CN109055488B - Method for preparing long probe by using annular single-chain probe and application of long probe in gene chip production - Google Patents
Method for preparing long probe by using annular single-chain probe and application of long probe in gene chip production Download PDFInfo
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Abstract
The invention discloses a method for preparing a long probe by using an annular single-chain probe and application of the long probe in gene chip production. The invention obtains the ring-shaped single-stranded probe by connecting the probe and the amplification joint into a ring or connecting the probe with the amplification joint into a ring; the circular single-stranded probe is amplified to obtain a long probe with a straight-chain single strand. The method has the following advantages: the length of the probe obtained by amplification is excellent and can reach more than 10000 nt; the amplification length and the amplification time of the probe are approximately in a linear relationship, and when batch operation is carried out, the length of the probe in the same batch is uniform, and hybridization signals are more uniform; the probe obtained by amplification has extremely high yield which is about dozens of times of that of a direct connection method, and the processes of repeated phosphorylation and connection reaction are saved, so that the use and preparation time of a large amount of biological enzyme is saved. Therefore, the present invention is particularly suitable for the application in the production of gene chips.
Description
Technical Field
The invention relates to a method for preparing a probe, in particular to a method for preparing a long probe by using an annular single-stranded probe and application thereof in gene chip production.
Background
A gene chip, also called DNA chip, is a biochip manufactured based on the principle of DNA molecular hybridization. Nucleic acid sequencing, qualitative and quantitative detection are performed by hybridization with a set of nucleic acid probes of known sequence. Target nucleotides of known sequence, immobilized on the substrate surface, are called probes. When the nucleic acid molecules with fluorescent labels in the solution generate complementary matching with the nucleic acid probes at corresponding positions on the gene chip, the sequence and content of the detection nucleic acid can be determined according to the intensity and position of the fluorescent signal. The gene chip can simultaneously realize high-efficiency, quick and low-cost detection and analysis on a large number of nucleic acid molecules.
The gene chip has wide application, including disease diagnosis and treatment, medicine screening, crop breeding optimization, judicial identification, food health supervision, environment detection, national defense, spaceflight and other fields. Helps human to know the origin, heredity, development and evolution process of life, opens up a brand new approach for diagnosis, treatment and prevention of human diseases, and provides a technical support platform for the brand new design of biomacromolecules, rapid screening of lead compounds in drug development and pharmacogenomics research.
Currently, there are two main types of gene chips, spotting and in situ synthesis. The spotting method is to spot a pre-prepared probe on a glass plate using an automated micro spotting device and fix the probe at a corresponding position by a chemical modification method. The lattice density of the method can range from hundreds to tens of thousands, and the bonding amount of each probe on the surface is consistent, so that the method is low in cost and flexible in customization. However, the lattice density of this method is still different from that of the in situ synthesis method. The in-situ synthesis method is an oligonucleotide probe array directly synthesized on hard surfaces such as glass and the like by applying a microelectronic photoetching technology and a DNA chemical synthesis technology. The method has extremely high probe density which can reach hundreds of thousands of levels, but the customization cost is extremely high, and the flexibility is not as good as that of a spotting method. Secondly, each probe molecule is bonded to the substrate by means of a chemical bond, so that the method also has the defects of low efficiency and high background signal.
Therefore, it is desirable to obtain a chip with high sensitivity at a low cost when preparing the chip. The national invention patent "gene chip with high specificity and high sensitivity and its preparation method and application" (CN 102168146B) provides a preparation method of gene chip, each probe molecule is combined with the surface of the substrate by a plurality of chemical bonds, thus greatly increasing the stability of the probe and reducing the influence of background noise. However, this method has a high requirement on the length of the probe, and on the one hand, if the length of the probe is too short, the immobilization of multiple chemical bonds can cause the probe to be fixed on the surface of the substrate, so that the hybridization efficiency is low or the hybridization cannot be performed; on the other hand, the probe length is short, in order to take the hybridization specificity and the hybridization efficiency into consideration, the concentration ratio requirement of the chemical modifier for fixing the probe is extremely strict, and if the probe length is long enough, a wider proportion range can be selected, thereby facilitating the operation in large batch. The probe preparation technique described in this patent is accomplished in two steps, first, chemically synthesized short probe sequences are 5' phosphorylated; ligation is then performed with the aid of a short auxiliary linker sequence, allowing the short probe sequences to be ligated head-to-tail into a long sequence. This approach still has certain drawbacks. Because all the bases of the probe molecules and the auxiliary joint molecules are open in a single-stranded form in the process of the connection reaction, the interaction between the probe molecules and the auxiliary joint molecules is difficult to avoid for a large number of different probe sequences, and a complex secondary structure is formed, thereby affecting the connection efficiency. This severely limits the wide application of the method. Meanwhile, because a plurality of ligation reactions are required, the probe molecules can be ligated to a sufficient length, which requires high activity and dosage of ligase, and high reaction time and temperature. The connected probe is directly used for chip production, and has higher consumption and higher cost for chemically synthesized short probes and enzyme dosage in each preparation link.
