CN114075598B - PCR detection kit for atrophic myotonic protein kinase gene CTG region and application thereof - Google Patents
PCR detection kit for atrophic myotonic protein kinase gene CTG region and application thereof Download PDFInfo
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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Abstract
The invention provides a PCR detection kit for a atrophic myotonic protein kinase gene CTG region, which comprises a reaction system, wherein the reaction system comprises: longAmp Taq DNA polymerase, 5x GC buffer, dNTPs, DNA sample, water, and a pair of specific primer pairs having nucleotide sequences as set forth in SEQ ID NOs: 1 and SEQ ID NO: 2. The method has the characteristics of strong amplification specificity and small amplification preference. The invention also provides an application of the PCR detection kit for the atrophic myotonic protein kinase gene CTG region. The PCR detection kit can amplify heterozygous allele samples carrying CTG repeated expansion, and can be used for detecting the CTG three-base repetition times of the 3' -UTR of the atrophic myotonic protein kinase gene.
Description
Technical Field
The invention belongs to the technical field of PCR amplification, and particularly relates to a PCR detection kit for a atrophic myotonic protein kinase gene CTG region and application thereof.
Background
Myotonic muscular dystrophy type 1 (Myotonic Dystrophy Type, dm 1) is a genetic disorder characterized by muscle weakness, myotonia, and muscular atrophy, with concomitant damage to other systems such as the endocrine, heart, ocular lens, and the like. DM1 is caused by three-base repeat expansion of CTG in the 3' -UTR region of DMPK gene located on chromosome 19q13.2-13.3, and is generally pathogenic only when the number of CTG repeats is greater than 50; the age of onset and the number of CTG repeats are inversely related, and the number of CTG repeats varies with the number of major complications and their severity. With the inheritance of the pathogenic gene, the number of CTG repeats in the DMPK gene of the next generation is generally larger than that of the previous generation, and the number of CTG repeats in the currently known DMPK gene varies from 5 times to 5000 times.
The determination of the number of times of CTG repetition in the DMPK gene of a patient is combined with clinical pathological manifestations, and helps doctors to know the relationship between genotype and pathological manifestations. Currently, the detection methods reported in the related art mainly include non-amplified Southern-blot and amplification-based PCR methods. Because DNA amplification is not involved, the Southern-blot requires a larger sample size, and in addition, the method is long in time consumption and high in cost, and is difficult to apply to clinical detection. The PCR amplification methods for determining the number of times of CTG repetition in the DMPK gene mainly comprise ordinary PCR, triple primer PCR (TP-PCR), thermal pulse PCR (HPE-PCR), small-pool-PCR (SP-PCR) and Flash-Small-pool PCR (FSP-PCR).
The method for measuring the CTG repetition number of the DMPK gene based on PCR amplification has the advantages of small sample consumption, short time consumption, controllable cost and the like compared with non-amplified southern-blot and three-generation sequencing. However, there is also a problem of preferential amplification, namely that in most cases, the number of CTG repeats in two alleles of DMPK of DM1 patient is one large and one small, while PCR technique is more prone to amplify small fragments, resulting in difficult detection of large fragments. The prior mature technology TP-PCR is stable and has good repeatability. However, it is only able to detect a clear value of the number of CTG repeats within 240 times, and it is not possible to accurately give the number of repeats for the case where the number of repeats is large (> 240 repeats) (for example, a set of primers for detecting CTG repeats and a detection kit therefor disclosed in chinese patent document 201410211499.0). In addition, some primers used in the TP-PCR method need fluorescent group modification, and the relative cost is high.
Whereas HPE-PCR, SP-PCR and FSP-PCR, although able to amplify DMPK gene fragments with larger CTG repeats, are time consuming longer than ordinary PCR and have poor stability. For example, HPE-PCR is not a constant temperature but a cyclic pulse wave of 70-83 ℃ in the extension stage of the PCR program, the setting of the program puts certain requirements on the instrument, and only the PCR instrument with nested cycles and the like can meet the requirements. The continuous and rapid heat pulse is more costly to the instrument and the procedure has a longer time between 70-83℃and places high demands on the thermal stability of the DNA polymerase. SP-PCR and FSP-PCR are complicated in operation and unsuitable for routine diagnosis.
