CN114480593A - Detection kit for detecting CGG amplification of 5' -UTR region of GIPC1 gene based on high GC PCR (polymerase chain reaction) - Google Patents
Detection kit for detecting CGG amplification of 5' -UTR region of GIPC1 gene based on high GC PCR (polymerase chain reaction) Download PDFInfo
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Abstract
The invention provides a PCR detection kit for detecting CGG amplification of 5' -UTR region of GIPC1 gene based on high GC PCR, wherein a reaction system comprises betaine, dimethyl sulfoxide, dNTP, 5 XLongAmp Taq reaction liquid, a specific primer pair, Taq DNA polymerase, a DNA template and water; wherein, the nucleotide sequences of the specific primer pair are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The PCR detection kit is used for detection, has the advantages of strong pertinence, simplicity and convenience in operation, lower cost and high accuracy, can effectively detect CGG amplification times of a 5' -UTR region of the GIPC1 gene, and has a good application prospect.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a PCR detection kit for detecting CGG amplification of 5' -UTR region of OPDM pathogenic mutation GIPC1 gene based on high GC PCR technology and application thereof.
Background
Oculopharyngeal type of distal myopathy (OPDM) is a genetic disease characterized by adult onset, ptosis, external ophthalmoplegia, facial muscle weakness, weakness and atrophy of the distal muscles of the extremities and pharyngeal involvement, leading to dysphagia and dysarthria. Skeletal muscle biopsies showed myopathy changes with increased vacuoles.
GIPC1 is a newly discovered pathogenic gene of OPDM, and because the GC content of a target region is more than 90%, the conventional PCR technology is difficult to amplify and cannot effectively detect.
The conventional PCR technology has certain requirements on the GC content of a target sequence, and is usually only suitable for samples with the GC content of 40-60 percent. In organisms, DNA sequences with high GC content (more than 70%) widely exist, and the sequences are easy to form complex secondary structures, prevent DNA denaturation, and make DNA polymerase difficult to combine and difficult to amplify. The high-GC PCR technology is a method for amplifying a target sequence with high GC content by using a PCR enhancer or special amplification conditions aiming at the problem.
Disclosure of Invention
The invention constructs a PCR detection kit for detecting CGG amplification of 5 ' -UTR region of GIPC1 gene based on high GC PCR aiming at CGG amplification of newly discovered OPDM pathogenic mutant GIPC1 gene 5 ' -UTR region, and the PCR detection kit has obvious advantages for detecting CGG amplification of 5 ' -UTR region of GIPC1 gene and has better adaptability to samples with higher repetition number. Therefore, it is a first object of the present invention to provide a PCR detection kit for detecting CGG amplification in the 5' -UTR region of GIPC1 gene based on high GC PCR. The second purpose of the invention is to provide an application of a PCR detection kit for detecting CGG amplification of 5' -UTR region of GIPC1 gene based on high GC PCR. It is a third object of the present invention to provide a method for detecting CGG amplification of the 5' -UTR region of the GIPC1 gene based on high GC PCR for non-disease diagnosis purposes.
In order to achieve the purpose, the invention adopts the following technical scheme:
as a first aspect of the present invention, a PCR detection kit for detecting CGG amplification of 5' -UTR region of GIPC1 gene based on high GC PCR comprises a reaction system, wherein the reaction system comprises betaine, dimethyl sulfoxide, dNTP, 5 × LongAmp Taq reaction solution, a specific primer pair, Taq DNA polymerase, a DNA template and water, wherein the nucleotide sequences of the specific primer pair are as set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
According to the invention, the reaction system is 25 mu L, and comprises 7 mu L of 5M betaine, 1.25 mu L of 100% dimethyl sulfoxide, 2 mu L of 2.5mM dNTP, 5 mu L of 5 XLongAmp Taq reaction solution, 1 mu L of specific primer pair, 1 mu L of Taq DNA polymerase 1 mu L, DNA template and the balance of water.
According to the invention, the PCR detection kit further comprises PCR reaction conditions: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 64 ℃ for 30s, extension at 68 ℃ for 1min, and total amplification for 35 cycles; extension at 68 ℃ for 10 min.
