CN112980938A - Method for capturing genetic deafness gene sequence in targeted manner and sequencing method - Google Patents
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Abstract
本申请涉及生物技术领域，具体提供一种靶向捕获遗传性耳聋基因序列的方法及测序方法，包括：制备靶向捕获遗传性耳聋基因外显子序列正负链的捕获膜，所述遗传性耳聋基因包括CDH23、COL11A1、DSPP、MYO7A、OTOF、PCDH15、MT‑RNR1、MT‑TL1、MT‑TS1、MYO15A、SLC26A4、DFNA5、GJB2、GJB3、KCNJ10、SOX10、TCOF1、WFS1、DFNB59、USH1G、TMC1、DFNB31；将片段化后的人基因组样品，构建为应用于高通量测序的核酸文库；将所述核酸文库进行变性处理，变性处理后的核酸文库与所述的捕获膜在杂交条件下进行杂交，形成目标核酸分子‑捕获膜复合物；用清洗液清洗所述目标核酸分子‑捕获膜复合物，然后再用洗脱液从捕获膜上洗脱、并纯化经富集后，获得遗传性耳聋基因序列。本发明能遗传性耳聋基因型鉴定准确率的提高，快速便捷。The present application relates to the field of biotechnology, and specifically provides a method and a sequencing method for targeted capture of a genetic deafness gene sequence, including: preparing a capture film that targets and captures the positive and negative strands of the exon sequence of the hereditary deafness gene. Deafness genes include CDH23, COL11A1, DSPP, MYO7A, OTOF, PCDH15, MT‑RNR1, MT‑TL1, MT‑TS1, MYO15A, SLC26A4, DFNA5, GJB2, GJB3, KCNJ10, SOX10, TCOF1, WFS1, DFNB59, USH1G, TMC1 , DFNB31; the fragmented human genome sample is constructed into a nucleic acid library for high-throughput sequencing; the nucleic acid library is denatured, and the denatured nucleic acid library and the capture membrane are subjected to hybridization conditions. Hybridize to form a target nucleic acid molecule-capture membrane complex; wash the target nucleic acid molecule-capture membrane complex with a cleaning solution, and then elute from the capture membrane with an eluent, purify and enrich to obtain a hereditary Deafness gene sequence. The invention can improve the accuracy rate of genotype identification of hereditary deafness, and is fast and convenient.
Description
Technical Field
The application relates to the technical field of biology, in particular to a method for capturing genetic deafness gene sequences in a targeted mode and a sequencing method.
Background
With the development of second-generation sequencing, genome sequencing becomes the most important means for identifying relevant site variation with high throughput and high depth so as to identify potential diseases, and has wide application in scientific research. The high-throughput sequencing can directly obtain a large amount of related site variation information, and has great significance for precise medical treatment.
The DNA targeting enrichment technology is applied to high-throughput sequencing, and can improve the proportion of a target region after a genome target region or interested target nucleic acid is enriched. Therefore, by using a DNA targeting enrichment technology, a specific disease-related target region, such as a human exome or a hereditary hearing loss gene, can be enriched, the sequencing cost can be greatly reduced, and the potential is huge. For example, exome sequencing only achieves sequencing of sequences which account for about 1% -2% of the full length of the human genome, and sequence information of exome regions is obtained by sequencing after capture, so that compared with whole genome sequencing, the cost is reduced by 50-100 times. And the genetic deafness gene region is captured, and genetic deafness gene sequences accounting for about 0.2 percent of the whole genome are enriched, so that the sequencing cost is greatly reduced.
Genetic deafness genotyping technology based on targeted capture currently comprises enrichment technology based on solid-phase carrier chips and enrichment technology based on solid-phase carrier magnetic beads. Solid phase carriers for nucleic acid hybridization, such as glass, plates, dishes, microspheres, and the like, need to be modified to specifically bind nucleic acids, such as magnetic beads, and need to be modified to specifically bind nucleic acids. However, the binding of nucleic acids is directional, and the amount of probes that can be bound is low, such as the current commercialized magnetic bead-based targeted nucleic acid molecule enrichment products, wherein a certain product has the characteristics that 20 micrograms of double-stranded DNA can be bound per milligram of magnetic bead, and the price per milligram of magnetic bead is about 240 yuan, which is high in cost. Meanwhile, the amount of the probe, the source of the probe, the preparation method of the probe, and the like are different due to different target enrichment techniques, which not only affects the diversity of the target nucleic acid, but also affects the cost.
