CN110225979B - Rolling circle amplification-based genome target region enrichment method and application thereof - Google Patents
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Abstract
The invention provides a genome target region enrichment method. The method comprises the following steps: (1) subjecting the DNA fragment sample to circularization treatment; (2) Binding the circularized product to a target region specific primer or target region specific probe; and (3) performing rolling circle amplification of the circularized product bound to the target region-specific primer or target region-specific probe.
Description
PRIORITY INFORMATION
Without any means for
Technical Field
The invention relates to the field of biological information, in particular to a genome target region enrichment method and application thereof. More specifically, the invention relates to methods for enriching genomic target regions based on rolling circle amplification, gene sequencing libraries and methods for gene sequencing.
Background
With the completion of the human genome project, technologies for enrichment of genomic target fragments have emerged. From enzyme digestion electrophoresis to various PCR technologies with various types, the DNA target fragment enrichment technology is widely applied to the fields of genome research, medical detection and the like. The rise of second generation high throughput sequencing technology has made it possible for researchers to analyze the entire genome at the same time, but it is also not affordable to many individuals due to its high price. The genome target region enrichment technology based on the NGS technology well solves the contradiction between price and demand, and is mainly represented by the Roche probe capture technology and the life company multiplex PCR technology, which have advantages and disadvantages.
Wherein, nimbleGen SeqCap technical scheme of the Roche company is as follows:
According to the scheme, a target region probe is designed, a biotin-labeled probe is used, the probe and a target region fragment are combined through a hybridization method by utilizing the characteristic of DNA complementary pairing, the probe with biotin is adsorbed through a magnetic bead carrying biotin antibody labeling, after multiple flushing, the DNA fragment captured by the probe is finally dissolved into ddH 2 O, and finally a small amount of captured target region fragment is amplified through PCR, so that enrichment of the target region is achieved.
The Apli-seq protocol of Life Inc. is as follows:
Apli-seq is used for amplifying a target region of a primer mainly by utilizing the amplification effect of PCR, so that the enrichment effect on the target region of a genome is achieved. The scheme mainly designs a plurality of pairs of primers for a target area, then connects together PCR primers pooling of all the target areas, and then carries out mixed PCR amplification of a plurality of primer sets to amplify the target area, thereby achieving enrichment effect for the target design area. Because the terminal of the primer is provided with a designed joint sequence, the products after PCR can be directly sequenced on a machine.
However, techniques for enrichment of genomic target regions remain to be developed and improved further.
Disclosure of Invention
The present application has been made based on the findings and knowledge of the inventors regarding the following facts and problems:
the NimbleGen SeqCap technical solution of the rogowski company at present has the following disadvantages: 1. the cost of the chip is high, and the cost of a single test is high due to the addition of the eluting reagent; 2. the test operation is complex and complicated, the stability is poor, and the industrialization popularization is not facilitated. 3. The number of PCR amplification cycles is high, and the introduced PCR error can affect the low frequency detection. 4. Capturing of smaller areas is not applicable, smaller areas capturing less effective. The Life company Apli-seq solution has the following disadvantages: 1. the primer capture area is small, and the most one reaction in the products provided by the company is 150 pairs of primers, and most of the primers are lower than 100 pairs of primers. 2. The multiplex PCR technology has stability defect, and the amplification efficiency difference of different primers is larger, so that the multiplex PCR technology has larger defect on CNV detection. 3. The method is not applicable to fragmented samples, and the sensitivity to the fragmented samples is greatly reduced. 4. Complex region primers are inefficient and difficult to capture. 5. The number of PCR amplification cycles is high, and the introduced PCR error can affect the low frequency detection.
