CN111793672A - Method for forming cross-linked inverted probe on gene chip - Google Patents
Method for forming cross-linked inverted probe on gene chip Download PDFInfo
- Publication number
- CN111793672A CN111793672A CN202010719525.6A CN202010719525A CN111793672A CN 111793672 A CN111793672 A CN 111793672A CN 202010719525 A CN202010719525 A CN 202010719525A CN 111793672 A CN111793672 A CN 111793672A
- Authority
- CN
- China
- Prior art keywords
- cross
- chip
- probe
- probes
- gene chip
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
Abstract
The invention provides a method for forming a cross-linked inverted probe on a gene chip by means of cross-chain cross-linking. The invention also relates to a gene chip with cross-linked inverted probes obtained by the method of the invention and the use of cross-chain cross-linking for forming cross-linked inverted probes on a gene chip.
Description
Technical Field
The invention provides a method for forming a cross-linked inverted probe on a gene chip, application thereof and the gene chip prepared by the method.
Background
The gene chip technology is the combination of micro processing technology and molecular biology in semiconductor industry technology, integrates a large number of densely arranged gene probes on the surface of a substrate, and obtains the number and sequence information of sample molecules by detecting the hybridization signal intensity of each probe, so that a large number of genes can be analyzed in a short time, and people can rapidly read and analyze the gene information of organisms. The gene chip technology not only provides an important means for the early completion of the human genome sequencing plan, but also becomes a core device of a DNA sequencing and diagnosis system, and the gene chip technology, a DNA information reading analyzer and analysis software of obtained data form an information system and a platform of modern genetics.
The preparation methods of gene chips are various, and there are three main methods according to the principle: in-situ synthesis, synthetic spotting and bead chips.
The in situ synthesis methods, including the in situ photolithography synthesis method and the in situ jet printing synthesis method, are all methods for synthesizing the required oligonucleotide fragments by sequentially connecting A, G, C, T four bases to the sequence of a substrate. Affymetrix, Centrilion and Agilent are the most prominent in situ synthesis chip manufacturers worldwide, with Affymetrix and Centrilion in situ synthesis chips being generated by in situ photolithographic synthesis and Agilent in situ synthesis chips being generated by in situ inkjet synthesis. The in-situ synthesized oligonucleotide chip has the advantages of high density, capability of synthesizing oligonucleotides with any sequence and the like, and is suitable for DNA sequence determination, SNP analysis and the like; however, the probes on the chip obtained by the in-situ synthesis method are not high in purity, many invalid/truncated probes are generated during synthesis, the length of the probes on the chip obtained by the photolithography synthesis method is limited, and the density of the probes on the chip obtained by the jet printing synthesis method is low. In addition, since all the current mature DNA synthesis techniques are synthesized from 3 ' end to 5 ' end, the 5 ' end of the DNA probe of the gene chip of the above 3 companies is outward, so that only a single hybridization can be performed, and the extension reaction cannot be directly performed.
The synthesis spotting method is to use a spotting instrument to directly contact a previously synthesized probe on a chip to form a microarray, and the connection between the probe and a medium is mainly completed by using a chemical bond formed between chemical groups. The principle of the spotting instrument manufactured by different manufacturers is similar, and the spotting mode is different. The method is simple and easy to realize because the probes are prepared in advance by a mature chemical method, but the number and the density of the probes of the microarray are limited due to the limitation of a spotting instrument, so that the realization of a high-density and high-throughput gene chip is difficult.
The microbead chip is a specific gene chip of Illumina, a synthesized probe is connected to microbeads through the 5 'end of the microbead chip, and the microbeads are randomly paved on the chip to obtain the chip with the 3' end facing outwards, but the chip can only reach medium probe density. Moreover, due to the random distribution of the microbeads, each core of the Illumina tablet is decoded and controlled in quality before delivery, which also increases the manufacturing cost.
The existing in-situ synthesized gene chip generated by a photoetching synthesis method has high density, but the length of probes on the chip is limited, a plurality of invalid probes exist, the 5' ends of the probes face outwards, only single hybridization detection can be carried out, and the chip can be loaded to carry out final test by complex sample pretreatment steps. The existing gene chip and Illumina microbead chip prepared by a synthetic spotting method can only reach low or medium probe density although the 3' end faces outwards, and high-throughput detection cannot be realized.
Therefore, the technical problem to be solved by the invention is to overcome the defect that the existing gene chip can not realize the performance of outward facing of the 3 'end of the probe and high probe density at the same time, and provide a high-density gene chip with the outward 3' end so as to realize higher-flux detection and simplify the processing procedure.
Cross-strand cross-linking (also known as "interchain cross-linking"; ICL) is a DNA damage that is abnormally biologically active. Cross-strand cross-linking, which endogenously produces genomic DNA, may lead to major diseases such as aging, neurodegenerative disorders and cancer. There are many factors that cause damage to DNA cross-strand cross-links, such as free radicals and other reactive intermediates produced during in vivo metabolism, ultraviolet and ionic radiation in the environment, some endogenous and exogenous chemicals, etc. The electrophilic alkylating agent can be decomposed into active ions in a cell body as an important chemical substance, the ions can perform alkylation reaction with a base on one DNA chain to form an active intermediate, and then perform alkylation reaction with another DNA chain to finally form DNA cross-chain connection in the cell. DNA cross-linking prevents DNA strand separation, completely blocks DNA replication or transcription, and if not repaired in a timely manner, the end result of DNA cross-linking is cell death. Various carcinogens and clinically used anticancer drugs can cause DNA cross-link damage. Nitrosamine compounds widely existing in the environment and food can generate alkyl positive ions after metabolic activation to react with DNA bases, so that DNA cross-strand cross-linking damage is caused and cancer is induced finally. Clinically used alkylating agents such as nitrosourea alkylating agents for treating brain tumors and leukemias, nitrogen mustard alkylating agents for treating malignant lymphomas, and the like also exert an anticancer effect by causing damage to the cross-chain junctions of DNA of cancer cells. However, there is no teaching or suggestion in the prior art to utilize the concept or technique of artificially generated cross-strand cross-linking in the sequencing art.