Disclosure of Invention
The primary object of the present invention is to overcome the disadvantages of the prior art and to provide a method for preparing a long probe from a circular single-stranded probe.
Another object of the present invention is to provide the use of the above-mentioned method for preparing a long probe from a circular single-stranded probe in the production of a gene chip.
The purpose of the invention is realized by the following technical scheme: a method for preparing a long probe from a circular single-stranded probe, comprising the steps of:
(1) obtaining a circular single-stranded probe by the scheme (A) or (B);
(A) connecting the probe and the amplification joint to form a ring;
(B) the probe is connected into a ring;
(2) preparing a linear single-chain long probe from the circular single-chain probe obtained in the step (1);
(A) amplifying the circular single-stranded probe obtained in the scheme (A) in the step (1) by using an amplification primer which can be complementary with the amplification joint to obtain a long probe;
(B) for the circular single-stranded probe obtained in the scheme (B) in the step (1), amplification is performed using a random primer or a primer specific to the probe to obtain a long probe.
The steps of scheme (a) are preferably as follows:
1) designing a composite probe: the structure of the composite probe is that a sequence 1, a short probe, a sequence 2 and a sequence 3 are connected in sequence; wherein, the sequence 1 and the sequence 2 are mutually reverse complementary sequences, so that a stable hairpin structure is formed in the molecule firstly, the internal base sequence of the short probe is protected, and only the sequence 3 is exposed, as shown in FIG. 1A;
2) designing a composite amplification site: the structure of the composite amplification site is that a sequence 4, a universal amplification primer site, a sequence 5 and a sequence 6 are connected in sequence, as shown in figure 1B; wherein, the sequence 4 and the sequence 5 are mutually reverse complementary sequences;
3) preparation of circular single-stranded Probe: and mixing the composite probe and the composite amplification site, phosphorylating, annealing, forming a hairpin structure in the molecule, and performing a ligation reaction to form a composite circular molecule containing one copy of the probe, as shown in FIG. 2.
The composite probe in the step 1) is one probe or a mixture of a plurality of probes.
When the composite probe is a mixture of multiple probes, the sequence 1 and the sequence 2 between different probes should not match with each other, so as to prevent the probes from forming a secondary structure with each other, and the sequence 3 is the same sequence.
The length of the short probe in the step 1) is preferably 20-60 nt.
The short probe is a target sequence to be detected selected by the gene chip or a reverse complementary sequence of the target sequence.
The length of the sequence 1 in the step 1) is preferably 5-10 nt.
The length of the sequence 2 in the step 1) is preferably 5-10 nt.
The length of the sequence 3 in the step 1) is preferably 1-8 nt.
The length of the sequence 4 in the step 2) is preferably 5-10 nt.
The length of the sequence 5 in the step 2) is preferably 5-10 nt.
The length of the sequence 6 in the step 2) is preferably 1-8 nt.
The length of the universal amplification primer site in the step 2) is preferably 10-30 nt, the GC content is preferably 40-60%, and the similarity with a chip detection target sequence is preferably lower than 50%.
The sequence 3 and the sequence 6 in the step 2) are reverse complementary matched sequences, and have good specificity when being non-palindromic structures.
The compound probe and the compound amplification site in the step 3) are preferably selected from the group consisting of a 1: 1-5.
The sequences 4 and 5 and the sequences 1 and 2 are completely mismatched sequences, so that the stable hairpin structure is formed by the sequences 1 and 2, and the stable hairpin structure is formed by the sequences 4 and 5.
The annealing conditions in step 3) are preferably: preserving the heat at 90-100 ℃ for 1-10 min, and then slowly cooling to room temperature, without being limited thereto; more preferably, the temperature is kept at 100 ℃ for 5min, and then the temperature is slowly reduced to room temperature.
The room temperature is 0-40 ℃; preferably 5-35 ℃; more preferably 20-30 ℃; most preferably 24 to 26 ℃.
The steps of scheme (B) are preferably as follows:
designing a composite probe: the structure of the composite probe is that a sequence 1, a short probe, a sequence 2 and a sequence 3 are connected in sequence; wherein, the sequence 1 and the sequence 2 are mutually reverse complementary sequences, so that a stable hairpin structure is formed in the molecule firstly, the internal base sequence of the short probe is protected, and only the sequence 3 is exposed, as shown in FIG. 1A, the sequence 3 is a palindrome structure;
preparing a circular single-stranded probe: phosphorylating and annealing the composite probe to form a hairpin structure in the molecule and perform a ligation reaction to form a composite cyclic molecule containing at least two copy probes; performing rolling circle amplification on the composite circular molecule, the amplification primer and the DNA polymerase to obtain a circular single-stranded probe, as shown in FIG. 3.
The composite probe in the step I is one probe or a mixture of a plurality of probes.
When the composite probe is a mixture of multiple probes, the sequence 1 and the sequence 2 between different probes should not match each other, so as to prevent the probes from forming a secondary structure with each other.
