RU158971U1 - BIOCHIP - Google Patents

Abstract

1. A biochip for detecting pathogenic mutations in coding regions of genes that are significant for the disease, which is a substrate with a set of oligonucleotides immobilized on it, characterized in that the substrate is made of a transparent material with a coating that provides strong binding of the oligonucleotide to the surface of the substrate, the set of immobilized oligonucleotides includes sequences 25-60 bases long, with and without a genetic marker (3 repeats of each oligonucleotide), as well as oligonucleotides from enes human housekeeping genes from other organisms that have no homology to the human genome.2. The biochip according to claim 1, characterized in that oligonucleotides containing a genetic marker are immobilized on a substrate with the sequence shown in Table. 1.3. The biochip according to claim 1, characterized in that oligonucleotides containing a genetic marker are immobilized on a substrate with the sequence shown in Table. 2.4. Biochip according to any one of paragraphs. 1, or 2, or 3, characterized in that oligonucleotides from the genes of the human household and from the genes of other organisms that do not have homology in the human genome in the sequence shown in Table 1 are immobilized on the substrate. 3.

Description

The utility model relates to molecular biology, microbiology and medicine, in particular, to methods for identifying and identifying known polymorphisms of genes responsible for the development of various diseases using a differentiating oligonucleotide biological microchip (biochip).

Accurate diagnosis in combination with a detailed analysis of the type of inheritance of a disease is crucial for the formation of risk groups, that is, the selection of patients in whom the likelihood of the disease is increased.

Currently, several methods are known for screening for various diseases.

For example, the biochip is known according to the patent of the Russian Federation for invention No. 2303634 (IPC C12N 15/11, publ. July 27, 2007), adopted as a prototype. The well-known biochip is based on an original version of the multiplex polymerase chain reaction (NDP) in two stages, followed by hybridization of a fluorescently labeled DNA fragment on a biological microchip containing an original set of differentiating oligonucleotides, as well as registration and interpretation of the results.

The disadvantage of the biochip according to the patent of the Russian Federation No. 2303634 is the use of multiplex polymerase chain reaction (PCR). PCR is rather laborious, it takes a lot of time, and therefore cannot cover a large number of pathogenic mutations, which reduces the specificity and reliability of the analysis.

The problem solved by the proposed utility model is to increase the specificity and reliability of the express analysis while simplifying its implementation. The utility model is also based on the task of creating a biochip that ensures the optimal functioning of biological macromolecules immobilized in it to detect pathogenic mutations in coding regions of genes and mutations that are significant for the disease outside coding regions, as well as in intergenic spaces as markers of the diagnosed disease.

The problem is solved in that in a biochip, which is a substrate with a set of oligonucleotides immobilized on it, according to the invention, the substrate is made of a transparent material with a coating that provides strong binding of the oligonucleotide to the surface of the substrate, the set of immobilized oligonucleotides includes sequences of 25-60 bases in length, containing and not containing a genetic marker (3 repeats of each oligonucleotide), as well as oligonucleotides from the genes of the human household and from the genes of each x organisms that have no homology to the human genome.

The immobilization of oligonucleotides with a length of 25-60 bases on a microchip increases the specificity of hybridization and, as a result, the probability of detecting a pathogenic mutation. In combination with 3 repetitions of each oligonucleotide, positive and negative controls, this will increase the specificity and reliability of the rapid analysis.

To diagnose a genetic predisposition to breast cancer on a biochip, it is advisable to immobilize oligonucleotides containing a genetic marker in the sequence shown in Table. one.

To diagnose a genetic predisposition to scoliosis on a biochip, it is advisable to immobilize oligonucleotides containing a genetic marker in the sequence shown in Table. 2.

Sequences of non-mutant oligonucleotides in both cases are taken from standard gene sequences published in the database.

The immobilization of oligonucleotides on the substrate, containing and not containing a genetic marker, allows you to simultaneously determine the presence of a pathogenic mutation and its status without additional operations that complicate the rapid analysis.

The presence on the biochip of oligonucleotides from genes in human households and from genes that do not have homology in the human genome provides control over the correctness of nucleic acid isolation from a patient’s blood sample, reverse transcription reaction, labeling, hybridization, and washing the microchip. Such control also increases the specificity and reliability of the rapid analysis.

For positive and negative controls, it is advisable to immobilize on a microchip oligonucleotides from genes of the human household and from genes of other organisms that do not have homology in the human genome in the sequence shown in Table. 3.

The invention is illustrated in Fig., Which schematically depicts the inventive biochip and tables, where in the table. 1 shows a list of oligonucleotide sequences for identifying pathogenic mutations in breast cancer, in table. 2 - a list of oligonucleotide sequences for identifying pathogenic mutations of scoliosis, in table. 3 is a listing of oligonucleotide sequences for positive and negative controls.

An example of a specific implementation of the inventive biochip for the diagnosis of susceptibility to breast cancer.

On a glass substrate of 25 × 75 × 1 mm in size coated with a material that provides strong adhesion of oligonucleotides to the surface (SuperEpoxy 3 from Arrayit), the oligonucleotides listed in Table 1 were immobilized by the method of robotic printing on a SpotBot® 2 microarray printer from Arrayit.

As positive and negative controls, confirming the correctness of all microchip processing operations and increasing the reliability of the analysis, the oligonucleotides listed in Table 3 are immobilized on the microchip.

A leukocyte fraction was isolated from a clinical sample, 5 ml, of venous blood by centrifugation, from which total RNA was isolated using the RIAeasy Protect Cell Mini Kit from QIAGEN.

Using the Invitrogen Superscript VILO Kit from Invitrogen, a reverse transcription reaction was carried out with simultaneous labeling when Perkin Elmer was added to the reaction mixture Su5 CTP.

After the cDNA production reaction and labeling, the mixture of labeled probes was cleared of the tag not included using the Fluorescent Probe Purification Kit, Single Column Format from Arrayit.

A labeled probe was mixed with hybridization buffer, placed on a glass-printed microchip under a coverslip, and hybridized in a sealed hybridization chamber at an appropriate temperature to discriminate mutant alleles from wild-type alleles.

After hybridization, the microarrays were washed in a series of buffers under conditions ensuring the washing of non-specifically bound probes.

The washed microchip was scanned with a Agilent Technologies SureScan Microaaray Scanner and analyzed using Agilent Microarray Control software. In the general analysis, all the positive control points on the microchip scan are lit, but there are no negative control points, it was concluded that the procedures for isolating RNA, obtaining cDNA, labeling, hybridization and washing were carried out correctly, and positive signals from printing points of mutant marker alleles can be trusted. Since oligonucleotides with mutant and normal marker alleles are printed under each other, the columns of the printing points of normal alleles should all shine and in all three repetitions, except when the carrier of any mutation is homozygous for this mutation. If the carrier is heterozygous for any mutation, the print points of both the mutant and normal variants will light up. A single individual can be a carrier of several mutations in one or different genes, in a homozygous or heterozygous state, and these facts provide the basis for calculating the risks of breast cancer occurrence and development in a mutation carrier before clinical symptoms of the disease appear.

An example of a specific implementation of the proposed method, a biochip and a set of oligonucleotides for the diagnosis of predisposition to scoliosis.

On a glass substrate of 25 × 75 × 1 mm in size coated with a material that provides strong adhesion of oligonucleotides to the surface (SuperEpoxy 3 from Arrayit), the oligonucleotides listed in Table 2 were immobilized by the method of robotic printing on a SpotBot® 2 microarray printer from Arrayit.

As positive and negative controls, confirming the correctness of all microchip processing operations and increasing the reliability of the analysis, the oligonucleotides listed in Table 3 are immobilized on the microchip.

A leukocyte fraction was isolated from a clinical sample, 5 ml, of venous blood from a patient with diagnosed idiopathic scoliosis by centrifugation, from which total DNA was isolated using the QIAamp DNA Mini Kit.

The isolated DNA was nick-transcribed using the PromoFluor-550 Nick Translation Labeling Kit. The labeled probe was cleaned using the Fluorescent Probe Purification Kit from Arrayit.

A labeled probe was mixed with hybridization buffer, placed on a glass-printed microchip under a coverslip, and hybridized in a sealed hybridization chamber at an appropriate temperature to discriminate mutant alleles from wild-type alleles.

After hybridization, the microarrays were washed in a series of buffers under conditions ensuring the washing of non-specifically bound probes.

The washed microchip was scanned on a SureScan Microaray Scanner from Agilent Technologies, and analyzed using Agilent Microarray Control software.

If during the general analysis all the positive control points on the scan of the microchip glow, but no negative control points, then it was concluded that DNA extraction, labeling using nick translation (or another way), hybridization and washing were carried out correctly, and the positive signals from printing points of mutant marker alleles can be trusted. Since oligonucleotides with mutant and normal marker alleles are printed under each other, the columns of the printing points of normal alleles should all shine and in all three repetitions, except when the carrier of any mutation is homozygous for this mutation. If the carrier is heterozygous for any mutation, the print points of both the mutant and normal variants will light up. A single individual can be a carrier of several mutations in one or different genes, in a homozygous or heterozygous state, and these facts provide the basis for calculating the risks of scoliosis in a patient even before the clinical symptoms of the disease appear.

Claims (4)

1. A biochip for detecting pathogenic mutations in coding regions of genes that are significant for the disease, which is a substrate with a set of oligonucleotides immobilized on it, characterized in that the substrate is made of a transparent material with a coating that provides strong binding of the oligonucleotide to the surface of the substrate, the set of immobilized oligonucleotides includes sequences 25-60 bases long, with and without a genetic marker (3 repeats of each oligonucleotide), as well as oligonucleotides from enes human housekeeping genes from other organisms that have no homology to the human genome. 2. The biochip according to claim 1, characterized in that oligonucleotides containing a genetic marker are immobilized on a substrate with the sequence shown in Table. one. 3. The biochip according to claim 1, characterized in that oligonucleotides containing a genetic marker are immobilized on a substrate with the sequence shown in Table. 2. 4. The biochip according to any one of paragraphs. 1, or 2, or 3, characterized in that oligonucleotides from the genes of the human household and from the genes of other organisms that do not have homology in the human genome in the sequence shown in Table 1 are immobilized on the substrate. 3.

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