RU2206615C1 - Method of partial dna sequencing for detection of mutations in short fragments of single-stranded dna using microchip - Google Patents

Abstract

FIELD: molecular biology, medicine. SUBSTANCE: mutations are detected by partial sequencing and using oligonucleotide microchip containing cells with immobilized taken sequences of synthetic oligonucleotides of definite length with hairpin structure. Method involves selection (theoretically) of oligonucleotides with length N (N-oligonucleotides) comprising self-closing hairpin structure. N-oligonucleotides are immobilized on micromatrix and nonself-complementary oligonucleotides with length n (n-oligonucleotides) are immobilized for hybridization butt. Melting point (T_m) of three variants of duplexes, namely: (1) DNA-immobilized N-oligonucleotide; (2) self-closing hairpin; (3) n- oligonucleotide immobilized N-oligonucleotide corresponds to inequality: T_m(1)>>T_m(2)>>T_m3. Test microchip is prepared with theoretically selected immobilized N-oligonucleotides and their hybridization is carried out with mixture of fluorescently labeled model DNA and each of theoretically selected n-oligonucleotides by selection of conditions for equilibration of energetics of AT- and GC-enriched oligonucleotides. Results are recorded by plotting melting curves using method of fluorescent analysis. Based on obtained data N-oligonucleotides are taken for preparing analytical microchip, n-oligonucleotides for hybridization butt and conditions for hybridization. EFFECT: simplified analysis, reduced time of analysis. 14 cl, 4 dwg, 2 ex

Description

 The invention relates to molecular biology and medicine, and contemplates a partial sequencing method for determining mutations in DNA sequences. Partial sequencing of mutations is detected using an oligonucleotide microchip, in the cells of which specially selected sequences of synthetic oligonucleotides of a certain length are immobilized.

 The invention includes an original method for selecting and modifying oligonucleotides immobilized in microchip cells and butt oligonucleotides, as well as a method for recording, analyzing and interpreting the results.

State of the art
A known hybridization approach for DNA sequencing using a microchip containing a complete set of oligonucleotides of a certain length [Dokl. USSR Academy of Sciences, 303 (1988) 1508-1511; USSR patent 1794088; USSR patent 2041261].

 The essence of the method lies in the fact that the DNA fragment is firmly bound in those microchip cells in which oligonucleotides that are fully complementary to the DNA fragment are immobilized. Knowing the sequence of these nucleotides, it is possible to restore the sequence of the original fragment or to determine the position and type of mutation. This approach is called "sequencing by hybridization on an oligonucleotide microchip."

 It is known that DNA sequencing by hybridization is possible only for DNA fragments of a limited length. For example, in the case of hybridization with an octanucleotide microchip, the length of the analyzed DNA fragment does not exceed approximately 200 nucleotides [J. Biomol. Struct. Dyn., 9 (1991) 399-410].

The most obvious way of developing this approach - increasing the length of the oligonucleotides in the hybridization matrix - leads to the fact that K cells should fit on the microchip in the analysis field (K = 4 ^N where N is the length of the oligonucleotide), which will make the microchip very bulky.

 A known approach to increasing the efficiency of sequencing using "continuous hybridization butt". Its essence lies in the fact that oligonucleotides of length N are immobilized on a microchip, DNA is hybridized with a matrix of immobilized oligonucleotides, then short fluorescently labeled oligonucleotides of length n are introduced and hybridization is carried out, which, in the case of formation of a perfect duplex with DNA and hybridization, increases the length of the oligonucleotide oligonucleotide oligonucleotide an oligonucleotide on a microchip to a value of N + n [Molecular. Biology, 27 (1993) 1126-1138].

 The increased stability of an n-oligonucleotide duplex (the so-called butt oligonucleotide) with DNA in the case of butt hybridization is due to the coaxial stacking interaction between N- and n-oligonucleotides [Nucleic Acids Res., 17 (1989) 4551-4565; Biochemistry, 29 (1990) 6017-6025; Nucleic Acids Res., 29 (2001) 2303-2313].

 When immobilizing octanucleotides (N-oligonucleotides) onto a matrix and hybridizing end-to-end with different sets of pentanucleotides (n-oligonucleotides), the length of a DNA fragment sequenced using an octanucleotide matrix increases from 200 to several thousand nucleotides [J. Biomol. Struct. Dyn., 11 (1994) 797-812].

 However, a method for reconstructing a DNA sequence using “continuous butt hybridization” has several disadvantages.

disadvantages
1. Steric difficulties in the gel lead to the fact that a lot of free places remain on the microchip. Butt oligonucleotides can hybridize on immobilized N-oligonucleotides of a microchip (the so-called parasitic cross hybridization), introducing false signals, worsening the technological properties of the microchip (reducing the working range of concentrations of an unknown sample and the reliability of sequence recovery and reducing the accuracy of determining the ratio between native and mutant forms in analyzed sample).