As a second aspect of the present invention, use of a PCR detection kit for detecting CGG amplification in the 5 '-UTR region of the GIPC1 gene based on high GC PCR for detecting the number of CGG amplifications in the 5' -UTR region of the OPDM causative mutation GIPC1 gene.
As a third aspect of the present invention, a method for detecting CGG amplification of the 5' -UTR region of the GIPC1 gene based on high GC PCR for non-disease diagnosis purposes, comprising the steps of:
step one, taking genome DNA of a sample to be detected as a template, and carrying out high GC PCR amplification to obtain an amplification product;
step two, carrying out electrophoresis on the amplification product to obtain CGG repeated information;
wherein the reaction system for amplification comprises: betaine, dimethyl sulfoxide, dNTP, 5 XLongAmp Taq reaction liquid, a specific primer pair, Taq DNA polymerase, a DNA template and water;
the nucleotide sequences of the specific primer pairs are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
According to the invention, the reaction system for PCR amplification is 25 μ L, comprising 5M betaine 7 μ L, 100% dimethyl sulfoxide 1.25 μ L, 2.5mM dNTP 2 μ L, 5 XLongAmp Taq reaction solution 5 μ L, specific primer pair 1 μ L each, Taq DNA polymerase 1 μ L, DNA template 1 μ L, and water in balance.
According to the present invention, the reaction conditions for PCR amplification are: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 64 ℃ for 30s, extension at 68 ℃ for 1min, and total amplification for 35 cycles; extension at 68 ℃ for 10 min.
According to the present invention, the electrophoresis method is at least one selected from agarose gel electrophoresis, capillary electrophoresis, fluorescence-labeled capillary electrophoresis, and the like.
The invention has the beneficial effects that: the PCR detection kit and the detection method have the advantages of strong pertinence, simple and convenient operation, lower cost and high accuracy, can effectively detect the CGG amplification times of the 5' -UTR region of the GIPC1 gene, can be used for medical auxiliary diagnosis, pre-pregnancy guidance, medical research and the like, and have better application prospects.
Drawings
FIG. 1 is a schematic flow chart of a method for detecting CGG amplification in the 5' -UTR region of GIPC1 gene.
Detailed Description
The present invention will be further described with reference to the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not specified, in the following examples are generally conducted under conventional conditions, or under conditions provided by the manufacturers.
Both DNA Polymerase and DNase-Free water in the examples of the present invention are commercially available.
In the embodiment of the invention, the length of the amplification product is detected by performing capillary electrophoresis on the amplification product, and the CGG amplification times of the 5' -UTR region of the GIPC1 gene are obtained by calculating the length of the expected fragment designed by the primer.
In the embodiment of the present invention, the source of the sample is not particularly limited. The genomic DNA sample is isolated from a human sample, and preferably, the genomic DNA nucleic acid sample is isolated from an anticoagulated blood sample of a healthy person or a patient, or tissue blood or the like which is separated from a human body.
In the embodiment of the present invention, the extracted genomic DNA is used as a template for amplification, wherein the polymerase used includes, but is not limited to, conventional taq enzyme, pfu high fidelity enzyme or other polymerase, preferably taq DNA polymerase.
In the embodiment of the present invention, the amplification product of step 2 is further purified and recovered by methods including, but not limited to, agarose gel recovery, magnetic bead purification and purification column purification.
In the embodiment of the invention, the detection is performed by a capillary electrophoresis apparatus or a Sanger sequencer, and the platform ABI3500 and ABI3730 are preferred.
Example 1 design of specific primer pairs
(1) The first primer pair and the second primer pair of this example are both primer pairs designed for CGG amplification sites in the 5' -UTR region of the GIPC1 gene, and can effectively detect the amplification region, and the PCR product can be detected using a capillary electrophoresis apparatus, a Sanger sequencer, or the like. The primer pair sequences are shown in Table 1.
TABLE 1 primer sequence information
(2) The PCR amplification system is shown in Table 2.