The length of the short probe in the step (i) is preferably 20-60 nt.
The length of the sequence 1 in the step (i) is preferably 5-10 nt.
The length of the sequence 2 in the step (i) is preferably 5-10 nt.
The length of the sequence 3 in the step (i) is preferably 2-8 nt.
The probes in the compound circular molecules in the step (II) are probes with the same sequence or probes with different sequences.
The amplification primer in the second step is a random primer or a specific primer aiming at the probe.
The length of the random primer is preferably 6-10 bases.
The specific primer aiming at the probe is preferably a primer with the following characteristics: the length is 10-30 nt, and the GC content is 40-60%.
When the composite probe is a plurality of probes, the specific primer aiming at the probe is a mixture formed by primers complementary to different probes.
The annealing conditions in the step (c) are preferably as follows: preserving the heat at 90-100 ℃ for 1-10 min, and then slowly cooling to room temperature, without being limited thereto; more preferably, the temperature is kept at 100 ℃ for 5min, and then the temperature is slowly reduced to room temperature.
The amplification in the second step is isothermal rolling circle amplification.
The composite circular molecule and the amplification primer in the second step are preferably mixed according to a molar ratio of 1: 2-7.
The method for preparing the long probe by the circular single-stranded probe is particularly suitable for being applied to the production of gene chips.
Compared with the technology of directly connecting and preparing the probe, the invention has the following advantages and effects: the invention only needs two molecules with two ends connected to form a ring, and then a macromolecular probe is amplified through the ring, therefore, the invention has the following advantages: firstly, each cyclic molecule only needs two ligation reactions, the amount of ligase and reaction conditions are easy to control, and long probes prepared by a direct ligation method need multiple ligation reactions; secondly, the average 300nt can be obtained by the traditional method for preparing the probe by direct connection, and the length of the probe obtained by amplification of the invention is excellent and can reach more than 10000 nt; thirdly, the amplification length of the probe and the amplification time are approximately in a linear relationship, so that the length of the probe in the same batch is uniform and hybridization signals are more uniform during batch operation; fourthly, the probe obtained by amplification has extremely high yield which is about dozens of times of that of a direct connection method, and the processes of repeated phosphorylation and connection reaction are saved, so that the use and preparation time of a large amount of biological enzyme is saved.
Drawings
FIG. 1 is a schematic diagram of the structure of a composite probe sequence and a composite amplification site sequence; wherein, Panel A is a schematic structural diagram of a composite probe sequence; FIG. B is a schematic diagram of the structure of the composite amplification site sequence.
FIG. 2 is a schematic diagram showing the preparation process of the probe of scheme (A).
FIG. 3 is a schematic diagram showing the preparation process of the probe of scheme (B).
FIG. 4 is an electrophoretogram of the results of the probe preparation in example 1; wherein, lanes 1, 6 and 11 are probes obtained by directly connecting salmonella, staphylococcus and vibrio probe sets respectively, lanes 2, 7 and 12 are circular probes obtained by connecting scheme (A), lanes 3, 8 and 13 are long probes obtained by amplifying scheme (A), lanes 4, 9 and 14 are circular probes obtained by connecting scheme (B), lanes 5, 10 and 15 are long probes obtained by amplifying scheme (B), and lane M is DNA Marker.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1
Bacterial infections are a common class of diseases in the clinic. The length of the 16s gene is about 1500 bases, and the diversity of the gene can be detected by a gene chip to distinguish the types of bacteria.
(1) Designing a probe: the following probe sequences are selected from unique sequences of 16S genes of salmonella, staphylococcus and vibrio, and the melting temperature of each probe is about 70 ℃; the same set of probes was designed and prepared using the direct ligation method and protocols (A), (B) herein, respectively.
The probe sequences for the direct ligation method, set I (5 '-3'), are as follows:
the probe sequence of scheme (A), group II (5 '-3'), is as follows:
the probe sequence of scheme (B), group III (5 '-3'), is as follows:
all sequences were prepared by chemical synthesis from a facilitator (synbio-tech).
(2) Probe preparation
1) Direct joining method
Mixing the probes of different strains in group I in equimolar amount, placing 5 μ g into 1.5mL centrifuge tube, adding 2 μ L10 XT 4 phosphorylase kinase buffer (pH 7.6, 0.5M Tris-HCl, 0.1M MgCl)220mM dithiothreitol), 2. mu.L of 10mM ATP, and water to a total volume of 20. mu.L. 5 units of T4 phosphorylase kinase (New England Biolabs) were added, mixed well and placed in an incubator at 37 ℃ for 2 hours.
Then, 20. mu.L of 2 × ligation buffer (400mM Tris-HCl, pH 7.8, 100mM MgCl)2100mM DTT), 1000 units of T4 ligase (New England Biolabs) and 2. mu.g of auxiliary linker were mixed well and placed in an incubator at 15 ℃ for 18 hours. After the reaction, 7. mu.l of 5M NaCl and 70. mu.l of isopropanol were added, mixed well, and placed in a refrigerator at-20 ℃ for 15 minutes or longer. Centrifugation was carried out at 14,000rpm for 10 minutes, the supernatant was removed, the precipitate was rinsed with an 80% ethanol solution by volume, the ethanol solution was aspirated, and air-dried to obtain a multimeric probe.