2. a) Butt oligonucleotides can form tandem structures (parasitic tandem hybridization) on the continuation of the analyzed single-stranded DNA and give a false signal; which will also lead to an error in DNA reconstruction;
b) butt oligonucleotides can dock at the other end of the N-oligonucleotide and also give a false signal.

 3. The different energies of AT- and GC-rich oligonucleotides (different dissociation temperatures) do not allow monitoring at the same temperature (ie, simultaneous determination of all mutations) and require several rounds of hybridization and construction of melting curves, which complicates the analysis and increases its duration, significantly complicating the hardware complex for monitoring.

4. The fluorescence method does not allow to unambiguously identify each of the 4 ⁵ pentanucleotides used for end-to-end hybridization due to the absence of the corresponding number of fluorescent labels differing in their parameters.

The basis of the invention is the creation of a method of partial DNA sequencing to determine mutations in short fragments of single-stranded DNA using a microchip, allowing:
1) increase the reliability of the interpretation of the results to restore the original DNA sequence in order to localize the mutation;
2) increase the working range of concentrations of the test sample and increase the signal-to-noise ratio during analysis;
3) to simplify the analysis and reduce its duration.

 The problem is solved by the invention.

SUMMARY OF THE INVENTION
A method is proposed for partial DNA sequencing to determine mutations in short fragments of single-stranded DNA using a microchip, including:
I. The choice of oligonucleotides of length N (N-oligonucleotides) for immobilization on a microarray and oligonucleotides of length n (n-oligonucleotides) for hybridization butt to immobilized N-oligonucleotides forming perfect duplexes with the analyzed DNA, moreover
the selection of N-oligonucleotides for immobilization and n-oligonucleotides for butt hybridization is carried out in accordance with the following criteria:
a) immobilized sequences on a microchip (N-oligonucleotides) must have a self-closing hairpin structure to prevent parasitic cross-hybridization,
b) n-oligonucleotides must be non-self-complementary and to prevent possible spurious tandem hybridization contain a stacking terminator at the non-seamless end,
c) the melting temperature of the three duplex variants, namely, (1) DNA - an immobilized N-oligonucleotide, (2) a self-closing hairpin, and (3) n-oligonucleotides - an immobilized N-oligonucleotide, must meet the inequality
T _{pl (1)} >> T _{pl (2)} >> T _{pl (3)} [1],
and the logic of choosing N- and n-oligonucleotides follows a certain algorithm that uses an empirical model for calculating the thermodynamic parameters of DNA duplexes [Biochemistry, 35 (1996) 3555-3562].

The selection of N-oligonucleotides for the manufacture of a microchip intended for determining mutations (analytical microchip) and n-oligonucleotides for hybridization with them is carried out using a microchip with theoretically selected immobilized N-oligonucleotides (test microchip) by hybridizing them with a fluorescence mixture DNA and each of the n-oligonucleotides separately under conditions of equalization of the energy of AT- and GC-rich oligonucleotides and registration of the results of hybridization constructing melting curves corresponding thermodynamic transitions duplexes formed, using the method of fluorescence analysis;
II. Immobilization on a microchip of N-oligonucleotides, moreover
the immobilization of N-oligonucleotides selected in accordance with claim 1, is carried out in a buffer solution at pH, ionic strength and temperature, providing a closed state of the hairpin.

III. Hybridization of the analyzed DNA and n-oligonucleotides with a microchip, moreover
for hybridization with an analytical microchip, a mixture of the analyzed single-stranded DNA and non-self-complementary butt n-oligonucleotides that serve as a mass label in mass spectrometric analysis or modified by introducing a mass label in case of degeneracy of their masses are used, and hybridization is carried out under conditions of equalization of the AT and GC- energies rich oligonucleotides;
IV. Registration of the results of hybridization, allowing the interpretation of the results in decoding the mutation, and
identification of n-oligonucleotides that have formed a perfect duplex with the analyzed DNA and hybridized end-to-end to N-oligonucleotides immobilized on an analytical microchip is carried out by mass spectrometry;
V. Interpretation of hybridization results, moreover
the reconstruction of the sequence of the analyzed DNA is carried out in stages, based on the structures of the immobilized N-oligonucleotide, which includes a fragment of length L, which forms a perfect duplex with the analyzed DNA, and an n-oligonucleotide, which hybridizes end-to-end to it, which forms a perfect duplex with DNA upon hybridization and detected on based on mass spectrometry data. The cell position of the analytical microchip determines the structure of an N-oligonucleotide containing an L-fragment complementary to a specific region of the analyzed DNA, and the mass of the informative peak in the mass spectrum recorded from this cell allows one to uniquely identify the n-oligonucleotide, and for unambiguous identification of n-oligonucleotides in the case of degeneracy of their structure, various linkers are used as mass tags.