TABLE 2 PCR amplification System for step 2
| Composition (I) | Volume (μ L) |
| 5M betaine | 7 |
| 100% DMSO (dimethyl sulfoxide) | 1.25 |
| 2.5mM dNTP (deoxyribonucleoside triphosphate) | 2 |
| 5 XLongAmp Taq reaction solution | 5 |
| Primer1(FAM Label) (Forward Primer) | 1 |
| Primer2 (reverse Primer) | 1 |
| Taq DNA Polymerase (Taq DNA Polymerase) | 1 |
| DNA template | 1 |
| DNase-Free water | 5.75 |
(3) The high GC PCR technology is combined with electrophoresis to detect CGG amplification of 5' -UTR region of GIPC1 gene, and the specific operation steps are as follows:
step 1, extracting genomic DNA from blood by using a blood DNA extraction kit (Axygen) according to the instructions.
And 2, respectively amplifying the samples by using the first primer pair and the second primer pair. The PCR amplification system of step 2 is shown in Table 2; the reaction conditions of the PCR of the step 2 are as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 64 ℃ for 30s, extension at 68 ℃ for 1min, and total amplification for 35 cycles; final extension at 68 ℃ for 10 min. Thus, a PCR product was obtained.
Step 3, performing capillary electrophoresis by using an ABI3500 Sanger sequencer and performing capillary electrophoresis by using the ABI3500 Sanger sequencer, wherein the results show that the sequence of SEQ ID NO: 1 and SEQ ID NO: 2, and the primer pair is successfully amplified, and the sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4 primer pairs failed amplification altogether. Samples successfully amplified were analyzed by GeneMarker software to obtain the length of PCR product fragments.
And 4, calculating the length of the segment to obtain the repetition times. Specific results are shown in table 3.
TABLE 3 allele number of repeats
Remarking: p7, P8, P9, P10, P11, P12, P13, P14 and P15 are anticoagulated blood of the sample of the non-eupharyngic type far-end myopathy; p95, P130 is the anticoagulant of the specimen with oculopharyngeal type distal myopathy. At present, amplification times of 40 or more are generally diseased.
And (4) conclusion: by amplifying the gene and the first primer pair according to the embodiment, the CGG amplification state of the 5' -UTR region of the GIPC1 gene in a sample, including a sample with increased repetition times due to abnormal amplification, can be effectively detected.
Example 2
The method adopts a high GC PCR technology and electrophoresis combined to detect CGG amplification of a 5' -UTR region of a GIPC1 gene, and comprises the following specific operation steps:
step 1, extracting genomic DNA from blood by using a blood DNA extraction kit (Axygen) according to the instructions.
And 2, respectively amplifying the samples by using the first primer pair and the second primer pair. The PCR amplification system in step 2 is shown in Table 4.
TABLE 4 PCR amplification System of step 2
The reaction conditions of the PCR of the step 2 are as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 64 ℃ for 30s, extension at 68 ℃ for 1min, and total amplification for 35 cycles; final extension at 68 ℃ for 10 min. Thus, a PCR product was obtained.
And 3, performing capillary electrophoresis by using an ABI3500 Sanger sequencer and performing capillary electrophoresis by using the ABI3500 Sanger sequencer, wherein the result shows that the first primer pair and the second primer pair fail to be amplified completely.
The results of example 1 and example 2 show that CGG amplification of the 5' -UTR region of the GIPC1 gene was efficiently performed by using the first primer set of example 1 and the reaction system shown in Table 2. Therefore, subsequent experiments were performed using the first primer set and the PCR reaction system of example 1.
Example 3 example verification
In this example, capillary electrophoresis was compared with a first generation sequencing protocol, and the specific procedures were as follows:
protocol one, example validation using the first primer set and capillary electrophoresis method of example 1
Step 1, extracting genomic DNA from blood by using a blood DNA extraction kit (Axygen) according to the instructions.
Step 2, using the nucleotide sequence shown as SEQ ID NO: 1 and SEQ ID NO: 2, amplifying the sample by the first primer pair shown in the figure. The PCR amplification system of step 2 is shown in Table 5.
TABLE 5 PCR amplification System of step 2
The reaction conditions of the PCR of the step 2 are as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 64 ℃ for 30s, extension at 68 ℃ for 1min, and total amplification for 35 cycles; final extension at 68 ℃ for 10 min. Thus, a PCR product was obtained.