Disclosure of Invention
Aiming at the defects of the existing DM1 gene diagnosis technology, the invention provides a PCR detection kit based on the atrophy myotonic protein kinase gene CTG region of the conventional PCR technology, the PCR detection kit has obvious advantages for detecting the repetition number of CTG in the DMPK gene, overcomes the defects of TP-PCR, can realize the amplification of samples with the repetition number of more than 240 times, and can calculate and give out the repetition number of CTG in the sample DMPK gene. Meanwhile, the method has the advantages of less sample quantity required by detection, relatively low detection cost, visual detection result, strong pertinence, high sensitivity, high accuracy and the like. For this purpose, the first object of the present invention is to provide a PCR detection kit for the CTG region of the atrophic myotonic protein kinase gene. The second object of the invention is to provide an application of the PCR detection kit for the CTG region of the atrophic myotonic protein kinase gene. The third object of the present invention is to provide a PCR detection method of the CTG region of the amyotrophic myotonic protein kinase gene for the purpose of non-disease diagnosis.
In order to achieve the above purpose, the invention adopts the following technical scheme:
as a first aspect of the present invention, a PCR detection kit for a CTG region of a amyotrophic myotonic protein kinase gene, comprising a reaction system comprising: longAmp Taq DNA polymerase, 5x GC buffer, dNTPs, DNA sample, water, and a pair of specific primer pairs, wherein,
the nucleotide sequences of the specific primer pairs are respectively shown in SEQ ID NO:1 and SEQ ID NO: 2.
According to the invention, the reaction system is 25. Mu.L, including 1. Mu.L of LongAmp Taq DNA polymerase, 5. Mu.L of 5 XGC buffer, 0.75. Mu.L of 10mM dNTPs, 2ng of DNA sample, 1. Mu.L of each specific primer pair of 10. Mu.M, and the balance of water.
According to the present invention, the PCR detection kit further comprises an amplification program: denaturation at 94℃for 30s; carrying out cycle, denaturation at 94 ℃ for 30s, annealing at 65 ℃ and extension for 1.5min, wherein the total cycle is 38; extending at 65 ℃ for 10min, and keeping the temperature unchanged at 4 ℃.
As a second object of the present invention, a PCR detection kit for a CTG region of a atrophic myotonic protein kinase gene is used for detecting the number of times of CTG three-base repeat of the CTG region of the atrophic myotonic protein kinase gene.
As a third object of the present invention, a method for amplifying a CTG region of a amyotrophic myotonic protein kinase gene, comprising the steps of:
firstly, taking DNA of a sample to be detected as a template, and carrying out PCR amplification to obtain a PCR amplification product;
secondly, observing an electrophoresis result of the amplified product, and if double bands exist in an electrophoresis chart, indicating that the PCR method can amplify a large fragment and a small fragment simultaneously;
wherein the reaction system comprises: longAmp Taq DNA polymerase, 5x GC buffer, dNTPs, DNA samples, water, and a pair of specific primer pairs having sequences shown in SEQ ID No.1 and SEQ ID No. 2.
According to the invention, the method further comprises a third step of calculating the repetition number of CTG in the sample to be tested.
According to the invention, the reaction system for PCR amplification is 25. Mu.L, including 1. Mu.L of LongAmp Taq DNA polymerase, 5. Mu.L of 5 xGC buffer, 0.75. Mu.L of 10mM dNTPs, 2ng of DNA sample, 1. Mu.L of 10. Mu.M specific primer pairs, and the balance of water.
According to the invention, the amplification procedure for PCR amplification is: denaturation at 94℃for 30s; carrying out cycle, denaturation at 94 ℃ for 30s, annealing at 65 ℃ and extension for 1.5min, wherein the total cycle is 38; extending at 65 ℃ for 10min, and keeping the temperature unchanged at 4 ℃.
The invention has the beneficial effects that: the method has the advantages of strong amplification specificity, small amplification preference, simple and convenient operation, lower cost and better application prospect.
Drawings
FIG. 1 is a flow chart of a method for detecting the number of CTG repeats of DMPK genes according to the present invention.
FIG. 2 is a comparison of amplification specificities of the different primers of example 1.
FIG. 3 shows the result of electrophoresis of the products under different amplification conditions in example 2. Wherein, lane M is a DNA marker; lanes S4, S5 and S6 are the results of PCR electrophoresis of three DNA samples, lanes S4 and S5 are homozygous allele samples, and lane S6 is heterozygous allele sample; lanes N are negative references.
FIG. 4 shows the forward sequencing result of the larger band of lane S6 of example 2.
FIG. 5 shows the reverse sequencing of the larger band in lane S6 of example 2.
FIG. 6 shows the amplification results of conventional PCR of example 4 and PCR performed under condition 6 of example 2.
FIG. 7 is a diagram showing the length of PCR products.
Detailed Description
The invention will be further illustrated with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally performed under conventional conditions, or conditions supplied by the manufacturer.
Example 1 design of specific primer pairs
(1) The main purpose of this example is to prefer a primer pair with higher specificity for CTG repeat region amplification of the 3' -UTR of the DMPK gene, and the DNA samples are homozygous allele samples selected without CTG expansion, and if the amplification result is a non-single band, the amplification specificity of the primer is not very good.
Two pairs of primers are preferred as shown in Table 1 by comparative analysis in the following literature. Then, PCR amplification was performed on 3 DNA samples using the two pairs of primers, respectively.
The document 1 is: novel Heat Pulse Extension-PCR Based Method for Detection of Large CTG-Repeat Expansions in Myotonic Dystrophy Type 1
Document 2 is: (CTG) n Expansion at DMPK locus seen only in muscle tissue A novel case
TABLE 1 primer names and sequence information
(2) The reaction system and the reaction procedure are shown in tables 2 and 3, respectively.
TABLE 2 reaction system
Remarks: the DNA samples S1, S2, S3 of this example are all: normal human blood DNA samples.
Table 3 reaction procedure
(3) The experimental results and the amplification results are shown in FIG. 2.
The results show that: when the primer pair 1 is used for PCR, the PCR results of all samples are single bands and negative reference is not provided with bands; when PCR was performed with primer set 2, the negative reference was free of bands, but the PCR result of sample S3 showed no single band. By comparing the PCR results of the two pairs of primers, it can be seen that the specificity of primer pair 1 is better than that of primer pair 2. Thus, subsequent experiments were performed using the primer pairs of example 1.
Example 2 no/Low preference experimental condition screening
The main objective of this example was to achieve a non/low bias amplification of the CTG repeat region of the 3' -UTR of the DMPK allele by optimizing the different experimental conditions using the specific primer pair 1 described above. The DNA samples included 2 homozygous allele samples without CTG expansion, 1 heterozygous allele sample carrying CTG repeat expansion. The expected PCR result was that 2 homozygous allele samples without CTG expansion were single bands, and 1 heterozygous allele sample carrying CTG repeat expansion were two bands of different sizes.
The DNA samples S4, S5, S6 of this example are respectively: s4 and S5 are normal human blood DNA samples; s6 is a blood DNA sample of the DM1 patient.
1. Experimental conditions
(1) The reaction system and the reaction conditions of condition 1 are shown in tables 4 and 5, respectively.
TABLE 4 reaction system
| Reagent(s) | Dosage of |
| I-5 TM 2X High-Fidelity Master Mix(TP001) | 12.5μL |
| DNA sample | 2ng |
| 10 mu M forward primer | 1μL |
| 10 mu M reverse primer | 1μL |
| Water and its preparation method | Supplement to 25 mu L |
Table 5 reaction procedure
(2) The reaction system and the reaction conditions of condition 2 are shown in tables 6 and 7, respectively.
TABLE 6 reaction system
| Reagent(s) | Dosage of |
| I-5 TM 2X High-Fidelity Master Mix(TP001) | 12.5μL |
| DNA sample | 2ng |
| 10 mu M forward primer | 1μL |
| 10 mu M reverse primer | 1μL |
| Water and its preparation method | Supplement to 25 mu L |
Table 7 reaction procedure
(3) The reaction system and reaction conditions of condition 3 are shown in tables 8 and 9, respectively.
TABLE 8 reaction system
Table 9 reaction procedure
(4) The reaction system and reaction conditions of condition 4 are shown in tables 10 and 11, respectively.
TABLE 10 reaction system
| Reagent(s) | Dosage of |
| Phusion U | 1μL |
| 5x buffer | 5μL |
| 10mM dNTPs | 0.75μL |
| DNA sample | 2ng |
| 10 mu M forward primer | 1μL |
| 10 mu M reverse primer | 1μL |
| Water and its preparation method | Supplement to 25 mu L |
Table 11 reaction procedure
(5) The reaction system and the reaction conditions of condition 5 are shown in tables 12 and 13, respectively.
TABLE 12 reaction system
TABLE 13 reaction procedure
(6) The reaction system and the reaction conditions of condition 6 are shown in tables 14 and 15, respectively.
TABLE 14 reaction system
| Reagent(s) | Dosage of |
| LongAmp Taq DNA polymerase | 1μL |
| 5x GC buffer | 5μL |
| 10mM dNTPs | 0.75μL |
| DNA sample | 2ng |
| 10 mu M forward primer | 1μL |
| 10 mu M forward primer | 1μL |
| Water and its preparation method | Supplement to 25 mu L |
Table 15 reaction procedure
(7) The reaction system of conditions 1 to 6 is shown in the summary table 16.