2) Scheme (a):
mixing the probes of different strains in group II in equal amount, placing 5 μ g into 1.5mL centrifuge tube, adding 2 μ L10 XT 4 phosphorylation kinase buffer (pH 7.6, 0.5M Tris-HCl, 0.1M MgCl)220mM dithiothreitol), 2. mu.L of 10mM ATP, 5 units of T4 phosphokinase (New England Biolabs) and 2. mu.g of the multiplex amplification sequence, supplemented with water to a total volume of 20. mu.L. Mixing, and reacting in a constant temperature box at 37 deg.C for 2 hr. Then, the mixture was heated to 100 ℃ for 5 minutes and naturally cooled to room temperature.
Then, 20. mu.L of 2 × ligation buffer (400mM Tris-HCl, pH 7.8, 100mM MgCl)2100mM DTT), 1000 units of T4 ligase (New England Biolabs), mixed well, and placed in a 15 ℃ incubator for 18 hours to obtain circularized DNA.
mu.L of each reaction product (i.e., cyclized DNA) was added to a 1.5mL centrifuge tube, and 10. mu.L of 10 XBst reaction buffer (200mM Tris-HCl pH 8.8, 100mM KCl, 100mM (NH)4)2SO4、20mM MgSO4) mu.L of 2.5mM dNTP, 1. mu.L of 10. mu.M amplification primer, 50 units of Bst enzyme (New England Biolabs), and water to 100. mu.L. Mixing, and reacting in a constant temperature box at 50 deg.C for 18 hr. After the reaction, 7. mu.l of 5M NaCl and 70. mu.l of isopropanol were added, mixed well, and placed in a refrigerator at-20 ℃ for 15 minutes or longer. Centrifuging at 14,000rpm for 10 minutes, removing the supernatantAnd (5) cleaning the solution, rinsing the precipitate by using an ethanol solution with the volume percentage of 80%, sucking the ethanol solution, and air-drying in the air to obtain the polymer probe.
3) Scheme (B):
mixing the probes of different strains in group III in equal amount, placing 5 μ g into 1.5mL centrifuge tube, adding 2 μ L10 XT 4 phosphorylation kinase buffer (pH 7.6, 0.5M Tris-HCl, 0.1M MgCl)220mM dithiothreitol), 2. mu.L of 10mM ATP, 5 units of T4 phosphokinase (New England Biolabs), and water supplemented to a total volume of 20. mu.L. Mixing, and reacting in a constant temperature box at 37 deg.C for 2 hr. Then, the mixture was heated to 100 ℃ for 5 minutes and naturally cooled to room temperature.
Then, 20. mu.L of 2 × ligation buffer (400mM Tris-HCl, pH 7.8, 100mM MgCl)2100mM DTT), 1000 units of T4 ligase (New England Biolabs), mixed well, and placed in a 15 ℃ incubator for 18 hours to obtain circularized DNA.
mu.L of each reaction product (i.e., cyclized DNA) was added to a 1.5mL centrifuge tube, and 10. mu.L of 10 XBst reaction buffer (200mM Tris-HCl pH 8.8, 100mM KCl, 100mM (NH)4)2SO4、20mM MgSO4) mu.L of 2.5mM dNTP, 1. mu.L of 10. mu.M universal amplification primer, 50 units of Bst enzyme (New England Biolabs), supplemented with water to 100. mu.L. Mixing, and reacting in a constant temperature box at 50 deg.C for 18 hr. After the reaction, 7. mu.l of 5M NaCl and 70. mu.l of isopropanol were added, mixed well, and placed in a refrigerator at-20 ℃ for 15 minutes or longer. Centrifugation was carried out at 14,000rpm for 10 minutes, the supernatant was removed, the precipitate was rinsed with an 80% ethanol solution by volume, the ethanol solution was aspirated, and air-dried to obtain a multimeric probe.
(3) Comparison of results
The amplification results were detected on a 2% agarose gel, and the results are shown in FIG. 4. Lanes 1, 6 and 11 are directly ligated probes with varying lengths of about 500bp, but insufficient ligation of about 100bp of the major band. Lanes 2, 7, 12 and 4, 9, 14 show the circular probes obtained by ligation according to schemes (A) and (B), respectively, with no obvious dragging and very sufficient ligation. Lanes 3, 8, 13 and 5, 10, 15 are the probes obtained from the protocol (A) and (B) amplification, respectively, and it can be seen that the smear extends to the glue well, and the length is significantly longer than that of the direct ligation method. As can be seen from the figure, the probes obtained by the rolling circle amplification of the two protocols are uniformly distributed, and the directly connected probes are concentrated in a shorter length. By adopting the scheme of the invention, the long polymer probe can be obtained by only one-step amplification of the ligation product circular probe during the second preparation. According to each connection, 40 mu L of the circular probe can be obtained, each amplification only needs 0.5 mu L, the short probe with the same quantity can be repeatedly amplified for 80 times theoretically, and a large amount of biological enzyme, materials and preparation time are saved.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> Zhongshan Kangyuan Gene technology, science and technology Co., Ltd
<120> a method for preparing a long probe by using a circular single-stranded probe and use thereof in the production of gene chips
<160> 91
<170> SIPOSequenceListing 1.0
<210> 1
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 1
<400> 1
gaggcgaagg tgttgtggtt aataaccgca gcaattgacg ttacgaggc 49
<210> 2
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 2
<400> 2
gaggcttagt tgccagcatt cagttgggca ctctaagggg actggaggc 49
<210> 3
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 3
<400> 3
gaggcagcga gaccgcgagg tcgagctaat ctccataagg aggc 44
<210> 4
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 4
<400> 4
gaggcgagga aggtgttgtg gttaataacc gcagcaattg acgttacgag gc 52
<210> 5
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 5
<400> 5
gaggcacaga aactttccag agaatggaat tggtgccctt cgggaacgag gc 52
<210> 6
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 6
<400> 6
gaggctttgt tgccagcggt taggccggga actcaaagga ggc 43
<210> 7
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 7
<400> 7
gaggcgacct cgcgagagca agcggacctc ataaagtatg gaggc 45
<210> 8
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 8
<400> 8
gaggctcact gggatgctta acgtcacaat tgaagggtaa tcagtcgagg c 51
<210> 9
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 9
<400> 9
gaggctttgt tgccagcgcg taatggcggg aactcaaagg agaggc 46
<210> 10
<211> 48
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 10
<400> 10
gaggcgatac aaagagaagc gacctcgcga gagcaagcgg aacgaggc 48
<210> 11
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 11
<400> 11
gaggcacggc tcaaccgtgg agggtcattg gaaactggaa aactgaggc 49
<210> 12
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 12
<400> 12
gaggcgtgct aagtgttagg gggtttccgc cccttagtgc tggaggc 47
<210> 13
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 13
<400> 13
gaggcctttg acaactctag agatagagcg ttccccttcg gaggc 45
<210> 14
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 14
<400> 14
gaggcgaagc cgcaaccctt atcttgtttg gccaagcgtc tggaggc 47
<210> 15
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 15
<400> 15
gaggcagtag ggaggaaagg gtgagtccta atacggctta tctgtgacga ggc 53
<210> 16
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 16
<400> 16
gaggctgggc tcaacctagg aatagcattt cgaactgaca aactagagga ggc 53
<210> 17
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 17
<400> 17
gaggctcgga gtttggtgtc ttgaacactg ggctctcaag ctaacgaggc 50
<210> 18
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 18
<400> 18
gaggcacaga agactgcaga gatgcggttg tgccttcgga ggc 43
<210> 19
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 19
<220>
<221> misc_feature
<222> (25)..(25)
<223> n is a, c, g, t or u
<400> 19
gaggctttgt tgccagcgat tcggncggga actcaaagga ggaggc 46
<210> 20
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 20
<400> 20
gaggcaaaga gaagcgacct cgcgagagca agcggaggc 39
<210> 21
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 21
<400> 21
gaggcgtcag ggaggaaatc cctagcgtta ataccgctgg gggatggagg c 51
<210> 22
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 22
<400> 22
gaggcgagct caacttggga actgcgtttg gaactgtcag actagaggag gc 52
<210> 23
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 23
<400> 23
gaggcaatta gctgttgggg gttagaatcc ctggtagcgt agctaacgag gc 52
<210> 24
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 24
<400> 24
gaggcgtacg gaacttgcca gagatggctt ggtgcccgaa aggaggc 47
<210> 25
<211> 48
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 25
<400> 25
gaggcgttct atgttgccag cgcgttatgg cggggactca taggaggc 48
<210> 26
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 26
<400> 26
gaggcgattt ggaggctgtg tccttgagac gtggcttccg aggc 44
<210> 27
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 27
<400> 27
gaggcgaatc ctgcagagat gcgggagtgc cttcgggaat cgaggc 46
<210> 28
<211> 48
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 28
<400> 28
gaggcgttgc cagcacgtca tggtgggaac tcaaaggaga ctggaggc 48
<210> 29
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 29
<400> 29
gaggcagata caaagtgaag cgaactcgcg agagcaagcg aggc 44
<210> 30
<211> 10
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> auxiliary joint
<400> 30
<210> 31
<211> 55
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 30
<400> 31
gctgacgaag gtgttgtggt taataaccgc agcaattgac gttacgtcag cgcag 55
<210> 32
<211> 56
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 31
<400> 32
gctgacctta gttgccagca ttcagttggg cactctaagg ggactggtca gcgcag 56
<210> 33
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 32
<400> 33
gctgaccagc gagaccgcga ggtcgagcta atctccataa ggtcagcgca g 51
<210> 34
<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 33
<400> 34
gctgacgagg aaggtgttgt ggttaataac cgcagcaatt gacgttacgt cagcgcag 58
<210> 35
<211> 59
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 34
<400> 35
gctgaccaca gaaactttcc agagaatgga attggtgccc ttcgggaacg tcagcgcag 59
<210> 36
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 35
<400> 36
gctgaccttt gttgccagcg gttaggccgg gaactcaaag gtcagcgcag 50
<210> 37
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 36
<400> 37
gctgacgacc tcgcgagagc aagcggacct cataaagtat ggtcagcgca g 51
<210> 38
<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 37
<400> 38
gctgacctca ctgggatgct taacgtcaca attgaagggt aatcagtcgt cagcgcag 58
<210> 39
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 38
<400> 39
gctgaccttt gttgccagcg cgtaatggcg ggaactcaaa ggagtcagcg cag 53
<210> 40
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 39
<400> 40
gctgacgata caaagagaag cgacctcgcg agagcaagcg gaacgtcagc gcag 54
<210> 41
<211> 56
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 40
<400> 41
gctgaccacg gctcaaccgt ggagggtcat tggaaactgg aaaactgtca gcgcag 56
<210> 42
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 41
<400> 42
gctgacgtgc taagtgttag ggggtttccg ccccttagtg ctggtcagcg cag 53
<210> 43
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 42
<400> 43
gctgaccctt tgacaactct agagatagag cgttcccctt cggtcagcgc ag 52
<210> 44
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 43
<400> 44
gctgacgaag ccgcaaccct tatcttgttt ggccaagcgt ctggtcagcg cag 53
<210> 45
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 44
<400> 45
gctgaccagt agggaggaaa gggtgagtcc taatacggct tatctgtgac gtcagcgcag 60
<210> 46
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 45
<400> 46
gctgacctgg gctcaaccta ggaatagcat ttcgaactga caaactagag gtcagcgcag 60
<210> 47
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 46
<400> 47
gctgacctcg gagtttggtg tcttgaacac tgggctctca agctaacgtc agcgcag 57
<210> 48
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 47
<400> 48
gctgaccaca gaagactgca gagatgcggt tgtgccttcg gtcagcgcag 50
<210> 49
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 48
<220>
<221> misc_feature
<222> (27)..(27)
<223> n is a, c, g, t or u
<400> 49
gctgaccttt gttgccagcg attcggncgg gaactcaaag gaggtcagcg cag 53
<210> 50
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 49
<400> 50
gctgaccaaa gagaagcgac ctcgcgagag caagcggtca gcgcag 46
<210> 51
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 50
<400> 51
gctgacgtca gggaggaaat ccctagcgtt aataccgctg ggggatggtc agcgcag 57
<210> 52
<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 51
<400> 52
gctgacgagc tcaacttggg aactgcgttt ggaactgtca gactagaggt cagcgcag 58
<210> 53
<211> 59
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 52
<400> 53
gctgaccaat tagctgttgg gggttagaat ccctggtagc gtagctaacg tcagcgcag 59
<210> 54
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 53
<400> 54
gctgacgtac ggaacttgcc agagatggct tggtgcccga aaggtcagcg cag 53
<210> 55
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 54
<400> 55
gctgacgttc tatgttgcca gcgcgttatg gcggggactc ataggtcagc gcag 54
<210> 56
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 55
<400> 56
gctgacgatt tggaggctgt gtccttgaga cgtggcttcc gtcagcgcag 50
<210> 57
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 56
<400> 57
gctgacgaat cctgcagaga tgcgggagtg ccttcgggaa tcgtcagcgc ag 52
<210> 58
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 57
<400> 58
gctgacgttg ccagcacgtc atggtgggaa ctcaaaggag actggtcagc gcag 54
<210> 59
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 58
<400> 59
gctgaccaga tacaaagtga agcgaactcg cgagagcaag cgtcagcgca g 51
<210> 60
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> multiplex amplification sequence
<400> 60
gtcagcgatc gatcgatcgc atctatgctg acctgc 36
<210> 61
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> amplification primers
<400> 61
atagatgcga tcgatcgatc 20
<210> 62
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 59
<400> 62
gctgacgaag gtgttgtggt taataaccgc agcaattgac gttacgtcag cgcatgc 57
<210> 63
<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 60
<400> 63
gctgacctta gttgccagca ttcagttggg cactctaagg ggactggtca gcgcatgc 58
<210> 64
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 61
<400> 64
gctgaccagc gagaccgcga ggtcgagcta atctccataa ggtcagcgca tgc 53
<210> 65
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 62
<400> 65
gctgacgagg aaggtgttgt ggttaataac cgcagcaatt gacgttacgt cagcgcatgc 60
<210> 66
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 63
<400> 66
gctgaccaca gaaactttcc agagaatgga attggtgccc ttcgggaacg tcagcgcatg 60
c 61
<210> 67
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 64
<400> 67
gctgaccttt gttgccagcg gttaggccgg gaactcaaag gtcagcgcat gc 52
<210> 68
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 65
<400> 68
gctgacgacc tcgcgagagc aagcggacct cataaagtat ggtcagcgca tgc 53
<210> 69
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 66
<400> 69
gctgacctca ctgggatgct taacgtcaca attgaagggt aatcagtcgt cagcgcatgc 60
<210> 70
<211> 55
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 67
<400> 70
gctgaccttt gttgccagcg cgtaatggcg ggaactcaaa ggagtcagcg catgc 55
<210> 71
<211> 56
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 68
<400> 71
gctgacgata caaagagaag cgacctcgcg agagcaagcg gaacgtcagc gcatgc 56
<210> 72
<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 69
<400> 72
gctgaccacg gctcaaccgt ggagggtcat tggaaactgg aaaactgtca gcgcatgc 58
<210> 73
<211> 55
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 70
<400> 73
gctgacgtgc taagtgttag ggggtttccg ccccttagtg ctggtcagcg catgc 55
<210> 74
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 71
<400> 74
gctgaccctt tgacaactct agagatagag cgttcccctt cggtcagcgc atgc 54
<210> 75
<211> 55
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 72
<400> 75
gctgacgaag ccgcaaccct tatcttgttt ggccaagcgt ctggtcagcg catgc 55
<210> 76
<211> 62
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 73
<400> 76
gctgaccagt agggaggaaa gggtgagtcc taatacggct tatctgtgac gtcagcgcat 60
gc 62
<210> 77
<211> 62
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 74
<400> 77
gctgacctgg gctcaaccta ggaatagcat ttcgaactga caaactagag gtcagcgcat 60
gc 62
<210> 78
<211> 59
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 75
<400> 78
gctgacctcg gagtttggtg tcttgaacac tgggctctca agctaacgtc agcgcatgc 59
<210> 79
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 76
<400> 79
gctgaccaca gaagactgca gagatgcggt tgtgccttcg gtcagcgcat gc 52
<210> 80
<211> 55
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 77
<220>
<221> misc_feature
<222> (27)..(27)
<223> n is a, c, g, t or u
<400> 80
gctgaccttt gttgccagcg attcggncgg gaactcaaag gaggtcagcg catgc 55
<210> 81
<211> 48
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 78
<400> 81
gctgaccaaa gagaagcgac ctcgcgagag caagcggtca gcgcatgc 48
<210> 82
<211> 59
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 79
<400> 82
gctgacgtca gggaggaaat ccctagcgtt aataccgctg ggggatggtc agcgcatgc 59
<210> 83
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 80
<400> 83
gctgacgagc tcaacttggg aactgcgttt ggaactgtca gactagaggt cagcgcatgc 60
<210> 84
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 81
<400> 84
gctgaccaat tagctgttgg gggttagaat ccctggtagc gtagctaacg tcagcgcatg 60
c 61
<210> 85
<211> 55
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 82
<400> 85
gctgacgtac ggaacttgcc agagatggct tggtgcccga aaggtcagcg catgc 55
<210> 86
<211> 56
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 83
<400> 86
gctgacgttc tatgttgcca gcgcgttatg gcggggactc ataggtcagc gcatgc 56
<210> 87
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 84
<400> 87
gctgacgatt tggaggctgt gtccttgaga cgtggcttcc gtcagcgcat gc 52
<210> 88
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 85
<400> 88
gctgacgaat cctgcagaga tgcgggagtg ccttcgggaa tcgtcagcgc atgc 54
<210> 89
<211> 56
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 86
<400> 89
gctgacgttg ccagcacgtc atggtgggaa ctcaaaggag actggtcagc gcatgc 56
<210> 90
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Probe 87
<400> 90
gctgaccaga tacaaagtga agcgaactcg cgagagcaag cgtcagcgca tgc 53
<210> 91
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Universal amplification primers
<220>
<221> misc_feature
<222> (1)..(1)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (2)..(2)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (3)..(3)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (4)..(4)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (5)..(5)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (6)..(6)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (7)..(7)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (8)..(8)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (9)..(9)
<223> n is a, c, g, t or u
<400> 91
Claims (8)
1. A method for preparing a long probe from a circular single-stranded probe, comprising the steps of:
(1) obtaining a circular single-stranded probe by the scheme (A) or (B);
(A) connecting the probe and the amplification joint to form a ring;
1) designing a composite probe: the structure of the composite probe is that a sequence 1, a short probe, a sequence 2 and a sequence 3 are connected in sequence; wherein, the sequence 1 and the sequence 2 are mutually reverse complementary sequences;
2) designing a composite amplification site: the structure of the composite amplification site is that a sequence 4, a universal amplification primer site, a sequence 5 and a sequence 6 are connected in sequence; wherein, the sequence 4 and the sequence 5 are mutually reverse complementary sequences; and the sequence 6 is a reverse complementary matched sequence with the sequence 3 in the step 1), and is a non-palindromic structure;
3) preparation of circular single-stranded Probe: mixing the composite probe and the composite amplification site, phosphorylating and annealing to form a hairpin structure in the molecule and perform a ligation reaction to form a composite circular molecule containing a copy probe;
(B) the probe is connected into a ring;
designing a composite probe: the structure of the composite probe is that a sequence 1, a short probe, a sequence 2 and a sequence 3 are connected in sequence; wherein, the sequence 1 and the sequence 2 are mutually reverse complementary sequences, and the sequence 3 is a palindrome;
preparing a circular single-stranded probe: phosphorylating and annealing the composite probe to form a hairpin structure in the molecule and perform a ligation reaction to form a composite cyclic molecule containing at least two copy probes; performing rolling circle amplification on the composite circular molecule, the amplification primer and the DNA polymerase to obtain a circular single-stranded probe;
(2) preparing a linear single-chain long probe from the circular single-chain probe obtained in the step (1);
(A) amplifying the circular single-stranded probe obtained in the scheme (A) in the step (1) by using an amplification primer which can be complementary with the amplification joint to obtain a long probe;
(B) amplifying the circular single-stranded probe obtained in the scheme (B) in the step (1) by using a specific primer aiming at the probe to obtain a long probe;
the sequences 4 and 5 and the sequences 1 and 2 are completely mismatched sequences.
2. The method for preparing a long probe by a circular single-stranded probe according to claim 1, wherein:
the composite probe in the step 1) is one probe or a mixture of a plurality of probes;
when the composite probe is a mixture of a plurality of probes, the sequences 1 and 2 among different probes should be not matched with each other, and the sequence 3 is the same sequence;
the short probe is a target sequence to be detected selected by the gene chip or a reverse complementary sequence of the target sequence;
the general amplification primer site in the step 2) is a sequence which has the length of 10-30 nt, the GC content of 40-60% and the similarity with a chip detection target sequence of less than 50%.
3. The method for preparing a long probe by a circular single-stranded probe according to claim 1, wherein:
the length of the short probe in the step 1) is 20-60 nt;
the length of the sequence 1 in the step 1) is 5-10 nt;
the length of the sequence 2 in the step 1) is 5-10 nt;
the length of the sequence 3 in the step 1) is 1-8 nt;
the length of the sequence 4 in the step 2) is 5-10 nt;
the length of the sequence 5 in the step 2) is 5-10 nt;
the length of the sequence 6 in the step 2) is 1-8 nt.
4. The method for preparing a long probe by a circular single-stranded probe according to claim 1, wherein:
the compound probe and the compound amplification site in the step 3) are mixed according to the molar ratio of 1: 1-5 parts by weight;
the annealing conditions in the step 3) are as follows: keeping the temperature at 90-100 ℃ for 1-10 min, and then slowly cooling to room temperature.
5. The method for preparing a long probe by a circular single-stranded probe according to claim 1, wherein:
the composite probe in the step I is one probe or a mixture of a plurality of probes;
when the composite probe is a mixture of a plurality of probes, the sequence 1 and the sequence 2 between different probes should not match with each other;
the probes in the compound circular molecules in the step II are probes with the same sequence or probes with different sequences;
the amplification primer in the second step is a specific primer aiming at the probe;
the specific primer aiming at the probe is a primer with the following characteristics: the length is 10-30 nt, and the GC content is 40-60%;
when the composite probe is a mixture of multiple probes, the specific primer for the probe is a mixture of primers complementary to different probes.
6. The method for preparing a long probe by a circular single-stranded probe according to claim 1, wherein:
the length of the short probe in the step I is 20-60 nt;
the length of the sequence 1 in the step I is 5-10 nt;
the length of the sequence 2 in the step I is 5-10 nt;
the length of the sequence 3 in the step I is 2-8 nt.
7. The method for preparing a long probe by a circular single-stranded probe according to claim 1, wherein:
the annealing conditions in the step II are as follows: preserving heat at 90-100 ℃ for 1-10 min, and then slowly cooling to room temperature;
the amplification in the second step is isothermal rolling circle amplification;
the compound circular molecule and the amplification primer in the step II are mixed according to the molar ratio of 1: 2-7.
8. The method for preparing a long probe from a circular single-stranded probe according to any one of claims 1 to 7, which is used in the production of a gene chip.
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| CN101415838A (en) * | 2003-09-26 | 2009-04-22 | 首科安普有限公司 | Amplification of polynucleotides by rolling circle amplification |
| CN102399853A (en) * | 2010-09-09 | 2012-04-04 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Padlock probe used for detecting transgenic soybean and detection method thereof |
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| CN101415838A (en) * | 2003-09-26 | 2009-04-22 | 首科安普有限公司 | Amplification of polynucleotides by rolling circle amplification |
| CN102399853A (en) * | 2010-09-09 | 2012-04-04 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Padlock probe used for detecting transgenic soybean and detection method thereof |
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