Disclosure of Invention
The inventor of the present invention has conducted a great deal of theoretical analysis and experimental research on the gene chip known in the prior art, and creatively thought of the improvement of the probe on the surface of the gene chip by cross-linking the probes on the gene chip to form the probe with the outward 3' end. The probe is fixed on a solid support through chemical crosslinking instead of hybridization, and can conveniently and directly carry out chain extension reaction because the 3' end faces outwards, so that the concentration of the effective probe can be greatly improved, the subsequent treatment of the chip is simplified, the probe is more widely suitable for in-situ chemical reaction on the surface of the chip, the application range of an in-situ synthesis gene chip is widened, the extracted genome can be subjected to reaction on the chip without complex pretreatment steps, and the probe has higher sensitivity and lower detection limit.
Therefore, the invention creatively applies the cross-chain connection to the sequencing field, and the probe with the 3' end facing outwards is formed on the gene chip. The probe is fixed on the solid phase support through chemical crosslinking rather than hybridization, and the defects of the traditional gene chip in the aspects of probe length, incapability of carrying out extension reaction and the like which are required to be overcome for a long time can be overcome.
In a first aspect, the present invention provides a method of forming cross-linked inverted probes on a gene chip by means of cross-strand cross-linking, the method comprising the steps of:
a) synthesizing U base after the last base of the 5' end of the probe on the gene chip to obtain a U-chip probe,
b) optionally, a wash is carried out,
c) hybridizing a synthetic probe to said U-chip probe, said synthetic probe comprising in order from the 5 'end to the 3' end at least a sequence that is reverse complementary to an adjacent sequence downstream of the U base on said U-chip probe, a U base and a protruding sequence, wherein the U bases in said U-chip probe and said synthetic probe form exactly a U base pair,
d) optionally, a wash is carried out,
e) adding UDG enzyme to cleave U bases in the U-chip probes and the synthetic probes to generate empty base pairs,
f) optionally, a wash is carried out,
g) adding a cross-linking agent for cross-linking the empty base pairs,
h) optionally, washing to remove synthetic probes not cross-linked to the U-chip probes,
thereby forming the cross-linked inverted probe on the gene chip.
In another aspect, the present invention provides a gene chip with a cross-linked inverted probe obtained by the method of the present invention.
The invention also relates to a gene chip with a cross-linking inverted probe, wherein a chip probe directly fixed on the gene chip and a synthetic probe not directly fixed on the gene chip have a cross-chain cross connecting point at a pair of base sites, the cross-chain cross connecting point is a 5 ' direction end point of the chip probe on the gene chip, and a sequence in the synthetic probe positioned in an upstream 5 ' direction of the cross-chain cross connecting point is reversely complementary with a downstream 3 ' direction sequence in the chip probe on the gene chip and close to the cross-chain cross connecting point.
The invention also relates to the use of cross-chain cross-linking for forming cross-linked inverted probes on gene chips.
Drawings
FIG. 1 shows the principle of using aoNao cross-linking to prepare a 3' end-outward chip. Wherein, the U base is synthesized at the 5' end of the chip probe, so that the synthesized probe is hybridized with the chip probe, the composition of the synthesized probe comprises a sequence which is reverse complementary with the chip probe, namely the U base-protruding sequence, and the U base of the chip probe and the synthesized probe just form a U base pair. And (3) using UDG enzyme to cut U base to generate empty base pairs, adding a cross-linking agent aoNao to cross-link the empty base pairs, and washing to remove synthetic probes on non-cross-linked parts to obtain the probe chip with the 3' end facing outwards.
FIG. 2 shows the effect of the reaction sequence on the crosslinking efficiency. The upper left drawing, the upper right drawing, the lower left drawing and the lower right drawing respectively adopt the following combination sequence: carrying out hybridization, cutting U, washing and crosslinking; cutting U, hybridizing, washing and crosslinking; washing after hybridization, cutting, washing with U, and crosslinking; and cross-linking the hybridized edges after U cutting.
FIG. 3 shows the stability results of cross-linked chips, where the upper part A shows the fluorescence at the position corresponding to AM1, and the line segment above the dark background indicates the position corresponding to the fluorescence intensity shown in the lower part B; part B shows the fluorescence intensity at the line segment in part A. Numbers 1, 2, 3, 4, 5, 6 correspond to the respective processing conditions in table 7, respectively.
FIG. 4 shows SAPE staining results of the chip 3' end-out chip obtained by simultaneous specific hybridization of AM1 probe and AM3 probe on the chip, followed by aoNao cross-linking, wherein the large boxes at the four corners represent the results for AM1 probe and the middle small boxes represent the results for AM3 probe.
FIG. 5 shows the extension test results obtained for the 3' end-out chip.
FIG. 6 shows ICL responses of AP pairs.
FIG. 7 shows the structural formula of aoNao and the reaction mechanism for cross-linking DNA using aoNao.
Detailed Description
Unless defined otherwise herein, scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
As used herein, the "gene chip" refers to a chip obtained by in situ synthesis of oligonucleotide probes on a solid support or by directly immobilizing a large number of probes prepared in advance on the surface of the support. Genetic information of a sample can be obtained by hybridizing a gene chip with the sample, and then detecting and analyzing the hybridization signal using a chip scanner and a computer.