Based on this, the present application aims to solve at least one of the technical problems in the related art to some extent. Therefore, the application provides a genome target region enrichment method. The method has the advantages that (1) through ingenious design and utilizing an amplification principle, the problems of low capturing efficiency, high cost and the like of NimbleGen SeqCap technology are solved, and the single reaction cost is lower; (2) The problems of small primer capturing area, poor multiplex amplification stability and reduced sensitivity of double-primer amplification in fragmented samples in the Apli-seq technical scheme are solved by utilizing single-primer amplification; (3) The application has simple experimental design, and the capture and amplification are completed within 12 hours after the machine is put on, so that the complex experimental operation process of the NimbleGen SeqCap technical scheme is solved; (4) The size of the capturing interval has strong compatibility, the capturing interval can be large or small, and the efficiency fluctuation is small. (5) The method can rapidly and efficiently enrich the target region of the genome, and further efficiently realize enrichment sequencing of hot spot mutation sites and mutation detection of the target region.
In a first aspect of the invention, the invention provides a method for enriching a genomic target region. According to an embodiment of the invention, the method comprises: (1) subjecting a sample of genomic DNA fragments to circularization; (2) Binding the circularized product to a target region specific primer or target region specific probe; and (3) performing rolling circle amplification of the circularized product bound to the target region-specific primer or target region-specific probe. According to the enrichment method for the target region of the genome, disclosed by the embodiment of the invention, the target region of the genome can be enriched rapidly and efficiently, and a sequencing library containing the target region of the genome is established, so that enrichment sequencing of hot spot variation sites and mutation detection of the target region are realized efficiently.
In a second aspect of the invention, the invention provides a method for enriching a genomic target region. According to an embodiment of the invention, the method comprises: 1) Subjecting a sample of genomic DNA fragments to a circularization treatment, the circularization treatment comprising adding a linker comprising a restriction endonuclease cleavage site of EcoR V; (2) performing enzyme digestion on the cyclization treatment product; (3) Performing rolling circle amplification treatment by taking a cyclization treatment product as a template and a target region specific primer as an amplification primer; (4) Fragmenting the rolling circle amplification product, wherein the fragmenting treatment is performed based on the arrangement of the enzyme cutting sites; and (5) subjecting the fragmented product to a purification treatment, which is performed by magnetic bead purification. By utilizing the enrichment method of the genome target region, the capturing efficiency of the genome target region is high and the cost is low; the single primer is utilized for amplification, the capture area is large, the stability is high, and the sensitivity is high; the capturing and amplifying are completed within 12 hours after the machine is put on, and the experimental operation process is simple; the size of the capturing interval has strong compatibility, the capturing interval can be large or small, and the efficiency fluctuation is small.
In a third aspect of the invention, the invention provides a method for enriching a genomic target region. According to an embodiment of the invention, the method comprises: (1) subjecting a sample of genomic DNA fragments to circularization; (2) performing enzyme digestion on the cyclization treatment product; (3) And (3) performing rolling circle amplification treatment by using the cyclization treatment product as a template and a target region specific primer as an amplification primer and utilizing Phi29 polymerase so as to obtain DNB. By utilizing the enrichment method of the genome target region, the capturing efficiency of the genome target region is high and the cost is low; the single primer is utilized for amplification, the capture area is large, the stability is high, and the sensitivity is high; the capturing and amplifying are completed within 12 hours after the machine is put on, and the experimental operation process is simple; the size of the capturing interval has strong compatibility, the capturing interval can be large or small, and the efficiency fluctuation is small.
In a fourth aspect of the application, the application provides a gene sequencing library. According to an embodiment of the application, the library is obtained by the genomic target region enrichment method proposed by the application. The gene sequencing library provided by the embodiment of the application can be effectively used for sequencing hot spot mutation sites and detecting mutation of a target region.
In a fifth aspect of the application, the application provides a method of gene sequencing. According to an embodiment of the application, the method comprises the step of sequencing and data analysis treatment of the sequencing library provided by the application. The method for sequencing the gene according to the embodiment of the application can be effectively used for analyzing the hot spot mutation sites and the mutation of the target region.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a schematic illustration of a method for enrichment of a genomic target region according to an embodiment of the present invention;
FIG. 2 is a schematic illustration of a method of enrichment of a genomic target region according to an embodiment of the present invention;
FIG. 3 is a graph showing capture efficiency 2100 quality control results according to an embodiment of the present invention;
FIG. 4 is a graph showing capture efficiency 2100 quality control results according to an embodiment of the present invention; and
FIG. 5 is a schematic diagram of RCA capture with a probe chip, according to an embodiment of the invention.
