CN116445594A - Sequencing method suitable for in-situ detection of continuous multiple nucleotide sites and application thereof - Google Patents

Sequencing method suitable for in-situ detection of continuous multiple nucleotide sites and application thereof Download PDF

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CN116445594A
CN116445594A CN202310290116.2A CN202310290116A CN116445594A CN 116445594 A CN116445594 A CN 116445594A CN 202310290116 A CN202310290116 A CN 202310290116A CN 116445594 A CN116445594 A CN 116445594A
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曹罡
张凌凯
吴小凤
戴金霞
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Kunyu Biotechnology Jiangmen Co ltd
Huazhong Agricultural University
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Huazhong Agricultural University
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Abstract

The invention discloses a sequencing method suitable for in-situ detection of continuous multiple nucleotide loci and application thereof. According to the design characteristics of the probe, the unknown nucleic acid sequence can be sequenced, and the method can be applied to tumor point mutation detection, continuous mutation detection and the like. Can realize the in-situ interpretation of single-cell, single-molecule and single-base resolution gene sequences. The method can perform high-flux tissue in-situ detection to obtain in-situ space spectrograms of target sequences of different cells of a tissue sample, and in-vitro oligos-specific cell enrichment library-building sequencing with high difficulty is not needed. And the signal interpretation is independent of a super-resolution imaging platform, and has good application prospects in the fields of individual development, genetic breeding, tumor microenvironment, clinical pathology detection and the like.

Description

Sequencing method suitable for in-situ detection of continuous multiple nucleotide sites and application thereof
Technical Field
The invention relates to the field of in-situ sequencing, in particular to a sequencing method suitable for in-situ detection of continuous multiple nucleotide sites and application thereof.
Background
Cells are the fundamental unit of organism structure and function. Since the establishment of the theory of cells in 1839, numerous scientists have been devoted to study the deconstructation, function and relationship of cells to each other in an effort to ascertain the nature of life. In 1958, watson and Crick put forward a central rule that states the order in which genetic information is transferred between biomacromolecules, and molecular biology time-lapse takes place. Based on the development, modern biological subjects such as genomics, transcriptomics, proteomics and the like are vigorous, and scientists search for sailing on the mystery research of life activities. Among them, mRNA is transcribed from DNA, synthesizes protein with the help of ribosome, is a transmitter of genetic information, and plays a vital role in life activity. To date, there are tens of thousands of researchers worldwide devoting to the study of mRNA.
Technological changes are fundamental to the development of the push research. Early studies of mRNA were limited by experimental techniques, requiring extraction of mRNA from cells or tissues, followed by Northern blot hybridization (Northern blot), fluorescent quantitative polymerase chain reaction (quantitative real time PCR, qPCR), first generation sequencing techniques, and the like. Such methods are time consuming and laborious and the information that can be obtained is limited. With the popularization of the second generation sequencing technology, scientists gradually realize the large-scale parallel sequencing of mRNA, and develop a series of RNA sequencing technologies (RNA-seq). In the last decade, RNA-seq has become an important tool for the analysis of differential gene expression and mRNA differential splicing in the whole transcriptome, including single cell gene expression, translation, RNA structure, and the like.
Traditional RNA sequencing (e.g., bulk RNA-seq) involves the extraction of RNA from a whole tissue, pooling and sequencing to obtain the average expression levels of the transcripts in the various cells in this region. However, the functions exerted by different cells in the tissue are different, the mRNA expression levels are also different, and the conventional RNA-seq homogenization strategy can mask a lot of important information. In order to search for the characteristics and functions of different cells in the tissue, the professor Shang Fu was improved based on the former technique, which forms the first truly single cell RNA sequencing technique (single cell RNAsequencing, scRNA-seq).