As used herein, a "chip probe" refers to a probe that is immobilized on a solid support, i.e., a chip, by in situ synthesis or by directly curing a large number of probes prepared in advance.
As used herein, the "U-chip probe" refers to a probe obtained by adding U base after the last base of the 5' end of a probe on a gene chip.
As used herein, a "synthetic probe" refers to a probe comprising, in order from the 5 'end to the 3' end, at least a sequence that is reverse complementary to an adjacent sequence downstream of the U base on the U-chip probe, the U base, and an overhang sequence, wherein the U bases in the U-chip probe and the synthetic probe form exactly a U base pair.
As used herein, "overhang sequence" refers to a sequence located 3' to the U base in a synthetic probe.
As used herein, an "inverted probe" refers to a probe that is located 3' of the gene chip outward.
As used herein, "U base" refers to a uracil (uracil) base.
As used herein, "cross-stranded cross-linking" refers to the covalent attachment of an empty base pair or a base-free pair (AP pair) between two complementary DNA strands, typically by means of a cross-linking agent, in vitro. FIG. 6 shows ICL responses to AP pairs, where "U" refers to deoxyuridine. "Cross-chain cross-linking", "interchain cross-linking" and "cross-linking" are used herein in the same sense and are used interchangeably. Empty base pair or abasic pair (AP pair) sites, which are covalently linked to each other between two complementary DNA strands, are referred to as "strand-spanning cross-linking points".
As used herein, the "gene chip with a cross-linked inverted probe" refers to a gene chip in which a chip probe directly immobilized on the gene chip has a cross-strand junction at a pair of base sites with a synthetic probe not directly immobilized on the gene chip, the cross-strand junction being the 5 ' -end of the chip probe on the gene chip, and a sequence in the synthetic probe located in the 5 ' -direction upstream of the cross-strand junction is reverse-complementary to a sequence in the chip probe on the gene chip immediately 3 ' -direction downstream of the cross-strand junction. "Gene chip with crosslinked inverted probes" and "crosslinked chip" have the same meaning herein and are used interchangeably. The probe generated by the above-described cross-linking reaction between the probe on the chip and the synthetic probe is collectively referred to as "cross-linked inverted probe".
As used herein, udg (uracil DNA glycosylase) refers to uracil DNA glycosylase that is capable of selectively breaking the glycosidic bond of deoxyuridine in single-and double-stranded DNA, releasing uracil, thereby creating an empty base or a base-free site.
As used herein, "crosslinker" refers to a substance used to effect covalent attachment of an AP pair, including, but not limited to, bifunctional alkylating agents, platinum compounds, psoralens, and unsaturated aldehydes, such as diamines, N' - (naphthalene-1, 5-diyl) bis [2- (aminooxy) acetamide ] (aoNao), benzene derivatives, other substances containing a bis (aminooxy) group, and the like. More preferred crosslinking agents are such as ethylenediamine, hexamethylenediamine, decamethylenediamine, aoNao, and the like. Examples of cross-linking agents commonly used in the art are described, for example, in Kohei Ichikawa et al, interaction cross-link of DNA by covalent linking a pair of immunological sites, chem. Commun.,2012,48, 2143-; ZHiyu Yang et al, interstrand cross-linking from strand and break at Nucleic Acids in duplex DNA, Nucleic Acids Research,2017, Vol.45, No. 116275-; yu Hirano et al, synthetic application of cross-linked duplex by scientific linking adapter of biological sites Current Protocols in Nucleic Acid Chemistry 63, Volume 75; todor Angelov et al, Generation of DNA Interstrand Cross by Post-Synthetic reduction amplification, organic Letters 2009,11(3),661-664, the entire contents of which are incorporated herein by reference in their entirety. FIG. 7 shows the structural formula of aoNao and the reaction mechanism for cross-linking DNA using aoNao.
In one aspect, the present invention provides a method for forming a cross-linked inverted probe on a gene chip by means of cross-linking across strands, the method comprising the steps of:
a) synthesizing U base after the last base of the 5' end of the probe on the gene chip to obtain a U-chip probe,
b) optionally, a wash is carried out,
c) hybridizing a synthetic probe to said U-chip probe, said synthetic probe comprising in order from the 5 'end to the 3' end at least a sequence that is reverse complementary to an adjacent sequence downstream of the U base on said U-chip probe, a U base and a protruding sequence, wherein the U bases in said U-chip probe and said synthetic probe form exactly a U base pair,
d) optionally, a wash is carried out,
e) adding UDG enzyme to cleave U bases in the U-chip probes and the synthetic probes to generate empty base pairs,
f) optionally, a wash is carried out,
g) adding a cross-linking agent for cross-linking the empty base pairs,
h) optionally, washing to remove synthetic probes not cross-linked to the U-chip probes,
thereby forming the cross-linked inverted probe on the gene chip.
It should be understood that the order of step c), step e) and step g) may be interchanged or may be performed simultaneously. For example, step e) may be performed before or after step c). For example, step c) may be performed after step e) and step g) may be performed after step c) or step c) and step g) may be performed simultaneously.
In some embodiments of the invention, step c) is performed before step e).
In some embodiments of the invention, step c) is performed after step e).
In some embodiments of the invention, step c) and step g) are performed simultaneously.