 This determines the sequence "L + n" in the cell. The analysis of the sequences of partially overlapping N-oligonucleotides immobilized in the microarray cells (tile method) in combination with the identification of n-oligonucleotides attached to them allows reconstructing the sequence of the analyzed DNA. Comparison of the reconstructed DNA sequences of wild and mutant types makes it possible to uniquely decipher the mutations.

 As a fluorescent label of n-oligonucleotides, the Texas Red dye is used to plot the melting curves on a test microchip.

 To introduce a fluorescent label into model DNA, fluoresceinisothiocyanate (FITC) is used to construct melting curves on a test microchip.

 As mass labels for analysis by mass spectrometry, combinations of standard aminolinkers of different lengths associated with n-oligonucleotides in their synthesis by the standard automated phosphoramidite method are used.

 As stacking terminators, FITC, Texas Red or their analogs or a ureide derivative of 2'-deoxyribose are used.

 The method involves the use of a microchip, which is a microarray with immobilized N-oligonucleotides having a hairpin structure, a fragment of each of which is complementary to a constant region of the analyzed gene in the region of mutations.

 A microchip with immobilized N-oligonucleotides, which selects N-oligonucleotides, n-oligonucleotides and hybridization conditions with model DNA by determining the melting points of duplexes using fluorescence analysis and thermodynamic parameters so that the melting points of the duplexes meet the inequality [1], is test.

 A microchip with immobilized N-oligonucleotides selected on a test microchip is analytical. The analysis of the results of hybridization on an analytical microchip is carried out using the method of mass spectrometry.

 The method involves the use of a microchip specially prepared for solving specific problems, containing a small number of cells with immobilized sequences selected for each specific case, in order to maximize the sensitivity and working range of the method.

Disclosure of the invention
The proposed method for determining mutations in a gene first involves the study of the region of the gene in which mutations will be determined using literature data. In this case, sections containing mutations are isolated.

 To determine the mutations, two types of microchips are used - test and analytical.

 A microchip is a polyacrylamide gel microarray containing cells with immobilized oligonucleotides.

 Using an empirical model for calculating the thermodynamic parameters of DNA duplexes [Biochemistry, 35 (1996) 3555-3562], select possible N-oligonucleotides and n-oligonucleotides that should participate in the formation of perfect duplexes with constant regions of the analyzed DNA and, accordingly, with its variable regions, in which all possible mutations ("hot spots" of the gene) are localized, and the number of necessary butt oligonucleotides depends on the number of mutations. In the case when the mutations are close and the distance between them is less than the length of the N-oligonucleotide sequence complementary to DNA (without taking into account the length of the hairpin part), another (neighboring) mutation (not detected in this cell) would fall into the N- hybridization zone oligonucleotide and would introduce additional instability, since a mismatch would possibly form, destabilizing the duplex of the N-oligonucleotide with DNA. The solution to the problem is to introduce one or several universal bases (mainly nitroindole) into the N-oligonucleotide opposite the mutation in DNA and to recalculate the entire thermodynamic scheme of oligonucleotides according to the NN1 model taking into account the thermodynamic coefficients for universal bases [see Biochemistry, 35 (1996) 3555-3562].

 The selected N-oligonucleotides that are intended to be immobilized on a test microchip are synthesized. Synthesize model fluorescently labeled target DNA and fluorescently labeled n-oligonucleotides, in which the fluorescent labels are different.

 N-oligonucleotides have a self-closing hairpin structure, they are immobilized on the matrix under conditions that provide a closed state of the hairpin. The advantage of using an immobilized N-oligonucleotide in the form of a self-closing hairpin is that this structure allows to suppress parasitic cross-hybridization.

 The butt n-oligonucleotides are non-self-complementary and contain a stacking terminator at the non-seamless end to prevent possible spurious tandem hybridization.

The melting points (T _PL ) of the three duplex variants, namely (1) DNA - an immobilized N-oligonucleotide, (2) a self-closing hairpin, and (3) n-oligonucleotides - an immobilized N-oligonucleotide, must meet the condition T _{mp (1)} >> T _{pl (2)} >> T _{pl (3)} [1], i.e. the thermodynamics of the hairpin must be selected so that it is completely open during hybridization with DNA, completely closed in its absence and not hybridized with n-oligonucleotides in the closed state. When a perfect duplex with DNA is formed, the hairpin must remain open, and the butt n-oligonucleotide must hybridize end-to-end to the end of the hairpin N-oligonucleotide. It is in this case that the stacking effect is manifested with the greatest force, and maximum discrimination is achieved in the method.

 N-oligonucleotides immobilized on a test microchip hybridize with fluorescently labeled DNA and n-oligonucleotides under conditions of equalization of the energy of AT- and GC-rich oligonucleotides, which is ensured by the use of equalization buffer solutions.

 By varying the length of the N-oligonucleotides and the design of the hairpins immobilized on the test microchip, the melting points of all three types of duplexes are selected in a known narrow range so that the above inequality [1] is observed for all cells of the microchip and all n-oligonucleotides of the mixture.