TABLE 16 reaction system
2. Test results.
The results of electrophoresis under different amplification conditions are shown in FIG. 3.
The results show that most conditions are not fully expected; only condition 6 was met, 2 homozygous allele samples (conditions 6:S4 and S5) were single bands, 1 heterozygous allele sample (condition 6:S6) was one large and 2 small bands, and the negative reference (condition 6:N) was no band.
Example 3 first generation sequencing validation of the amplification results of condition 6
To further confirm that the larger band in S6 amplified in condition 6 was the target band, it was subjected to a first generation sequencing, and the results are shown in FIGS. 4 and 5.
FIG. 4 shows that the CTG repeats are present in the forward sequencing results, while the 5' specific sequences are also identical; FIG. 5 shows that the result of reverse sequencing has a CAG (reverse complement of CTG) repeat sequence, while the 5' specific sequence is also identical; analysis showed that the larger fragment in lane S6 of condition 6 was the band of interest.
Example 4 example verification of amplification preference
The first object of this example was to conduct PCR amplification using the reaction system and amplification procedure of condition 6 of example 2, and to observe the amplification preference of this PCR amplification method for detecting the CTG region of the atrophic myotonic protein kinase gene.
The second object of this example is to directly compare the amplification preference of the CTG region of the amyotrophic myotonic protein kinase gene by the conventional method and the PCR amplification method of condition 6 of example 2 of the present invention by selecting the same sample.
(1) Sample: 3 heterozygous allele samples (samples 1/2/3) carrying CTG repeat expansions were all blood DNA samples from DM1 patients.
(2) The results of the amplification using the conventional PCR method on each of the 3 samples are shown in FIG. 6.
(3) The results of the amplification by the PCR amplification method of condition 6 of example 2 are shown in FIG. 6 for 3 samples, respectively.
(4) The preferred amplification condition 6 of example 2 was applied to the measurement of the number of CTG repeats in the 3' utr region of DMPK gene, 3 heterozygous allele samples carrying CTG repeat expansion were amplified according to condition 6, and the fragment size L1 was calculated based on the migration distance of the larger fragment, and the number of CTG repeats was further calculated (as shown in fig. 7: number of repeats n=l3/3, l3=l1- [ l2+l4], l2+l4=327), and the result was shown in fig. 6.
The results show that (1) for sample 1, the traditional PCR method has preference, the amplification efficiency of large fragments is extremely low, and the agarose gel electrophoresis result shows that only one small fragment exists; the method of the present invention has small preference, and the agarose gel electrophoresis result can show large fragment and small fragment, and the large fragment repetition number of sample 1 can be detected to be 176 times by the measurement of CTG repetition number.
(2) Similarly, for sample 2, the traditional PCR method can only give the size of a small fragment; the method has small preference, the agarose gel electrophoresis result can display large fragments and small fragments, and the large fragment repetition number can be detected to be 292 times by measuring the CTG repetition number; for sample 3, the conventional PCR method can only give the size of a small fragment, and the method has small preference, the agarose gel electrophoresis result can show the large fragment and the small fragment, and the large fragment repetition number can be detected to be 425 times by measuring the CTG repetition number.
Conclusion: the PCR amplification was performed according to the specific primer pairs of condition 6 and example 1 of example 2, and the amplification method has the characteristics of small amplification preference, simple operation, and low cost, and can give the number of repetitions.
In conclusion, the reaction system and the amplification conditions designed by the invention are used for PCR detection, and can be used for simultaneously detecting the large fragment and the small fragment of the CTG region of the atrophic myotonic protein kinase gene, and the amplification preference, the detection cost are relatively low, the detection result is visual, the pertinence is strong, the sensitivity is high, and the accuracy is high. Therefore, the PCR amplification method has good application prospect in the detection of the CTG three-base repetition number in the atrophic myotonic protein kinase gene (DMPK) of the myotonic muscular dystrophy type 1 (DM 1). The specific primer pairs and reaction conditions may be further formulated into PCR detection kits using methods known in the art, as will be apparent to those skilled in the art.
The foregoing is merely illustrative of embodiments of this invention and it will be appreciated by those skilled in the art that changes and modifications may be made without departing from the principles of the invention, which is also intended to be within the scope of the invention.
Sequence listing
<110> university of double denier affiliated Huashan Hospital
SHANGHAI ANGPU BIOTECHNOLOGY Co.,Ltd.