In one embodiment of the method according to the first aspect of the present invention, the present invention provides a method for forming a cross-linked inverted probe on a gene chip by means of cross-chain cross-linking, the method comprising the following steps in order:
1) synthesizing U base after the last base of the 5' end of the probe on the gene chip to obtain a U-chip probe,
2) optionally, a wash is carried out,
3) hybridizing a synthetic probe to said U-chip probe, said synthetic probe comprising in order from the 5 'end to the 3' end at least a sequence that is reverse complementary to an adjacent sequence downstream of the U base on said U-chip probe, a U base and a protruding sequence, wherein the U bases in said U-chip probe and said synthetic probe form exactly a U base pair,
4) optionally, a wash is carried out,
5) adding UDG enzyme to cleave U bases in the U-chip probes and the synthetic probes to generate empty base pairs,
6) optionally, a wash is carried out,
7) adding a cross-linking agent for cross-linking the empty base pairs,
8) optionally, washing to remove synthetic probes not cross-linked to the U-chip probes,
thereby forming the cross-linked inverted probe on the gene chip.
In another embodiment of the method according to the first aspect of the present invention, the present invention provides a method for forming a cross-linked inverted probe on a gene chip by means of cross-chain cross-linking, the method comprising the following steps in order:
1) synthesizing U base after the last base of the 5' end of the probe on the gene chip to obtain a U-chip probe,
2) optionally, a wash is carried out,
3) adding UDG enzyme to the U-chip probes and synthetic probes to cleave the U bases in the U-chip probes and the synthetic probes, thereby generating empty base pairs, the synthetic probes comprising, in order from the 5 'end to the 3' end, a sequence that is reverse complementary to at least the contiguous sequence downstream of the U bases on the U-chip probes, a U base, and an overhang sequence, wherein the U bases in the U-chip probes and the synthetic probes just form a U base pair,
4) optionally, a wash is carried out,
5) hybridizing the synthesized probes to the U-chip probes,
6) optionally, a wash is carried out,
7) adding a cross-linking agent for cross-linking the empty base pairs,
8) optionally, washing to remove synthetic probes not cross-linked to the U-chip probes,
thereby forming the cross-linked inverted probe on the gene chip.
In another embodiment of the method according to the first aspect of the present invention, the present invention provides a method for forming a cross-linked inverted probe on a gene chip by means of cross-chain cross-linking, the method comprising the following steps in order:
1) synthesizing U base after the last base of the 5' end of the probe on the gene chip to obtain a U-chip probe,
2) optionally, a wash is carried out,
3) adding UDG enzyme to the U-chip probes and synthetic probes to cleave the U bases in the U-chip probes and the synthetic probes, thereby generating empty base pairs, the synthetic probes comprising, in order from the 5 'end to the 3' end, a sequence that is reverse complementary to at least the contiguous sequence downstream of the U bases on the U-chip probes, a U base, and an overhang sequence, wherein the U bases in the U-chip probes and the synthetic probes just form a U base pair,
4) optionally, a wash is carried out,
5) adding a cross-linking agent for cross-linking the empty base pairs while hybridizing the synthetic probes to the U-chip probes,
6) optionally, washing to remove synthetic probes not cross-linked to the U-chip probes,
thereby forming the cross-linked inverted probe on the gene chip.
In an embodiment of the present invention, the crosslinking agent used in the crosslinking step may be a crosslinking agent known to those skilled in the art to be capable of being used for interchain crosslinking. In a preferred embodiment of the invention, the crosslinking agent used is a diamine, aoNao, a benzene derivative or another substance containing a bis (aminooxy) group. In a more preferred embodiment of the invention, the crosslinking agent used is ethylenediamine, hexamethylenediamine, decamethylenediamine, aoNao. In the most preferred embodiment of the invention, the crosslinking agent used is aoNao.
In some embodiments of the invention, crosslinking is performed at 0 ℃ to 37 ℃. In some embodiments of the invention, crosslinking is performed at 4 ℃ to 37 ℃. In some embodiments of the invention, crosslinking is performed at 4 ℃ to 25 ℃. In some embodiments of the invention, crosslinking is performed at 25 ℃. In some embodiments of the invention, crosslinking is performed at 37 ℃.
In some embodiments of the invention, crosslinking is performed for 0.5 hours to overnight. In some embodiments of the invention, crosslinking is performed for 1 hour to overnight. In some embodiments of the invention, crosslinking is performed for 2 hours to overnight. In some embodiments of the invention, crosslinking is performed for 4 hours to overnight. In some embodiments of the invention, crosslinking is performed for 6 hours to overnight. In some embodiments of the invention, crosslinking is performed for 8 hours to overnight. In some embodiments of the invention, crosslinking is performed for 10 hours to overnight. In some embodiments of the invention, crosslinking is performed for 0.5 hours to 8 hours. In some embodiments of the invention, crosslinking is performed for 0.5 hours to 6 hours. In some embodiments of the invention, crosslinking is performed for 0.5 hours to 4 hours. In some embodiments of the invention, crosslinking is performed for 0.5 hours to 2 hours. In some embodiments of the invention, crosslinking is performed for 0.5 hours to 1 hour. In some embodiments of the invention, crosslinking is performed for 1 hour to 8 hours. In some embodiments of the invention, crosslinking is performed for 1 hour to 6 hours. In some embodiments of the invention, crosslinking is performed for 1 hour to 4 hours. In some embodiments of the invention, the crosslinking is performed for 1 hour to 2 hours. In some embodiments of the invention, crosslinking is performed for 2 hours to 8 hours. In some embodiments of the invention, crosslinking is performed for 2 hours to 6 hours. In some embodiments of the invention, crosslinking is performed for 2 hours to 4 hours. In some embodiments of the invention, crosslinking is performed for 4 hours to 8 hours. In some embodiments of the invention, crosslinking is performed for 4 hours to 6 hours. In some embodiments of the invention, crosslinking is performed for 6 hours to 8 hours. In some embodiments of the invention, crosslinking is performed overnight. In some embodiments of the invention, crosslinking is performed for 10 hours. In some embodiments of the invention, crosslinking is performed for 8 hours. In some embodiments of the invention, crosslinking is performed for 6 hours. In some embodiments of the invention, crosslinking is performed for 4 hours. In some embodiments of the invention, crosslinking is performed for 2 hours. In some embodiments of the invention, the crosslinking is performed for 1 hour. In some embodiments of the invention, crosslinking is performed for 0.5 hours.
