Primer combination and application thereof in genetic typing of red and green blindness genes OPN1LW and OPN1MW

Abstract

The invention discloses a primer combination and application of the primer combination in genetic typing of red and green color blindness genes OPN1LW and OPN1MW. Wherein the primer combination comprises at least one of the following components: OPN1L / MW-1-F, OPN1L / MW-1-R, OPN1L / MW-2-F, OPN1L / MW-2-R, OPN1L / MW-3-F, OPN1L / MW-3-R, OPN1L / MW-4-F, OPN1L / MW-4-R, OPN1L / MW-5-F, OPN1L / MW-6-F and OPN1L / MW-7-R. The invention further discloses a kit for detecting the content of the OPN1L / MW-7-R. When the primer combination or the kit containing the primer combination is used for carrying out OPN1LW and OPN1MW genetic typing, the result is more accurate, and the sensitivity is higher.

Description

Technical Field

[0001] The present application relates to the technical field of genotyping detection, and in particular to a primer combination and its use in genotyping of red-green color blindness genes OPN1LW and OPN1MW. Background Art

[0002] Red-green color blindness is a common X-linked recessive genetic disease that mainly affects an individual's ability to discern red and green colors. It is relatively common in clinical practice. The prevalence of red-green color blindness in men is 2% to 8%, and the prevalence in women is 0.4% to 1.7%. Red-green color blindness is mainly caused by dysfunction or loss of a specific type of cone cells in the retina. Cones in the human retina are responsible for color perception and central vision in bright light, while rods are mainly responsible for dim light vision. Under normal circumstances, the human retina contains three types of cone cells, which are sensitive to short-wavelength (blue), medium-wavelength (green), and long-wavelength (red) light. The cone opsins in these cone cells are encoded by different genes, among which the medium-wavelength and long-wavelength sensitive cone opsins are encoded by the OPN1MW (red opsin gene) and OPN1LW (green opsin gene) genes, respectively.

[0003] The OPN1LW and OPN1MW genes are located on the X chromosome. Abnormalities in the genes or their promoter regions can lead to red-green color blindness, that is, the photosensitive pigments that are sensitive to long and medium wavelengths cannot produce corresponding color vision signals. The two genes are adjacent, arranged in tandem and have a high degree of similarity (about 98% of the DNA sequences are identical), and both contain 6 exons. The sequences of exons 1, 3, and 6 are completely identical, while there are only a few base differences in exons 2, 4, and 5. These two genes usually exist in the form of a single OPN1LW and up to five OPN1MW genes arranged in tandem. This arrangement is called the OPN1LW / OPN1MW gene cluster. Their high similarity makes it easy for the two genes to recombine during the inheritance process, thereby increasing the risk of red-green color blindness. At the same time, there is a locus control region (LCR) about 3.5 kilobase pairs (kb) upstream of the OPN1LW gene. This regulatory region only binds to the promoters of the first two gene copies and activates the proximal gene copy (OPN1LW or OPN1MW) for expression, while the distal OPN1MW gene copy is not expressed. Common mutation types include gene deletion, gene rearrangement, gene insertion, missense mutation, nonsense mutation, small fragment insertion / deletion, splice site variation, etc. These mutations affect the production and signal transduction of photosensitive pigments, which in turn causes patients to be unable to correctly identify red and green. For example, certain specific nucleotide variation combinations, such as the LIAVA combination in OPN1LW and the MVVVA combination in OPN1MW, have been shown to cause Bornholm ophthalmopathy (BED), a more severe color vision disorder.

[0004] With the rapid development of gene diagnosis technology, the diagnosis, classification, severity and pathological development of red and green color blindness through gene sequencing have been gradually carried out. Gene therapy refers to the introduction of exogenous normal genes into target cells to replace non-functional or missing abnormal genes, thereby achieving the purpose of treatment.

[0005] In recent years, researchers have performed gene therapy on adult red-green color blind male squirrel monkeys, effectively restoring their three primary color vision function. Some studies have also shown that gene therapy can partially restore the cone function of patients with achromatopsia. These studies provide new hope for the treatment of patients with red-green color blindness. Gene detection for red-green color blindness / color weakness is not a simple detection of point mutations and insertions and deletions. For gene fusion, due to the homology of the OPN1LW and OPN1MW gene sequences and the fact that the OPN1MW gene itself has multiple copies, it is not easy to locate and analyze with traditional gene detection methods, especially for multiple copies of the OPN1MW gene. The detected mutations and recombination cannot be accurately located to the expressed genes or the unexpressed genes. Therefore, a method for detecting the OPN1LW / OPN1MW gene with accurate positioning, low cost and high throughput is urgently needed in clinical practice, which is particularly important for gene therapy.

[0006] In recent years, with the innovation of technology, genotyping has become increasingly common. Currently, the representative OPN1LW and OPN1MW gene detection and typing technologies are mainly the following categories: 1) Nucleic acid sequencing technology (PCR-Sequencing-Based Typing; PCR-SBT), namely Sanger first-generation sequencing technology, which mainly PCR amplifies the exon-specific fragments of OPN1LW and OPN1MW genes, and performs DNA sequencing on the PCR products to analyze their mutations. Since Sanger first-generation sequencing technology can only obtain 600-800bp of sequence information at a time, and the full length of OPN1LW and OPN1MW genes exceeds 14.2Kb, in order to obtain all OPN1LW and OPN1MW gene information, primers and sequencing are required for multiple positions, which leads to high costs and complicated operations. Due to the limitations of this method, it is impossible to distinguish the haplotype, gene deletion, gene rearrangement and copy number variation of the sample, which will lead to ambiguous results for heterozygous samples and cannot be accurately typed. 2) Biochip detection of SNP sites: This method can detect specific single nucleotide polymorphism (SNP) sites, such as rs2315122, rs2315123, and rs121918344. However, due to the high homology of OPN1LW and OPN1MW genes, the accuracy of biochips and conventional library construction / high-throughput sequencing may be affected, especially when designing chip probes, the influence of homologous genes needs to be considered. Therefore, this method only designs probes for known mutation sites, but cannot detect unknown mutation sites. At the same time, this method cannot distinguish the haplotype, gene deletion, gene rearrangement, and copy number variation of the sample, which will lead to ambiguous results for heterozygous samples and cannot be accurately typed. 3) Multiplex ligation probe amplification (MLPA): MLPA is a technology used to quantify the copy number of pre-selected targets, which can be used to determine the copy number of a specific gene in a sample. This method can analyze the copy number of OPN1LW and OPN1MW genes, but it cannot locate the position of different copies, that is, it cannot accurately locate the mutation that affects the phenotype to the expressed copy or the unexpressed copy of the OPN1MW gene. At the same time, other gene rearrangements, gene deletions, point mutations, etc. cannot be detected at one time, especially new mutations cannot be accurately obtained. 4) Next Generation Sequencing (NGS): Currently, the market is mainly dominated by Illumina's NGS sequencing platform. This technology connects specific oligonucleotide sequences (i.e., adapters) at both ends of the OPN1LW and OPN1MW gene products, and loads the products onto a flow cell with a complementary adapter sequence on the surface. DNA replication is performed through a bridge polymerase chain reaction, the signal is amplified, and the optical signal is converted into a base sequence to achieve high-speed and high-throughput sequencing.This method has a high throughput, so the cost per sample is relatively low, and it can also discover new alleles; however, the technology can only obtain a length of 150-250bp, resulting in the need for further assembly of the original sequence obtained by sequencing to obtain complete OPN1LW and OPN1MW gene information, and this process needs to be inferred by statistical methods, which cannot distinguish haplotypes well, and therefore cannot completely solve exon joint mutations, copy number variations, gene deletions, homologous gene exchanges, etc., and therefore often results in some ambiguous and erroneous results.

[0007] Due to the complexity of OPN1LW and OPN1MW genotyping and the limitations of existing common detection technologies, it is impossible to quickly and accurately type them. The inaccurate and ambiguous results may mislead clinical diagnosis of the disease and personalized gene therapy.

[0008] Therefore, there is an urgent need in the art to develop new methods for OPN1LW and OPN1MW genotyping. Summary of the invention

[0009] Based on this, it is necessary to provide at least one primer combination and its use in the genotyping of red-green color blindness genes OPN1LW and OPN1MW.

[0010] In the first aspect of the present application, a primer combination is provided, which includes one or more of the following primers:

[0011] OPN1L / MW-1-F having a nucleotide sequence as shown in SEQ ID NO: 1;

[0012] OPN1L / MW-1-R having a nucleotide sequence as shown in SEQ ID NO:2;

[0013] OPN1L / MW-2-F having a nucleotide sequence as shown in SEQ ID NO:3;

[0014] OPN1L / MW-2-R having a nucleotide sequence as shown in SEQ ID NO:4;

[0015] OPN1L / MW-3-F having a nucleotide sequence as shown in SEQ ID NO:5;

[0016] OPN1L / MW-3-R having a nucleotide sequence as shown in SEQ ID NO:6;

[0017] OPN1L / MW-4-F having a nucleotide sequence as shown in SEQ ID NO:7;

[0018] OPN1L / MW-4-R having a nucleotide sequence as shown in SEQ ID NO:8;

[0019] OPN1L / MW-5-F having a nucleotide sequence as shown in SEQ ID NO:9;

[0020] OPN1L / MW-6-F having a nucleotide sequence as shown in SEQ ID NO: 10; and

[0021] The nucleotide sequence of OPN1L / MW-7-R is shown in SEQ ID NO:11.

[0022] In some embodiments, the primer combination includes the following primer combination 1:

[0023] OPN1L / MW-1-F having a nucleotide sequence as shown in SEQ ID NO: 1;

[0024] OPN1L / MW-1-R having a nucleotide sequence as shown in SEQ ID NO:2;

[0025] OPN1L / MW-3-F having a nucleotide sequence as shown in SEQ ID NO:5;

[0026] OPN1L / MW-3-R having a nucleotide sequence as shown in SEQ ID NO:6;

[0027] OPN1L / MW-5-F having a nucleotide sequence as shown in SEQ ID NO:9; and

[0028] The nucleotide sequence of OPN1L / MW-7-R is shown in SEQ ID NO:11.

[0029] In some embodiments, the primer combination includes the following primer combination 2:

[0030] OPN1L / MW-2-F having a nucleotide sequence as shown in SEQ ID NO:3;

[0031] OPN1L / MW-2-R having a nucleotide sequence as shown in SEQ ID NO:4;

[0032] OPN1L / MW-4-F having a nucleotide sequence as shown in SEQ ID NO:7;

[0033] OPN1L / MW-4-R having a nucleotide sequence as shown in SEQ ID NO:8; and

[0034] The nucleotide sequence of OPN1L / MW-6-F is shown in SEQ ID NO:10.

[0035] In a second aspect of the present application, a kit is provided, wherein the kit comprises the primer combination as described in the first aspect.

[0036] In some embodiments, the kit further comprises one or more of a PCR buffer, dNTPs, a DNA polymerase, and deionized water.

[0037] In a third aspect of the present application, a method for genotyping and detecting red-green color blindness genes OPN1LW and OPN1MW is provided, the method comprising:

[0038] PCR amplify genes OPN1LW and OPN1MW using the primer combination described in the first aspect or the kit described in the second aspect to obtain an amplified product;

[0039] Constructing a third-generation sequencing library based on the amplified product;

[0040] Purifying the third-generation sequencing library using magnetic beads to obtain a purified library;

[0041] Mixing the purified library to obtain a mixed library; and,

[0042] The pooled library was sequenced.

[0043] In some embodiments, in the step of constructing a third-generation sequencing library based on the amplified product, PCR amplification is performed using PCR reaction system 1 and PCR reaction system 2 respectively; wherein:

[0044] The PCR reaction system 1 includes: primer combination 1, 2×PCR buffer of KOD FX Neo, dNTPs, KOD NeoFX, deionized water, and gDNA;

[0045] The PCR reaction system 2 includes: primer combination 2, 2×PCR buffer of KOD FX Neo, dNTPs, KOD NeoFX, deionized water, and gDNA.

[0046] In some embodiments, in the PCR reaction system 1 or the PCR reaction system 2, the concentration of the primers in the primer combination 1 or the primer combination 2 is independently 100 μM.

[0047] In some embodiments, PCR amplification is performed using the following PCR program:

[0048] 94°C, 2 min;

[0049] 98°C, 12 s, 68°C, 12 min, 26 cycles;

[0050] 68℃、10min.

[0051] In some embodiments, the step of constructing a third-generation sequencing library based on the amplification product includes:

[0052] Barcode preparation;

[0053] connector connections; and,

[0054] Exonuclease digestion.

[0055] In some embodiments, the reaction system for linker ligation includes:

[0056] Amplification products, barcode adapters, and ligase mixture.

[0057] In some embodiments, the ligase mixture includes: 10×T4 DNA ligase buffer, dNTPs, dATP, T4 DNA polymerase, T4 polynucleotide kinase, and T4 DNA ligase.

[0058] In some embodiments, the reaction system comprises 2.5 μL of Barcode adapter, 3.55 μL of ligase mixture, and 8.95 μL of amplification product.

[0059] In some embodiments, the reaction procedure for linker ligation includes:

[0060] 37°C, 25 min;

[0061] 25℃, 20min;

[0062] 65℃、10min.

[0063] This application has developed a new technology to overcome the defects and bottlenecks of the existing OPN1LW and OPN1MW genotyping methods, making OPN1LW and OPN1MW genotyping more accurate and more sensitive. According to the accurate genotyping results, the clinic can be guided to choose the appropriate treatment plan to maximize the safety of the treatment plan. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] In order to more clearly illustrate the technical solutions in the implementation methods and examples of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for use in the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in explaining the present application.

[0065] Figure 1 This is a quality inspection diagram of the PCR product of reaction 1 in one embodiment of the present application.

[0066] Figure 2 This is a quality inspection diagram of the PCR product of reaction 2 in one embodiment of the present application.

[0067] Figure 3 It is the haplotype type sequenced in one embodiment of the present application.

[0068] Figure 4 This is an exemplary sequencing result in one embodiment of the present application.

[0069] Figure 5 This is an exemplary sequencing result in one embodiment of the present application. DETAILED DESCRIPTION

[0070] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0071] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present application belongs. The terms used herein in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0072] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and the like, when used in other ways to mean "one or more", are also understood in the same way unless otherwise specified.

[0073] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0074] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method", etc. is based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0075] In this application, "further", "further", "particularly", "for example", "such as", "example", "for example", etc. are used for descriptive purposes, indicating that the previous and subsequent different technical solutions are related in terms of the content covered, but should not be understood as a limitation on the previous technical solution, nor can it be understood as a limitation on the protection scope of this application. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0076] In this application, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent. Unless otherwise specified, the descriptions of "optionally include", "optionally contain", etc. in this application, taking "optionally include" as an example, mean "may include or not include".

[0077] The terms "contain", "include" and "include" used in this application are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members or features. Members or features include materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, states, etc.

[0078] In this application, the technical features or technical solutions described in an open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0079] In the present application, exemplary descriptions such as "in some implementation modes (or examples)" and "in one implementation mode (or example)" may include but are not limited to the following meanings: these solutions may be combined with other solutions in a suitable manner to form new technical solutions.

