Ultralow frequency mutation nucleic acid fragment detection method, library construction method, primer design method and reagent
阅读说明：本技术 一种超低频突变核酸片段检测方法、文库构建方法、引物设计方法和试剂 (Ultralow frequency mutation nucleic acid fragment detection method, library construction method, primer design method and reagent ) 是由 陆利 唐薇 朱丽芳 曹曼曼 徐根明 潘艺 赵谦 于 2019-10-14 设计创作，主要内容包括：本发明公开了一种超低频突变核酸片段检测方法、突变核酸片段文库构建方法、引物设计方法和试剂。首先对待检样本的片段化核酸进行末端修复补平加A；进行分子标签接头连接；采用分子标签引物与单分子特异性引物组合,对连接接头产物进行扩增子法靶向富集；最后进行PCR扩增富集。本发明设计的单分子特异性引物使得可利用的有效模板数明显提高、其带有的调节序列可明显降低特异性引物之间形成引物二聚体的概率,还可明显提高模板利用率。本发明超低频突变核酸片段检测方法,使用单侧单引物扩增子法富集和分子标签技术,提高了有效模板利用率,降低了噪音源,降低假阳性,提高了检测的灵敏度和特异性,特别适合痕量超低频突变样本检测。同时本发明还能进行高效文库构建。(The invention discloses an ultralow frequency mutation nucleic acid fragment detection method, a mutation nucleic acid fragment library construction method, a primer design method and a reagent. Firstly, performing end repairing and filling-in addition A on fragmented nucleic acid of a sample to be detected; performing molecular label joint connection; combining a molecular label primer and a single-molecule specific primer to perform targeted enrichment on the connecting joint product by an amplicon method; and finally, carrying out PCR amplification enrichment. The designed single molecule specific primer obviously increases the number of available effective templates, and the regulating sequence can obviously reduce the probability of primer dimer formation between specific primers and also can obviously increase the utilization rate of the templates. The method for detecting the ultralow-frequency mutation nucleic acid fragment improves the utilization rate of an effective template, reduces a noise source, reduces false positive, improves the sensitivity and specificity of detection and is particularly suitable for detecting trace ultralow-frequency mutation samples by using a single-side single-primer amplicon method enrichment and molecular labeling technology. Meanwhile, the invention can also carry out high-efficiency library construction.)
1. A design method of single molecule specific primer for detecting ultra-low frequency mutation nucleic acid fragment is characterized in that the design method comprises a targeting sequence and a sequencing primer sequence, or also comprises a regulating sequence, wherein the regulating sequence divides the targeting sequence into a targeting upstream sequence and a targeting downstream sequence; the target sequence is a sequence which is specifically complementary with the upstream or downstream of the mutation region to be detected, so as to obtain an upstream monomolecular specific primer or a downstream monomolecular specific primer.
2. The method of claim 1, wherein the targeting sequence satisfies the following conditions:
1) the length of the targeting sequence is 20-100 base pairs;
2) the annealing temperature of the targeting sequence is 50-80 ℃;
3) the 3' end base of the targeting sequence is C or G.
3. The method of designing a single-molecule-specific primer for the detection of an ultralow frequency mutant nucleic acid fragment according to claim 1, wherein the regulatory sequence satisfies the following conditions:
1) the regulatory sequence consists of 4-30 base pairs;
2) the bases of the regulatory sequence consist of natural bases, or unnatural bases, or natural bases and unnatural bases;
3) the 3 'end of the regulatory sequence is 10-30 base pairs from the 3' end of the targeted downstream sequence.
4. The method of claim 1, wherein the distance from the 3' end of the primer to the site or region of the mutation to be detected is 1-30 bp.