The various embodiments and preferences described above for the individual steps of the method according to the invention can be combined with one another, as long as they are not inherently contradictory to one another, and the various embodiments formed by this combination are considered part of the disclosure of the present application.
The invention also relates to a gene chip with a cross-linked inverted probe, wherein a chip probe directly fixed on the gene chip and a synthetic probe not directly fixed on the gene chip have a cross-chain cross-connecting point at a pair of base sites, the cross-chain cross-connecting point is a 5 ' direction end point of the chip probe on the gene chip, and a sequence in the synthetic probe positioned in an upstream 5 ' direction of the cross-chain cross-connecting point is reversely complementary with a sequence in a downstream 3 ' direction of the cross-chain cross-connecting point in the chip probe on the gene chip.
The invention also relates to a gene chip with the cross-linked inverted probe obtained by the method.
The invention also relates to the use of cross-chain cross-linking for forming cross-linked inverted probes on gene chips.
In addition, the present invention also provides the following embodiments:
a) synthesizing U base after the last base of the 5' end of the probe on the gene chip to obtain a U-chip probe,
b) optionally, a wash is carried out,
c) hybridizing a synthetic probe to said U-chip probe, said synthetic probe comprising in order from the 5 'end to the 3' end at least a sequence that is reverse complementary to an adjacent sequence downstream of the U base on said U-chip probe, a U base and a protruding sequence, wherein the U bases in said U-chip probe and said synthetic probe form exactly a U base pair,
d) optionally, a wash is carried out,
e) adding UDG enzyme to cleave U bases in the U-chip probes and the synthetic probes to generate empty base pairs,
f) optionally, a wash is carried out,
g) adding a cross-linking agent for cross-linking the empty base pairs,
h) optionally, washing to remove synthetic probes not cross-linked to the U-chip probes,
thereby forming the cross-linked inverted probe on the gene chip.
Embodiment 7, the method of any one of the preceding embodiments, wherein the reverse complement sequence is 5-30 bases, 10-25 bases, or 15-25 bases in length.
Embodiment 8, a gene chip with a cross-linked inverted probe obtained by the method of any one of the preceding embodiments.
Embodiment 9 a gene chip with a cross-linked inverted probe, wherein a chip probe directly immobilized on the gene chip and a synthetic probe not directly immobilized on the gene chip have a cross-strand junction at a pair of base sites, the cross-strand junction being the 5 ' -end of the chip probe on the gene chip, and a sequence in the synthetic probe located in the 5 ' -end upstream of the cross-strand junction is reverse-complementary to a sequence in the chip probe on the gene chip immediately downstream of the cross-strand junction in the 3 ' -end.
Embodiment 10 the gene chip with the crosslinked inverted probe according to embodiment 9, wherein the length of the reverse complementary sequence is 5 to 30 bases, 10 to 25 bases, or 15 to 25 bases.
Embodiment 11, use of cross-chain cross-linking to form cross-linked inverted probes on a gene chip.
The technical solution of the present invention will be more clearly and clearly illustrated by way of example in the accompanying drawings and examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way. The scope of the invention is only limited by the claims.
Examples
Unless otherwise indicated, the probes used in the following examples were purchased from Shanghai Czeri bioengineering, Inc., and the enzymes and their buffers from New England Biolabs (NEB). The chips used in the examples are Banff chips available from Shengjie technology (Hangzhou) Inc., which are gene chips having 5 'end-outward probes synthesized by in situ synthesis, but it will be understood by those skilled in the art that any gene chip having 5' end-outward probes is suitable for use in the present invention. The general procedures in molecular biology can be found, for example, in the molecular cloning guidelines. Sequence information of the probes or DNA fragments used in the examples is shown in table 1 below. The probe or DNA fragment is only for illustrative purposes and is not intended to limit the probe or DNA fragment of the present invention. It will be appreciated by those skilled in the art that any probe or DNA sequence which complies with the principles of probe design and pairing of the present invention is suitable for use in the present invention.
Table 1: sequence information of the probes or DNA fragments used
Example 1: preparation of Gene chip with Cross-Linked inverted Probe
1.1 use of diamines as crosslinking agents
1.1.1 determination of the influence of diamine species on the crosslinking efficiency
Methods for adding a U base to the 5 '-end of a chip probe on a Banff chip and methods for synthesizing a synthetic probe containing a U base and complementarily hybridizing to the 5' -end of a chip probe are well known in the art. The U-base-added Banff chip was hybridized with the synthetic probe, cleaved with 1. mu.L of UDG enzyme (M0280S, from NEB) at 37 ℃ for 1h, and then transferred to 50. mu. LpH ═ 6.03. mu.L of 5mM diamine and 3. mu.L of 0.5M NaCNBH were added to the phosphate buffer (9)3Crosslinking at room temperature overnight. For the control, only the hybridization step of the chip probe and the synthetic probe was performed but the crosslinking step was not performed, and other reaction conditions were the same as those of the experimental group using diamine. Washing was performed under the washing conditions described in table 2, wherein for the experimental group using diamine, washing was performed in 1mL of 0.2m naoh for 5min (strong base washing); for control, wash in 1mL of 4 XSSC for 5min (gentle wash, not disrupting hybridization of chip probes to synthetic probes). The chip was then placed in a PCR tube containing 1. mu.L of 1. mu.M AMT-P and 50. mu.L of 4 XSSC and hybridized at 50 ℃ for 30 min. The hybridized chip was rinsed once with 1mL of 4 XSSC and then stained in 50. mu.L of SAPE (streptavidin-phycoerythrin) solution for 30min in the dark. After rinsing once with 1mL of 4 XSSC, the fluorescence intensity was measured using a SUMMIT chip scanner (available from Promega technologies, Hangzhou) to determine the crosslinking efficiency. The results show that: the fluorescence intensities obtained when different diamines are used are similar, wherein the ethylenediamine is optimal, and the crosslinking efficiency can reach about 40%.