Detailed Description
Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative only and are not to be construed as limiting the invention.
It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.
Method for enriching genome target region
In one aspect, the invention provides a method for enriching a genomic target region. According to an embodiment of the invention, the method comprises: (1) subjecting a sample of genomic DNA fragments to circularization; (2) combining the cyclization treatment product with a target region specific primer or a target region specific probe, and (3) performing rolling circle amplification (Rolling circle amplification, RCA) on the cyclization treatment product combined with the target region specific primer or the target region specific probe.
According to the examples of the present invention, the DNA fragments were amplified by PCR in advance, and the inventors found that PCR was required to meet the requirements of library construction when the initial amount was insufficient.
According to an embodiment of the invention, the cyclisation comprises at least one selected from double-stranded cyclisation and single-stranded cyclisation.
According to a specific embodiment of the invention, the target region specific probes are immobilized on a solid phase sequencing chip.
According to a specific embodiment of the present invention, further comprising subjecting the circularized product, to which the target region-specific primer or target region-specific probe is not bound, to an elution treatment.
According to an embodiment of the present invention, in step (1), further comprising subjecting the DNA fragment sample to an addition of a linker, the linker comprising a specific site. Specifically, the specific site comprises at least one selected from the group consisting of a restriction endonuclease site, a DNA sequence-specific hydrolase site, a chemical sequence-specific degradation site, a transcription activation-like effector nuclease site, a zinc finger nuclease site, a CRISPR/Cas site, and a NgAgo-gDNA site. According to a specific example of the present invention, the restriction enzyme site is EcoRV. The inventor skillfully introduces specific sites in the cyclization treatment process, and then can carry out fragmentation treatment on the cyclization library, so that sequencing is realized, the experimental operation is simple, the size compatibility of a capturing interval is strong, the capturing interval can be large or small, and the efficiency fluctuation is small.
According to the embodiment of the invention, the joint is a Y-shaped joint.
According to an embodiment of the present invention, the above-mentioned method for enriching a genomic target region further comprises: (1) Subjecting the rolling circle amplification product to a fragmentation treatment, the fragmentation treatment being based on the setting of the specific sites; and (2) subjecting the fragmented product to a purification treatment, optionally by magnetic bead purification. Furthermore, the capturing efficiency of the genome target area is further improved, the stability and the sensitivity are also further improved, the size compatibility of the capturing interval is stronger, and the efficiency fluctuation is smaller.
According to a further embodiment of the invention, the amplification is performed using a strand displacing enzyme, preferably a Phi29 polymerase, resulting in a DNA nanosphere (DNA Nano Ball, DNB).
According to still another embodiment of the present invention, the rolling circle amplification treatment is preceded by further comprising subjecting the circularized treatment product to an enzymatic cleavage treatment. Furthermore, the non-circular fragments in the circularized product can be removed, the purity of the circularized nucleic acid library can be further improved, and the rolling circle amplification efficiency can be further improved.
According to a specific example of the invention, the target region specific primer or target region specific probe has the sequence of SEQ ID NO:1 to 23.
