used. When labeling the antibody with enzyme 3, the binding of the antibody to probe 3 is accomplished by means of substrate β.

Substrate β is a reactant in enzyme catalyzed reactions. For an alkaline phosphatase labeled antibody, it may consist of 0.33 mg nitrotetrazolium blue and 0.17 mg 5-bromo-4-chloro-3-indolylphosphate per milliliter dissolved in 100 mM Tris HCl - 0.1 M NaCl - 5 mm MgCl ₂ / Leary, JJ, DJ Brigati, and DC Ward, Proc. Natl, Acad. Sci. USA 80: 4,045-4,079, 1983]. When added, a color reaction is formed 2 · The result can be read.

It can be used in human and veterinary medicine, in determining the presence of a gene on the chromosome, in biotechnological procedures, in microbiological diagnostics, in the search for homologous stretches of nucleic acids in the evolutionary context of organs.

Claims (3)

Example 26 Denatured single-stranded deoxyribonucleic acid 1 was transferred to nitrocellulose filter 2 at 5-30 µl, which was left under vacuum at 80 degrees Celziana for 2 hours. Subsequently, hybridization with the pre-determined probe 2 is initiated at 1 to 1000 ng / ml for 3 to 20 hours in hybridization buffer in the presence of 45 to 55% formamide at 42 degrees Celsius. The probe used is a single-stranded nucleic acid segment. The ability to hybridize to an unknown acid at a site that is complementary to the base (Maniatis, Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982). A single-stranded probe or a complex may be used where the single strand forms a specific strand and the other strand may be a double stranded strand. An example of a single-stranded region that is part of a vector (Hu over Messing, Gene, 17, 271-277, 1982). The vector is a small portion of dfoxyribonucleic acid that serves as a foreign DNA carrier, phages, plasmids, and others. After hybridization, nucleic acids 4 are formed at the site of the homologous regions to form a double strand. The sample is overflowed and treated with monoclonal antibody 5 against double-stranded nucleic acid, its ability to bind only to double-stranded structures will form a complex. Binding can be accomplished by labeling the antibody. Enzymatic 5, or radioisotopes 6 are preferred. When the antibody is labeled with enzyme 5, binding of the antibody to probe 3 can be accomplished using substrate f 1. Substrate 8 is a reactant in enzyme catalyzed reactions. For an alkaline phosphatase-labeled antibody, 0.33 mg of nitrotetrazolium blue and 0.17 mg of 5-bromo-4-chloro-3-indolylphosphate per milliliter can be dissolved in 100 mM Tris HCl - 0.1 M NaCl - 5 mm MgCl 2 / Leary, JJ, DJ Brigati, and DC Ward, Proc. Natl, Acad. Sci. USA 80: 4045-4449 (1983). When it is added, a color reaction is created 2 · The result can be read. It is possible to use it in human and veterinary medicine, in the presence of chromosome presence, in biotechnological procedures, in microbiological diagnostics, in the search for homologous regions of nucleic acids in the evolutionary context of organisms. SUBJECT MATTER A method for determining homologous regions of nucleic acids, for example a genome on a chromosome in which a double-stranded nucleic acid is first denatured to a single-stranded nucleic acid to which a probe comprising at least 8 phages is used, characterized in that the reaction system containing the carrier, e.g. nylone-fixed investigated single-stranded nucleic acid and the probe itself, optionally incorporated into the vector nucleic acid, adds specific DNA-antibody and evaluates, for example, by adding a substrate to form a color reaction. 2. Process according to claim 1, characterized in that a considerable antibody specific to the DNA antibody is added to the reaction system and the evaluation is performed. 3 drawings