<120> PCR detection kit for atrophic myotonic protein kinase gene CTG region and application thereof
<130> 201090
<141> 2020-08-12
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
gccagttcac aaccgctccg agcgtgggtc 30
<210> 2
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
acgctcccca gagcagggcg tcatgc 26
<210> 3
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
ggtcggggtc tcagtgcatc ca 22
<210> 4
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
cacagggctg aagtggcagt tcca 24
Claims (6)
1. The PCR detection kit for the CTG region of the atrophic myotonic protein kinase gene is characterized by comprising a reaction system, wherein the reaction system is as follows: longAmp Taq DNA polymerase, 5 xgc buffer, dNTPs, DNA sample, water, and a pair of specific primer pairs, wherein,
the nucleotide sequences of the specific primer pairs are respectively shown in SEQ ID NO:1 and SEQ ID NO: 2.
2. The PCR detection kit for the CTG region of the amyotrophic myotonic protein kinase gene according to claim 1, wherein the reaction system is 25 μl, and comprises a LongAmp Taq DNA polymerase 1 μl, a 5 xgc buffer 5 μl,10mm dNTPs 0.75 μl, a DNA sample 2ng, a specific primer pair of 10 μΜ each 1 μl, and the balance water.
3. A method for amplifying a CTG region of a myotonic protein kinase gene for non-disease diagnosis, comprising the steps of:
firstly, taking DNA of a sample to be detected as a template, and carrying out PCR amplification to obtain a PCR amplification product;
secondly, observing whether double bands exist in the electrophoresis result of the amplified product;
wherein, the reaction system is: longAmp Taq DNA polymerase, 5 xgc buffer, dNTPs, DNA samples, water, and a pair of specific primer pairs having sequences shown in SEQ ID No.1 and SEQ ID No. 2.
4. The method for amplifying the CTG region of the amyotrophic myotonic protein kinase gene according to claim 3, further comprising a third step of calculating the number of repetitions of CTG in the sample to be tested.
5. The method for amplifying the CTG region of the atrophic myotonic protein kinase gene according to claim 3, wherein the PCR amplification reaction system is 25 [ mu ] L, and comprises 1 [ mu ] L of LongAmp Taq DNA polymerase, 5 [ mu ] L of 5 XGC buffer, 0.75 [ mu ] L of 10mM dNTPs, 2ng of DNA samples, 1 [ mu ] L of each specific primer pair of 10 [ mu ] M, and the balance of water.
6. The method for amplifying the CTG region of the atrophic myotonic protein kinase gene according to claim 3, wherein the PCR amplification is performed by: denaturation at 94℃for 30s; carrying out cycle, denaturation at 94 ℃ for 30s, annealing at 65 ℃ and extension for 1.5min, wherein the total cycle is 38; extending at 65 ℃ for 10min, and keeping the temperature unchanged at 4 ℃.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103167883A (en) * | 2010-07-19 | 2013-06-19 | F·C·贝内特 | Modulation of dystrophia myotonica-protein kinase (dmpk) expression |
| CN103966332A (en) * | 2014-05-19 | 2014-08-06 | 复旦大学 | Primers for detecting CTG repetitive sequence and detection kit of primers |
| CN105164278A (en) * | 2013-04-18 | 2015-12-16 | 三星生命公众福利基金 | Method for diagnosing myotonic dystrophy type 1 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012031021A2 (en) * | 2010-09-01 | 2012-03-08 | Orion Genomics Llc | Improved detection of nucleic acid sequences adjacent to repeated sequences |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103167883A (en) * | 2010-07-19 | 2013-06-19 | F·C·贝内特 | Modulation of dystrophia myotonica-protein kinase (dmpk) expression |
| CN105164278A (en) * | 2013-04-18 | 2015-12-16 | 三星生命公众福利基金 | Method for diagnosing myotonic dystrophy type 1 |
| CN103966332A (en) * | 2014-05-19 | 2014-08-06 | 复旦大学 | Primers for detecting CTG repetitive sequence and detection kit of primers |
Non-Patent Citations (3)
| Title |
|---|
| Analysis of CTG repeat length variation in the DMPK gene in the general population and the molecular diagnosis of myotonic dystrophy type 1 in Malaysia;Kathlin K Ambrose 等;《BMJ Open . 》;第7卷(第3期);e010711 * |
| DM1发病机制及分子诊断方法研究进展;李娜 等;陇东学院学报;27(03);69-73 * |
| Robust and accurate detection and sizing of repeats within the DMPK gene using a novel TP-PCR test;Maike Leferink 等;SCIENTIFIC REPORTS;第9卷;8280 * |
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