Table 2: effect of diamine species on crosslinking efficiency
1.1.2 determination of the Effect of ethylenediamine concentration on crosslinking efficiency
Crosslinking was performed according to the procedure and conditions described in 1.1.1 using ethylenediamine as crosslinking agent. The concentration of ethylenediamine and the corresponding fluorescence intensity are shown in table 3. The results show that: the fluorescence intensities obtained with different concentrations of ethylenediamine were similar, with 50mM ethylenediamine being the best, and cross-linking efficiencies around 70% were achieved.
Table 3: effect of ethylenediamine concentration on crosslinking efficiency
1.1.3 determination of the Effect of crosslinking time on crosslinking efficiency
Crosslinking was performed according to the procedure and conditions described in 1.1.1, using ethylenediamine as crosslinking agent, using the concentrations and crosslinking times as listed in table 4. The results show that: the fluorescent intensity is not greatly influenced by increasing the concentration of the ethylenediamine, and the fluorescent intensity is greatly influenced by prolonging the crosslinking time. Among them, the fluorescence intensity at 37 ℃ is low, and it is presumed that the cause is the detachment of the hybridization probe.
Table 4: effect of crosslinking time on crosslinking efficiency
| Concentration of ethylene diamine | Crosslinking time | Intensity of fluorescence |
| 5mM | Room temperature, 5h | 1800 |
| 50mM | Room temperature, 5h | 1800 |
| 5mM | At room temperature, overnight | 4400 |
| 5mM | At 37 ℃ overnight | 2500 |
1.2 crosslinking with aoNao as crosslinker
1.2.1 determination of the influence of the crosslinking temperature and crosslinking time on the crosslinking efficiency
Crosslinking was performed according to the procedure and conditions described in 1.1.1, using aoNao as crosslinking agent, using aoNao final concentration, temperature and crosslinking time as listed in table 5. The results show that: the fluorescence intensity at 37 ℃ is slightly higher than that at 25 ℃ and the fluorescence intensity at 4 ℃ is very low; the fluorescence intensity was low at 2 hours of crosslinking and the crosslinking reached approximately saturation at 6 hours.
Table 5: effect of crosslinking temperature and crosslinking time on crosslinking efficiency
| Final concentration of aoNao | Temperature of | Reaction time | Intensity of fluorescence |
| 0.22 |
4℃ | 8h | 800 |
| 0.22mM | 25℃ | 8h | 7000 |
| 0.22mM | 37℃ | 8h | 7600 |
| 0.22mM | 25℃ | 2h | 3000 |
| 0.22mM | 25℃ | 4h | 6400 |
| 0.22mM | 25℃ | 6h | 8200 |
| 0.22mM | 25℃ | 8h | 8100 |
1.2.1 determination of the influence of the reaction sequence on the crosslinking efficiency
Under otherwise identical conditions, the combined sequence of hybridization (hybridization) of the chip probe to the synthetic probe, cleavage of the U base by the UDG enzyme (U cleavage), washing, and crosslinking (crosslinking) of the chip probe to the synthetic probe was adjusted to determine the effect of the reaction sequence on the crosslinking efficiency. The following combination sequences were used, respectively: firstly, hybridizing, cutting U, washing and crosslinking; cutting U, washing and crosslinking; thirdly, washing after hybridization, cutting, washing and crosslinking; fourthly, cross-linking the hybridized edges after cutting U. The results are shown in table 6 and fig. 2, where the experimental background obtained when using reaction sequence c, which will be used in the examples below, is the cleanest.
Table 6: effect of reaction sequence on crosslinking efficiency
Example 2: stability of chips on which AM1 Probe has been crosslinked with AM1-U chain
To a 200. mu.L PCR tube, 49. mu.L of 4 XSSC, 1. mu.L of synthetic probe AM1-U, and U-Banff chip were added. Placing in 50 deg.C oven for 30 min. The chip was removed and rinsed once in 1mL of 4 XSSC, then 44. mu.L of ddH was added to a 200. mu.L PCR tube2O, 5. mu.L of UDG buffer and 1. mu.L of UDG enzyme, placing the chip in an oven at 37 ℃ for 1 h. To a 200. mu.L PCR tube, 41. mu.L of pH 6 PBS buffer and 6.5. mu.L of 2M aoNao were added, and the chip was placed and crosslinked at room temperature overnight. The chip was washed in 1mL of 0.2M NaOH for 3min and rinsed once with 1mL of 4 XSSC, resulting in a chip on which the AM1 probe had been intrachain crosslinked.
The chip obtained by the above method and the chip without any treatment were subjected to the treatment conditions described in the following Table 7, and then put into a PCR tube containing 1. mu.L of 1. mu.M AMT-P and 50. mu.L of 4 XSSC, and hybridized at 50 ℃ for 30 min. The hybridized chip was rinsed once with 1mL of 4 XSSC and then stained in 50. mu.L of SAPE solution in the dark for 30 min. After rinsing once with 1mL of 4 XSSC, fluorescence signal detection was performed using a SUMMIT chip scanner (available from Shengjie technology, Hangzhou, Ltd.). The results are shown in table 7 and fig. 3. The results show that: the cross-linked chip has good stability under strong alkaline condition and certain high temperature resistance.
Table 7: stability of the crosslinked chips
| Numbering | Chip and method for manufacturing the same | Conditions of treatment | Intensity of |
| 1 | Cross-linked chip | 95℃5min | 4800 |
| 2 | Cross-linked chip | 95℃20min | 3300 |
| 3 | Cross-linked chip | 0.2M NaOH 5min | 8000 |
| 4 | |
4×SSC 5min | 8800 |
| 5 | Uncrosslinked chip | 95 |
0 |
| 6 | Uncrosslinked chip | 0.2 |
0 |
Example 3: preparation of chips on which AM1 and AM3 probes had been intrachain-crosslinked with AM1-U, AM3-U, respectively
To a 200. mu.L PCR tube were added 48. mu.L of 4 XSSC, 1. mu.L of synthetic probe AM1-U, 1. mu.L of synthetic probe AM3-U, and U-Banff chip. Placing in 50 deg.C oven for 30 min. Core of the utility modelThe slide was removed and rinsed once in 1mL of 4 XSSC, then 44. mu.L of ddH was added to a 200. mu.L PCR tube2O, 5. mu.L of UDG buffer and 1. mu.L of UDG enzyme, placing the chip in an oven at 37 ℃ for 1 h. To a 200. mu.L PCR tube, 41. mu.L of pH 6 PBS buffer and 6.5. mu.L of 2M aoNao were added, and the chip was placed and crosslinked overnight at room temperature. The chip was washed in 1mL of 0.2M NaOH for 3min and rinsed once with 1mL of 4 XSSC.
The prepared cross-linked chip was put into a PCR tube containing 1. mu.L of 1. mu.M AMT-P and 50. mu.L of 4 XSSC, and hybridized at 50 ℃ for 30 min. The hybridized chip was rinsed once with 1mL of 4 XSSC and then stained in 50. mu.L of SAPE solution in the dark for 30 min. After rinsing once with 1mL of 4 XSSC, fluorescence signal detection was performed using a SUMMIT chip scanner (available from Shengjie technology, Hangzhou, Ltd.). The results are shown in FIG. 4. The results show that: by using the method of the invention, specific hybridization crosslinking can be simultaneously carried out on a plurality of probes on the chip, thereby preparing the chip with the outward 3' end.
Example 4: chip extension test
To 49. mu.L of 4 XSSC was added the chip on which the AM1 probe had been crosslinked with the AM1-U strand prepared as described above and 1. mu.L of 1. mu.M extension template (AM1-T), followed by hybridization at 50 ℃ for 0.5h, washing with 1mL of 4 XSSC for 5min, followed by addition of Klenow exo- (M0212S, available from NEB) and dNTP (containing biotin-dUTP) (dCTP, dATP, dGTP concentration of 1mM, available from Bioengineering (Shanghai) Co., Ltd.; biotin-dUTP is R0081 available from ThermoFisher, concentration of 1mM) for extension testing (37 ℃, 30 min). After completion of the extension, the chip was rinsed once with 1mL of 4 XSSC and then stained in 50. mu.L of SAPE solution in the dark for 30 min. After rinsing once with 1mL of 4 × SSC, fluorescence signal detection was performed using a sumit chip scanner (purchased from seikaga technologies, hangzhou) and the results showed: only the AM1 site of the chip had a fluorescent signal. This indicates that: the gene chip with the cross-linked inverted probe obtained by the method can directly carry out extension reaction. Therefore, the method not only can simplify the SNP detection process, reduce the reagent cost and improve the accuracy, but also can use the cross-linked chip of the invention in the application fields of targeted sequencing and the like.
While particular embodiments of the present invention have been illustrated and described, it is not intended that these embodiments illustrate and describe all possible forms of the invention. Rather, the words used in the specification are words of description rather than limitation. It will be apparent to those skilled in the art that various other changes and modifications can be made without departing from the general scope of the disclosure. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
Claims (11)
1. A method of forming cross-linked inverted probes on a gene chip by means of cross-strand cross-linking, the method comprising the steps of:
a) synthesizing U base after the last base of the 5' end of the probe on the gene chip to obtain a U-chip probe,
b) optionally, a wash is carried out,
c) hybridizing a synthetic probe to said U-chip probe, said synthetic probe comprising in order from the 5 'end to the 3' end at least a sequence that is reverse complementary to an adjacent sequence downstream of the U base on said U-chip probe, a U base and a protruding sequence, wherein the U bases in said U-chip probe and said synthetic probe form exactly a U base pair,
d) optionally, a wash is carried out,
e) adding UDG enzyme to cleave U bases in the U-chip probes and the synthetic probes to generate empty base pairs,
f) optionally, a wash is carried out,
g) adding a cross-linking agent for cross-linking the empty base pairs,
h) optionally, washing to remove synthetic probes not cross-linked to the U-chip probes,
thereby forming the cross-linked inverted probe on the gene chip.
2. The method of claim 1, wherein the cross-linking agent for cross-linking across strands is selected from aoNao, diamines.
3. The method of any one of the preceding claims, wherein said step c) is performed before or after said step e).
4. The method of any one of the preceding claims, wherein said step c) and said step g) are performed simultaneously.
5. The method of any one of the preceding claims, wherein step g) is performed at room temperature or 37 ℃.
6. The method of any one of the preceding claims, wherein step g) is performed for 2 hours to overnight, 4 hours to overnight, 6 hours to overnight, 8 hours to overnight, 6 hours, 8 hours, or overnight.
