RU2617936C2 - Method for genetic polymorphism rapid analysis for detection of genetic predisposition to breast cancer - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method is for rapid analysis of genetic polymorphism to detect genetic predisposition to breast cancer (BC). 40 bases long oligonucleotides containing the genetic marker and not containing the genetic marker shown in Table 1, are immobilized on the microchip, 3 repetitions of each oligonucleotide, together with those shown in Table 3 from human housekeeping gene as a positive control and from genes of other organisms that have no homology in the human genome, as a negative control. Nucleic acids are selected from the blood sample, using the total RNA or DNA. A labeled probe is obtained for hybridization by reverse transcription reaction with simultaneous fluorescent labeling for RNA or nick translation labeling of DNA. The obtained probe is hybridized with oligonucleotides immobilized on the microchip. The non-specifically bound probe is washed under conditions discriminating against mutant genotypes. The microchip is scanned and the results evaluated.

EFFECT: increased specificity and reliability of the analysis and improved breast cancer risk assessment reliability.

3 tbl, 1 dwg

Description

The invention 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 following can be distinguished.

A known method for diagnosing a genetic predisposition to myocardial infarction in men (RF patent for the invention No. 2182175, IPC C12Q 1/68, publ. 05/10/2002), including the identification of genotypes of a genomic locus containing a gene allele, characterized in that genotyping is performed in the studied patients the V64I polymorphism of the CCR2 gene and the identification of the genotype of the 3q21 genomic locus containing the allele of the chemokine receptor CCR2 gene with G replaced by A at the 190th position of the coding part, leading to the replacement of V by I at the 64th position of the amino acid sequence of the CC protein R2 641, a genetic predisposition to myocardial infarction in men is diagnosed.

The disadvantage of this method is the lack of specificity of the rapid analysis, due to the fact that this method evaluates only one factor out of many that affect the risk of developing cardiovascular diseases and therefore cannot provide complete information about the patient.

Known analysis and use of polymorphic forms of PARI to assess the risk of cardiovascular disease (Application No. 2005136430, 2006.04.20), based on the detection of point mutations in the PARI gene. The isolated polynucleotide sequence of the PARI gene is characterized in that at position 3090 of the sequence according to NM_001992 there is a substitution of T for C.

The disadvantage of this method is the limited information content and the inability to predict the risks of various pathologies, the cardiovascular system, independent of the PARI gene.

A common drawback of the above methods: they are impractical to apply for mass screening, because they are laborious and allow you to diagnose only one disease.

Closest to the proposed methods are a rapid analysis of genetic polymorphism to determine the pharmacogenetic status of a person and identify a genetic predisposition to cancer, a biochip and a set of oligonucleotides according to the RF patent for invention No. 2303634 (IPC C12N 15/11, published on July 27, 2007). The method is based on carrying out an original version of a multiplex polymerase chain reaction (PCR) 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 this method and biochip 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 invention is to increase the specificity and reliability of the express analysis while simplifying its implementation. The invention 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 a diagnosed disease.

The problem is solved in that in the method of rapid analysis of genetic polymorphism to identify a genetic predisposition to the diagnosed disease, including verification of pathogenic mutations in the clinical sample of NK by differentiating hybridization of labeled probes from the patient's NK, comprising the following stages:

- immobilization of oligonucleotides on a microchip;

- isolation of nucleic acids from a patient’s blood sample;

- obtaining a labeled probe for hybridization;

- hybridization of the obtained labeled probe with oligonucleotides immobilized on a microchip;

- washing of a non-specifically bound probe under conditions that discriminate mutant genotypes;

- scanning a microchip and obtaining results;

- an overall assessment of the results of the analysis;

according to the invention:

- oligonucleotides of 25-60 bases in length containing and not containing a genetic marker (3 printed dots for each oligonucleotide), as well as oligonucleotides from genes of human households and from genes of other organisms that do not have homology in the human genome, are immobilized on a microchip;

- a labeled probe is obtained by the reverse transcription reaction with simultaneous labeling with a fluorescent label for RNA or nick-translation labeling for DNA; moreover, total RNA or DNA from cells of a clinical sample is used.

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.

Obtaining a labeled probe from total RNA or DNA of a clinical sample also increases the specificity of hybridization and the reliability of the results. In addition, it allows to simplify the rapid analysis by eliminating additional operations.

The inventive method allows to obtain a more comprehensive assessment of the genetic predispositions of the patient to the diagnosed disease, and more accurately assess the risks of developing this disease.