CCTCCCAGTGCCTGAATACATAAAC(SEQ ID NO：1)。
TGGCTCTGTGCAGAATCCTGTCTAT(SEQ ID NO：2)。
CCTGACTACCAGCAGGACTT(SEQ ID NO：3)。
CAGTGAATTTATTGGAGCATGACCACGGA(SEQ ID NO：4)。
GGAGGATAGTATGAGCCCTA(SEQ ID NO：5)。
CCGACTAGCCAGGAAGTACT(SEQ ID NO：6)。
GCACATTTTGGGAAGTTGCA(SEQ ID NO：7)。
TTCAATGGGCTCTTCCAACA(SEQ ID NO：8)。
CAGGCCCAACTGTGAGCAAG(SEQ ID NO：9)。
CTGCTTCAAGGCTTCCACTG(SEQ ID NO：10)。
TGGGCAAAGAAGAAACGGAG(SEQ ID NO：11)。
GCATCAGGTCCTTTGGGGCATAGAT(SEQ ID NO：12)。
ATATGTTCCCTCCAGGTCAG(SEQ ID NO：13)。
CTCTGCCTTGAGTCATCTAT(SEQ ID NO：14)。
CTAGTTAGGAGCCCACCTTT(SEQ ID NO：15)。
CTCCTAGTCAATATCCACCCCATCC(SEQ ID NO：16)。
CAGCTCAAAGCAATTTCTACACGAGATCCT(SEQ ID NO：17)。
GCTTGGTGGGTAGGCAAGAGTGCCTTGACGAT(SEQ ID NO：18)。
CTTCGGCTGCCTCCTGGACTATGTCCGGGAACAC(SEQ ID NO：19)。
ACTGCTGGGTGCGGAAGAGAAAGAATACCATGC(SEQ ID NO：20)。
CTCCGAAAGCCAACAAGGAAATCCTCGATG(SEQ ID NO：21)。
CTGGGCATCTGCCTCACCTCCACCGTGCAGCTCAT(SEQ ID NO：22)。
CAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTAT(SEQ ID NO：23)。
Wherein, SEQ ID NO: 1-16 is a specific primer for EGFR C.3518 … C.5600 region, SEQ ID NO: 17-23 are specific primers for the mutation site region of the hot spot tumor. The specific primer or probe can be used for realizing efficient enrichment of EGFR target region or hot spot tumor mutation hot spot region.
According to a specific embodiment of the present invention, the method for enriching a genomic target region provided by the present invention includes: (1) Performing cyclization treatment on the genome DNA fragment sample, wherein the cyclization treatment comprises adding a linker to introduce an enzyme cutting site, and the enzyme cutting site is EcoR V; (2) performing enzyme digestion on the cyclization treatment product; (3) Performing rolling circle amplification treatment by taking a cyclization treatment product as a template and a target region specific primer as an amplification primer; (4) Fragmenting the rolling circle amplification product, wherein the fragmenting treatment is performed based on the arrangement of the enzyme cutting sites; and (5) subjecting the fragmented product to a purification treatment by magnetic bead purification, in particular, the target region specific primer has the sequence of SEQ ID NO:1 to 23.
See table 1 and fig. 1 for specific process procedures.
Table 1:
By utilizing the enrichment method of the genome target region, the capturing efficiency of the genome target region is high and the cost is low; the single primer is utilized for amplification, the capture area is large, the stability is high, and the sensitivity is high; the capturing and amplifying are completed within 12 hours after the machine is put on, and the experimental operation process is simple; the size of the capturing interval has strong compatibility, the capturing interval can be large or small, and the efficiency fluctuation is small.
According to another embodiment of the present invention, the method for enriching a genomic target region according to the present invention comprises: (1) subjecting the DNA fragment sample to circularization treatment; (2) performing enzyme digestion on the cyclization treatment product; (3) Performing rolling circle amplification treatment by using a cyclization treatment product as a template and a target region specific primer as an amplification primer by using Phi29 polymerase so as to obtain DNB, wherein the target region specific primer has a nucleotide sequence shown in SEQ ID NO:1 to 23.
See table 2 and fig. 2 for specific process procedures.
Table 2:
According to another embodiment of the present invention, the method for enriching a genomic target region according to the present invention comprises: (1) subjecting the DNA fragment sample to circularization treatment; (2) Fixing target region specific probes on a solid-phase sequencing chip, and hybridizing cyclization treatment products with the probes on the sequencing chip; (3) eluting the circularized product not bound to the probe; (4) Rolling circle amplification is performed on circularized products bound to probes on a sequencing chip.