And 3, performing capillary electrophoresis by using an ABI3500 Sanger sequencer to obtain the length of the PCR product fragment.
And 4, calculating the length of the segment to obtain the repetition times. Specific results are shown in table 6.
TABLE 6 allele number of repeats
| Sample(s) | Whether or not the disease is affected | Number of allele 1 repeats | Allele 2 number of repeats |
| P8 | Whether or not | 12 | 12 |
| P13 | Whether or not | 11 | 11 |
| P15 | Whether or not | 11 | 11 |
| P58 | Whether or not | 12 | 12 |
| P79 | Whether or not | 12 | 12 |
| P100 | Whether or not | 11 | 11 |
| P106 | Whether or not | 10 | 10 |
| P109 | Whether or not | 11 | 11 |
| P113 | Whether or not | 11 | 11 |
| P95 | Is that | 11 | 78 |
| P130 | Is that | 10 | 81 |
Remarking: p8, P13, P15, P58, P79, P100, P106, P109 and P113 are anticoagulated blood of a sample which is not suffered from the oculopharyngeal type distal myopathy; p95, P130 is anticoagulant of the sample with eye pharyngeal type far-end myopathy.
And (4) conclusion: the first primer set of example 1 in combination with the reaction system of Table 2 enables efficient detection of CGG amplification of the 5' -UTR region of GIPC1 gene of a sample using a method of capillary electrophoresis.
Scheme two, first generation sequencing method verification
Step 1, extracting genomic DNA from blood by using a blood DNA extraction kit (Axygen) according to the instructions.
Step 2, using the nucleotide sequence shown as SEQ ID NO: 1 and SEQ ID NO: 2, amplifying the sample by the first primer pair shown in the figure. The PCR amplification system of step 2 is shown in Table 7.
TABLE 7 PCR amplification System of step 2
| Composition (I) | Volume (μ L) |
| 5M betaine | 7 |
| 100％DMSO | 1.25 |
| 2.5mM dNTP | 2 |
| 5×LongAmp Taq Reaction Buffer | 5 |
| Primer1(SEQ ID NO：1) | 1 |
| Primer2(SEQ ID NO：2) | 1 |
| Taq DNA Polymerase | 1 |
| DNA Template | 1 |
| DNase-Free water | 5.75 |
The reaction conditions of the PCR of the step 2 are as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 64 ℃ for 30s, extension at 68 ℃ for 1min, and total amplification for 35 cycles; final extension at 68 ℃ for 10 min. Thus, a PCR product was obtained.
Step 3, recycling PCR product by AxyPrepTMThe PCR Clean-up Kit (Axygen) Kit was recovered. The procedure is shown in Table 8 below.
TABLE 8 recovery of PCR products
And 4, performing Sanger sequencing by using an ABI3500 Sanger sequencer to obtain a PCR product sequence, and confirming the repetition times. Specific results are shown in table 9.
TABLE 9 allele number of repeats
| Sample(s) | Whether or not the disease is affected | Number of allele 1 repeats | Allele 2 number of repeats |
| P8 | Whether or not | 12 | 12 |
| P13 | Whether or not | 11 | 11 |
| P15 | Whether or not | 11 | 11 |
| P58 | Whether or not | 12 | 12 |
| P79 | Whether or not | 12 | 12 |
| P100 | Whether or not | 11 | 11 |
| P106 | Whether or not | 10 | 10 |
| P109 | Whether or not | 11 | 11 |
| P113 | Whether or not | 11 | 11 |
| P95 | Is that | -- | -- |
| P130 | Is that | -- | -- |
Among them, Sanger sequencing cannot be achieved due to the large number of repetitions of P95 and P130.
Taken together, the results obtained using the high GC PCR technique are in full agreement with those obtained by Sanger sequencing. But the technique of scheme one has better adaptability to samples with higher repetition times. The primer pair can be further prepared into a detection kit by a method known in the art, which is obvious to those skilled in the art.