Other reported methods for identifying hereditary deafness genes by enrichment technology all need to synthesize modified single-stranded nucleic acid probes and prepare modified solid phase carriers simultaneously by means of artificial synthesis. The enrichment process is also mostly to form nucleic acid probes firstly: target nucleic acid complex, and then nucleic acid complex: and (5) enriching the carrier to obtain the enriched target nucleic acid sequence. Since the binding of the complex to the carrier passes through the modifying groups of the nucleic acid probe and the solid phase carrier, the probe modification and the coating of the solid phase carrier with a specific group are inevitably carried out, which increases the capturing cost and complicates the steps.
Disclosure of Invention
The embodiment of the application aims to provide a method for targeted capture of genetic deafness gene sequences and a sequencing method, so as to solve the problems of high cost and low capture diversity of targeted capture in the related technology.
To achieve the above object, according to a first aspect of embodiments of the present invention, a method for targeted capture of genetic deafness gene sequences, the method comprising:
1) preparing a capture membrane for capturing the positive and negative chains of exon sequences of genetic deafness genes, wherein the genetic deafness genes comprise CDH23, COL11A1, DSPP, MYO7A, OTOF, PCDH15, MT-RNR1, MT-TL1, MT-TS1, MYO15A, SLC26A4, DFNA5, GJB2, GJB3, KCNJ10, SOX10, TCOF1, WFS1, DFNB59, USH1G, TMC1 and DFNB 31;
2) constructing a fragmented human genome sample into a nucleic acid library applied to high-throughput sequencing;
3) carrying out denaturation treatment on the nucleic acid library, and hybridizing the denatured nucleic acid library with the capture membrane under hybridization conditions to form a target nucleic acid molecule-capture membrane complex;
4) and (3) washing the target nucleic acid molecule-capture membrane complex by using a washing solution, then eluting from the capture membrane by using an eluent, purifying and enriching to obtain the genetic deafness gene sequence.
Optionally, the capture membrane is a nylon-based membrane or a derivative thereof, a cellulose acetate membrane or a derivative thereof, or a cellulose paper membrane or a derivative thereof; preferably a neutral or positively charged nylon membrane, cellulose acetate membrane, or cellulose paper membrane.
Optionally, a nucleic acid probe library with similarity of more than 90% with the exon sequence of the genetic deafness gene is combined on the capture membrane.
Optionally, the corresponding nucleic acid probes are present on the capture membrane in the positive and negative chains of the exon sequences of the genetic deafness gene, and can be combined under hybridization conditions.
Alternatively, the preparation of the library of nucleic acid probes is made by:
obtaining and purifying nucleic acid sequences with the same, complementary and similar hereditary hearing loss genes, and fragmenting to form a fragment range of 20bp-10kb, preferably a fragment of 150bp-800 bp;
and (3) carrying out fragmentation treatment and then carrying out physical or chemical denaturation to obtain the nucleic acid probe library.
Optionally, the genetic deafness gene exon sequence positive and negative chains are combined with the membrane through one or more of physical, chemical and photochemical means to form a capture membrane.
Optionally, the nucleic acid library contains linker sequences at both ends of the nucleic acid fragments, and has sequence characteristics in the form of nucleic acid linker-insert nucleic acid-nucleic acid linker, which are consistent with the fragment characteristics required by a high-throughput sequencing platform, and can be directly used for high-throughput sequencing.
Optionally, the nucleic acid library after denaturation treatment is a single-stranded nucleic acid sample generated after double-stranded nucleic acid is denatured before hybridization.
Alternatively, the hybridization conditions are hybridization in a hybridization solution with a temperature ranging from 40 degrees centigrade to 70 degrees centigrade, wherein the temperature is preferably 55 to 65 degrees centigrade, so as to achieve the binding of the target nucleic acid molecule and the nucleic acid probe in the hybridization solution at the temperature.
Optionally, the hybridization solution is a solution containing sodium phosphate buffer solution, EDTA, BSA and SDS, or a solution containing one or more of sodium citrate buffer solution (SSC), Denhardt solution, SDS and formamide, or a solution containing one or more of SSPE, Denhardt solution, SDS and formamide; preferably, the hybridization solution is a solution containing 0.5M sodium phosphate buffer solution, 1mM EDTA, 1% (w/v) BSA and 7% (w/v) SDS, or a solution containing 5 XSSC, 5 XDenhardt's solution and 1% (w/v) SDS.
Optionally, the cleaning solution is a solution containing sodium phosphate buffer solution and SDS, or a solution containing 0.2-2 XSSC and 1% (w/v) SDS; preferably, the wash solution is a solution containing 0.04M sodium phosphate buffer solution and 1.6% (w/v) SDS, or a solution containing 2 XSSC and 0.1% (w/v) SDS, or a solution containing 0.2 XSSC and 0.1% (w/v) SDS, or a solution containing 0.1 XSSC and 0.1% (w/v) SDS.
Optionally, the eluent is water, a TE buffer solution, a 0.4M NaOH solution, or a 0.1% (w/v) SDS solution.
Optionally, the enriching further comprises: eluting in an eluent at 80-100 ℃, or eluting the enriched nucleic acid in an eluent with the pH of 10-12, wherein the eluent at 80-100 ℃ is an aqueous solution, a TE buffer solution or a 0.1% (w/v) SDS solution, and the eluent with the pH of 10-12 is a 0.4M NaOH solution.
According to a second aspect of the embodiments of the present invention, there is also provided a sequencing method for targeted capture of genetic deafness gene sequences, the method comprising:
preparing a capture membrane for capturing the positive and negative chains of exon sequences of genetic deafness genes, wherein the genetic deafness genes comprise CDH23, COL11A1, DSPP, MYO7A, OTOF, PCDH15, MT-RNR1, MT-TL1, MT-TS1, MYO15A, SLC26A4, DFNA5, GJB2, GJB3, KCNJ10, SOX10, TCOF1, WFS1, DFNB59, USH1G, TMC1 and DFNB 31;
obtaining a fragmented human genome sample, and constructing the human genome sample into a nucleic acid library applied to high-throughput sequencing;
carrying out denaturation treatment on the nucleic acid library, and hybridizing the denatured nucleic acid library with the capture membrane under hybridization conditions to form a target nucleic acid molecule-capture membrane complex;
washing the target nucleic acid molecule-capture membrane complex with a washing solution, then eluting from the capture membrane with an eluent, purifying and enriching to obtain a genetic deafness gene sequence;
and (3) carrying out high-throughput sequencing on the genetic deafness gene sequence.