The sequence of oligonucleotides containing a genetic marker determines which disease will be diagnosed. So, to diagnose a genetic predisposition to breast cancer (BC), it is advisable to use oligonucleotides with the sequence shown in Table 1 as oligonucleotides containing a genetic marker. one.

The proposed set of genetic markers sufficiently comprehensively covers known pathogenic mutations causing a predisposition to the development of breast cancer, and the length and sequence of oligonucleotides containing pathogenic mutations allows one to reliably discriminate mutant alleles from wild-type alleles, even if they differ only by a single nucleotide substitution.

To diagnose a predisposition to scoliosis, it is advisable to use oligonucleotides with the sequence shown in Table 1 as oligonucleotides containing a genetic marker. 2

The set of genetic markers developed by the authors, as well as the set of markers for diagnosing a predisposition to breast cancer, provides high specificity and reliability of the express analysis.

If necessary, the diagnosis of other diseases is the sequence of oligonucleotides, including the optimal number of known pathogenic mutations characteristic of each specific 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.

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.

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 problem is solved in that for the diagnosis of susceptibility to breast cancer, a set of oligonucleotides is used to identify pathogenic mutations with the sequences shown in table. one.

To diagnose a predisposition to scoliosis, a set of oligonucleotides is used to identify genetic markers with the sequences shown in table. 2.

The sequences of non-mutant oligonucleotides in both cases are taken from standard gene sequences published in the database http://www.ncbi.nlm.nih.gov.

The invention is illustrated by a figure, which schematically depicts the inventive biochip and tables where table 1 shows a list of oligonucleotide sequences for diagnosing a genetic predisposition to breast cancer, table 2 shows a list of oligonucleotide sequences for diagnosing a genetic predisposition to scoliosis, table 3 shows a list of oligonucleotide sequences for positive and negative controls.

The proposed biochip is a substrate 1 made of a transparent material with a coating that provides strong binding of the oligonucleotide to the surface of the substrate. On substrate 1, immobilized sequences of oligonucleotides 25-60 bases long, not containing 2 and containing 3 genetic markers (3 repeats of each oligonucleotide), as well as oligonucleotides 4 from genes of the human household and 5 oligonucleotides from genes of other organisms that do not have homology in human genome.

An example of a specific implementation of the proposed method, a biochip and a set of oligonucleotides for the diagnosis of susceptibility to breast cancer.

On a substrate 1 of glass 25 × 75 × 1 mm in size coated with a material that provides strong adhesion of the oligonucleotides to the surface (SuperEpoxy 3 from Arrayit), the oligonucleotides 2 and 3 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, oligonucleotides 4 and 5 are listed on the microchip, listed in Table 3.

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.

Invitrogen's SuperScript VILO Kit from Invitrogen performed a reverse transcription reaction with simultaneous labeling while adding Perkin Elmer CTP to the reaction mixture.

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.

The labeled probe was mixed with hybridization buffer, placed on a substrate 1 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 4 control points on the scan of the microchip glow, and the negative 5 control points are not, from which it was concluded that the procedures for isolating RNA, obtaining cDNA, labeling, hybridization and washing were carried out correctly, and for positive signals from the printing points of mutant marker alleles can trust. Since oligonucleotides with mutant 3 and normal 2 marker alleles are printed under each other, the columns of the print points of the normal 2 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 the mutant 3 and normal 2 options 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-translated 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 (11)

A method for the rapid analysis of genetic polymorphism to detect a genetic predisposition to breast cancer, comprising verifying pathogenic mutations in a clinical nucleic acid (NK) sample by differentiating hybridization of labeled probes from a patient's NK, comprising the following steps: - immobilization of oligonucleotides on a microchip; - isolation of nucleic acids from a patient’s blood sample; - obtaining a labeled probe for hybridization; - hybridization of the obtained labeled probe with oligonucleotides immobilized on a microchip; - washing of a non-specifically bound probe under conditions that discriminate mutant genotypes; - scanning a microchip and obtaining results; - an overall assessment of the results of the analysis; characterized in that - on a microchip, 40 bases long oligonucleotides containing the genetic marker are shown in the table. 1, and not containing a genetic marker, 3 repetitions of each oligonucleotide, as well as presented in table. 3 oligonucleotides from the genes of the human household as a positive control and from the genes of other organisms that do not have homology in the human genome, as a negative control; - a labeled probe is obtained by a reverse transcription reaction with simultaneous labeling with a fluorescent label for RNA as a clinical sample, or by nick-translation for DNA as a clinical sample, using total RNA or DNA.

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