The foregoing is merely an example of the embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
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Claims (8)
1. A PCR detection kit for detecting CGG amplification of 5' -UTR region of GIPC1 gene based on high GC PCR is characterized by comprising a reaction system, wherein the reaction system comprises betaine, dimethyl sulfoxide, dNTP, 5 XLongAmp Taq reaction liquid, a specific primer pair, Taq DNA polymerase, a DNA template and water; wherein, the nucleotide sequences of the specific primer pair are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
2. The PCR detection kit of claim 1, wherein the reaction system is 25 μ L, and comprises 7 μ L of 5M betaine, 1.25 μ L of 100% dimethyl sulfoxide, 2 μ L of 2.5mM dNTP, 5 μ L of 5 XLongAmp Taq reaction solution, 1 μ L of each specific primer pair, 1 μ L of Taq DNA polymerase 1 μ L, DNA template, and the balance water.
3. The PCR assay kit of claim 1, further comprising reaction conditions for PCR amplification, the reaction conditions being: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 64 ℃ for 30s, extension at 68 ℃ for 1min, and total amplification for 35 cycles; extension at 68 ℃ for 10 min.
4. The application of the PCR detection kit for detecting CGG amplification of the 5 '-UTR region of the GIPC1 gene based on high GC PCR is characterized in that the PCR detection kit is used for detecting the CGG amplification times of the 5' -UTR region of the OPDM pathogenic mutation GIPC1 gene.
5. A method for detecting CGG amplification in the 5' -UTR region of the GIPC1 gene based on high GC PCR for non-disease diagnosis purposes, which is characterized by comprising the following steps:
step one, taking genome DNA of a sample to be detected as a template, and carrying out high GC PCR amplification to obtain an amplification product;
step two, carrying out electrophoresis on the amplification product to obtain CGG repeated information;
wherein the reaction system for amplification comprises: betaine, dimethyl sulfoxide, dNTP, 5 XLongAmp Taq reaction liquid, a specific primer pair, Taq DNA polymerase, a DNA template and water;
the nucleotide sequences of the specific primer pairs are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
6. The method of claim 5, wherein the reaction system for PCR amplification is 25 μ L, comprising 5M betaine 7 μ L, 100% dimethyl sulfoxide 1.25 μ L, 2 μ L of 2.5mM dNTP 2 μ L, 5 μ L of 5 XLongAmp Taq reaction solution, 1 μ L of each specific primer pair, 1 μ L of Taq DNA polymerase 1 μ L, DNA template, and the balance water.
7. The method of claim 5, wherein the PCR amplification is performed under the following reaction conditions: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 64 ℃ for 30s, extension at 68 ℃ for 1min, and total amplification for 35 cycles; extension at 68 ℃ for 10 min.
8. The method of claim 5, wherein the electrophoresis method is at least one selected from agarose gel electrophoresis, capillary electrophoresis, fluorescence-labeled capillary electrophoresis, and the like.
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| CN107034211A (en) * | 2017-05-09 | 2017-08-11 | 广州海力特生物科技有限公司 | A kind of PCR reaction systems and kit for high GC fragment amplifications |
| CN110577987A (en) * | 2019-06-24 | 2019-12-17 | 胜亚生物科技(厦门)有限公司 | Detection method of CGG (glutamic acid G) repetitive sequence of FMR1 gene and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107034211A (en) * | 2017-05-09 | 2017-08-11 | 广州海力特生物科技有限公司 | A kind of PCR reaction systems and kit for high GC fragment amplifications |
| CN110577987A (en) * | 2019-06-24 | 2019-12-17 | 胜亚生物科技(厦门)有限公司 | Detection method of CGG (glutamic acid G) repetitive sequence of FMR1 gene and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| JIANWEN DENG等: "Expansion of GGC Repeat in GIPC1 Is Associated with Oculopharyngodistal Myopathy", AM J HUM GENET, vol. 106, no. 6, pages 796 - 800 * |
| 朱平: "PCR基因扩增实验操作手册", 31 January 1992, 北京：中国科学技术出版社, pages: 92 - 96 * |
| 王健: "第三届中国畜牧科技论坛论文集", 31 October 2007, 北京：中国农业出版社, pages: 627 - 629 * |
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