 The oligonucleotide duplexes immobilized on the test microchip are melted using a controlled thermostol, and the melting is recorded using an automated fluorescence microscope equipped with a highly sensitive CCD camera connected to a computer. An array of experimental data obtained during melting is processed using a computer program that calculates the melting temperatures and thermodynamic parameters of duplexes.

 N-oligonucleotides selected in this way, ensuring the fulfillment of the above inequality, are used to prepare an analytical microchip.

 Then carry out the determination of mutations in the analyzed DNA. For this, the analytical microchip is hybridized with a mixture of the prepared sample and n-oligonucleotides, which are also selected using a test microchip and are intended for analysis by mass spectrometry. Oligonucleotides that differ in molecular masses are used without further modification, while in the case of mass degeneracy they are modified by the introduction of standard aminolinkers.

 At the end of hybridization, the microchip is washed from non-specifically bound oligonucleotides and heated in the presence of a mass spectral matrix to destroy duplexes of n-oligonucleotides that hybridize end-to-end to N-oligonucleotides with the analyzed DNA. Released butt n-oligonucleotides recorded using mass spectrometry. The cell position of the analytical microchip determines the structure of the N-oligonucleotide containing a fragment complementary to a certain region of the analyzed DNA, and the mass of the informative peak in the mass spectrum recorded from this cell allows us to uniquely identify the n-oligonucleotide.

 When hybridizing a mixture of target and butt oligonucleotides, only one of them hybridizes end-to-end to the immobilized oligonucleotide with the formation of a perfect duplex. It is also possible the formation of a mismatch duplex of the butt oligonucleotide with the open part of the hairpin, which, however, does not interfere with detection.

 Mutations are determined by comparing sequences of n-oligonucleotides complementary to the variable regions of wild-type DNA and the test sample.

The invention is illustrated by the following examples and figures, in which
figure 1 depicts a schematic diagram of the hybridization of DNA and butt oligonucleotides with immobilized oligonucleotides, both with and without hairpin structure;
FIG. 2 represents the mass spectra of pentanucleotides: hybridized end-to-end to the immobilized oligonucleotide and “parasitic”;
figure 3 represents the principle of operation of the analytical microchip;
FIG. 4 presents the results of determining point mutations in the rpoB gene of mycobacterium Mycobacterium tuberculosis.

 Example 1. The use of self-closing hairpin structures to increase the reliability of determining the mutation in the analyzed DNA.

Theoretically selected 5-, 10-, and 21-membered oligonucleotides and target nucleotides are synthesized on an ABI-394 DNA RNA synthesizer (Applied Biosystems, Foster City, CA, USA). The 3′-amino group is introduced into 10- and 21-membered oligonucleotides using a 3 ′ amino modifier C ₇ CPG (Glen Research Corp., Sterling, VA, USA) during the synthesis. Oligonucleotides are purified by reverse phase high performance liquid chromatography on C-18 Nucleosil (Sigma, St Louis, MO, USA) or by electrophoresis in denaturing polyacrylamide gel.

 Mass spectra were recorded on a Kompact MALDI 4 mass spectrometer (Kratos Analytical, Chestnut Ridge, NY, USA).

 Microchips containing 10- and 21-membered oligonucleotides immobilized in a polyacrylamide gel are manufactured using standard technology [Proc. Natl Acad. Sci. USA, 93 (1996) 4913-4118].

 Microchips for mass spectral monitoring are made on an electrically conductive silicon substrate with a size of 50 • 10 • 0.3 mm, they consist of 2 gel cells with a size of 1 • 1 • 0.02 mm, which are 1.5 mm apart. Each gel cell is fragmented and consists of an array of microcells measuring 0.1 • 0.1 • 0.02 mm.

Model microchip contains 2 cells (figure 1). In one of them, a regular (linear) 10-membered oligonucleotide (L) is immobilized, and in the second a self-closing hairpin (21-membered oligonucleotide, N), both oligonucleotides are able to form duplexes of the same length (10 bp) with the analyzed DNA. Both cells hybridize with DNA containing a mutation in the stacking zone of a 5-membered n-pentanucleotide with immobilized L- or N-oligonucleotide in 1 M TEAA buffer at pH 7.0 and 15 ^{° C.} for a day. Before hybridization, the microchip with the hybridization solution is heated at 60 ^o C for 5 minutes, after which the temperature is sharply reduced to 15 ^o C.

After hybridization, the microchip is washed from non-specifically bound oligonucleotides at 5 ^{° C.} for 10 minutes. Then, 0.7 μl of a solution of the mass spectral matrix (0.3% ammonium citrate and a saturated solution of 2-amino-5-nitropyridine (2A5NP, Sigma)) is applied to each cell of the microchip and the microchip is incubated under sealed conditions at a dew point of 55 ^° C in within 20 minutes Then the microchip is quickly dried, placed in a mass spectrometer, and mass spectra are recorded from each cell (Fig. 2). In order to verify the reliability and accuracy of the proposed method of direct mass spectral analysis of the microchip, the oligonucleotides from the cells are eluted on a standard slide for analysis by KRATOS. Mass spectra are obtained in a linear mode with pulsed extraction and registration of negative ions.