5. A method for detecting an ultralow frequency mutant nucleic acid fragment is characterized by comprising the following steps:
(1) designing an upstream single-molecule specific primer and a downstream single-molecule specific primer by the method of any one of claims 1 to 4;
(2) carrying out end repair filling-up addition A on fragmented nucleic acid of a DNA sample to be detected;
(3) performing molecular tag linker connection on the fragmented nucleic acid with the end repairing and filling-in addition A;
(4) combining a molecular label primer with an upstream monomolecular specific primer and a downstream monomolecular specific primer respectively to perform targeted enrichment on a connecting joint product by an amplicon method; the upstream single molecule specific primer is a single primer or a primer set designed according to the upstream of more than two mutation regions, and the downstream single molecule specific primer is a single primer or a primer set designed according to the downstream of more than two mutation regions;
(5) performing PCR enrichment on the target enrichment product by the amplicon method; and detecting the PCR enrichment product.
6. The method of claim 5, wherein the molecular tag adaptor of step (3) is a double strand that is fully or partially complementary; the molecular tag linker comprises a molecular tag sequence and a sequencing primer sequence.
7. The method according to claim 5, further comprising any one or more of the following processing modes:
a, combining the two reactions of the step (2) and the step (3) into a one-step reaction and simultaneously carrying out the two reactions;
b, after the step (3), purifying the reaction product after the linker is linked, and removing the excessive linker;
and C, purifying the upstream single-molecule specific primer enrichment product and the downstream single-molecule specific primer enrichment product after the step (4) to remove excessive upstream single-molecule specific primer, downstream single-molecule specific primer and molecular label primer.
8. The method according to claim 5 or 7, further comprising any one or more of the following processing modes:
a, performing pre-amplification on the reaction product which is connected with the joint in the step (3), and then performing targeted enrichment by an amplicon method;
and b, combining the two enrichment steps of the step (4) and the step (5) into one PCR reaction enrichment step.
9. The method of claim 8, wherein the pre-amplification comprises two modes:
the method comprises the steps of carrying out pre-amplification by using a molecular label primer and a universal primer, and then carrying out targeted enrichment by using a single-molecule specific primer and the universal primer, or carrying out pre-amplification by using the universal primer, and then carrying out targeted enrichment by using the single-molecule specific primer and the molecular label primer.
10. The method of claim 5, wherein the ultra-low frequency mutation comprises one or more of a point mutation, a deletion, an insertion, a gene amplification, a gene methylation, and a gene fusion.
11. The method of claim 10,
when detecting the fusion gene, combining a molecular label primer with an upstream monomolecular specific primer or a downstream monomolecular specific primer to perform targeted enrichment and PCR reaction enrichment by an amplicon method;
when it is determined that the gene fusion occurs in a known region and a specific site of the gene fusion in the region needs to be detected, primer sets are designed at intervals of 30-50bp in sequence in the region, and targeted enrichment and PCR (polymerase chain reaction) enrichment are performed by an amplicon method.
12. The method of claim 5, wherein the nucleic acid fragments comprise DNA or RNA; sources include animals, plants, or microorganisms.
13. A method for constructing a library of ultra-low frequency mutant nucleic acid fragments, which comprises constructing a library using the PCR amplification product obtained in step 5) of the method according to any one of claims 5 to 12.
14. An ultralow frequency mutation nucleic acid fragment detection kit is characterized in that: the method comprises the following steps: a reagent for repairing a fragmented nucleic acid at the end and adding A, a reaction reagent for molecular tag linker ligation of nucleic acid, and a single-molecule specific primer for detecting ultra-low frequency gene mutation designed by the method according to any one of claims 1 to 4.
15. An ultra-low frequency mutation nucleic acid fragment library construction kit is characterized by comprising: a reagent for repairing a fragmented nucleic acid at the end and adding A, a reaction reagent for molecular tag linker ligation of nucleic acid, and a single-molecule specific primer for detecting ultra-low frequency gene mutation designed by the method according to any one of claims 1 to 4.
16. A reagent for end-repair-filling-up a of a fragmented nucleic acid, comprising: a reaction activator, a reaction enzyme and a reaction stabilizer;
the reactive agents include: 5-500mM Tris-HCl pH 8.0, 50-1000mM NaCl, 50-200 mM dNTPs, 0.5-4mM ATP, 1-100mM DTT, 50-250mM MgCl2(ii) a Wherein the percentage is calculated by taking the volume of the sterile water as a reference;
the reaction enzyme is an enzyme mixture consisting of functional enzymes with 5' -3' DNA polymerase activity, 5' -3' exonuclease activity, 3' -5 ' exonuclease activity and 3' end base A added to the product; preferably comprising: 1-50U of Taq DNA polymerase, 10-50U of 3 '-5' exo klenow fragment, 2-100U of T4DNA polymerase;
the reaction stabilizer is a reagent for stabilizing the activity of the enzyme; preferably comprising: 0.05-1% Tween 20, 1-50% glycerol, 20-100. mu.g/mL BSA, 0.02-0.1% Triton X-100, 0.1-1% beta-mercaptoethanol, wherein the percentages are based on the volume of sterile water.
17. A reagent for performing molecular tag linker ligation of nucleic acids, comprising: a connection reinforcing agent, a high-efficiency ligase and a molecular label joint;
the ligation enhancer comprises 1-4mM ATP, 1-100mM NAD +, 1-200mM DTT, 0.05-1% Tween 20, 5-50% PEG polymer, wherein the percentages are calculated by using sterile water volume as a reference;
the efficient ligase is a ligase which catalyzes the combination of the 5 '-P end and the 3' -OH end of the sticky end or the blunt end double-stranded or single-stranded DNA or RNA by a phosphodiester bond; the high-efficiency ligase consists of one or more ligases;
preferred high efficiency ligases include: 500-2000U T4-DNA ligase, 50-500U Taq DNA ligase and 1-500U E. coli DNA ligase;
the molecular label joint is a double chain which is completely or partially complementary; the molecular tag linker comprises a molecular tag sequence and a sequencing primer sequence.
Technical Field
The invention belongs to the technical field of molecular biology detection, and particularly relates to an ultralow-frequency mutant nucleic acid fragment detection method, a mutant nucleic acid fragment library construction method, a primer design method and a reagent.
Background
The second Generation Sequencing technology (Next Generation Sequencing) is used as a means for detecting somatic mutation, and plays an important role in tumor research and tumor diagnosis. When the method is applied to the aspects of rare tumor mutation gene detection, tumor mutation drug resistance identification, treatment response effect evaluation and the like, the mutation frequency needs to be detected to be 0.1 percent or lower. Because the standard library construction process adopts a DNA polymerase method to introduce background noise, the ultralow frequency mutation signal can be covered by the background noise and is difficult to distinguish. While the main noise source can be subdivided into three parts: the first moiety is from a targeted capture technique; the second part is generated by PCR polymerase when the library amplification is enriched; finally, the sequencing error of the sequencer is detected; aiming at the first two noise sources, the targeted enrichment technology is mainly used for noise reduction at present. There are two main types of targeted enrichment techniques currently in use: one is amplicon and one is hybrid capture; two obvious defects exist in hybridization capture, 1) experiment operation is complicated, the flow is long, and 2) the probe hybridization process can damage DNA, so that base mutation and noise are increased. The amplicon technology has no hybridization process, no extra noise, simple operation and convenient flow.
The traditional amplicon enrichment technology is based on bilateral enrichment, the problem of cfDNA template loss exists, and trace ctDNA is rapidly removed. The single-side enrichment technology can effectively solve the problems, and the current main single-side enrichment technology has two types, one is a single-side enrichment-chain looping (back-to-back primer) mode; a single-sided enrichment-nested PCR primer (nested PCR primer) mode; the single-detection enrichment-strand looping mode has no single molecular label technology to reduce noise, and the single-detection enrichment-nested PCR primer mode adopts double primers to cause that the 3' end of the primer and a site to be detected have to keep a certain distance, so that a small amount of template loss exists, and the detection limit is seriously influenced. In the invention, a single-side enrichment-single molecule specific primer (single primer) mode is adopted for the first time to carry out an amplicon enrichment technology (as shown in figure 1); the application range of the primer is enlarged, the usage amount of the effective template is increased, and particularly, the detection limit of detecting the low-frequency mutation sample is greatly improved. Secondly, the single molecule specific primer of the invention consists of a specific sequence, a regulating sequence and a sequencing sequence, and the combination of the specific sequence and the regulating sequence can effectively provide the specificity and the primer utilization rate of the multiple PCR. Thirdly, the YEA and YLA reaction solution provided by the invention optimizes the library construction process, realizes that pre-amplification (PCR-Free technology) is not needed before amplicon enrichment, and reduces the error probability introduced due to PCR amplification. Finally, the invention provides a linker technology with a single molecular label, which can further correct the errors of the primer for library construction and sequencing.
Disclosure of Invention
The invention aims to provide a design method of a single-molecule specific primer for detecting ultralow-frequency gene mutation. The applicant summarizes the method through a large number of previous explorations and verifications, and the single-molecule specific primer designed and synthesized by the method can effectively improve the specificity and the template utilization rate of the multiplex PCR.
The design method of the single-molecule specific primer for detecting the ultralow-frequency mutation nucleic acid fragment comprises two modes: the primer only contains a targeting sequence and a sequencing primer sequence, wherein the targeting sequence is a sequence which is specifically complementary with the upstream or downstream of a mutation region to be detected, and then an upstream single-molecule specific primer or a downstream single-molecule specific primer is obtained. Or in addition to the targeting sequence and the sequencing primer sequence, a regulatory sequence that divides the targeting sequence into two parts, a targeted upstream sequence and a targeted downstream sequence. Preferred are designs containing regulatory sequences.
The target sequence is at the 3 'end of the single molecule specific primer, and the sequencing primer sequence is at the 5' end of the single molecule specific primer.
Further, in the above design method, the conditions to be satisfied by the targeting sequence specifically include:
1) the length of the targeting sequence is 20-100 base pairs;
2) the annealing temperature of the targeting sequence is 50-80 ℃;
3) the 3' end base of the targeting sequence is C or G.
Further, in the above design method, the condition to be satisfied by the regulatory sequence specifically includes:
1) the regulatory sequence consists of 4-30 base pairs;
2) the bases of the regulatory sequence consist of natural bases, or unnatural bases, or natural bases and unnatural bases;
3) the 3 'end of the regulatory sequence is 10-30 base pairs from the 3' end of the targeted downstream sequence.
The regulatory sequence can obviously reduce the probability of forming primer dimers among specific primers and can obviously improve the utilization rate of the primers.
Further, the above design method needs to satisfy: the distance from the 3' end of the single molecule specificity primer to the mutation site or the region to be detected is 1-30 base pairs.
The second purpose of the invention is to provide a method for detecting ultralow frequency mutation nucleic acid fragments, which solves the technical problems of low specificity of DNA fragment capture and low accuracy of ultralow frequency gene detection in the existing detection technology.
The method for detecting the ultralow frequency mutation nucleic acid fragment comprises the following steps:
(1) designing an upstream single molecule specific primer and a downstream single molecule specific primer by the method;
(2) carrying out end repair filling-up addition A on fragmented nucleic acid of a DNA sample to be detected;
(3) performing molecular tag linker connection on the fragmented nucleic acid with the end repairing and filling-in addition A;
(4) combining a molecular label primer with an upstream monomolecular specific primer and a downstream monomolecular specific primer respectively to perform targeted enrichment on a connecting joint product by an amplicon method; the upstream single molecule specific primer is a single primer or a primer set designed according to the upstream of more than two mutation regions, and the downstream single molecule specific primer is a single primer or a primer set designed according to the downstream of more than two mutation regions;
(5) performing PCR enrichment on the target enrichment product by the amplicon method; and detecting the PCR enrichment product.