In 2013, nature Method granted annual technology to single cell sequencing. The technology can provide RNA expression profile of each cell and identify rare cells in heterogeneous cell groups, so that research on developmental biology, neurobiology, cancer mechanism and the like is greatly promoted. The scRNA-seq method and bulk RNA-seq technology jointly function, provide highly detailed data about tissues or cell populations for researchers, and improve the cognition of people on the identity information and the state of cells in the tissues. However, a common disadvantage of such methods is the loss of spatial information of the mRNA in the cell. Such information is important because specific cell types and tissues are closely related to vital activities.
To study the spatial information of transcripts to analyze the close relationship between cellular environment and gene expression, scientists have proposed spatial transcriptomics (spatial transcriptomics, ST). The imaging-based RNA in-situ sequencing method (in situ sequencing, ISS) can directly sequence mRNA in cells in a tissue environment, so that specific sequences of specific transcripts of single cells can be obtained, the spatial positions and corresponding expression levels of the specific transcripts can be obtained, and the obtained mRNA information can be calibrated to the real places of the specific transcripts, thus being a relatively ideal mode.
Currently, commonly used methods for in situ sequencing of mRNA can be divided into two categories based on their method of transcript capture:
1) One class of methods for non-targeted capture of transcripts is represented by FISSEQ, INSTA-Seq. The method can theoretically capture all transcripts and detect information of all transcripts in cells, but in practice, the technology has poor specificity, more rRNA signals in the obtained data, lower mRNA detection efficiency, more complicated steps and few application at present.
2) Another class of methods is the targeted capture of transcripts, a representative technique is STARmap (spatial-resolved Transcript Amplicon Readout Mapping). The method generally relies on a lock probe (padlock probe), uses the probe to capture a specific transcript in situ, is generally sensitive and specific, but has lower sequencing flux, one lock probe can only detect one mutation of one site, and a plurality of probes are required to be designed for slightly long sequencing, so that the method has complex operation and higher cost.
As shown in fig. 1: in situ sequencing of classical lock-in probes, once mutations exist in the sequence to be tested, a variety of probes need to be designed and synthesized based on a priori knowledge. When a point mutation exists in a site to be detected, because the mutation result can be any one of A\T\C\G, at least 4 corresponding probes are respectively synthesized, and the specific position of a mutation base is also needed to be known to capture by using a classical lock-type probe. Once there are several mutations in succession, even if multiple mutations occur simultaneously, a large number of probes need to be designed for detection. The use of multiple classical probes in one experiment greatly increases the cost, reduces the hybridization efficiency and increases the non-specificity.
How to directly, targeted and simply sequence nucleic acids in situ is an urgent need in the development of current technology.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a sequencing method suitable for in-situ detection of continuous multiple nucleotide loci and application thereof. The method can measure the sequence of continuous 4 bases on the nucleic acid by only 4 rounds of connection sequencing. The common lock probe is used, and 4 is required to be arranged for measuring 4 base sequences 4 256 probes are adopted, only 1 probe is needed in the method, and the design workload and the preparation cost of the probes are greatly reduced. The invention can directly sequence RNA in situ, does not need to reversely transcribe the RNA into cDNA, reserves space information, reduces operation steps and improves detection efficiency. The method can detect a section of unknown sequence without priori knowledge, and has strong specificity and high fault tolerance.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
the invention provides a sequencing method suitable for in-situ detection of a plurality of continuous nucleotide sites, which comprises the following steps:
1) Design of probes
Screening out a target area with high hybridization efficiency, good specificity and no advanced structure according to a sequence to be detected, and setting a hybridization probe; wherein the hybridization probe comprises a semi-closed lock-type probe and an RCA initiation probe;