Due to steric difficulties in the gel, 5% of the immobilized oligonucleotides are involved in hybridization with DNA (Fig. 1, conditional numbers), moreover, in accordance with the different resistance of duplexes (see inequality [1] above), hybridization with a hairpin is accompanied by its opening. Then the microchip is hybridized with a mixture of butt n-oligonucleotides, one of which (informative, n ₁ ) is complementary to DNA in the mutation zone and mates with the immobilized sequence; its content corresponds to the content of hybridized DNA (not more than 5%) in both cells.

The other n-oligonucleotide of the mixture (n ₂ ) is able to hybridize with an immobilized L-oligonucleotide that has not participated in hybridization with DNA (up to 95%, cell 1), or with the free half of the immobilized hairpin (N-oligonucleotide), revealed as a result of hybridization with DNA (no more than 5%, cell 2). Such parasitic cross hybridization leads to the appearance of a “parasitic” peak in the mass spectrum. As can be seen from the above diagram (Fig. 1) and experimentally obtained mass spectra (Fig. 2), the spurious peak is higher in intensity than the informative peak in the case of immobilization of a conventional oligonucleotide (cell 1) and is practically absent in cell 2 containing an immobilized hairpin structure. This significantly increases the reliability of determining the mutation in the analyzed DNA.

 The results of mass spectral analysis of the microchip and slide are the same.

 The method proposed by this invention was applied to a real model - the detection of mutations leading to rifampicin resistance in Mycobacterium tuberculosis.

 The main problem encountered in the treatment of tuberculosis is the resistance of mycobacterial strains to antibiotics. Rifampicin is the most common drug for the treatment of tuberculosis, so a quick determination of resistance to it is of great medical and economic importance.

 Example 2. Determination of mutations in the rpoB gene of mycobacteria Mycobacterium tuberculosis.

 The vast majority of mutations that cause rifampicin resistance in mycobacteria are point-wise and concentrated in a rather small (~ 80 bp) variable region of the rpoB gene encoding the β-subunit of RNA polymerase.

 The sequence of this region of the gene is shown in FIGS. 3 and 4, and the M1-M4 mutations are localized in only four “hot spots” separated by constant regions.

 The definition of mutations involves the use of two types of microchips - test and analytical.

 The first of them serves to select n- and N-oligonucleotides so that the melting points of the corresponding duplexes correspond to inequality [I]. Selection is made by constructing melting curves on a microchip using a fluorescence microscope [J. Opt. Technol., 65 (1998) 938-941]. N-oligonucleotides selected in this way are used to prepare an analytical microchip.

 To select the N- and n-oligonucleotides, five 81-membered target nucleotides were synthesized, containing the variable part of the rpoB gene and corresponding to one wild type of mycobacteria and four mutant, mutations in which are responsible for the following amino acid substitutions: 1) 516 Asp-Val, 2 ) 522 Ser-Leu; 3) 526 His-Leu; and 4) 531 Ser-Leu. To select the thermodynamic parameters by the method of fluorescence analysis and the subsequent manufacture of an analytical microchip, fluorescent labels were introduced into all targets.

Theoretically selected 5-membered and 27-membered oligonucleotides (n- and N-oligonucleotides) and 81-membered target nucleotides are synthesized on an ABI-394 DNA RNA synthesizer (Applied Biosystems, Foster City, CA, USA). The 3'-amino group is introduced into the N-oligonucleotides using the 3'-amino C ₇ CPG modifier (Glen Research Corp., Sterling, VA, USA) during the synthesis. The introduction of FITC tags into and 81-membered target nucleotides and TR tags into n-oligonucleotides is carried out using the standard protocol [Haugland, RP, (1996) Hadnbook of Fluorescent Probes and Research Chemicals, 6th Edn., Molecular Probes Inc., Eugene, OR, USA]. Unlabeled and fluorescently labeled oligonucleotides are purified by C-18 Nucleosil reverse phase high performance liquid chromatography (Sigma, St Louis, MO, USA) or by denaturing polyacrylamide gel electrophoresis.

 Mass spectra were recorded on a Kompact MALDI 4 mass spectrometer (Kratos Analytical, Chestnut Ridge, NY, USA).

 Microchips for mass spectral monitoring are made on an electrically conductive silicon substrate with a size of 50 • 10 • 0.3 mm, they have linear ordering, consist of 4 gel cells with a size of 1 • 1 • 0.02 mm, which are separated by 1 5 mm. Each gel cell was fragmented and consisted of an array of microcells measuring 0.1 • 0.1 • 0.02 mm.