Further, in the method for detecting the ultralow frequency mutant nucleic acid fragment, the molecular tag adaptor in the step (3) is a double strand which is completely or partially complementary; the molecular label joint comprises a molecular label sequence and a sequencing primer sequence; the molecular tag sequence consists of 8-12 random nucleotides.
Further, the method for detecting the ultralow frequency mutation nucleic acid fragment also comprises any one or more of the following treatment modes:
a, combining the two reactions of the step (2) and the step (3) into a one-step reaction and simultaneously carrying out the two reactions; namely, the operations of performing end repairing and filling-in addition A and molecular tag joint connection on the fragmented nucleic acid are completed in the same reaction system;
b, after the step (3), purifying the reaction product after the linker is linked, and removing the excessive linker;
and C, purifying the upstream single-molecule specific primer enrichment product and the downstream single-molecule specific primer enrichment product after the step (4) to remove excessive upstream single-molecule specific primer, downstream single-molecule specific primer and molecular label primer.
Further, the method for detecting the ultralow frequency mutation nucleic acid fragment also comprises any one or more of the following processing modes:
a, performing pre-amplification on a purified reaction product linked with a joint, and then performing targeted enrichment by an amplicon method;
and b, combining the two enrichment steps of the step 4) and the step 5) into one PCR reaction enrichment step.
Further, the pre-amplification comprises: the method comprises the steps of firstly carrying out pre-amplification by adopting a molecular label primer and a universal primer combination, and then carrying out targeted enrichment by adopting a single-molecule specific primer and the universal primer combination by adopting an amplicon method, or firstly carrying out pre-amplification by adopting the universal primer combination, and then carrying out targeted enrichment by adopting the single-molecule specific primer and the molecular label primer combination by adopting the amplicon method.
Furthermore, the ultra-low frequency mutation nucleic acid fragment detection method comprises one or more of point mutation, deletion insertion, gene amplification, gene methylation and gene fusion.
Furthermore, when detecting the fusion gene, a molecular label primer and an upstream monomolecular specific primer or a downstream monomolecular specific primer are combined to carry out targeted enrichment and PCR reaction enrichment by an amplicon method.
Furthermore, when it is determined that the gene fusion occurs in a known region and a specific site of the gene fusion in the region needs to be detected, primer sets are designed at intervals of 30-50bp in sequence in the region, and the targeted enrichment by an amplicon method and the enrichment by a PCR reaction are carried out.
Furthermore, the method for detecting the ultralow frequency mutation nucleic acid fragment comprises DNA or RNA, and can be in a complete or fragmented form.
Furthermore, fragmentation is required for the complete DNA or RNA before filling in the end repair. RNA requires conversion to cDNA.
Further, the source of the nucleic acid fragment includes an animal, a plant or a microorganism.
Further, the nucleic acid source includes plasma, urine, sweat, saliva, semen, pleural fluid, ascites, stool, fossil, paraffin embedded (FFPE), or criminal investigation samples.
The third purpose of the invention is to provide a method for constructing an ultralow frequency mutant nucleic acid fragment library, so as to solve the technical problems of low specificity of DNA fragment capture, introduction of error mutation in the library construction process and low accuracy of ultralow frequency gene detection in the existing detection technology.
The process of the method for constructing the ultra-low frequency mutant nucleic acid fragment library is basically consistent with the steps of the method for detecting the ultra-low frequency mutant nucleic acid fragment, and the difference is that the PCR amplification product obtained in the last step 5) in the detection method is constructed by the library.
The fourth purpose of the invention is to provide a kit matched with the method for detecting the ultralow frequency mutation nucleic acid fragment. The kit comprises: a reagent for repairing and adding A at the tail end of the fragmented nucleic acid, a reaction reagent for connecting molecular label joints of the nucleic acid and a single-molecule specific primer for detecting the ultra-low frequency gene mutation.
The fifth purpose of the invention is to provide a kit matched with the construction method of the ultralow frequency mutation nucleic acid fragment library. The kit comprises: a reagent for repairing and adding A at the tail end of the fragmented nucleic acid, a reaction reagent for connecting molecular label joints of the nucleic acid and a single-molecule specific primer for detecting the ultra-low frequency gene mutation.
Furthermore, the reaction reagent for connecting the molecular tag joints of the nucleic acids contains the molecular tag joints, and the molecular tag joints comprise single-chain joints, bubble-shaped double-chain joints, Y-shaped joints or irregular Y-shaped joints.
Furthermore, the kit matched with the construction method of the ultralow frequency mutation nucleic acid fragment library also comprises three universal primers, namely a molecular tag primer and two sequencing universal primers; the molecular label primer is used for distinguishing different samples.
It is a sixth object of the present invention to provide a reagent for end-repair-addition of A to a fragmented nucleic acid, referred to herein as YEA reaction solution. The YEA reaction solution provided by the invention optimizes the library establishment process, realizes that pre-amplification (PCR-Free technology) is not required before amplicon enrichment, and reduces the error probability caused by PCR amplification.
The YEA reaction solution comprises: a reaction activator, a reaction enzyme and a reaction stabilizer; 1L of YEA reaction liquid is prepared, and the concentration of each component is the concentration in the whole reagent system:
the reactive agents include: 5-500mM Tris-HCl pH 8.0, 50-1000mM NaCl, 50-200 mM dNTPs, 0.5-4mM ATP, 1-100mM DTT, 50-250mM MgCl2(ii) a Wherein the percentage is calculated by taking the volume of the sterile water as a reference;
the reaction enzyme is an enzyme mixture consisting of functional enzymes with 5' -3' DNA polymerase activity, 5' -3' exonuclease activity, 3' -5 ' exonuclease activity and 3' end base A added to the product; preferably comprising: 1-50U of Taq DNA polymerase, 10-50U of 3 '-5' exo klenow fragment, and 2-100U of T4DNA polymerase;
the reaction stabilizer is a reagent for stabilizing the activity of the enzyme; preferably comprising: 0.05-1
The seventh object of the present invention is to provide a reaction reagent for molecular tag linker ligation of nucleic acids, referred to as YLA reaction solution in the present invention. The YLA reaction solution provided by the invention optimizes the library construction process, realizes that pre-amplification (PCR-Free technology) is not required before amplicon enrichment, and reduces the error probability caused by PCR amplification.
Further, the YLA reaction solution comprises: a connection reinforcing agent, a high-efficiency ligase and a molecular label joint; preparing 1L of YLA reaction liquid, wherein the concentration of each component is the concentration in the whole reagent system:
the connection enhancer comprises 1-4mM ATP, 1-100mM NAD +, 1-200mM DTT, 0.05-1
the efficient ligase is a ligase which catalyzes the combination of the 5 '-P end and the 3' -OH end of the sticky end or the blunt end double-stranded or single-stranded DNA or RNA by a phosphodiester bond; the high-efficiency ligase consists of one or more ligases;
preferred high efficiency ligases include: 500-2000U T4-DNA ligase, 50-500U Taq DNA ligase and 1-500U E. coli DNA ligase;
the molecular label joint is a double chain which is completely or partially complementary; the molecular tag linker comprises a molecular tag sequence and a sequencing primer sequence.
FIG. 2 shows the detection principle of the mutation site of the present invention. Wherein the content of the first and second substances,
Fig. 3 shows several variations of the library building process of the present invention, which specifically include the following forms:
the No.1 process comprises the following steps: pre-library amplification, single-side single-primer PCR amplification and enrichment and general PCR amplification and enrichment;
the No.2 process comprises the following steps: single-sided single-primer PCR amplification enrichment + universal PCR amplification enrichment, but no pre-library amplification is required before amplification enrichment;
the No.3 process comprises the following steps: the two steps of combined one-step PCR of pre-library amplification + single-side single-primer PCR amplification enrichment and general PCR amplification enrichment;
the No.4 process comprises: single-sided single-primer PCR amplification enrichment and universal PCR amplification enrichment are combined to form one-step PCR, but pre-library amplification is not needed before amplification enrichment.
Wherein the process No.4 is a classical library construction process of the invention.
The designed single molecule specific primer obviously increases the number of available effective templates, and the regulating sequence can obviously reduce the probability of primer dimer formation between specific primers and also can obviously increase the utilization rate of the templates.
The invention can correct the noise introduced by the detection process by using the joint with the molecular label.
The method for detecting the ultralow-frequency mutation nucleic acid fragment improves the utilization rate of an effective template, reduces a noise source, reduces false positive, improves the sensitivity and specificity of detection and is particularly suitable for detecting trace ultralow-frequency mutation samples by using a single-side single-primer amplicon method enrichment and molecular labeling technology.
The method for constructing the ultralow-frequency mutant nucleic acid fragment library has the advantages of the detection method and can also avoid introducing error mutation in the library construction process.
The invention also has the advantages that:
1. the library is constructed by a one-step method, the template loss is reduced, the operation steps are reduced, the detection cost is reduced, and the detection period is shortened;
2. the method is suitable for detection by taking DNA as a template and is also suitable for detection by taking RNA as a template.
The high-throughput library construction method can be used for high-throughput sequencing platforms such as Roche, Illumina, ThermoFisher, Pacific Biosciences, Huada genes, Oxford Nanopore Technologies, Huanyang and Hanhai genes.
Drawings
FIG. 1 is a comparison of the enrichment capture principle of the amplicon according to the present invention with other enrichment capture methods of the prior art.
FIG. 2 shows the detection principle of the mutation site of the present invention.
FIG. 3 is several variations of the library building process of the present invention;
the No.1 process comprises the following steps: pre-library amplification, single-side single-primer PCR amplification and enrichment and general PCR amplification and enrichment;
the No.2 process comprises the following steps: single-sided single-primer PCR amplification enrichment + universal PCR amplification enrichment, but no pre-library amplification is required before amplification enrichment;
the No.3 process comprises the following steps: the two steps of combined one-step PCR of pre-library amplification + single-side single-primer PCR amplification enrichment and general PCR amplification enrichment;
the No.4 process comprises: single-sided single-primer PCR amplification enrichment and universal PCR amplification enrichment are combined to form one-step PCR, but pre-library amplification is not needed before amplification enrichment.
FIG. 4 shows the results of the electrophoretic detection analysis of the library of example 1.
Note: m is 100bp DNA ladder (Takara Bio Inc.);
1: library of sample No.1
2: library of sample No.1
3: library of sample PCR reaction tube No.21
4: library of sample
FIG. 5 shows the results of the electrophoretic detection analysis of the library of example 2;
note: m is 100bp DNA ladder (Takara Bio Inc.);
1: the library of the construction method of the ultra-low frequency mutation DNA fragment library.
FIG. 6 shows the results of the electrophoretic detection analysis of the library of example 3;
note: m is 100bp DNA ladder (Takara Bio Inc.);
1: library of sample No.1
2: library of sample No.1
3: library of sample PCR reaction tube No.21
4: library of sample
5: library of sample No.3
6: library of sample
FIG. 7 shows the results of the electrophoretic detection analysis of the library of example 4;
note: m is 100bp DNA ladder (Takara Bio Inc.);
1: library of RNA library construction methods.
FIG. 8 shows the results of the electrophoretic detection analysis of the library of example 5;
note: m is 100bp DNA ladder (Takara Bio Inc.);
1: the library constructed by the total RNA one-step method library.
FIG. 9 is a schematic diagram of the EML4-ALK gene fusion detection;
FIG. 10 shows the results of the electrophoretic detection analysis of the library of example 6;
note: m is 100bp DNA ladder (Takara Bio Inc.);
1: library of FP001 sample
2: library of FP001 sample
3: library of FP002 sample
4: library of FP002 sample
5: library of FP No. 003 sample
6: FP No. 003 sample library from
Detailed Description
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention.