Primer group and kit for detecting pathogenic mutation of DNAAF3 gene

Abstract

The invention discloses a primer group for detecting pathogenic mutation of a DNAAF3 gene, a kit containing the primer group and application of the primer group and relates to the technical field of biology, and the primer group for detecting the pathogenic mutation of the DNAAF3 gene comprises a first primer group and a second primer group; according to the technical scheme provided by the invention, the detection of the pathogenic mutation of the DNAAF3 gene can be completed by utilizing Sanger sequencing, that is, whether the pathogenic mutation exists in the DNAAF3 gene or not can be determined through Sanger sequencing, so that the detection period is shortened, and the detection cost is reduced.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a primer set and a kit for detecting pathogenic mutations of a DNAAF3 gene. Background Art

[0002] Primary Ciliary Dyskinesia (PCD) is a serious genetic disease that affects the function of cilia in the body. Cilia are tiny structures on the surface of cells that are found in many parts of the human body, including the respiratory tract, reproductive system, and ears. They are responsible for moving and clearing mucus and foreign matter outside the cells. PCD is caused by genetic mutations that lead to defects in the structure or function of cilia.

[0003] The full name of the DNAAF3 gene is Dynein Axonemal Assembly Factor 3, which is located on chromosome 19 and contains 12 exons. The DNAAF3 protein it encodes is a vital protein whose main function is to participate in the assembly of cilia and maintain the normal movement function of cilia. DNAAF3 protein is an important component in cilia assembly, especially closely related to the movement function of cilia. Defects in the DNAAF3 gene can lead to ciliary motility disorders, such as primary ciliary dyskinesia (PCD).

[0004] In addition, mutations in the DNAAF3 gene affect the normal function of cilia, causing a variety of clinical symptoms including Kartagener syndrome. Kartagener syndrome patients often show symptoms such as chronic respiratory infections and situs inversus.

[0005] Currently, pathogenic mutations in the DNAAF3 gene can only be detected through whole exome sequencing (WES) or whole genome sequencing (WES) technology. Both methods are second-generation sequencing technologies, which have high requirements for detection technology and equipment, are expensive, and have a long cycle. Summary of the invention

[0006] The main purpose of the present invention is to propose a primer set for detecting pathogenic mutations in the DNAAF3 gene, aiming to determine whether the DNAAF3 gene has pathogenic mutations by Sanger sequencing, thereby shortening the detection cycle and reducing the detection cost.

[0007] To achieve the above object, the primer set for detecting pathogenic mutations of DNAAF3 gene proposed in the present invention comprises a first primer set and a second primer set;

[0008] The first primer set includes a first upstream primer and a first downstream primer, the nucleotide sequence of the first upstream primer is shown in SEQ ID NO.9, and the nucleotide sequence of the first downstream primer is shown in SEQ ID NO.10;

[0009] The second primer set includes a second upstream primer and a second downstream primer, the nucleotide sequence of the second upstream primer is shown in SEQ ID NO.37, and the nucleotide sequence of the second downstream primer is shown in SEQ ID NO.38.

[0010] Optionally / in one embodiment, the first primer set is used to detect the c.192G>A mutation site of the DNAAF3 gene, and the second primer set is used to detect the c.842G>A mutation site of the DNAAF3 gene.

[0011] Optionally / in one embodiment, the first primer set and the second primer set are used for Sanger sequencing.

[0012] Optionally / in one embodiment, the PCR reaction system for Sanger sequencing includes the following components: 10×PCR Buffer; dNTP (2.5 mM); genomic DNA; upstream primer; downstream primer; Taq DNA polymerase; ddH2O.

[0013] Optionally / in one embodiment, the added concentration of the genomic DNA is 100 ng / μL.

[0014] Optionally / in one embodiment, the volumes of the components of the PCR reaction system for Sanger sequencing are as follows: 10×PCRBuffer: 1.0 μL; dNTP (2.5 mM): 0.8 μL; genomic DNA (100 ng / μL): 0.5 μL; upstream primer: 0.4 μL; downstream primer: 0.4 μL; Taq DNA polymerase: 0.1 μL; ddH2O: 6.8 μL; total volume: 10 μL.

[0015] Optionally / in one embodiment, the first primer set is used in PCR amplification m The values ​​ranged from 58.2°C to 58.8°C.

[0016] Optionally / in one embodiment, the second primer set is used in PCR amplification m The values ​​ranged from 58.9°C to 59.4°C.

[0017] The present invention also provides a kit for detecting pathogenic mutations of DNAAF3 gene, comprising the above primer set.

[0018] The present invention also proposes the use of the kit in detecting pathogenic mutations of the DNAAF3 gene.

[0019] The technical solution of the present invention adopts two sets of primers to detect two pathogenic mutations of the DNAAF3 gene, so that Sanger sequencing can be used to complete the detection of pathogenic mutations of the DNAAF3 gene, that is, Sanger sequencing can be used to determine whether the DNAAF3 gene has a pathogenic mutation, thereby shortening the detection cycle and reducing the detection cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0021] Figure 1 The band result diagrams of primer set 1 to primer set 3 of the present invention; Figure 2 The band result diagrams of primer set 4 to primer set 6 of the present invention; Figure 3 The band result diagram of primer set 7 to primer set 9 of the present invention; Figure 4 The band result diagram of primer set 10 to primer set 12 of the present invention; Figure 5 Graph showing the band results of primer set 13 to primer set 15 of the present invention; Figure 6 Graph showing the band results of primer set 16 to primer set 18 of the present invention; Figure 7 The band result diagram of primer set 19 to primer set 21 of the present invention; Figure 8 Graph showing the band results of primer set 22 to primer set 24 of the present invention; Fig. 9 Graph showing the band results of primer set 25 to primer set 27 of the present invention; Fig.10 The band result diagram of primer set 28 to primer set 30 of the present invention; Fig.11 This is a pedigree diagram of Application Example 1 of the present invention; Fig.12 Schematic diagram of the c.192G>A mutation site in the present invention; Fig.13 Schematic diagram of the c.842G>A mutation site in the present invention.

[0022] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0023] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] In addition, if there are descriptions involving "first", "second", etc. in the embodiments of the present invention, the descriptions of "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, if the meaning of "and / or" appearing in the full text is to include three parallel schemes, taking "A and / or B" as an example, it includes scheme A, or scheme B, or a scheme that satisfies both A and B. In addition, the technical solutions between the various embodiments can be combined with each other, but it must be based on the ability of ordinary technicians in this field to implement. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0025] Primary Ciliary Dyskinesia (PCD) is a serious genetic disorder that affects the function of cilia in the body. Cilia are tiny structures on the surface of cells that are found in many parts of the body, including the respiratory tract, reproductive system, and ears. They are responsible for pushing and clearing mucus and foreign matter from the outside of cells. PCD is caused by genetic mutations that cause defects in the structure or function of cilia. These genes usually encode components of cilia, such as the motor arm and other related proteins, which are essential for the normal movement of cilia. PCD can be caused by mutations in multiple genes, and at least 30 genes are currently known to be associated with this disease. The main symptoms of PCD include: chronic respiratory infections, such as bronchitis and pneumonia, because cilia cannot effectively clear mucus and bacteria from the respiratory tract; sinusitis and ear infections, which frequently occur due to the same reason, that is, impaired ciliary cleaning function; and fertility problems, such as sperm amotility in men and difficulty conceiving in women due to dysfunction of fallopian tube cilia. PCD has a significant impact on the patient's quality of life. Due to the complexity and severity of PCD, patients and families need to seek professional genetic counseling and long-term specialist care.

[0026] Kartagener syndrome is a special form of primary ciliary dyskinesia (PCD). In addition to the various symptoms of PCD, the most notable feature of Kartagener syndrome is situs inversus, which means that the heart and other major organs are located in mirror-image positions in the body. The syndrome is usually inherited in an autosomal recessive manner, meaning that the patient inherits a mutated gene from each parent.

[0027] The full name of the DNAAF3 gene is Dynein Axonemal Assembly Factor 3, which is located on chromosome 19 and contains 12 exons. The DNAAF3 protein it encodes is a vital protein whose main function is to participate in the assembly of cilia and maintain the normal movement function of cilia. DNAAF3 protein is an important component in cilia assembly, especially closely related to the movement function of cilia. In the ciliary structure, DNAAF3 is mainly involved in the correct assembly of the inner and outer power fiber arms of cilia. These power fiber arms are the driving force for ciliary movement, and defects can lead to ciliary motility disorders, such as primary ciliary dyskinesia (PCD).

[0028] In addition, mutations in the DNAAF3 gene are common in hereditary diseases that cause respiratory and reproductive system diseases. These mutations affect the normal function of cilia, causing a variety of clinical symptoms including Kartagener syndrome. Kartagener syndrome patients often show characteristics such as chronic respiratory infections and situs inversus. The mutated DNAAF3 gene affects the occurrence of these symptoms by affecting the structure and movement of cilia.

[0029] Currently, there is no primer set specifically targeting the pathogenic mutation of the DNAAF3 gene, and it can only be detected through whole exome sequencing (WES) or whole genome sequencing (WES) technology. Both methods are second-generation sequencing technologies, which have high requirements for detection technology and equipment, are expensive, and have a long cycle.

[0030] The inventors of the present invention identified two new pathogenic mutations of DNAAF3 gene by Trio-WES technology in clinical work. These two mutations were inherited from parents respectively. They are NM_178837.4: c.192G>A (p.Trp64*) and NM_178837.4: c.842G>A (p.Arg281Gln). There is no report of this mutation site in ESP database, 1000 database, EXAC database, GnomAD and other databases.

[0031] The alignment information of the c.192G>A and c.842G>A mutation sites of DNAAF3 gene with the human genome reference sequence GRCh37 are as follows:

[0032] 1.c.192G>A mutation site: The base G at the physical position 55677731 of chromosome 19 mutates to A, i.e. c.192G>A mutation, located in exon 2 of DNAAF3 gene. This mutation will cause the 64th codon TGG encoding Trp (tryptophan) in mRNA to change to the termination codon TGA, i.e. the amino acid changes to p.Trp64*. It directly leads to premature termination, which is a loss-of-function mutation.

[0033] 2.c.842G>A mutation site: The base G at the physical position 55672749 of chromosome 19 mutates to A, i.e., c.842G>A mutation, located in exon 7 of DNAAF3 gene. This mutation will cause the codon encoding Arg (arginine) at position 281 in mRNA to change to the codon encoding Gln (glutamic acid), i.e., the amino acid changes to p.Arg281Gln.

[0034] The wild-type DNAAF3 gene is coded as ENSG00000167646 in the Ensemble database (https: / / useast.ensembl.org / index.html). Currently, there is no detection method for the c.192G>A and c.842G>A mutation sites of the DNAAF3 gene. Genetic etiology detection is generally performed by whole exome sequencing (WES) or whole genome sequencing (WES) technology. Based on the advantages and disadvantages of the second-generation sequencing technology, the present invention hopes to determine whether the DNAAF3 gene has a pathogenic mutation by a simpler detection method, thereby shortening the detection cycle and reducing the detection cost.

[0035] Sanger sequencing, the full name of which is Sanger chain termination sequencing, is a DNA sequencing method invented by British scientist Frederick Sanger in 1977. The basic principle of Sanger sequencing is to use DNA polymerase and end-labeled ddNTPs (dideoxynucleotide triphosphates) to terminate the synthesis of the DNA chain. This method has long been the gold standard in genomics and molecular biology research due to its high accuracy and reliability. After years of development, Sanger sequencing has mature kits and automated equipment, which have the advantages of high accuracy, easy result reading and low cost.

[0036] In summary, the present invention hopes to propose a primer set for detecting pathogenic mutations in the DNAAF3 gene, which can determine whether the DNAAF3 gene has pathogenic mutations through Sanger sequencing, thereby shortening the detection cycle and reducing the detection cost.

[0037] Accordingly, in the present invention, the normal human DNAAF3 gene coding sequence is shown as SEQ ID NO.61; the normal human DNAAF3 protein amino acid sequence is shown as SEQ ID NO.62; the DNAAF3 gene coding sequence of the c.192G>A pathogenic mutation is shown as SEQ ID NO.63; the DNAAF3 protein amino acid sequence of the c.192G>A pathogenic mutation is shown as SEQ ID NO.64; the DNAAF3 gene coding sequence of the c.842G>A pathogenic mutation is shown as SEQ ID NO.65; the DNAAF3 protein amino acid sequence of the c.842G>A pathogenic mutation is shown as SEQ ID NO.66.

[0038] Specifically, the primer set for detecting pathogenic mutations in the DNAAF3 gene proposed in the present invention comprises a first primer set and a second primer set;

[0039] The first primer set includes a first upstream primer and a first downstream primer, the nucleotide sequence of the first upstream primer is shown in SEQ ID NO.9, and the nucleotide sequence of the first downstream primer is shown in SEQ ID NO.10; the second primer set includes a second upstream primer and a second downstream primer, the nucleotide sequence of the second upstream primer is shown in SEQ ID NO.37, and the nucleotide sequence of the second downstream primer is shown in SEQ ID NO.38. The first primer set is used to detect the c.192G>A mutation site of the DNAAF3 gene, and the second primer set is used to detect the c.842G>A mutation site of the DNAAF3 gene.

[0040] Among them, when performing Sanger sequencing, the volumes of each component of the PCR reaction system are as follows: 10×PCR Buffer: 1.0 μL; dNTP (2.5 mM): 0.8 μL; genomic DNA (100 ng / μL): 0.5 μL; upstream primer: 0.4 μL; downstream primer: 0.4 μL; Taq DNA polymerase: 0.1 μL; ddH2O: 6.8 μL; total volume: 10 μL.

[0041] The following is a description of the specific embodiments.

[0042] Example 1 Design of primer sets: The present invention designs 15 pairs of candidate primer sets for the c.192G>A and c.842G>A mutation sites of the DNAAF3 gene, respectively. The designed primer sets follow the design principles of primers.

[0043] It should be noted that the primers in the following table have different binding sites. To show the difference, the bases of the binding sites on the exons in the primers are represented by capital letters, and the bases of the binding sites on the introns are represented by lowercase letters. In addition, the primer sets numbered 1-15 are candidate primer sets for detecting the c.192G>A mutation site, and the primer sets numbered 16-30 are candidate primer sets for detecting the c.842G>A mutation site.

[0044] Table 1 Candidate primer set sequence list

[0045]

[0046]

[0047]

[0048] For the above 30 primer sets, each primer set was set up with a 5-tube PCR reaction system to verify the feasibility of the primers. The reaction settings of the 5-tube PCR reaction system are as follows:

[0049] Table 2 5-tube reaction system

[0050] Components Reaction 1 Reaction 2 Reaction 3 Reaction 4 Reaction 5 10×PCR Buffer 1.0 1.0 1.0 1.0 1.0 dNTP(2.5mM) 0.8 0.8 0.8 0.8 0.8 Genomic DNA (100 ng / μL) 0.5 0 0.5 0.5 0.5 Upstream primer 0 0.4 0.4 0 0.4 Downstream primer 0 0.4 0 0.4 0.4 Taq DNA polymerase 0.1 0.1 0.1 0.1 0.1 <![CDATA[ddH2O]]> 7.6 7.3 7.4 7.4 6.8 Total volume 10 10 10 10 10

[0051] Referring to the table above, the design idea of ​​the 5-tube PCR reaction system is as follows: Reaction 1 is a negative control with only template DNA and no primers; Reaction 2 is a negative control with only upstream and downstream primers and no template DNA; Reaction 3 is to test the upstream primer; Reaction 4 is to test the downstream primer; Reaction 5 is a complete PCR system test. In this way, the feasibility of each primer set is verified.

[0052] Among them, a thermal cycler is used in PCR amplification, and the settings of the thermal cycler are shown in the following table:

[0053] Table 3 PCR amplification setting table

[0054] Temperature(℃) time Number of cycles 94 3 minutes 1 40 30 seconds 32 Tm 30 seconds 32 72 1 minute 32 72 5 minutes 1 10 ∞ 1

[0055] In the above table, T m The value is set according to the T in each primer set in Table 1 m It should be noted that, if only the upstream primer or the downstream primer is used in reaction 3 and reaction 4, the T value of each primer is used. m For example, reaction 2 and reaction 5 have both upstream primers and downstream primers, then the T values ​​of the upstream primers and downstream primers in each primer set can be taken as m The mean of the values; it should be noted that the T m The values ​​are sometimes quite different, so you can choose T according to the actual situation. m The specific value of the value.

[0056] Reference Figures 1 to 10 From the table below, it can be found that the first primer set (primer set 5) and the second primer set (primer set 19) have better reaction results than other primer sets.

[0057] Table 4 Reaction results of each primer set

[0058]

[0059]

[0060]

[0061] The first primer set and the second primer set will be used for subsequent detection.

[0062] Application example 1: Reference Figures 11 to 13 , a non-consanguineous couple, pregnant 3 and gave birth 2 times. NT ultrasound showed fetal neck cystic hygroma and abnormal fetal heart sonographic changes. The stomach was located on the right side of the abdominal cavity, the fetal ductus venosus was absent, and complete situs inversus was not ruled out. Mid-pregnancy color Doppler ultrasound showed fetal heart sonographic changes, complete situs inversus and mirrored dextrocardia. Fetal heart sonographic changes were consistent with congenital heart disease: functional single atrium, complete septal defect, functional single ventricle, ectopic great arteries, and severe stenosis of the aortic arch. The pregnant woman had given birth to two children, the first of whom was a phenotypically normal girl. The second child had complete situs inversus combined with mirrored dextrocardia, anal atresia, and frequent pneumonia and respiratory infections. Heart surgery, anal surgery, and nasal surgery were performed. Two weeks later, the pregnant couple chose to terminate the pregnancy.

[0063] After extracting fetal abortion tissue and peripheral blood genomic DNA from pregnant women and their spouses, WES technology was used to find that the fetus had two mutations in the primary ciliary dyskinesia (PCD)-related gene DNAAF3: NM_178837.4: c.192G>A (p.Trp64*) and NM_178837.4: c.842G>A (p.Arg281Gln). PCR amplification was performed using the primer set of the present invention, and the amplified products were successfully verified by Sanger sequencing. After family verification, it was found that the husband of the pregnant woman was a heterozygous carrier of NM_178837.4: c.192G>A (p.Trp64*), and the pregnant woman was a heterozygous carrier of NM_178837.4: c.842G>A (p.Arg281Gln). Therefore, the fetus is a compound heterozygous case of two mutations.

[0064] We further used the primer set of the present invention to verify the two elder sisters of the proband and found that the first child carried the mother's NM_178837.4: c.842G>A (p.Arg281Gln) mutation, while the second child was compound heterozygous like the proband.

[0065] The present invention also proposes a kit for detecting pathogenic mutations in the DNAAF3 gene, which includes the above-mentioned primer set. The specific sequence and usage of the primer set refer to the above description and embodiments. Since the present kit adopts all the technical solutions of all the above-mentioned embodiments, it has at least all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, which will not be repeated here one by one.

[0066] The present invention also proposes the use of the above-mentioned kit in detecting pathogenic mutations of the DNAAF3 gene. The above-mentioned kit can be used as one of the means for rapid screening of pathogenic mutations of the DNAAF3 gene; and because the present invention uses Sanger sequencing, the experimental conditions are very low and can be used in conventional biological laboratories. The cost is very low and the detection cycle is short.

[0067] The above descriptions are only optional embodiments of the present invention, and are not intended to limit the patent scope of the present invention. All equivalent structural changes made using the contents of the present invention's specification and drawings, or directly / indirectly applied in other related technical fields, are included in the patent protection scope of the present invention.

Claims

1. A primer set for detecting pathogenic mutations in the DNAAF3 gene, characterized in that: comprising a first primer set and a second primer set; The first primer set includes a first upstream primer and a first downstream primer, the nucleotide sequence of the first upstream primer is shown in SEQ ID NO.9, and the nucleotide sequence of the first downstream primer is shown in SEQ ID NO.10; The second primer set includes a second upstream primer and a second downstream primer, the nucleotide sequence of the second upstream primer is shown in SEQ ID NO.37, and the nucleotide sequence of the second downstream primer is shown in SEQ ID NO.

38.

2. The primer set according to claim 1, characterized in that The first primer set is used to detect the c.192G>A mutation site of the DNAAF3 gene, and the second primer set is used to detect the c.842G>A mutation site of the DNAAF3 gene.

3. The primer set according to any one of claims 1 or 2, characterized in that The first primer set and the second primer set are used for Sanger sequencing.

4. The primer set according to claim 3, characterized in that The PCR reaction system of the Sanger sequencing includes the following components: 10×PCR Buffer; dNTPs (2.5 mM); Genomic DNA; Upstream primer; Downstream primer; Taq DNA polymerase; ddH2O.

5. The primer set according to claim 4, characterized in that The added concentration of the genomic DNA was 100 ng / μL.

6. The primer set according to claim 5, characterized in that The volumes of the components of the PCR reaction system for Sanger sequencing are as follows: 10×PCR Buffer: 1.0 μL; dNTP (2.5 mM): 0.8 μL; Genomic DNA (100 ng / μL): 0.5 μL; Upstream primer: 0.4 μL; Downstream primer: 0.4 μL; Taq DNA polymerase: 0.1 μL; ddH2O: 6.8 μL; Total volume: 10 μL.

7. The primer set according to claim 3, characterized in that The first primer set is used in PCR amplification m The values ​​ranged from 58.2°C to 58.8°C.

8. The primer set according to claim 3, characterized in that The second primer set is used in PCR amplification m The values ​​ranged from 58.9°C to 59.4°C.

9. A kit for detecting pathogenic mutations in the DNAAF3 gene, characterized in that: Comprising the primer set according to any one of claims 1 to 8.

10. Use of the kit according to claim 9 in detecting pathogenic mutations in the DNAAF3 gene.

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