 A standard microchip for fluorescence monitoring consists of cells fixed on a glass substrate measuring 0.1 • 0.1 • 0.02 mm, located in a two-dimensional array and 0.2 mm apart.

 A microchip containing 27-membered oligonucleotides complementary to constant regions of a gene near mutations immobilized in a polyacrylamide gel is manufactured using standard technology [Proc. Natl Acad. Sci. USA, 93 (1996) 4913-4118].

(27 нуклеотидов), в которой фрагмент 5^'-TGAATTGGCTCAG-3' (13 нуклеотидов) комплементарен последовательности 3'-ACTTAACCGAGTC-5' (13 нуклеотидов) мишени;
2) фрагмент олигонуклеотида длиной в 13 нуклеотидов выбирают для того, чтобы он а) был уникальным в гене и не повторялся; б) температура плавления его дуплекса с мишенью была выше, чем температура плавления шпилечных структур, образующихся в самой мишени (63^oС); в) температура плавления его дуплекса с мишенью была гораздо выше температуры плавления дуплексов со стыковыми n-олигонуклеотидами (15-20^oС) и выше температуры плавления его шпилечной самозакрывающейся структуры (30^oС). Дальнейшее наращивание длины указанного фрагмента приводит к повышению температуры плавления до 80^oС и выше, что затрудняет отмывку микрочипа для повторных экспериментов и уменьшает специфичность гибридизации;
3) фрагменты 5'-TGAATTGGCTCAG-3' и

в составе 27-членного олигонуклеотида способны образовать самозакрывающуюся шпильку, а последовательность tttt вводят для образования головки шпильки, поскольку известно, что остатки t представляют собой самые гибкие основания [Biochemistry, 33 (1994) 12715-12719);
4) в участок

вводят мисматч

для того, чтобы температура плавления шпильки отвечала неравенству [1] и была в соответствии с теоретическими расчетами [Biochemistry, 35 (1996) 3555-3562]. Тем самым ее понижают с 55^oС (без мисматчей) до 30^oС.A synthetic oligonucleotide N1 for determining the Ml mutation (see FIGS. 3 and 4) is selected as follows:
1) it has a structure

(27 nucleotides), wherein the fragment 5 ^{'-TGAATTGGCTCAG-3'} (13 nucleotides) complementary to the sequence 3'-ACTTAACCGAGTC-5 '(13 nucleotides) of the target;
2) the oligonucleotide fragment with a length of 13 nucleotides is selected so that it a) is unique in the gene and is not repeated; b) the melting temperature of its duplex with the target was higher than the melting temperature of the hairpin structures formed in the target itself (63 ^o C); c) the melting temperature of its duplex with the target was much higher than the melting temperature of duplexes with butt n-oligonucleotides (15-20 ^° C) and higher than the melting temperature of its hairpin self-closing structure (30 ^° C). A further increase in the length of the indicated fragment leads to an increase in the melting temperature to 80 ^o C and higher, which complicates the washing of the microchip for repeated experiments and reduces the specificity of hybridization;
3) fragments 5'-TGAATTGGCTCAG-3 'and

as part of a 27-membered oligonucleotide, they can form a self-closing hairpin, and the tttt sequence is introduced to form the hairpin head, since t residues are known to be the most flexible bases [Biochemistry, 33 (1994) 12715-12719);
4) to the site

introduce mismatch

so that the melting point of the stud meets the inequality [1] and is in accordance with theoretical calculations [Biochemistry, 35 (1996) 3555-3562]. Thus, it is lowered from 55 ^o C (without mismatch) to 30 ^o C.

5) Aminolink-C ₇ -NH ₂ at the 3'-end is introduced for immobilization in a gel [Anal. Biochemistry, 250 (1997) 203-211; Nucleic Acids Res., 24 (1996) 3142-3148)].

 The selection of immobilized and butt oligonucleotides is carried out by calculation and subsequent verification by taking the melting curves on a test microchip using the method of fluorescence analysis and in solution (for hairpin structures, UV spectroscopy).

 Hybridization of the analyzed sample and one of the butt pentanucleotides is carried out in one cycle. 100 μl of a mixture of a 1 μM solution of a FITC-labeled 81-membered oligonucleotide and 5 μM solution of a TR-labeled 5-membered n-oligonucleotide in 1 M triethylammonium acetate buffer (TEAA, Fluka) was placed in a hybridization chamber.

The hybridization cycle consists of two stages. Initially, the hybridization chamber is placed on a thermostat having a temperature of 20 ^{° C.} for 20 hours. Then the camera was kept for 2 hours at -5 ^° C. Using a fluorescence microscope equipped with a CCD camera, the fluorescence of FITC and TR was recorded, i.e. microchip hybridization is detected.