after the semi-closed lock probe is complemented with a target sequence of a sequence to be detected, a gap is formed between the 5 'end and the 3' end of the semi-closed lock probe,
the gap is filled by a single-stranded deoxyribonucleic acid random fragment (5 '-NNNN-3') with X bases and is complementary with a target sequence (namely a sequencing target) of a sequence to be detected; wherein X is 2, 3, 4, 5, 6, 7, 8 and … …;
2) Probe pretreatment
Phosphorylating the 5' end of a semi-closed lock probe and a single-stranded deoxyribonucleic acid random fragment of X bases in the hybridization probe;
3) Sample hybridization
Preparing a reaction chamber on a sample to be detected, fixing the sample by using 4% Paraformaldehyde (PFA), dehydrating and denaturing the sample by using methanol, and adding a system containing a hybridization probe into the reaction chamber for incubation for a period of time;
4) Ligation reaction
Adding a connection system containing single-stranded deoxyribonucleic acid random fragments with X bases into a reaction chamber, filling gaps (Gap) on a semi-closed lock-type probe complementarily paired with a sequence to be detected, and forming a closed annular structure;
5) Rolling circle amplification
The RCA initiating probe is complementary with the targeting sequence of the sequence to be detected and the semi-closed lock probe at the same time, and under the action of Phi29DNA polymerase, rolling circle amplification is carried out by taking the RCA initiating probe as a primer and the annular structure formed by the semi-closed lock probe as a template, so that a target signal is amplified;
6) In situ sequencing of target sequences (applying the principle of ligation sequencing)
In situ sequencing
Adding a sequencing system containing an anchor primer and a sequencing primer into a reaction chamber for in-situ sequencing reaction, sequencing and imaging a sequence corresponding to a first base in a Gap between semi-closed lock probes, and reading the spatial position and sequence information of a target sequence;
sequentially reading information of base sequences at other positions in a Gap between semi-closed lock probes by the same method;
and III, matching the obtained multiple rounds of sequencing information, and reading a targeting sequence corresponding to a Gap formed between the semi-closed lock probes.
Further, in the step 1), the target sequence of the sequence to be detected consists of 4 segments of continuous target sequences A, D, B and C, the semi-closed lock type probe is A 'sequence from 5' end to 3 'end, loop sequence and B' sequence,
the A 'sequence and the B' sequence are respectively complementary with the target sequence A and the target sequence B, and the semi-closed lock type probe and the target sequence are complementary and paired to form a semi-closed annular structure; the loop sequence consists of an anchor sequence and a barcode sequence respectively;
the empty sequence D' is complementary to the target sequence D.
Still further, in the step 1), the sequence D' of the void is composed of a sequence of Y segments, Y is less than X and Y is 1, 2, 3, 4, 5, 6, 7, 8 … … (i.e.: x=x 1 +x 2 +……+x Y X is the number of all bases in the gap, Y is the number of segments that make up the random sequence in the gap, and X is the number of bases in each segment of sequence).
Still further, in the vacant sequence D', X is 2 to 8 and Y is 1 to 6.
Still further, in the step 1), X is 4 (the vacant sequence D' is composed of a single-stranded deoxyribonucleic acid random fragment of 4 bases), and Y is 1.
Still further, in the step 2), 5' -ends of the random fragments of single-stranded deoxyribonucleic acid are phosphorylated.
Still further, in the step 2), the enzyme used in the phosphorylation process is T4 polynucleotide kinase (PNK).
Still further, in the step 4), the ligase of the ligase reaction system is a Splint R ligase.
Still further, in step 6), the target sequence is sequenced in situ
In situ sequencing
Adding a sequencing system containing an anchor primer and a sequencing primer into a reaction chamber for in-situ sequencing reaction, sequencing and imaging a sequence corresponding to a first base in a Gap between semi-closed lock probes, and reading the number, the spatial position and the sequence information of a target sequence;
sequentially reading the information of the base sequences of the second position, the third position and the fourth position in the Gap between the semi-closed lock probes by the same method;
and III, matching the obtained four-wheel sequencing information, and reading a targeting sequence corresponding to a Gap formed between the semi-closed lock probes.
The invention also provides an application of the sequencing method suitable for in-situ detection of continuous multiple nucleotide sites in point mutation detection.