[0080] In the present application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0081] In the present application, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to an integer in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0082] In this application, if there are multiple steps involved in the method flow, unless there is a clear different description in this document, there is no strict order restriction for the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0083] The method of the present application may choose to use the sequencing technology platform of Pacific Biosciences, whose HiFi sequencing mode can achieve an offline data accuracy of QV30 (99.9%), and the length of a single subread can reach 15-50kb. It effectively solves the shortcomings of traditional sequencing methods, such as low accuracy and short sequencing length, and can obtain complete OPN1LW and OPN1MW gene sequences or deleted and recombined sequences without assembly. In particular, it can distinguish the haplotypes of OPN1LW and OPN1MW and the arrangement positions of multiple copies of OPN1MW, effectively improving resolution and accuracy. Currently, there are no products based on Pacific Biosciences technology on the market that are applied to OPN1LW and OPN1MW genotyping detection. Therefore, the present application uses its technical advantages to develop a series of OPN1LW and OPN1MW genotyping processes.

[0084] The method in this application targets two haploids and can detect any single SNP mutation, multiple SNP mutation, new allele, copy number variation, gene deletion, LCR deletion, homologous gene exchange and other types of variation at one time, taking the following types as examples:

[0085] 1) Under normal copy number conditions: For samples with single copies of OPN1LW and OPN1MW on haploid 1 and single copies of OPN1LW and OPN1MW on haploid 2, any single SNP mutation, multiple SNP mutation, new allele, LCR deletion, homologous gene exchange and other types of variation can be detected at one time, and haplotypes can be distinguished to clarify the arrangement of LCR, OPN1LW and OPN1MW genes on different haploids.

[0086] 2) In the case of gene deletion: For samples with OPN1LW or OPN1MW deletion on both haploids, or OPN1LW or OPN1MW deletion on only one haploid and the other haploid is normal, various types of variations such as single SNP mutations, multiple SNP mutations, new alleles, LCR deletions, gene deletions, and homologous gene exchanges occurring on both haploids can be detected at one time, and haplotypes can be distinguished. The positions of existing genes and deleted genes can be accurately located on the same haploid at one time, and the arrangement of LCR, OPN1LW, OPN1MW deleted genes and existing genes on different haploids can be clarified.

[0087] 3) In the case of copy number variation: For the existence of multiple copies of OPN1MW on two haploids alone or simultaneously, various types of variation, including single SNP mutations, multiple SNP mutations, new alleles, LCR deletions, gene deletions, copy number variations, and homologous gene exchanges, occurring on the two haploids can be detected at one time, and haplotypes can be distinguished. The arrangement of LCR, OPN1LW, the first expressed OPN1MW, and other non-expressed OPN1MW on different haploids can be clarified at one time.

[0088] In the first aspect of the present application, a primer combination is provided, which includes one or more of the following primers:

[0089] OPN1L / MW-1-F having a nucleotide sequence as shown in SEQ ID NO: 1;

[0090] OPN1L / MW-1-R having a nucleotide sequence as shown in SEQ ID NO:2;

[0091] OPN1L / MW-2-F having a nucleotide sequence as shown in SEQ ID NO:3;

[0092] OPN1L / MW-2-R having a nucleotide sequence as shown in SEQ ID NO:4;

[0093] OPN1L / MW-3-F having a nucleotide sequence as shown in SEQ ID NO:5;

[0094] OPN1L / MW-3-R having a nucleotide sequence as shown in SEQ ID NO:6;

[0095] OPN1L / MW-4-F having a nucleotide sequence as shown in SEQ ID NO:7;

[0096] OPN1L / MW-4-R having a nucleotide sequence as shown in SEQ ID NO:8;

[0097] OPN1L / MW-5-F having a nucleotide sequence as shown in SEQ ID NO:9;

[0098] OPN1L / MW-6-F having a nucleotide sequence as shown in SEQ ID NO: 10; and

[0099] The nucleotide sequence of OPN1L / MW-7-R is shown in SEQ ID NO:11.

[0100] It should be understood that the term "primer combination" in the present application can refer to different primers existing separately but combined in form, for example, different primers exist simultaneously in a specific space as independent units (in the form of dry powder or liquid storage); it can also exist in the form of a composition, such as a combination of dry powders or simultaneously dissolved in the same solvent (such as ionized water, etc.) to exist in the form of liquid storage.

[0101] In some embodiments, the primer combination comprises at least 2 of the above 11 primers. Without wishing to be bound by any theory, it should be understood that the upstream and downstream primers of the 11 primers will be simultaneously included in the primer combination. For example, it at least comprises OPN1L / MW-1-F with a nucleotide sequence as shown in SEQ ID NO: 1 and OPN1L / MW-1-R with a nucleotide sequence as shown in SEQ ID NO: 2, and / or OPN1L / MW-3-F with a nucleotide sequence as shown in SEQ ID NO: 5 and OPN1L / MW-3-R with a nucleotide sequence as shown in SEQ ID NO: 6.

[0102] In some embodiments, the primer combination includes the following primer combination 1: OPN1L / MW-1-F having a nucleotide sequence as shown in SEQ ID NO:1; OPN1L / MW-1-R having a nucleotide sequence as shown in SEQ ID NO:2; OPN1L / MW-3-F having a nucleotide sequence as shown in SEQ ID NO:5; OPN1L / MW-3-R having a nucleotide sequence as shown in SEQ ID NO:6; OPN1L / MW-5-F having a nucleotide sequence as shown in SEQ ID NO:9; and OPN1L / MW-7-R having a nucleotide sequence as shown in SEQ ID NO:11.

[0103] In some embodiments, the primer combination includes the following primer combination 2: OPN1L / MW-2-F having a nucleotide sequence as shown in SEQ ID NO:3; OPN1L / MW-2-R having a nucleotide sequence as shown in SEQ ID NO:4; OPN1L / MW-4-F having a nucleotide sequence as shown in SEQ ID NO:7; OPN1L / MW-4-R having a nucleotide sequence as shown in SEQ ID NO:8; and OPN1L / MW-6-F having a nucleotide sequence as shown in SEQ ID NO:10.

[0104] In a second aspect of the present application, a kit is provided, wherein the kit comprises the primer combination as described in the first aspect.

[0105] In some embodiments, the kit further comprises one or more of a PCR buffer, dNTPs, a DNA polymerase, and deionized water.

[0106] In the present application, the kit may include different units. Each unit may be contained in an independent small package or in a separate partitioned space. In some embodiments, each unit includes different primers. Each unit may also include different subunits, and the subunits may be, for example, primers. For example, a primer with a sequence as shown in SEQ ID NO: 1 is in the form of a dry powder or a liquid storage as one of the subunits. A primer with a sequence as shown in SEQ ID NO: 2 is in the form of a dry powder or a liquid storage as another subunit, etc.

[0107] In a third aspect of the present application, a method for genotyping and detecting red-green color blindness genes OPN1LW and OPN1MW is provided, the method comprising:

[0108] PCR amplify genes OPN1LW and OPN1MW using the primer combination described in the first aspect or the kit described in the second aspect to obtain an amplified product;

[0109] Constructing a third-generation sequencing library based on the amplified product;

[0110] Purifying the third-generation sequencing library using magnetic beads to obtain a purified library;

[0111] Mixing the purified library to obtain a mixed library; and,

[0112] The pooled library was sequenced.

[0113] In some embodiments, the sequencing method is third-generation sequencing, such as the single-molecule instant sequencing method of Pacific Biosciences.

[0114] It should be understood that Pacific Biosciences' Single Molecule Real-Time Sequencing (SMRT) method mainly utilizes several apertures in a zero-mode waveguide (ZMW) to detect the fluorescence generated during DNA polymerization and repeat sequencing through circular DNA.

[0115] In some embodiments, in the step of constructing a third-generation sequencing library based on the amplified product, PCR amplification is performed using PCR reaction system 1 and PCR reaction system 2 respectively; wherein:

[0116] The PCR reaction system 1 comprises: the primer combination 1 as described above, 2×PCR buffer of KOD FX Neo, dNTPs, KOD Neo FX, deionized water, and gDNA;

[0117] The PCR reaction system 2 includes: the primer combination 2 described above, 2×PCR buffer of KOD FX Neo, dNTPs, KOD Neo FX, deionized water, and gDNA.

[0118] In some embodiments, in the PCR reaction system 1 or the PCR reaction system 2, the concentration of the primers in the primer combination 1 or the primer combination 2 is independently 100 μM. In some embodiments, the volume ratio of each primer in the primer combination 1 can be, for example, as shown in Table 2 below. In some embodiments, the volume ratio of each primer in the primer combination 2 can be, for example, as shown in Table 3 below.

[0119] In some embodiments, the PCR reaction system 1 is a 25 μL system. For example, it may include 12.5 μL 2×PCR Buffer for KOD FX Neo, 5 μL dNTPs (2 mM), 0.32 μL primer combination 1, 0.5 μL KOD Neo FX, 50 ng gDNA, and DNase / RNase-Free Deionized Water. The total volume of DNase / RNase-Free Deionized Water and gDNA is 6.68 μL.

[0120] In some embodiments, the PCR reaction system 2 is a 25 μL system. For example, it may include 12.5 μL 2×PCR Buffer for KOD FX Neo, 5 μL dNTPs (2 mM), 0.4 μL primer combination 2, 0.5 μL KOD Neo FX, 50 ng gDNA, and DNase / RNase-Free Deionized Water. The total volume of DNase / RNase-Free Deionized Water and gDNA is 6.6 μL.

[0121] In some embodiments, PCR amplification is performed using the following PCR program:

[0122] 94°C, 2 min;

[0123] 98°C, 12 s, 68°C, 12 min, 26 cycles;

[0124] 68℃、10min.

[0125] In some embodiments, the hot cover temperature of the PCR program is 105° C., and the rate of temperature rise and fall may be, for example, 6.0° C. / S.

[0126] In some embodiments, the step of constructing a third-generation sequencing library based on the amplification product includes:

[0127] Barcode preparation;

[0128] connector connections; and,

[0129] Exonuclease digestion.

[0130] The process of preparing the barcode can refer to the conventional barcode preparation process in the art, for example, including barcode adapter dilution and barcode annealing.

[0131] The sequence information of the Barcode adapter can be found on a sequencing platform such as Pacific Biosciences. The sequence type can be found on Table 7.

[0132] The process of barcode annealing, for example, includes: diluting the 100 μM barcode adapter solution 5 times using 10×annealing buffer and RNAase Free Water, mixing, and annealing the single-stranded barcode adapter into a double-stranded barcode adapter in a PCR instrument according to the annealing program shown in Table 10.

[0133] In some embodiments, the reaction system for linker ligation includes:

[0134] Amplification products, barcode adapters, and ligase mixture.

[0135] In some embodiments, the ligase mixture includes: 10×T4 DNA ligase buffer, dNTPs, dATP, T4 DNA polymerase, T4 polynucleotide kinase, and T4 DNA ligase.

[0136] In some embodiments, the ligase mixture can be found in Table 11.

[0137] In some embodiments, the reaction system comprises 2.5 μL of Barcode adapter, 3.55 μL of ligase mixture, and 8.95 μL of amplification product.

[0138] In some embodiments, the reaction procedure for linker ligation includes:

[0139] 37°C, 25 min;

[0140] 25℃, 20min;

[0141] 65℃、10min.

[0142] Some examples are provided below.

[0143] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance given in the present application, and can also be based on the experimental manual or normal conditions in the art, or can also be based on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0144] Example 1

[0145] According to the complex gene structure of OPN1LW and OPN1MW, multiple pairs of amplification primers were designed, which can accurately detect the arrangement order of the two genes on the haploid regardless of any mutation. Multiple amplification in one tube was achieved by developing the most appropriate primer ratio, screening reagent raw materials, and amplification reaction conditions; the PCR product used a self-developed library construction system to obtain a third-generation sequencing library; finally, Pacific Biosciences third-generation sequencing technology was used, combined with self-developed analysis processes and software, to achieve high coverage, sensitivity, and accuracy of OPN1LW and OPN1MW genotyping.

[0146] The entire process is divided into five parts, namely PCR amplification, library construction, library purification, pooling, and sequencing.

[0147] 1. PCR amplification

[0148] 1. Primer sequences: Specific amplification primers for OPN1LW and OPN1MW genes were designed. The primer sequences are shown in Table 1 below.

[0149] Table 1

[0150]

[0151] 2. Primer Mix System

[0152] The synthesized primer powder was centrifuged at high speed before use, and diluted to the working solution concentration using EB buffer. The diluted primers were prepared into primer MIX according to the system shown in Table 2 and Table 3 below.

[0153] Table 2

[0154]

[0155] Table 3

[0156]

[0157] 3. After preparing primer MIX, add the reagents listed in the PCR amplification system shown in Table 4 and Table 5 below into the reaction tube in sequence, cover the tube, mix well, and centrifuge.

[0158] Table 4

[0159]

[0160] Table 5

[0161]

[0162]

[0163] 3. Set the PCR instrument program according to the parameters shown in Table 6 below for amplification: hot cover: 105°C, heating and cooling rate: 6.0°C / S.

[0164] Table 6

[0165]

[0166] 5. PCR product quality inspection: Use 5200 Fragment Analyzer System to detect the size of library fragments. Figure 1 and Figure 2 .

[0167] 2. Library Construction

[0168] 1. Barcode preparation

[0169] 1) Barcode adapter dilution

[0170] The new Barcode adapter dry powder tube was centrifuged at 10000rpm for 5min, a certain volume of TE buffer was added to dilute the dry powder Barcode to a concentration of 100μM, vortexed and mixed, and centrifuged instantly. A total of 96 Barcode adapters, the sequence types are shown in the following Table 7.

[0171] Table 7

[0172]

[0173] Note: a. The Barcode adapter sequence information is provided by Pacbio. / 5Phos / indicates that the 5' base of the Barcode is phosphorylated during synthesis); b. The bold part above is the Barcode sequence.

[0174] 2) Barcode annealing

[0175] Prepare 10×annealing buffer according to the table below, and use 10×annealing buffer and RNAaseFree Water to dilute the diluted 100μM Barcode adapter solution 5 times, vortex to mix and centrifuge instantly, then place the mixed solution on a PCR instrument and anneal the single-stranded Barcode adapter into a double-stranded one according to the following annealing program. After the annealing program is completed, immediately place the product on ice for 5 minutes and then store it at -20℃.

[0176] a. 10× annealing buffer composition (as shown in Table 8 below)

[0177] Table 8

[0178] Tris-HCl, pH 7.5 100mM NaCl 1M RNAase Free Water Supplement system

[0179] b. Annealing system (as shown in Table 9 below)

[0180] Table 9

[0181] 10×annealing buffer 10μL Barcode adapter(100uM) 20μL RNAase Free Water 70μL

[0182] c. Annealing program: The annealing temperature is gradually reduced by 50°C, see Table 10.

[0183] Table 10

[0184] 80℃ 2min 80℃ 0.1℃ / s decrease 2min 75℃ 0.1℃ / s decrease 2min 70℃ 0.1℃ / s decrease 2min 65℃ 0.1℃ / s decrease 2min ... 0.1℃ / s decrease 2min 30℃ 0.1℃ / s decrease 2min 25℃ 0.1℃ / s decrease 2min 4℃ 2min

[0185] 2. Adapter ligation reaction

[0186] 1) Prepare the linker ligation reaction MIX on ice according to Table 11 below.

[0187] Table 11

[0188]

[0189] 2) Table 12 below is a connection system: Add the listed materials in order, cover the tube, mix well, and centrifuge.

[0190] Table 12

[0191]

[0192] 3) Table 13 below shows the ligation reaction program. After the parameters are pre-set, the ligation reaction is performed. The heated cover is set to 75°C and the heating and cooling rate is 6°C / s.

[0193] Table 13

[0194]

[0195] 4. Exonuclease digestion reaction

[0196] 1) Exonuclease system: Incorrect ligation may occur during the ligation process, so the incorrect ligation product is digested by exonuclease. Prepare exonuclease MIX on ice according to the table below. After the ligation reaction is completed, place it on ice, add 2μL exonuclease MIX to each tube, close the tube cap, mix well, and centrifuge.

[0197] Table 14

[0198]

[0199] 2) Exotomy procedure: Table 15 below shows the digestion reaction parameters, which are set in advance for digestion reaction, heated cover: 45°C, heating and cooling rate: 6°C / s.

[0200] Table 15

[0201]

[0202] After the digestion reaction is completed, magnetic bead purification must be performed as soon as possible.

[0203] 3. Library Purification

[0204] 1. Take out the VAHTSTM DNA Clean Beads (hereinafter referred to as magnetic beads) from the refrigerator half an hour in advance, place them on a vertical mixer and slowly mix them at room temperature for 30 minutes;

[0205] 2. Add 1 volume of DNase / RNase-Free Deionized Water to the digested product, then add 15.3 μL magnetic beads (0.45x), flick to mix or shake at low speed, and leave at room temperature for 10 minutes, flicking 2-3 times to mix;

[0206] 3. Separate the PCR tube instantly, then place it on a magnetic rack to adsorb the magnetic beads for 10 minutes. During this time, prepare 70% alcohol. The alcohol needs to be prepared before use.

[0207] 4. Discard the supernatant, then add 200 μL of 70% alcohol along the opposite side of the tube where the magnetic beads are adsorbed, and be sure not to disperse the magnetic beads;

[0208] 5. Repeat the previous step and perform alcohol cleaning for the second time;

[0209] 6. Discard the alcohol, centrifuge the PCR tube, put it back on the magnetic rack, and discard the residual liquid;

[0210] 7. Open the PCR tube and dry it for no more than 30 seconds, then add 12μL EB Buffer;

[0211] 8. Gently flick the beads to mix them evenly. Keep the beads in a mixed state and place them at room temperature for 10 minutes, flicking them 2-3 times during the process.

[0212] 9. Separate the PCR tube instantly, then place it on a magnetic rack to adsorb the magnetic beads for 10 minutes;

[0213] 10. Transfer 10 μL of supernatant to a new PCR tube;

[0214] 11. Use Qubit to quantify the single sample library, and take 6ng pooling of the same mass for each single sample library as a mixed library.

[0215] 4. Mixed Database

[0216] 1. Use a gun to measure the exact volume x μL (> 400 ng) of the mixed library, and add x*0.6 μL magnetic beads (0.6x) to it.

[0217] The purification process was the same as the library purification process, and finally 50 μL EB was used to elute the library;

[0218] 2. Take x μL (>200 ng) of the library obtained in the previous process and add x*0.6 μL magnetic beads (0.6x) to it.

[0219] The purification process was the same as the library purification process, and finally 20 μL EB was used to elute the library;

[0220] 3. Use Qubit to quantify the final library. Three replicates are required and the average is taken as the final library concentration. The final library concentration is required to be higher than 3ng / μL.

[0221] 5. Sequencing

[0222] The recommended loading volume is 150pM, using HiFi reads mode, and the average fragment size is 10000bp.

[0223] 6. Sequencing results

[0224] The amplified product is used to obtain a library through the library construction process. The data obtained after the library is sequenced on the machine is converted into high-accuracy HiFi data. Then, through data analysis, the mutation site information and structural variation information are obtained. For the haplotype type of sequencing, see Figure 3 , where only a few representative haplotype types are illustrated. The present application can detect various types of variations as described in the technical solution.

[0225] Sequencing example:

[0226] Hap1 sample sequencing sequence:

[0227] Hap1 sample haplotype: LCR is normal, OPN1LW gene is present, OPN1MW gene is missing. Figure 4 .

[0228] Hap5 sample sequencing sequence:

[0229] Haplotype of Hap5 sample: LCR is normal, OPN1LW gene is present, and OPN1MW gene has 2 copies. Figure 5 .

[0230] In summary, this embodiment designs and implements a set of technologies that can amplify the complete haploid of OPN1LW and OPN1MW genes by 2-tube multiplex PCR, making the experimental process simpler. Through primer design, the amplification region extends to the upstream of the LCR region of the OPN1LW front regulatory region and the middle region of the TKTL1 gene after OPN1MW, and the gene information on the LCR-OPN1LW-OPN1MW-TKTL1 haploid can be effectively detected. And due to the characteristics of this technology, all OPN1LW and OPN1MW information is obtained (including exon regions and intron regions). At present, most OPN1LW and OPN1MW genotyping only needs to use exon region information, and this embodiment can provide more comprehensive intron information, as well as the flanking sequences of the gene deletion region or the region where OPN1LW and OPN1MW are recombined, clarifying the common copy number variation, perfectly distinguishing haplotypes, and providing a complete solution for further studying the functions of OPN1LW and OPN1MW genes in the future, which is not available in traditional technologies.

[0231] On the other hand, due to the technical characteristics of the Pacific Biosciences sequencing platform, the raw data obtained only requires minimal sequence assembly to obtain complete information, avoiding the inability of the first and second generation sequencing technologies to distinguish sister chromosome information (phasing) and possible erroneous information caused by assembly, etc. The entire OPN1LW and OPN1MW typing technology has been brought to a new level.

[0232] Comparative Example

[0233] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0234] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims

1. A primer combination, characterized in that: It includes one or more of the following primers: OPN1L / MW-1-F having a nucleotide sequence as shown in SEQ ID NO: 1; OPN1L / MW-1-R having a nucleotide sequence as shown in SEQ ID NO:2; OPN1L / MW-2-F having a nucleotide sequence as shown in SEQ ID NO:3; OPN1L / MW-2-R having a nucleotide sequence as shown in SEQ ID NO:4; OPN1L / MW-3-F having a nucleotide sequence as shown in SEQ ID NO:5; OPN1L / MW-3-R having a nucleotide sequence as shown in SEQ ID NO:6; OPN1L / MW-4-F having a nucleotide sequence as shown in SEQ ID NO:7; OPN1L / MW-4-R having a nucleotide sequence as shown in SEQ ID NO:8; OPN1L / MW-5-F having a nucleotide sequence as shown in SEQ ID NO:9; OPN1L / MW-6-F having a nucleotide sequence as shown in SEQ ID NO: 10; and The nucleotide sequence of OPN1L / MW-7-R is shown in SEQ ID NO:

11.

2. The primer combination according to claim 1, characterized in that It includes the following primer combination 1: OPN1L / MW-1-F having a nucleotide sequence as shown in SEQ ID NO: 1; OPN1L / MW-1-R having a nucleotide sequence as shown in SEQ ID NO:2; OPN1L / MW-3-F having a nucleotide sequence as shown in SEQ ID NO:5; OPN1L / MW-3-R having a nucleotide sequence as shown in SEQ ID NO:6; OPN1L / MW-5-F having a nucleotide sequence as shown in SEQ ID NO:9; and OPN1L / MW-7-R having a nucleotide sequence as shown in SEQ ID NO:11; Alternatively, it comprises the following primer combination 2: OPN1L / MW-2-F having a nucleotide sequence as shown in SEQ ID NO:3; OPN1L / MW-2-R having a nucleotide sequence as shown in SEQ ID NO:4; OPN1L / MW-4-F having a nucleotide sequence as shown in SEQ ID NO:7; OPN1L / MW-4-R having a nucleotide sequence as shown in SEQ ID NO:8; and The nucleotide sequence of OPN1L / MW-6-F is shown in SEQ ID NO:

10.

3. A kit, characterized in that: It comprises the primer combination as claimed in claim 1 or 2.

4. The kit according to claim 3, characterized in that The kit further comprises one or more of a PCR buffer, dNTPs, a DNA polymerase and deionized water.

5. A method for detecting the genotyping of red-green color blindness genes OPN1LW and OPN1MW, characterized in that: The method comprises: PCR amplify genes OPN1LW and OPN1MW using the primer combination as described in claim 1 or 2 or the kit as described in claim 3 or 4 to obtain amplified products; Constructing a third-generation sequencing library based on the amplified product; Purifying the third-generation sequencing library using magnetic beads to obtain a purified library; Mixing the purified library to obtain a mixed library; and, The pooled library was sequenced.

6. The method according to claim 5, characterized in that In the step of constructing a third-generation sequencing library based on the amplified product, PCR amplification is performed using PCR reaction system 1 and PCR reaction system 2 respectively; wherein: The PCR reaction system 1 includes: primer combination 1, 2×PCR buffer of KOD FX Neo, dNTPs, KOD Neo FX, deionized water, and gDNA; The PCR reaction system 2 includes: primer combination 2, 2×PCR buffer of KOD FX Neo, dNTPs, KOD Neo FX, deionized water, and gDNA.

7. The method according to claim 5 or 6, characterized in that The step of constructing a third-generation sequencing library based on the amplification product includes: Barcode preparation; connector connections; and, Exonuclease digestion.

8. The method according to claim 7, characterized in that The reaction system for linker ligation includes: Amplification products, barcode adapters, and ligase mixture.

9. The method according to claim 8, characterized in that The ligase mixture includes: 10×T4 DNA ligase buffer, dNTPs, dATP, T4 DNA polymerase, T4 polynucleotide kinase, and T4 DNA ligase.

10. The method according to claim 7, characterized in that The reaction procedure for performing adapter ligation includes: 37°C, 25 min; 25℃, 20min; 65℃、10min.

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