In the method of the present embodiment, as one embodiment, the present invention provides a sequencing method for targeted capture of genetic deafness gene sequence, the method comprising the steps of:
(1) obtaining and purifying nucleic acid sequence with the same, complementary or similar sequence as genetic deafness gene sequence and sequence similarity higher than 90% to the cDNA of genetic deafness gene through biological synthesis mode and/or biological enzyme reaction. The nucleic acid sequence can be a nucleic acid segment obtained by genome DNA, plasmid, cosmid, bacterial artificial chromosome, yeast artificial chromosome, artificial synthetic sequence, enzyme digestion product, PCR product, DNA transcription and RNA reverse transcription;
(2) preparing the nucleic acid fragments to generate a nucleic acid probe library, and if the nucleic acid fragments are longer, such as more than 10kb, fragmenting; if the nucleic acid fragment is between 20bp and 10kb, directly performing the step (4), or transcribing the nucleic acid fragment to generate a nucleic acid probe library for enriching the genetic deafness gene sequence;
(3) fragmenting the longer nucleic acid fragment obtained in the step (2) by means of enzyme digestion, ultrasonic waves and other technical means, wherein the fragmentation range is 20bp-10 kp; preferably 150bp-800 bp;
(4) denaturing the nucleic acid fragments by a heating/quenching denaturation method or an alkaline denaturation method for 15 minutes to obtain nucleic acid fragments as a nucleic acid probe library;
(5) transferring a solution comprising probe nucleic acid directly onto the solid phase support membrane;
(6) putting the solid phase carrier membrane obtained in the step (5) into a vacuum box, and naturally drying the solid phase carrier membrane at room temperature under vacuum for about 40 minutes or till the solid phase carrier membrane is dried, so that the probe nucleic acid is fixed on the solid phase carrier membrane;
(7) baking the solid phase carrier membrane of the step (6) in a vacuum box at 80 ℃ for 1-2 hours, preferably 1 hour, so that the single-stranded nucleic acid probe is firmly combined with the solid phase carrier membrane;
(8) transferring the solid phase carrier membrane of the step (7) into a hybridization bottle containing a hybridization solution, placing the hybridization bottle in a hybridization box at 65 ℃ for pre-hybridization, wherein the time depends on the solid phase carrier membrane used, and the time is 30 minutes for a nylon membrane and 6 hours for an acetate fiber membrane as an illustrative example;
(9) denaturing a nucleic acid sample containing a genetic deafness gene sequence and a blocker probe at 98 ℃, wherein if the nucleic acid library contains a linker sequence and/or a tag nucleic acid sequence and has sequence characteristics of high-throughput sequencing, the blocker probe is a single-stranded nucleic acid which is complementary to the linker sequence in the nucleic acid library, and immediately placing the nucleic acid library on ice after being taken out, so that the nucleic acid denaturation state is maintained; (10) transferring the specific nucleic acid library denatured in the step (9) to the hybridization bottle in the step (8), and rotating at 65 ℃ in a hybridization box overnight;
(11) transferring the solid phase carrier membrane hybridized in the step (10) to a cleaning solution sealed tube containing 10 times of the volume of the solid phase carrier membrane, placing the solid phase carrier membrane in a hybridization box at 63 ℃, cleaning for 15 minutes, and cleaning for three times in total;
(12) transferring the solid-phase carrier membrane washed three times in the step (11) into a 1.5ml centrifuge tube containing 500ul of 1xTE buffer solution, placing the centrifuge tube in a water bath at 100 ℃, boiling for 5 minutes, and eluting the enriched DNA;
(13) taking out the solid phase carrier membrane obtained in the step (12), and then placing the liquid in a centrifugal tube at room temperature for cooling, wherein the obtained solution contains the enriched nucleic acid library containing the genetic deafness gene sequence;
(14) purifying and processing the nucleic acid library of the step (13) into a library capable of high-throughput sequencing, and then using the library for high-throughput sequencing.
The technical scheme provided by the embodiment of the application can have the following beneficial effects:
(1) the nucleic acid probe library on the capture membrane is obtained and prepared by a biosynthesis mode and/or a biological enzymatic reaction, and can be used for enriching the gene of hereditary hearing loss only by complementing the gene sequence of the hereditary hearing loss or having the similarity of more than 80 percent.
(2) Compared with the existing nucleic acid enrichment technology, the targeted nucleic acid enrichment technology adopted by the invention is easier to operate and has lower cost. Due to the characteristics of the capture membrane, the solid phase carrier membrane does not need to be excessively modified to enable the solid phase carrier membrane to be specifically combined with a specific nucleic acid modification group, so that the probe on the capture membrane can be obtained by a biosynthesis mode and/or a biological enzymatic reaction.
(3) The genetic deafness gene sequence enriched by the invention is finally applied to high-throughput sequencing service, and the specific information of the enriched genetic deafness gene sequence is obtained through high-throughput sequencing, so that the genetic deafness gene sequence enriched by the invention can be applied to genetic deafness genotyping.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the application.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and together with the description, serve to explain the principles of the application.