The principle of the invention is as follows:
in the invention, after the semi-closed lock probe is complemented with the target sequence of the sequence to be detected, a gap is formed between the 5 'end and the 3' end of the semi-closed lock probe,
the gap can be filled by a single-stranded deoxyribonucleic acid random fragment with the length of X bases, and a lock-type probe is connected to form a complete annular structure. The process relies on recognition of precise complementary pairing of the bases at the 5 'and 3' ends of the random fragment and the target sequence by the ligase, and any end which is not matched cannot be connected by the ligase to form a closed loop. Meanwhile, when the middle part of the random fragment is not completely matched with the target sequence, the random fragment is easily dissociated from the target sequence due to weak hydrogen bond force formed by hybridization. Only fragments of the complete complementary pair stably fill the Gap and are ligated to the padlock probes by the ligase. Target sequences include, but are not limited to, DNA or RNA.
The invention has the beneficial effects that:
1. the specificity is high: the present invention uses semi-closed lock probes to capture specific transcripts and single-stranded deoxyribonucleic acid random fragments to fill Gap, and the signal amplification depends on two parts: accurate complementary pairing of the capture zone of the lock-type probe and the target sequence and accurate filling of random fragments. Only the target fragments meeting the conditions can be finally detected, thereby improving the specificity of the method.
2. The probe is simple in design and low in cost. The traditional in-situ sequencing technology is used for measuring continuous 4 base sequences, and 4 base sequences are required to be arranged 4 The invention only needs 1 locking probe and matches with a random fragment of 4 base single-stranded deoxyribonucleic acid to measure the sequence of 4 continuous bases on RNA. The probe design is simplified and the cost is greatly reduced.
3. Can be used for detecting gene mutation. The invention can directly sequence the point mutation existing on the sequence or continuous mutation with a certain length, and has better application prospect in tumor mutation detection. Deletion mutations at a single site are also detectable by the present invention, such as: a lock-type probe expected to form a Gap with 4 bases is used for detecting a certain site possibly having deletion mutation, a 3-base single-stranded deoxyribonucleic acid random fragment is additionally added into a connecting system, if the deletion mutation occurs, the lock-type probe forms the 3-base Gap and can be filled, and the information of the deletion mutation can be obtained through subsequent sequencing.
4. The method can directly detect a segment of nucleic acid with unknown sequence, and only the fragments at the two sides of the probe are needed to be known when the probe is designed.
5. Imaging conditions are highly universal: the confocal imaging system can realize signal interpretation. The invention amplifies the signal by the rolling circle amplification method, and the result has high brightness and can be detected by a confocal imaging system.
6. The invention can realize in-situ sequencing at single cell level.
7. The invention directly targets mRNA without reversely transcribing the mRNA into cDNA, thereby improving accuracy, saving cost and having simple and convenient operation.
8. The linking step utilizes the SplingR enzyme, which is more specific and efficient.
In conclusion, the invention uses the semi-closed lock probe to accurately fill the random fragment of the 4-base single-stranded deoxyribonucleic acid in Gap under the action of the SplingR enzyme to form a complete annular structure, and then amplifies signals through rolling circle amplification to read the sequencing result. Compared with the traditional method, the probe provided by the invention has the advantages of simpler design, time and labor saving, low cost, capability of directly capturing the sequence on the mRNA, and good application prospect for detecting mutation. In addition, the confocal imaging system is used for capturing signals, a super-resolution imaging system is not needed, the experimental cost and difficulty are reduced, and the method is easy to popularize.
Drawings
FIG. 1 is a schematic diagram of a classical lock-in probe;
FIG. 2 is a schematic diagram of a hybridization probe according to the present invention;
FIG. 3 is a technical flow chart of the Fill-in method of the present invention;
FIG. 4 is a diagram showing the sequencing imaging effect of the Fill-in method of the present invention;
in the figure, the red dot represents the measured sequence guanine G and the indigo dot represents cytosine C.
Detailed Description
The present invention is described in further detail below in conjunction with specific embodiments for understanding by those skilled in the art.
The invention provides a sequencing method suitable for in-situ detection of a plurality of continuous nucleotide sites, which comprises the following steps:
1) Design of probes
Screening out a target area with high hybridization efficiency, good specificity and no advanced structure according to a sequence to be detected, and setting a hybridization probe; wherein the hybridization probe comprises a semi-closed lock-type probe and an RCA initiation probe;
after the semi-closed lock probe is complemented with a target sequence of a sequence to be detected, a gap is formed between the 5 'end and the 3' end of the semi-closed lock probe,
the gap is filled by a single-stranded deoxyribonucleic acid random fragment (5 '-NNNN-3') with X bases and is complementary with a target sequence (namely a sequencing target) of a sequence to be detected; wherein X is 2, 3, 4, 5, 6, 7, 8 and … …;
2) Probe pretreatment
Phosphorylating the 5' end of a semi-closed lock probe and a single-stranded deoxyribonucleic acid random fragment of X bases in the hybridization probe;
3) Sample hybridization
Preparing a reaction chamber on a sample to be detected, fixing the sample by using 4% Paraformaldehyde (PFA), dehydrating and denaturing the sample by using methanol, and adding a system containing a hybridization probe into the reaction chamber for incubation for a period of time;
4) Ligation reaction
Adding a connection system containing single-stranded deoxyribonucleic acid random fragments with X bases into a reaction chamber, filling gaps (Gap) on a semi-closed lock-type probe complementarily paired with a sequence to be detected, and forming a closed annular structure;
5) Rolling circle amplification
The RCA initiating probe is complementary with the targeting sequence of the sequence to be detected and the semi-closed lock probe at the same time, and under the action of Phi29DNA polymerase, rolling circle amplification is carried out by taking the RCA initiating probe as a primer and the annular structure formed by the semi-closed lock probe as a template, so that a target signal is amplified;
6) In situ sequencing of target sequences (applying the principle of ligation sequencing)
In situ sequencing
Adding a sequencing system containing an anchor primer and a sequencing primer into a reaction chamber for in-situ sequencing reaction, sequencing and imaging a sequence corresponding to a first base in a Gap between semi-closed lock probes, and reading the spatial position and sequence information of a target sequence.
And II, sequentially reading information of base sequences at other positions in the Gap between the semi-closed lock probes by the same method.
And III, matching the obtained multiple rounds of sequencing information, and reading a targeting sequence corresponding to a Gap formed between the semi-closed lock probes.
Based on the method, the following detection is carried out according to the actual conditions:
example 1
Sequencing the sequence of 4 bases on beta-actin transcript in situ using the sequencing method for in situ detection of multiple nucleotide sites
1. Design of hybridization probes
According to the target sequence of the sequence to be detected, a section with high hybridization efficiency, good specificity and no advanced structure is screened out, and a hybridization probe is designed, wherein the hybridization probe targets a section of sequence on mRNA. TABLE 1
2. Probe pretreatment
Phosphorylating the 5' end of a single-stranded deoxyribonucleic acid random fragment of 4 bases with a semi-closed lock probe by using T4 polynucleotide kinase (PNK);
3. sample hybridization
(1) The method performs an assay on a cell sample. To culture the cells to a sufficient number for the test and to facilitate subsequent handling, first, the cells are cultured to 80% full in a glass bottom dish, the medium is discarded before the test, and a reaction chamber is prepared on the glass bottom dish. PBST (PBS containing 0.1% Tween-20) was washed 3 times to remove residual medium and other impurities.
(2) To maintain the cells in morphology and to allow the mRNA therein to remain in its original spatial position, the cells were fixed with 4% paraformaldehyde for 10 minutes, the reaction was completed, the liquid was discarded, and PBST was washed 3 times at room temperature for 5 minutes each to remove excess PFA.
(3) In order to remove the original water in the cells, the reagent is convenient to enter and react, pre-cooled 100% methanol is added on the fixed cells, and the cells are immediately placed at-80 ℃ for incubation for 15 minutes, so that tissue dehydration and denaturation are carried out. After the reaction was completed, the samples were taken out from-80℃and allowed to equilibrate at room temperature for 5 minutes, after which the liquid was discarded and PBST was washed 3 times at room temperature for 5 minutes each.
(4) In order to increase the cell permeability, macromolecular reagents in subsequent reagents are easy to enter cells, and 0.1M hydrochloric acid solution is used for permeabilizing cell membranes, so that a hole structure is formed on the cell membranes. The cells were treated for 5 minutes at room temperature and after the reaction was completed, the liquid was discarded, and PBST was washed 3 times for 5 minutes at room temperature.
(5) After the cell treatment was completed, the hybridization solution containing the lock-in probe was added to the cell sample at a final concentration of 100nM. After overnight incubation at 37 ℃, the padlock probes bind to the target mRNA, forming a complementary structure. After the reaction was completed, the liquid was discarded, and PBST was washed 3 times at room temperature for 5 minutes each.
4. Ligation reaction
After hybridization of the lock-in probe to the mRNA, the gap formed needs to be filled. At this point, a ligation system containing a 4 base random fragment was added to the reaction chamber, the final concentration of random fragment was 100nM. Incubation is carried out at 25 ℃ for 2 hours, and random fragments complementarily paired with RNA are connected with the upper semi-closed lock-type probes under the action of ligase to form a complete circular structure. After the reaction was completed, the liquid was discarded, and PBST was washed 3 times at room temperature for 5 minutes each.
5. Rolling circle amplification
After the lock-in probe is looped, in order to read the target sequence information, the captured information is amplified, and the lock-in probe is amplified in a rolling circle amplification mode. Preparing a rolling circle amplification system containing Phi29DNA polymerase, taking an RCA priming probe as a primer, taking a semi-closed lock type probe for completing a connection reaction as a template, adding the template into a reaction chamber after uniformly mixing, and incubating at 30 ℃ for 2 hours. After the reaction was completed, the liquid was discarded, and PBST was washed 3 times at room temperature for 5 minutes each.
6. In situ sequencing (applying the principle of ligation sequencing)
(1) First round sequencing
a. The ligation sequencing system containing the Anchor primer Anchor-1 and the 4 sequencing primers was added to the reaction chamber for in situ sequencing reaction and incubated at 25℃for 2 hours. The sequences of the anchor primer and the sequencing primer are shown in Table 2.
TABLE 2
After the reaction was completed, the liquid was discarded, and PBST was washed 3 times at room temperature for 5 minutes each. DAPI-stained nuclei were added at 0.1. Mu.g/mL and incubated for 5 minutes at room temperature. After the reaction was completed, the liquid was discarded, and PBST was washed 3 times at room temperature for 5 minutes each.
b. Imaging by using Leica TCS SP8 laser confocal, and reading a sequencing result of a first base to be detected, and simultaneously reading the quantity, the spatial position and the sequence information of target mRNA. Channels corresponding to different fluorescence are set, the range of cell signals is observed and set, the sample is swept layer by layer at a step diameter of 0.4 mu m, and then images of all layers are overlapped to obtain an image of the whole signal.
(2) Second round of sequencing
a. 70% formamide elution buffer was prepared, added to the reaction chamber, incubated at room temperature for 5 minutes, and the sequencing signal from the first round eluted. After the reaction was completed, the liquid was discarded, and PBST was washed 3 times at room temperature for 5 minutes each.
b. And adding a connection sequencing system containing an Anchor primer Anchor-2 and 4 sequencing primers into the reaction chamber, and reading a sequencing result of a second base to be detected according to the reaction time and the flow of the first round of sequencing. In the second imaging pass, the same position and parameters must be maintained as in the first imaging pass.