 Fluorescence measurements of the thermodynamic parameters of duplexes on a microchip are carried out in automatic mode and in real time. The setup consists of a two-wave fluorescence microscope, a CCD camera (Princeton Instruments, Trenton, NJ, USA), a Peltier thermostat, a temperature controller and a computer with a data acquisition board and software. The fluorescence microscope has interchangeable emission and absorption filters for FITC and TR.

The hybridized DNA is washed at 90 ^{° C.} for 5 minutes and the butt hybridization cycle is repeated - melting with another 5-membered n-oligonucleotide.

 All melting points of the butt n-oligonucleotides are equalized so that they all lie in the half-width of the temperature zone of the optimal discrimination of mismatches, which is achieved using the equalization buffer (1M TEAA), and those joints (combinations of two bases in the joint zone) that give the maximum energy the effect.

 Selected synthetic butt oligonucleotides are represented by a mixture of 13 pentanucleotides complementary to the "hot" sections of the gene (figure 3). Each pentanucleotide is complementary to a constant region of the target adjacent to a constant region forming a duplex with a hairpin structure of the N nucleotide and “covers” a “hot spot” in the target, each oligonucleotide of the mixture is complementary to its “hot region” in the target DNA containing a certain type substitutions, and the entire mixture covers all possible combinations of substitutions, i.e. mutations, as well as the wild type.

 The above methodology is applied to the selection of immobilized and butt oligonucleotides designed to determine the mutations M2, M3 and M4.

 One or two universal bases (nitroindole) are introduced into the immobilized oligonucleotides N3 and N4 opposite the mutation in DNA to avoid the formation of a mismatch that destabilizes the duplex of the N-oligonucleotide with DNA, since the M3 and M4 mutations are close, the distance between them is less than the length complementary to The DNA of the N-oligonucleotide sequence (without taking into account the length of the hairpin part), and the adjacent mutation (not detected in this cell) may be in the hybridization zone of the N-oligonucleotide. Accordingly, the entire thermodynamic scheme of oligonucleotides is recalculated according to the NN1 model taking into account the thermodynamic coefficients for universal bases [Biochemistry, 35 (1996) 3555-3562].

 The analytical microchip consists of four cells in which N-oligonucleotides are immobilized, complementary to four different constant parts of the rpoB gene near the mutation sites, selected using a test microchip (Fig. 4).

 Three independent experiments are carried out for each of the four mutation zones, in each of which the microchip is hybridized 1) with wild-type DNA, 2) with mutant-type DNA (with the characterized location and type of nucleotide substitution) 3) with an equimolar mixture of wild-type and mutant DNA (for differential diagnostics) and simultaneously with the same mixture of 13 butt oligonucleotides selected using a test microchip complementary to the “hot” regions of the gene, each n-oligonucleotide of the mixture was chosen complementary to a certain type of substitution us in a certain cell of the chip, and the entire mixture encompasses wild-type, as well as all possible combinations of substitutions that have mutations.

 After hybridization, the microchip is prepared for mass spectrometric analysis, as described in example 1, and mass spectra are recorded from each cell (FIG. 4).

 The formation of a mismatch duplex of the butt oligonucleotide with the open part of the hairpin is also possible, which is visible in the spectra from the 3rd and 4th cells, when a spurious peak is formed. This peak does not interfere with detection.

 The peak in the mass spectrum determines which pentanucleotide has hybridized end-to-end to the immobilized oligonucleotide. In the case of the M1 “hot spot”, 1a (m / z = 1463) turned out to be such a pentanucleotide during hybridization of wild-type DNA, 1b turned out to be such a pentanucleotide (m / z = 1472). Comparison of pentanucleotides, which hybridized end-to-end to an immobilized oligonucleotide, shows the nature of the mutation, namely: in the mutant type DNA at point M1, A replaced by T. The presence of mutant type DNA is also detected when it is present together with wild-type DNA: in mass peaks with m / z = 1465 and 1474 are recorded in the spectrum.

 In each of the 12 cases depicted in Fig. 4, the type of DNA that was used is clearly defined, that is, the absence of mutations is detected (wild type of DNA), or the single nucleotide mutation is determined, namely for M1 - T instead of A, for M2 - T instead of C, for M3 - T instead of A, for M4 - T instead of C, or a mixture of mutant and wild types of DNA is detected.

 The latter circumstance is used to determine the resistance of mycobacterial samples to rifampicin in clinical trials.

Claims (14)

1. A method of partial DNA sequencing to determine mutations in short fragments of single-stranded DNA using a microchip, which consists in sequentially selecting oligonucleotides for immobilization on a microarray and oligonucleotides for hybridization butt to immobilized oligonucleotides forming perfect duplexes with the analyzed DNA; in immobilization of oligonucleotides per microarray; in hybridization of a microchip with a single-stranded DNA sequence being analyzed and butt oligonucleotides; in recording the results of hybridization and in interpreting the results of hybridization, characterized in that (theoretically) N-oligonucleotides are selected (N-oligonucleotides) having a self-closing hairpin structure, for their subsequent immobilization on a microarray and non-self-complementary oligonucleotides of length n (n-oligonucleotides) end-to-end, such that the melting temperatures of the three duplex variants, namely: (1) DNA is an immobilized N-oligonucleotide, (2) a self-closing hairpin, and (3) an n-oligonucleotide is immobilized this N-oligonucleotide, met the inequality T _{pl (1)} >> T _{pl (2)} >> T _{pl (3)} ; a test microchip is prepared with theoretically selected immobilized N-oligonucleotides, they are hybridized with a mixture of fluorescently labeled model DNA and each of the theoretically selected n-oligonucleotides, choosing conditions for balancing the energy of AT- and GC-rich oligonucleotides; hybridization results are recorded by constructing melting curves corresponding to the thermodynamic transitions of the resulting duplexes using the fluorescence analysis method and, based on the results obtained, N-oligonucleotides for the manufacture of the analytical microchip, n-oligonucleotides for butt hybridization and hybridization conditions are selected; immobilization of the selected N-oligonucleotides to the microarray for the manufacture of the analytical microarray is carried out in a buffer solution at pH, ionic strength and temperature, ensuring the closed state of the hairpin; hybridization of the analytical microchip is carried out with a mixture of the analyzed DNA and non-self-complementary butt oligonucleotides (n-oligonucleotides) under conditions of equalization of the energy of AT- and GC-rich oligonucleotides; hybridization results are recorded by mass spectrometry to identify n-oligonucleotides that have formed a perfect duplex with the analyzed DNA and have hybridized end-to-end to N-oligonucleotides immobilized on an analytical microchip; interpretation of the results of hybridization includes a phased reconstruction of the sequence of the analyzed DNA, based on the structures of the immobilized N-oligonucleotide, which includes a fragment of length L, which forms a perfect duplex with the analyzed DNA, and an n-oligonucleotide, which hybridizes end-to-end to it, also forms a perfect duplex with DNA, detected on the basis of mass spectrometry data, and the position of the cell of the analytical microchip determines the structure of the N-oligonucleotide containing the L-fragment is complementary tare to a specific region of the analyzed DNA, and the mass of the main peak in the mass spectrum recorded from this cell allows us to uniquely identify the n-oligonucleotide, and the uniqueness of identification of n-oligonucleotides in the case of degeneracy of their structure is due to the use of different mass labels, thereby restoring the sequence the analyzed DNA fragment with a length of L + n nucleotides; analyzing the sequences of partially overlapping N-oligonucleotides immobilized in the microarray cells (tiling method) in combination with the identification of n-oligonucleotides attached to them, reconstruct the sequence of variable regions of the analyzed DNA; comparing the reconstructed DNA sequences of wild and mutant types, mutations are uniquely deciphered.  2. The method according to claim 1, characterized in that the butt oligonucleotides (n-oligonucleotides) contain a stacking terminator and mass tags.  3. The method according to claim 2, characterized in that F1TS or its analogue is used as a stacking terminator.  4. The method according to claim 2, characterized in that Texas Red or its analogue is used as a stacking terminator.  5. The method according to claim 2, characterized in that as a stacking terminator use a ureide derivative of 2 '-deoxyribose.  6. The method according to claim 2, characterized in that standard linkers of different lengths are used as mass tags.  7. The method according to p. 1, characterized in that to obtain fluorescently labeled n-oligonucleotides use Texas Red.  8. The method according to p. 1, characterized in that to obtain a fluorescently labeled model DNA using F1TC.  9. The method according to claim 1, characterized in that the alignment of the energy of AT- and GC-rich oligonucleotides is ensured by the use of equalizing buffer solutions.  10. The method according to claim 9, characterized in that as a equalizing buffer solution using a 1 M solution of triethylammonium acetate (TEAA). 11. The method according to claim 1, characterized in that the immobilization of N-oligonucleotides for the manufacture of the analytical microchip is carried out in a buffer solution with a pH of 7.0 in the presence of 0.1 M NaCl at 20 ^o C.  12. A microchip for implementing the partial DNA sequencing method according to claim 1, which is a microarray with immobilized N-oligonucleotides having a hairpin structure, a fragment of each of which is complementary to a constant region of the analyzed gene in the region of mutations. 13. The microchip according to claim 12, characterized in that the N-oligonucleotides immobilized on it are selected on the basis of theoretical calculations and are intended to select the length and design of the hairpin and hybridization conditions with theoretically selected butt n-oligonucleotides and model DNA so that the melting temperature three types of duplexes, namely: (1) DNA - an immobilized N-oligonucleotide, (2) a self-closing hairpin, and (3) an n-oligonucleotide - an immobilized N-oligonucleotide, met the inequality T _{m (1)} >> T _{m (2)} >> T _{pl (3)} (test microchip).  14. The microchip according to claim 12, characterized in that the N-oligonucleotides immobilized on it are selected in accordance with paragraph 13 (analytical microchip).

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