FIG. 1 is a graph showing fragment range sizes of nucleic acid probes after fragmentation, according to an exemplary embodiment.
FIG. 2 illustrates a library of nucleic acid probes whose probes are single base shifted according to an exemplary embodiment.
Detailed Description
Reference will now be made in detail to the exemplary embodiments, examples of which are illustrated in the accompanying drawings. When the following description refers to the accompanying drawings, like numbers in different drawings represent the same or similar elements unless otherwise indicated. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present application. Rather, they are merely examples of apparatus and methods consistent with certain aspects of the present application, as detailed in the appended claims.
Example 1 preparation of Capture Membrane for Targeted Capture of exon sequences of genetic deafness Gene plus and minus chains
Obtaining nucleic acid molecules for preparing capture probes
1. The cDNA plasmids of genetic deafness genes CDH23, COL11A1, DSPP, MYO7A, OTOF, PCDH15, MT-RNR1, MT-TL1, MT-TS1, MYO15A, SLC26A4, DFNA5, GJB2, GJB3, KCNJ10, SOX10, TCOF1, WFS1, DFNB59, USH1G, TMC1 and DFNB31 in the laboratory are collected.
2. After transformation, identification and plasmid extraction, a large number of cDNA plasmids are obtained for preparing capture probes.
Secondly, preparing a capture probe:
1. mixing 22 kinds of plasmid solutions according to the same molecular weight, including CDH23, COL11A1, DSPP, MYO7A, OTOF, PCDH15, MT-RNR1, MT-TL1, MT-TS1, MYO15A, SLC26A4, DFNA5, GJB2, GJB3, KCNJ10, SOX10, TCOF1, WFS1, DFNB59, USH1G, TMC1 and DFNB 31;
2. taking 5 mu g of the mixed solution, and fragmenting nucleic acid by using an ultrasonic instrument (Bioruptor), wherein the conditions are that the opening is carried out for 30 s/the closing is carried out for 90s,6 cycles are carried out, and the range of the generated fragments is 150bp-800 bp;
3. taking a proper amount of the solution after ultrasonic treatment, and detecting the size of the fragment after ultrasonic treatment by an Agilent bioanalyzer 2200, wherein the size is in the range of 150bp-800bp (shown in figure 1);
4. the above-mentioned nucleic acid fragments were denatured by an alkaline denaturation method, and prepared at room temperature:
24 μ l 500 ng/. mu.l DNA library
12μl 3M NaCl
1.44μl 10N NaOH
5. Standing at room temperature for 15 minutes to obtain the nucleic acid probe.
6. Cutting with scissors to about 0.3cm2With positive electricityNylon membrane loaded with lotus (NG0312, RPN3038, GE);
7. transferring the solution of the denatured single-stranded nucleic acid probe in the step 5 onto a nylon membrane by using a pipette;
8. transferring the nylon membrane into a vacuum box, and drying for 40 minutes at room temperature in a vacuum environment or until the nylon membrane is dried;
9. and transferring the nylon membrane into a vacuum box, adjusting the temperature to 80 ℃, and baking the nylon membrane for 1 hour in a vacuum environment to firmly combine the single-stranded nucleic acid probe with the nylon membrane.
10. After cooling, the capture membrane is the capture membrane for capturing the positive and negative chains of the exon sequences of the hereditary hearing loss genes in a targeted manner.
Thirdly, controlling the quality of the plasmid fragment for forming the probe:
1. 500ng of the fragmented plasmid fragment was used
UltraTMDNA Library construction of sample DNA Library PrepKit for Illumina (E7645, NEB).
2. Quantifying the constructed library (Agilent bioanalyzer 2200);
3. high-throughput sequencing and analysis were performed, the number of sequences per gene was substantially identical, and the fragmented nucleic acids, in the form of single base shifts, showed high diversity (FIG. 2).
Example 2 construction of DNA sample library
1. The DNA samples were 3 samples in total.
2. Taking 500ng of DNA sample, fragmenting the DNA by using an ultrasonic instrument (Bioruptor), wherein the conditions are that the opening is performed for 30 s/the closing is performed for 90s,5 cycles are performed, and the range of the generated fragments is 300bp-600 bp;
3. taking 5 mul of the solution after ultrasonic treatment, adding 1 mul of 6x loading Buffer, and detecting the size of the fragment after ultrasonic treatment by agarose gel electrophoresis, wherein the size is within the range of 300bp-600 bp;
4. for the rest of the sample, use
UltraTMDNA Library Prep Kit for IlluConstructing a sample DNA library by mina (E7645, NEB), wherein the size range of the insert is 300-600 bp.
Example 3 enrichment of genetic deafness Gene sequences
Firstly, denaturing a DNA sample library:
1. the DNA sample library is the DNA sample library in example 2.
2. Mixing the DNA sample library with components that help to increase the efficiency of hybridization:
mu.l of 50 ng/. mu.l DNA sample library, total 1. mu.g
5ul 1μg/μl human Cot-1 DNA
8ul Blocker Probe
3. After mixing uniformly, heating at 98 ℃ for 5 minutes to denature the double-stranded DNA of the library into single strands;
4. immediately after removal, the DNA was kept single-stranded by placing on ice.
II, enriching the deafness gene nucleic acid sequence:
1. preparing a hybridization solution:
mixing to clarify;
2. placing the capture membrane in a hybridization bottle containing 1 ml of hybridization solution, placing the hybridization bottle in a hybridization box at 65 ℃, pre-hybridizing for 0.5 hour, then replacing fresh hybridization solution and preheating to 65 ℃;
3. transferring the mixed solution in the step one to a hybridization bottle containing a capture membrane and a hybridization solution, and hybridizing for 24 hours at 65 ℃;
4. preparing a cleaning solution:
sodium phosphate buffer 0.04M
SDS 1.6％
Uniformly mixing until the mixture is clear, and preheating to 63 ℃;
5. transferring the capture membrane into a 5ml centrifuge tube containing 4 ml of preheated cleaning solution, cleaning for 15 minutes at 63 ℃, and cleaning for three times;
6. the capture membrane after three washes was transferred to a 1.5ml centrifuge tube containing 500. mu.l TE buffer;
7. heating a 1.5ml centrifuge tube to 98 ℃, eluting the enriched nucleic acid, taking out the capture membrane after 5 minutes, and cooling the residual solution to a greenhouse.
Thirdly, purifying the enriched nucleic acid sample library:
1. the remaining liquid from step two was purified (QIAquick PCR Purification Kit) and eluted with 30. mu.l of TE buffer;
2. and obtaining the purified DNA.
Fourthly, (optional) capturing for a second time:
1. the capture is repeated for the second time, so that the proportion of the target nucleic acid molecules in the nucleic acid sample can be further improved;
2. preparing a PCR system by amplification:
20. mu.l of TE buffer containing the target nucleic acid
2.5. mu.l of 10. mu. m P5 primer
(5’-AATGATACGGCGACCACCGAGATCTACAC-3’)
2.5 μ l 10 μm P7 primer (5'-CAAGCAGAAGACGGCATACGAGAT-3')
25 μ l Pre-mix Taq enzyme (Takara)
Mixing uniformly, and the total volume is 50 mu l;
PCR conditions were as follows:
4. and repeating the first step to the fourth step.
Example 4 high throughput sequencing and data analysis
1. The product in example 3 was quantified by an Agilent bioanalyzer 2200;
2. high throughput sequencing of 3 samples from example 3, 2x150bp sequencing using the illumina sequencing platform Hiseq 2500;
3. raw data is obtained, and data is processed and analyzed to obtain statistical results of key parameters, which are shown in table 1.
Table 1:
example 5 sample mutation analysis
Based on the data in example 4, 3 samples were taken and the mutation sites were found by a second generation sequencing data analysis procedure, see table 2.
Table 2:
other embodiments of the present application will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the application being indicated by the following claims.
It will be understood that the present application is not limited to the precise arrangements described above and shown in the drawings and that various modifications and changes may be made without departing from the scope thereof. The scope of the application is limited only by the appended claims.
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