By utilizing the enrichment method of the genome target region, the capturing efficiency of the genome target region is high and the cost is low; the single primer is utilized for amplification, the capture area is large, the stability is high, and the sensitivity is high; the capturing and amplifying are completed within 12 hours after the machine is put on, and the experimental operation process is simple; the size of the capturing interval has strong compatibility, the capturing interval can be large or small, and the efficiency fluctuation is small.
Gene sequencing library
In yet another aspect, the application provides a gene sequencing library. According to an embodiment of the application, the library is obtained by the genomic target region enrichment method proposed by the application. The gene sequencing library provided by the embodiment of the application can be effectively used for sequencing hot spot mutation sites and detecting mutation of a target region.
Method of gene sequencing
In yet another aspect, the application features a method of gene sequencing. According to an embodiment of the application, the method comprises the step of sequencing and data analysis treatment of the sequencing library provided by the application. The method for sequencing the gene according to the embodiment of the application can be effectively used for analyzing the hot spot mutation sites and the mutation of the target region.
Specifically, the sequencing is performed by the BGI-SEQ500, proton, pacbio, qiagen, hiseq2500, illuminea or SEQ500 platform.
Wherein the library of target regions obtained by the method of enriching the target regions by introducing cleavage sites during the cyclization treatment can be performed by at least one of a second generation sequencing platform or a third generation sequencing platform, in particular, the second generation sequencing platform comprises at least one selected from Illumina, proton, pacbio and Qiagen sequencing platforms. The target region library in the form of DNB nanospheres obtained by the method of enriching the target region which requires amplification of the hybrid of the strand displacing enzyme Phi29 after cyclization can be subjected to high-efficiency sequencing through a BGI-seq500 platform.
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1: EGFR Capture (2 k)
1. EGFR C.3518 … C.5600 interval primer with interval distance of 50bp is designed, and partial sequence is shown in table 3 (synthesized by Shanghai Biotechnology Co., ltd.);
Table 3:
2. constructing a cyclization library;
3. Performing RCA capture by using the primer designed in the step 1;
4. The library after capture is not different from the library before capture in fragment distribution form, which indicates that the capture preference is within a controllable range (see the result of FIG. 3), FIG. 3 shows the quality control result of capture efficiency 2100, 442cut-444cut is a reaction without capture probe, and the detection ratio is 3:3;41b1cut-44b1cut is the RCA probe capture result. 2100 results showed that the non-captured product averaged 2.71 ng/. Mu.L; the capture product averaged 22.02 ng/. Mu.L; the RCA enrichment energy efficiency ratio is 8.13 times;
5. sequencing the single-stranded library on an Illumina sequencing platform after quantitative analysis by QPCR;
6. The machine-down data 1M reads, the effective data 0.58M reads, and the target area depth is about 0.79×. The analysis results showed a 41.58-fold enrichment over the NGS predicted depth of 0.58M x 100/3 x 1000 = 0.019 x. Experimental results show that the method has a large application space in the aspect of enrichment of the target area.
Based on the method described in example 1, the inventors performed enrichment on the test samples, and the sequencing results after enrichment are shown in table 4.
Table 4:
example 2: hot spot mutation capture (interval size 1 k)
1. Hot spot tumor mutation site capture primers were designed as shown in table 5 (synthesized by Shanghai Biotechnology Co., ltd.);
Table 5:
2. constructing a cyclization library;
3. Performing an RCA capture experiment by using the primer designed in the step 1;
4. The library after capture is not different from the library before capture in fragment distribution form, which shows that the capture preference is in a controllable range (see fig. 4 for specific results), fig. 4 shows the quality control result of capture efficiency 2100, 442cut-444cut shows the reaction without capture probe, and the detection ratio is 3:3;41b1cut-44b1cut is the RCA probe capture result. 2100 results showed that the non-captured product averaged 1.38 ng/. Mu.L; the capture product averaged 16.38 ng/. Mu.L; the RCA enrichment energy efficiency ratio is 11.88 times;
5. Sequencing the single-stranded library on an Illumina platform after QPCR quantification;
6. The machine-down data is 1M reads, the effective data is 0.98M reads, and the depth of the target area is about 1.93×. The analysis results showed that the NGS predicted depth was 0.98M x 100/3 x 1000 = 0.033 x, enriched by 59.1 fold. The preliminary experiment shows that the method has a larger application space in the aspect of enrichment of the target area.
Example 3 Simultaneous enrichment of genomic target region and DNB production Using solid phase sequencing chip
As shown in fig. 5, a DNA fragment sample is first subjected to circularization; fixing a target region specific probe on a solid phase sequencing chip; hybridizing the cyclization treatment product with a probe on a sequencing chip; eluting the circularized product not bound to the probe by an elution step; and (3) performing rolling circle amplification on the cyclization treatment product combined with the probe on the sequencing chip to form DNB, and sequencing by using a BGI-SEQ 500 sequencing platform.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
SEQUENCE LISTING
<110> Shenzhen Hua big Gene Co., ltd
All companies of Tianjin Huada medical inspection
Guangzhou Hua big Gene medicine inspection all Limited
<120> Rolling circle amplification-based genome target region enrichment method and application thereof
<130> PIOC3168035PCN
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<213> Artificial
<220>
<223> Specific primers for EGFR C.3518 … C.5600 region
<400> 7
gcacattttg ggaagttgca 20
<210> 8
<211> 20
<212> DNA
<213> Artificial
<220>
<223> Specific primers for EGFR C.3518 … C.5600 region
<400> 8
ttcaatgggc tcttccaaca 20
<210> 9
<211> 20
<212> DNA
<213> Artificial
<220>
<223> Specific primers for EGFR C.3518 … C.5600 region
<400> 9
caggcccaac tgtgagcaag 20
<210> 10
<211> 20
<212> DNA
<213> Artificial
<220>
<223> Specific primers for EGFR C.3518 … C.5600 region
<400> 10
ctgcttcaag gcttccactg 20
<210> 11
<211> 20
<212> DNA
<213> Artificial
<220>
<223> Specific primers for EGFR C.3518 … C.5600 region
<400> 11
tgggcaaaga agaaacggag 20
<210> 12
<211> 25
<212> DNA
<213> Artificial
<220>
<223> Specific primers for EGFR C.3518 … C.5600 region
<400> 12
gcatcaggtc ctttggggca tagat 25
<210> 13
<211> 20
<212> DNA
<213> Artificial
<220>
<223> Specific primers for EGFR C.3518 … C.5600 region
<400> 13
atatgttccc tccaggtcag 20
<210> 14
<211> 20
<212> DNA
<213> Artificial
<220>
<223> Specific primers for EGFR C.3518 … C.5600 region
<400> 14
ctctgccttg agtcatctat 20
<210> 15
<211> 20
<212> DNA
<213> Artificial
<220>
<223> Specific primers for EGFR C.3518 … C.5600 region
<400> 15
ctagttagga gcccaccttt 20
<210> 16
<211> 25
<212> DNA
<213> Artificial
<220>
<223> Specific primers for EGFR C.3518 … C.5600 region
<400> 16
ctcctagtca atatccaccc catcc 25
<210> 17
<211> 30
<212> DNA
<213> Artificial
<220>
<223> Specific primers for Hot tumor mutation site region
<400> 17
cagctcaaag caatttctac acgagatcct 30
<210> 18
<211> 32
<212> DNA
<213> Artificial
<220>
<223> Specific primers for Hot tumor mutation site region
<400> 18
gcttggtggg taggcaagag tgccttgacg at 32
<210> 19
<211> 34
<212> DNA
<213> Artificial
<220>
<223> Specific primers for Hot tumor mutation site region
<400> 19
cttcggctgc ctcctggact atgtccggga acac 34
<210> 20
<211> 33
<212> DNA
<213> Artificial
<220>
<223> Specific primers for Hot tumor mutation site region
<400> 20
actgctgggt gcggaagaga aagaatacca tgc 33
<210> 21
<211> 30
<212> DNA
<213> Artificial
<220>
<223> Specific primers for Hot tumor mutation site region
<400> 21
ctccgaaagc caacaaggaa atcctcgatg 30
<210> 22
<211> 35
<212> DNA
<213> Artificial
<220>
<223> Specific primers for Hot tumor mutation site region
<400> 22
ctgggcatct gcctcacctc caccgtgcag ctcat 35
<210> 23
<211> 34
<212> DNA
<213> Artificial
<220>
<223> Specific primers for Hot tumor mutation site region
<400> 23
cagaaggtga gaaagttaaa attcccgtcg ctat 34
Claims (9)
1. A method for enriching a genomic target region, comprising:
(1) Cyclizing the genome DNA fragment sample;
(2) Binding the circularized product to a target region specific primer or target region specific probe; and
(3) Performing enzyme digestion treatment and rolling circle amplification on a circularized treatment product combined with a target region specific primer or a target region specific probe; and
(4) Subjecting the rolling circle amplification product to a fragmentation treatment, the fragmentation treatment being based on the setting of the specific sites; and
(5) Subjecting the fragmented product to a purification treatment, the purification treatment being performed by magnetic bead purification;
the DNA fragment sample comprises the steps of adding a joint, wherein the joint is a Y joint;
the enzyme digestion treatment comprises opening a notch of the cyclization treatment product;
The linker comprises a specific site comprising a restriction enzyme site EcoR V;
The target region specific primer or the target region specific probe is SEQ ID NO:1 to 23.
2. The method of claim 1, wherein the DNA fragments are previously amplified by PCR.
3. The method of claim 1, wherein the cyclizing comprises at least one selected from the group consisting of double-stranded cyclizing and single-stranded cyclizing.
4. The method of claim 1, further comprising eluting the circularized product not bound to the target region specific primer or target region specific probe.
5. The method according to any one of claims 1 to 4, wherein the rolling circle amplification is performed using a strand displacing enzyme in order to obtain DNA nanospheres.
6. The method of claim 5, wherein the strand displacing enzyme is Phi29 polymerase.
7. A method for enriching a genomic target region, comprising:
(1) Subjecting a sample of genomic DNA fragments to a circularization treatment, the circularization treatment comprising adding a linker comprising a restriction endonuclease cleavage site of EcoR V;
(2) Performing enzyme digestion on the cyclization treatment product;
(3) Performing rolling circle amplification treatment by taking a cyclization treatment product as a template and a target region specific primer as an amplification primer;
(4) Fragmenting the rolling circle amplification product, wherein the fragmenting treatment is performed based on the arrangement of the enzyme cutting sites; and
(5) Subjecting the fragmented product to a purification treatment by magnetic bead purification,
The target region specific primer is SEQ ID NO:1 to 23.
8. A method for enriching a genomic target region, comprising:
(1) Cyclizing the genome DNA fragment sample;
(2) Performing enzyme digestion on the cyclization treatment product;
(3) Taking the cyclization treatment product as a template, taking a target region specific primer as an amplification primer, performing rolling circle amplification treatment by utilizing Phi29 polymerase so as to obtain the DNA nanospheres,
The target region specific primer is SEQ ID NO:1 to 23.
9. A library of genetic sequencing, characterized in that it is obtained by the method according to any one of claims 1 to 8.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2017/085594 WO2018214036A1 (en) | 2017-05-23 | 2017-05-23 | Enrichment method for genomic target region based on rolling circle amplification and application thereof |
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| CN110225979A CN110225979A (en) | 2019-09-10 |
| CN110225979B true CN110225979B (en) | 2024-05-31 |
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