(3) Third round of sequencing
a. 70% formamide elution buffer was prepared, added to the reaction chamber, incubated at room temperature for 5 minutes, and the second round of sequencing signal eluted. After the reaction was completed, the liquid was discarded, and PBST was washed 3 times at room temperature for 5 minutes each.
b. And adding a connection sequencing system containing Anchor primers Anchor-3 and 4 sequencing primers into the reaction chamber, and reading a sequencing result of a third base to be detected according to the reaction time and the flow of the first round of sequencing. In the third imaging, the same position and parameters as in the imaging must be maintained.
(3) Fourth round of sequencing
a. 70% formamide elution buffer was prepared, added to the reaction chamber, incubated at room temperature for 5 minutes, and the third round of sequencing signal eluted. After the reaction was completed, the liquid was discarded, and PBST was washed 3 times at room temperature for 5 minutes each.
b. And adding a connection sequencing system containing an Anchor primer Anchor-4 and 4 sequencing primers into the reaction chamber, and reading a sequencing result of a fourth base to be detected according to the reaction time and the flow of the first round of sequencing. In the fourth imaging, the same position and parameters as in the imaging must be maintained.
7. Data analysis
The four rounds of sequencing information obtained above were matched to read the mRNA sequence captured in the Gap formed by the lock probe.
As shown in fig. 4: the target sequence is located on mRNA transcribed from beta-Actin gene, the part to be detected is the sequence with the length of 4 bases, and the sequence is 5'-GGCC-3' according to the existing data. In the test process, the sequencing probes used emit different fluorescent colors, and respectively sequence different bases. The corresponding relation is as follows: green-sequencing to A (adenine); red-sequencing to C (cytosine); magenta-sequencing to T (thymine); indigo-sequencing to G (guanine). Sequencing from the 5 'end to the 3' end of the sequence to be tested, and obtaining the image with the following colors according to our experimental results: red-indigo, i.e. the result of the corresponding sequencing is: 5'-GGCC-3', in complete agreement with the expectations.
Other parts not described in detail are prior art. Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims (10)

1. A sequencing method suitable for in situ detection of a plurality of consecutive nucleotide sites, characterized by: the method comprises the following steps:
1) Design of probes
Screening out a target area with high hybridization efficiency, good specificity and no advanced structure according to a sequence to be detected, and setting a hybridization probe; wherein the hybridization probe comprises a semi-closed lock-type probe and an RCA initiation probe;
after the semi-closed lock probe is complemented with a target sequence of a sequence to be detected, a gap is formed between the 5 'end and the 3' end of the semi-closed lock probe,
the gap is filled by a single-stranded deoxyribonucleic acid random fragment with X bases and is complementary with a targeting sequence of a sequence to be detected; wherein X is 2, 3, 4, 5, 6, 7, 8 and … …;
2) Probe pretreatment
Phosphorylating the 5' end of a semi-closed lock probe and a single-stranded deoxyribonucleic acid random fragment of X bases in the hybridization probe;
3) Sample hybridization
Preparing a reaction chamber on a sample to be detected, fixing the sample by using 4% Paraformaldehyde (PFA), dehydrating and denaturing the sample by using methanol, and adding a system containing a hybridization probe into the reaction chamber for incubation for a period of time;
4) Ligation reaction
Adding a connection system containing single-stranded deoxyribonucleic acid random fragments with X bases into a reaction chamber, filling gaps (Gap) on a semi-closed lock-type probe complementarily paired with a sequence to be detected, and forming a closed annular structure;
5) Rolling circle amplification
The RCA initiating probe is complementary with the targeting sequence of the sequence to be detected and the semi-closed lock probe at the same time, and under the action of Phi29DNA polymerase, rolling circle amplification is carried out by taking the RCA initiating probe as a primer and the annular structure formed by the semi-closed lock probe as a template, so that a target signal is amplified;
6) In situ sequencing of target sequences
In situ sequencing
Adding a sequencing system containing an anchor primer and a sequencing primer into a reaction chamber for in-situ sequencing reaction, sequencing and imaging a sequence corresponding to a first base in a gap between semi-closed lock probes, and reading the spatial position and sequence information of a target sequence of a sequence to be detected;
sequentially reading information of base sequences at other positions in the gaps between the semi-closed lock probes by the same method;
and III, matching the obtained multiple rounds of sequencing information, and reading a target sequence corresponding to the gap formed between the semi-closed lock probes.
2. The sequencing method of claim 1 adapted for in situ detection of a plurality of consecutive nucleotide sites, wherein: in the step (1) of the above-mentioned process,
the targeting sequence of the sequence to be detected consists of 4 segments of continuous target sequences A, D, B and C, the semi-closed lock probe is A 'sequence from 5' end to 3 'end, loop sequence and B' sequence,
the A 'sequence and the B' sequence are respectively complementary with the target sequence A and the target sequence B, and the semi-closed lock type probe and the target sequence are complementary and paired to form a semi-closed annular structure; the loop sequence consists of an anchor sequence and a barcode sequence respectively;
the empty sequence D' is complementary to the target sequence D.
3. The sequencing method suitable for in situ detection of a plurality of consecutive nucleotide sites according to claim 2, wherein: in step 1), the vacant sequence D' is composed of a Y segment sequence, Y is smaller than X and is 1, 2, 3, 4, 5, 6, 7 and 8 … ….
4. A sequencing method suitable for in situ detection of a plurality of consecutive nucleotide sites according to claim 3, wherein: in the vacant sequence D', X is 2-8 and Y is 1-6.
5. The sequencing method of claim 4 adapted for in situ detection of a plurality of consecutive nucleotide sites, wherein: in the step 1), X is 4, and Y is 1.
6. The sequencing method of claim 1 adapted for in situ detection of a plurality of consecutive nucleotide sites, wherein: in the step 2), 5' -end of the random fragment of single-stranded deoxyribonucleic acid is phosphorylated.
7. The sequencing method of claim 1 adapted for in situ detection of a plurality of consecutive nucleotide sites, wherein: in the step 2), the enzyme used in the phosphorylation process is T4 polynucleotide kinase.
8. The sequencing method of claim 1 adapted for in situ detection of a plurality of consecutive nucleotide sites, wherein: in the step 4), the ligase of the ligase reaction system is a Splint R ligase.
9. The sequencing method of claim 5 adapted for in situ detection of a plurality of consecutive nucleotide sites, wherein: in step 6), the target sequence is sequenced in situ
In situ sequencing
Adding a sequencing system containing an anchor primer and a sequencing primer into a reaction chamber for in-situ sequencing reaction, sequencing and imaging a sequence corresponding to a first base in a gap between semi-closed lock probes, and reading the number, the spatial position and the sequence information of a target sequence;
sequentially reading the information of the base sequences of the second position, the third position and the fourth position in the gap between the semi-closed lock probes by the same method;
and III, matching the obtained four-wheel sequencing information, and reading a targeting sequence corresponding to the gap formed between the semi-closed lock probes.
10. Use of a sequencing method suitable for in situ detection of consecutive nucleotide sites according to claim 1 in point mutation detection.
CN202310290116.2A 2023-03-23 2023-03-23 Sequencing method suitable for in-situ detection of continuous multiple nucleotide sites and application thereof Pending CN116445594A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117343993A (en) * 2023-12-01 2024-01-05 中国科学院遗传与发育生物学研究所 Method for in situ detection of target nucleic acid sequences in a sample

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117343993A (en) * 2023-12-01 2024-01-05 中国科学院遗传与发育生物学研究所 Method for in situ detection of target nucleic acid sequences in a sample

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