7. The method of any one of the preceding claims, wherein the reverse complement sequence is 5-30 bases, 10-25 bases, or 15-25 bases in length.
8. A gene chip with cross-linked inverted probes obtained by the method of any one of the preceding claims.
9. A gene chip with cross-linked inverted probes, wherein a chip probe directly immobilized on the gene chip and a synthetic probe not directly immobilized on the gene chip have a cross-chain cross-linking point at a pair of base sites, the cross-chain cross-linking point being the 5 ' -side end point of the chip probe on the gene chip, and the sequence of the synthetic probe in the 5 ' -side upstream of the cross-chain cross-linking point is reverse-complementary to the sequence of the chip probe on the gene chip in the 3 ' -side immediately downstream of the cross-chain cross-linking point.
10. The gene chip with crosslinked inverted probe according to claim 9, wherein the length of the reverse complementary sequence is 5-30 bases, 10-25 bases or 15-25 bases.
11. Use of cross-chain cross-linking to form cross-linked inverted probes on a gene chip.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010719525.6A CN111793672A (en) | 2020-07-23 | 2020-07-23 | Method for forming cross-linked inverted probe on gene chip |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010719525.6A CN111793672A (en) | 2020-07-23 | 2020-07-23 | Method for forming cross-linked inverted probe on gene chip |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111793672A true CN111793672A (en) | 2020-10-20 |
Family
ID=72828664
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010719525.6A Pending CN111793672A (en) | 2020-07-23 | 2020-07-23 | Method for forming cross-linked inverted probe on gene chip |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111793672A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113215223A (en) * | 2021-05-28 | 2021-08-06 | 生捷科技(杭州)有限公司 | SNP typing detection method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170349623A1 (en) * | 2014-12-12 | 2017-12-07 | National Institute Of Advanced Industrial Science And Technology | Nucleic acid complex, method for forming nucleic acid hybridization, pharmaceutical composition, nucleic acid probe, and complementary-strand nucleic acid complex |
| CN110249057A (en) * | 2016-11-17 | 2019-09-17 | 空间转录公司 | Method for the nucleic acid in free token and analysis biological sample |
-
2020
- 2020-07-23 CN CN202010719525.6A patent/CN111793672A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170349623A1 (en) * | 2014-12-12 | 2017-12-07 | National Institute Of Advanced Industrial Science And Technology | Nucleic acid complex, method for forming nucleic acid hybridization, pharmaceutical composition, nucleic acid probe, and complementary-strand nucleic acid complex |
| CN110249057A (en) * | 2016-11-17 | 2019-09-17 | 空间转录公司 | Method for the nucleic acid in free token and analysis biological sample |
Non-Patent Citations (1)
| Title |
|---|
| LONGXING FAN等: "A tri-functional probe mediated exponential amplification strategy for highly sensitive detection of Dnmt1 and UDG activities at single-cell level" * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113215223A (en) * | 2021-05-28 | 2021-08-06 | 生捷科技(杭州)有限公司 | SNP typing detection method |
| CN113215223B (en) * | 2021-05-28 | 2022-10-18 | 生捷科技(杭州)有限公司 | SNP typing detection method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11767557B2 (en) | Single cell analyses | |
| US20200370105A1 (en) | Methods for performing spatial profiling of biological molecules | |
| CN109797201B (en) | In-situ multiple nucleic acid detection method | |
| WO2000077260A8 (en) | Genomic profiling: a rapid method for testing a complex biological sample for the presence of many types of organisms | |
| WO2003080645A2 (en) | Amplification of nucleic acid fragments using nicking agents | |
| CN104254620A (en) | Method for amplifying nucleic acid and method for detecting amplified nucleic acid | |
| JP7296409B2 (en) | Compositions for use in polynucleotide sequencing | |
| JP2008502367A (en) | Fast generation of oligonucleotides | |
| EP1956089B1 (en) | Method for detecting target nucleic acid with specific base sequence and set of nucleic acids for detection | |
| CN109852667B (en) | Method for detecting nucleic acid terminal structure based on single molecule force spectrum | |
| CN108424844B (en) | Single-molecule sequencing chip and preparation method thereof | |
| WO2023011628A1 (en) | High-resolution spatial omics detection method for tissue sample | |
| CN111808928A (en) | SNP typing detection method | |
| CN111793672A (en) | Method for forming cross-linked inverted probe on gene chip | |
| WO2004096986A2 (en) | Method for quantitative detection of nucleic acid molecules | |
| CN112639126A (en) | Reducing phasing with unlabeled nucleotides during sequencing | |
| JP2005520130A (en) | Method for complex detection of biological material in a sample | |
| CN100590204C (en) | Method for preparing three-dimensional gel micro array chip without excitant | |
| KR101319104B1 (en) | Method for fixing a supercoiled dna and the use for analysing the dna repair | |
| CN109439751A (en) | It is a kind of for detecting the fluorescence in situ hybridization probe kit of KIAA1549-BRAF fusion | |
| EP3447154A1 (en) | Method for detection of mutations, polymorphisms and specific dna sequences on dna matrices with dna imaging techniques for the use in medical diagnostics and forensic genetics | |
| CA2442438C (en) | Oligonucleotide-immobilized substrate for detecting methylation | |
| CN103966320A (en) | Gene chip for integrated detection and on-substrate amplification method | |
| KR20030020232A (en) | A method of preparing a hydrogel biochip by using satar-like PEG derivatives | |
| WO2009107614A2 (en) | Method for detection of target nucleic acid and device for detection of target nucleic acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |