Primer group, MYH3 gene promoter region DNA methylation level analysis method and application

Abstract

The invention relates to the field of molecular biology, in particular to a primer group, an MYH3 gene promoter region DNA methylation level analysis method and application. The primer group comprises a first primer pair and a second primer pair, the upstream sequence of the first primer pair is as shown in SEQ ID NO. 1; the downstream sequence of the first primer pair is as shown in SEQ ID NO. 2; the upstream sequence of the second primer pair is as shown in SEQ ID NO.3; and the downstream sequence of the second primer pair is as shown in SEQ ID NO.4. The method for analyzing the methylation level of the DNA in the MYH3 gene promoter region relates to PCR reaction conditions for obtaining qualified target DNA fragments for MSP sequencing, the primer group and the PCR reaction conditions are combined to have the advantage of high amplification efficiency, the qualified DNA fragments are obtained through amplification, the DNA fragments are provided for analyzing the methylation level of the DNA in the MYH3 gene promoter region, and the methylation level analysis efficiency and accuracy are improved.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and in particular to a primer set and a method and application for analyzing DNA methylation levels in the promoter region of a MYH3 gene. Background Art

[0002] Promoter DNA methylation analysis is a method for analyzing the degree of gene DNA methylation, also known as bisulfite-modified sequencing. The basic process is: genomic DNA extraction → bisulfite conversion → PCR amplification of the target fragment using methylated primers → ligation, conversion, and sequencing of the target gene fragments → methylation ratio analysis. Bisulfite conversion targets the entire genomic DNA. Bisulfite conversion kits have limited conversion capacity, with a single reaction yielding less than 1000 ng, and the final recovered conversion product is 10-20 μL. Furthermore, after bisulfite treatment of the DNA template, the 5'-terminal cytosine (C) of CpG sites within CpG islands in methylated gene promoter regions remains unchanged, while the 5'-terminal cytosine of CpG sites within unmethylated CpG islands is converted to uracil (U), or CU. A challenge encountered with methylation-specific PCR (MS-PCR, or MSP) on bisulfite-converted genomic DNA is that the target sequences are promoters or portions of the first exon, which have relatively high (C+G) content, making MSP amplification more challenging. The key to analyzing DNA methylation in gene promoter regions is obtaining PCR-amplified fragments of these regions. Therefore, the quality of primer design and amplification conditions are crucial to the success of amplification. MSP primer design differs from conventional PCR primer design, so optimized amplification conditions should be employed to improve PCR amplification efficiency.

[0003] The myosin heavy chain 3 (MYH3) gene, encoding an embryonic myosin heavy chain protein, is a key marker gene for muscle differentiation. It regulates muscle development and energy metabolism, playing a crucial role in the entire muscle development process in animals. The MYH3 gene is highly conserved across species and expressed in multiple tissues in animals, with higher expression levels in embryonic muscle tissue and during muscle regeneration. DNA methylation at CpG sites within the MYH3 gene promoter region is crucial for epigenetic inheritance, and the degree of methylation may influence muscle and fat differentiation and tissue formation. Gene expression and the degree of DNA promoter methylation may vary between pig breeds due to differences in lean meat percentage and fat content.

[0004] The length of genomic DNA from animal tissues is approximately tens of thousands or even more bases. Bisulfite conversion requires a large amount of reagents, and the amount of recovered bisulfite-converted genomic DNA is small. In multiple experiments, it was found that the PCR amplification effect of bisulfite-converted genomic DNA is not ideal. Therefore, it is crucial to obtain ordinary PCR amplification products for bisulfite sequencing.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The present invention aims to provide a primer set and a method and application for analyzing the DNA methylation level in the MYH3 gene promoter region. PCR amplification primers for the outer and inner sides of the MYH3 gene are designed according to a methylation primer design website, and touchdown PCR is combined with conventional PCR for amplification to improve the PCR amplification efficiency of the methylation primers, obtain qualified MYH3 gene DNA fragments, and overcome the low efficiency of PCR amplification of bisulfite-converted genomic DNA.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0008] One aspect of the invention relates to a primer set, comprising: a first primer pair and a second primer pair;

[0009] Wherein, the upstream sequence of the first primer pair is shown as SEQ ID NO.1;

[0010] The downstream sequence of the first primer pair is shown in SEQ ID NO.2;

[0011] The upstream sequence of the second primer pair is shown in SEQ ID NO.3;

[0012] The downstream sequence of the second primer pair is shown in SEQ ID NO.4.

[0013] The primer set is used to amplify genomic DNA after bisulfite conversion, has the advantage of high amplification efficiency, and can obtain qualified DNA fragments through amplification, thereby providing a sufficient amount of DNA fragments for analyzing the DNA methylation level in the MYH3 gene promoter region, thereby improving the efficiency and accuracy of methylation level analysis.

[0014] Another aspect of the present invention also relates to a method for analyzing DNA methylation levels in the promoter region of the MYH3 gene, comprising the following steps:

[0015] (a) using the bisulfite-converted genomic DNA as a DNA template and the first primer pair as primers to perform a first PCR amplification reaction;

[0016] (b) performing a second PCR amplification reaction using the product of the first PCR amplification reaction as a DNA template and the second primer pair as primers.

[0017] The method for analyzing the DNA methylation level in the promoter region of the MYH3 gene can obtain qualified PCR amplification products and has high efficiency and accuracy in methylation level analysis.

[0018] Another aspect of the present invention also relates to a kit for detecting the DNA methylation level of the MYH3 gene promoter region, comprising the primer set.

[0019] Another aspect of the present invention also relates to a method for analyzing the quality of animal meat, including the method for analyzing the DNA methylation level in the promoter region of the MYH3 gene.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The primer set provided by the present invention is used to amplify genomic DNA after bisulfite conversion, and has the advantage of high amplification efficiency. Qualified DNA fragments are amplified, providing a sufficient amount of DNA fragments for analyzing the DNA methylation level in the MYH3 gene promoter region, thereby improving the efficiency and accuracy of methylation level analysis.

[0022] (2) The method for analyzing DNA methylation levels in the MYH3 gene promoter region provided by the present invention has high efficiency and accuracy in methylation level analysis. This method first uses a first primer pair to perform a first PCR amplification reaction, thereby increasing the DNA template concentration required for a second PCR amplification reaction, providing a qualified DNA template for the second amplification, and improving the efficiency of PCR amplification directly using bisulfite-converted gene DNA as a template, thereby improving the efficiency and accuracy of DNA methylation level analysis in the MYH3 gene promoter region.

[0023] (3) The present invention can realize the analysis of the degree of DNA methylation in the promoter region of the MYH3 gene, which is specific and sensitive to pig fat differentiation and production. It can analyze the role of the MYH3 gene in the process of fat production and differentiation, and provide a powerful means for pork quality analysis and molecular genetic marker-assisted selection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 The CpG island status of MYH3 gene;

[0026] Figure 2 The figure shows the results of agarose gel electrophoresis of genomic DNA extracted from pig adipose tissue;

[0027] Figure 3 This is the result of agarose gel electrophoresis of the amplified MYH3 gene target fragment;

[0028] Figure 4 is the truncated partial sequencing result;

[0029] Figure 5 Graph showing the agarose gel electrophoresis results of the PCR amplification products of the first primer pair in the comparative example and the example;

[0030] Figure 6 This is a graph showing the results of PCR amplification and agarose gel electrophoresis using the second primer pair in the comparative example and the embodiment;

[0031] Figure 7 The figure shows the agarose gel electrophoresis detection results of PCR amplification in the comparative example and the example using bisulfite-converted DNA as a template. DETAILED DESCRIPTION

[0032] The technical scheme of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and specific embodiments, but it will be understood by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. Those who do not specify specific conditions in the embodiments are carried out according to normal conditions or the conditions recommended by the manufacturer. Those whose reagents or instruments are not specified by the manufacturer are conventional products that can be purchased commercially.

[0033] One aspect of the present invention relates to a primer set, comprising: a first primer pair and a second primer pair;

[0034] Wherein, the upstream sequence of the first primer pair is shown as SEQ ID NO.1;

[0035] The downstream sequence of the first primer pair is shown in SEQ ID NO.2;

[0036] The upstream sequence of the second primer pair is shown in SEQ ID NO.3;

[0037] The downstream sequence of the second primer pair is shown in SEQ ID NO.4.

[0038] The primer set is used to amplify genomic DNA after bisulfite conversion, has the advantage of high amplification efficiency, and obtains qualified DNA fragments through amplification, providing DNA fragments for analyzing the DNA methylation level in the MYH3 gene promoter region, thereby improving the efficiency and accuracy of methylation level analysis.

[0039] Further, SEQ ID NO. 1: TTGTATTTATTTTTGGGTATTTTGTT.

[0040] Further, SEQ ID NO. 2: ACCTTCCCATCCTAAATACTCTTAATT.

[0041] Further, SEQ ID NO. 3: GTTAGGGTTGGAGTTTGGTT.

[0042] Further, SEQ ID NO. 4: TAAATACTCTTAATTTTCCCCTTAAC.

[0043] The present invention obtained the NCBI reference sequence of the MYH3 gene: NC_010454.4 in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ).

[0044] The CpG islands in the gene promoter region were predicted based on the methylation primer design website (http: / / www.urogene.org / methprimer2 / ). Figure 1 ), and design methylation primers near the CpG island, Figure 1 The blue shaded area in the figure represents a CpG island, and the primer length ranges from 18 to 30 bp. Two pairs of primers were designed for two PCR amplifications: one pair of outer primers (first primer pair: SEQ ID NO. 1 and SEQ ID NO. 2) and the other pair of inner primers (second primer pair: SEQ ID NO. 3 and SEQ ID NO. 4). The start and end points of the inner primer amplification should be at least 20 bp away from the product amplified by the outer primers.

[0045] The present invention designs two pairs of primers: one pair of outer primers for PCR amplification (first primer pair: upstream primer: SEQ ID NO. 1, downstream primer: SEQ ID NO. 2), and one pair of inner primers (second primer pair: upstream primer: SEQ ID NO. 3, downstream primer: SEQ ID NO. 4). During PCR amplification, the outer primers are used for the first PCR amplification, followed by the inner primers for the second PCR amplification. The template for the first PCR amplification is bisulfite-converted DNA; the template DNA for the second amplification is the product obtained from the first PCR amplification. The designed primers are shown in Table 1.

[0046] Table 1 Primer sequences designed for MSP amplification of the MYH3 gene

[0047]

[0048] Another aspect of the present invention also relates to a method for analyzing DNA methylation levels in the promoter region of the MYH3 gene, comprising the following steps:

[0049] (a) using bisulfite-converted genomic DNA as a DNA template and the first primer pair as primers to perform a first PCR amplification reaction by touchdown PCR;

[0050] (b) using the product of the first PCR amplification reaction as a DNA template and the second primer pair as primers to perform a second PCR amplification reaction using conventional PCR.

[0051] The described method for analyzing DNA methylation levels in the MYH3 gene promoter region has high efficiency and accuracy in methylation level analysis. The method first uses a first primer set to perform a first PCR amplification reaction (touchdown PCR), with the resulting product serving as the template DNA for a second PCR amplification. This increases the concentration of the template DNA required for the latter, providing a qualified template for the second amplification. This improves the efficiency of direct PCR amplification using bisulfite-converted genomic DNA as a template, and increases the efficiency of obtaining DNA fragments for sequencing, thereby enhancing the efficiency and accuracy of analyzing DNA methylation levels in the MYH3 gene promoter region.

[0052] The first PCR amplification reaction of the present invention is a touchdown PCR, which amplifies the target fragment. Because the Tm value at the beginning of amplification is higher than the estimated primer Tm value, as the cycles proceed, the Tm value gradually decreases to the primer Tm value and eventually falls below this level, ensuring that outer primer-template hybridization occurs between the most complementary reactants, i.e., those that produce the target amplification product. Although the Tm value eventually drops to the Tm value of nonspecific hybridization, the target amplification product has already begun geometric amplification at this point, and remains superior to nonspecific PCR products for the remaining cycles.

[0053] After designing the synthetic primer set, the amplification conditions of the first PCR amplification reaction and the second PCR amplification reaction need to be optimized to improve the PCR amplification efficiency.

[0054] The present invention adopts Ex PCR amplification was performed using DNA Polymerase (Code No. RR001Q) according to the instructions.

[0055] Furthermore, the concentration of the genomic DNA is above 50 ng / μL.

[0056] Furthermore, the OD of the genomic DNA 260 / OD 280 The ratio is 1.8 to 2.0.

[0057] Furthermore, the reaction system of the first PCR amplification reaction is shown in Table 2.

[0058] Table 2 Reaction system of the first PCR amplification reaction

[0059]

[0060]

[0061] Furthermore, the conditions of the first PCR amplification reaction are as follows:

[0062] Pre-denaturation at 95°C for 3–5 minutes; denaturation at 95°C for 30 seconds–1 minute, followed by annealing at 64.3°C for 30 seconds–1 minute in the first cycle. The annealing temperature for each subsequent cycle was lowered by 0.5°C relative to the previous cycle, with each cycle annealing for 30 seconds–1 minute and extension at 72°C for 30 seconds–1 minute, for a total of 20 cycles; denaturation at 95°C for 30 seconds–1 minute, annealing at 53.3–55.3°C for 30 seconds–1 minute, and extension at 72°C for 30 seconds–1 minute, for a total of 20 cycles. Finally, a final extension at 72°C for 5–10 minutes, with a hold at 4–12°C. These amplification conditions offer high amplification efficiency, increasing the concentration of the specific target DNA product in the first PCR amplification reaction and providing sufficient template DNA for the second PCR amplification reaction.

[0063] Furthermore, the reaction system of the second PCR amplification reaction is shown in Table 2. The difference is that the template is the product after the first PCR amplification reaction, and the primers used are SEQ ID NO. 3 and SEQ ID NO. 4.

[0064] Furthermore, the conditions of the second PCR amplification reaction are as follows:

[0065] Pre-denaturation at 95°C for 3-5 min; denaturation at 95°C for 30s-1 min, annealing at 54-56°C for 30s-1 min, extension at 72°C for 30s-1 min, for a total of 35 cycles, and finally total extension at 72°C for 5-10 min, holding at 4-12°C.

[0066] Furthermore, the method for analyzing the DNA methylation level in the promoter region of the MYH3 gene further includes: step (c) recovering the product obtained by the second PCR amplification reaction by agarose gel, ligating, transforming, screening positive clones, and culturing and PCR identifying them in LB liquid culture medium to obtain a positive bacterial solution.

[0067] Furthermore, the bacterial solution identified as positive is sequenced, and the methylation level is analyzed based on the sequencing results.

[0068] The present invention does not limit the specific methods of agarose gel recovery, ligation, transformation, screening of positive clones, bacterial culture and PCR identification, and the technical solutions of the present invention can be implemented by conventional methods in the art.

[0069] The present invention does not specifically limit the method for converting genomic DNA with bisulfite, and any conventional conversion method in the art can be used to implement the technical solution of the present invention.

[0070] The present invention does not specifically limit the method for calculating the DNA methylation level of the MYH3 gene promoter region, and conventional calculation methods in the art can be used to implement the technical solution of the present invention.

[0071] Another aspect of the present invention also relates to a kit for detecting the DNA methylation level of the MYH3 gene promoter region, comprising the primer set.

[0072] Another aspect of the present invention also relates to a method for analyzing the quality of animal meat, including the method for analyzing the DNA methylation level in the promoter region of the MYH3 gene.

[0073] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all commercially available conventional products.

[0074] Example Analysis of DNA methylation levels in the MYH3 gene promoter region of Landrace pig adipose tissue

[0075] 1.1 Genomic DNA extraction and bisulfite conversion

[0076] Subcutaneous fat tissue from the neck of adult pigs was collected immediately after slaughter. Genomic DNA was extracted using the Blood / Cell / Tissue Genomic DNA Extraction Kit (Catalog No. DP304-02) produced by Tiangen Biochemical Technology (Beijing) Co., Ltd. The results of 1.5% agarose gel electrophoresis were shown in Table 1. Figure 2 The concentration test results are shown in Table 3. Figure 2 It can be seen that the extracted genomic DNA bands are single, the position is correct, the concentration is above 160ng / μL, and the OD 260 / OD 280 The ratios were all between 1.8 and 2.0, which met the experimental conditions. The extracted genomic DNA was converted using the DNA Bisulfite Conversion Kit (spin column type) (Catalog No. DP215-02) produced by Tiangen Biochemical Technology (Beijing) Co., Ltd. according to the instructions. The total amount of DNA used for conversion was 900 ng.

[0077] Table 3 Extracted genomic DNA concentration test results

[0078]

[0079] Note: Sample 1, sample 2, sample 3 and sample 4 are genomes extracted from different individual pigs

[0080] DNA.

[0081] 1.2 Obtaining the target fragment

[0082] The first PCR amplification reaction was carried out using the bisulfite-converted genomic DNA as a template using the first primer pair (SEQ ID NO.1 and SEQ ID NO.2); the second PCR amplification reaction was carried out using the product of the first PCR amplification reaction as a DNA template using the second primer pair (SEQ ID NO.3 and SEQ ID NO.4). The amplified target fragment was detected by agarose gel electrophoresis. Figure 3 As shown, Figure 3 The M on the top represents Marker D2000 [D2000 molecular weight standard (catalog number: MD114-02) produced by Tiangen Biochemical Technology (Beijing) Co., Ltd., the same below], 1, 2, 3 and 4 represent the amplified MYH3 gene DNA fragments of different individual pigs. Figure 3 It can be seen that after the second round of amplification, the target band is located at 223 bp, which is consistent with the theoretical value.

[0083] The conditions for the first PCR amplification reaction were as follows:

[0084] Pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 64.3°C for 30 s in the first cycle, the Tm value of each subsequent cycle is reduced by 0.5°C relative to the previous cycle, the annealing time of each subsequent cycle is 30 s, extension at 72°C for 30 s, for a total of 20 cycles; denaturation at 95°C for 30 s, annealing at 54.3°C for 30 s, extension at 72°C for 30 s, for a total of 20 cycles, and finally a total extension at 72°C for 5 min, hold at 4°C.

[0085] The conditions for the second PCR amplification reaction were as follows:

[0086] Pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, for a total of 35 cycles, and a final extension at 72°C for 5 min, followed by a hold at 4°C.

[0087] 1.3 Target DNA fragment cloning and sequencing

[0088] The target band agarose gel block obtained by the second PCR amplification reaction was cut out and the target DNA fragment was recovered using a common agarose gel DNA recovery kit (spin column type) (catalog number: (DP209-02) produced by Tiangen Biochemical Technology (Beijing) Co., Ltd. and then compared with the vector pMD TM The MYH3 gene was cloned and transformed with the -19T Vector Cloning Kit (Code No. 6013) into competent E. coli DH5α Competent Cells (Code No. 9057) produced by Bio-Rad Biotechnology (Beijing) Co., Ltd. Positive clones were screened by blue-white staining and cultured in LB liquid medium for PCR identification. Those with results consistent with the theoretical values ​​were sent to Jilin Kumei Biotechnology Co., Ltd. for sequencing. The DNA methylation level in the MYH3 gene promoter region was analyzed based on the sequencing results.

[0089] PCR identification of positive clones: Take 2 μL of positive clones as a DNA template and use the second primer pair for PCR amplification. The system and reaction conditions are the same as those of the second PCR amplification reaction in 1.2.

[0090] Sequencing results analysis: Figure 4 is the intercepted partial sequencing result. Figure 4 The arrows above indicate the methylation status of CpG islands, where C represents methylated and T represents unmethylated. Statistical analysis showed that the DNA methylation level of the MYH3 gene promoter region in Landrace pig adipose tissue was 83.9%.

[0091] Comparative Example 1

[0092] The difference between this comparative example and the embodiment is only that the conditions of the first PCR amplification reaction are different, which are as follows:

[0093] Pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 54.3°C for 30 s, extension at 72°C for 30 s, for a total of 35 cycles; final extension at 72°C for 5 min, hold at 4°C.

[0094] Comparative Examples and Examples The results of agarose gel electrophoresis of the target fragment amplified by the first primer pair PCR are as follows: Figure 5 As shown, Figure 5 The M on the left represents Marker D2000, and 1 to 8 represent MYH3 gene DNA fragments of different individual pigs amplified by the first primer pair: 1 to 4 represent MYH3 gene DNA fragments amplified by the example; 5 to 8 represent MYH3 gene DNA fragments amplified by the comparative example. As can be seen from the results, the amplification efficiency of the comparative example is not high, and the target band is almost invisible; while the amplification efficiency of the example is also not high, the target band can be amplified. The results of agarose gel electrophoresis after the comparative example and the example were amplified for the second time using the second primer pair are as follows: Figure 6 As shown, Figure 6 The "M" in the figure represents Marker D2000, and "1-8" represent MYH3 gene DNA fragments from different pigs amplified using the second primer pair. Numbers 1-4 represent MYH3 gene DNA fragments amplified in the Example, located at 223 bp, consistent with the theoretical value. Numbers 5-8 represent MYH3 gene DNA fragments amplified in the comparative example, which did not amplify the target band. This indicates that touchdown PCR performed using the first primer pair in the Example combined with PCR amplification using the second primer pair greatly improves the efficiency of obtaining the target DNA fragment. PCR amplification using the first primer pair provides a highly concentrated or qualified DNA template for the second primer pair. In contrast, conventional PCR using the first primer pair fails to provide a highly concentrated or qualified DNA template for PCR amplification using the second primer pair, resulting in low amplification efficiency.

[0095] Comparative Example 2

[0096] The difference between this comparative example and the embodiment is that only one PCR reaction is performed. The details are as follows:

[0097] The primers used are shown in Table 4. The PCR reaction conditions were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 54-58°C for 30 s, and extension at 72°C for 30 s for a total of 35 cycles, with a final extension at 72°C for 5 min and a hold at 4°C.

[0098] Table 4 Primer sequences designed for MSP amplification of the MYH3 gene in porcine adipose tissue

[0099]

[0100] The amplified target fragment was detected by agarose gel electrophoresis. Figure 7 shown. Figure 7 The M on the top represents Marker D2000, and 1 to 5 represent DNA fragments of the MYH3 gene of different individual pig fat obtained by performing a PCR reaction using bisulfite-converted genomic DNA as a template and different MSP primers; Figure 7 A represents the product of PCR amplification reaction using primers SEQ ID NO.5 and SEQ ID NO.6; Figure 7 B represents the product of PCR amplification reaction using primers SEQ ID NO.7 and SEQ ID NO.8; Figure 7 C represents the product of PCR amplification reaction using primers SEQ ID NO.9 and SEQ ID NO.10; Figure 7 D represents the product of PCR amplification reaction with primers SEQ ID NO.3 and SEQ ID NO.4. As can be seen from the results, the amplification efficiency of the comparative examples is not high ( Figure 7 C, D), some are even difficult to amplify ( Figure 7 A, B); Although the target DNA fragment can be amplified using primers SEQ ID NO.3 and SEQ ID NO.4, the amplification efficiency is very low. However, after the second PCR amplification reaction (the first one uses touchdown PCR and the second one uses ordinary PCR), the amplification efficiency is greatly improved ( Figure 6 , 1-4).

[0101] Although the present invention has been illustrated and described using specific embodiments, it should be appreciated that the above embodiments are merely intended to illustrate the technical solutions of the present invention rather than to limit them. Those skilled in the art should understand that the technical solutions described in the above embodiments may be modified, or some or all of the technical features thereof may be replaced by equivalents, without departing from the spirit and scope of the present invention. However, these modifications or replacements do not deviate from the essence of the corresponding technical solutions within the scope of the technical solutions of the embodiments of the present invention. Therefore, this means that all such replacements and modifications within the scope of the present invention are included in the appended claims.

Claims

1. A primer set, characterized in that: The primer set includes: a first primer pair and a second primer pair; Wherein, the upstream sequence of the first primer pair is shown as SEQ ID NO.1; The downstream sequence of the first primer pair is shown in SEQ ID NO.2; The upstream sequence of the second primer pair is shown in SEQ ID NO.3; The downstream sequence of the second primer pair is shown as SEQ ID NO.

4.

2. A method for analyzing DNA methylation level in the promoter region of MYH3 gene, characterized in that: The following steps are involved: (a) using the bisulfite-converted genomic DNA as a DNA template and the first primer pair described in claim 1 as a primer to perform a first PCR amplification reaction; (b) using the product of the first PCR amplification reaction as a DNA template and the second primer pair described in claim 1 as a primer to perform a second PCR amplification reaction.

3. The method for analyzing DNA methylation level in the promoter region of MYH3 gene according to claim 2, characterized in that: The conditions of the first PCR amplification reaction are as follows: Pre-denaturation at 95℃ for 3-5min; denaturation at 95℃ for 30s-1min, annealing at 64.3℃ for 30s-1min in the first cycle, and the annealing temperature of each subsequent cycle is reduced by 0.5℃ relative to the previous cycle, and the annealing time of each cycle is 30s-1min, extension at 72℃ for 30s-1min, for a total of 20 cycles; denaturation at 95℃ for 30s-1min, annealing at 53.3-55.3℃ for 30s-1min, extension at 72℃ for 30s-1min, for a total of 20 cycles, finally extension at 72℃ for 5-10min, hold at 4-12℃.

4. The method for analyzing DNA methylation level in the promoter region of MYH3 gene according to claim 2, characterized in that: The conditions of the second PCR amplification reaction are as follows: Pre-denaturation at 95℃ for 3-5min; denaturation at 95℃ for 30s-1min, annealing at 54-56℃ for 30s-1min, extension at 72℃ for 30s-1min, for a total of 35 cycles, and finally total extension at 72℃ for 5-10min, holding at 4-12℃.

5. The method for analyzing DNA methylation level in the promoter region of MYH3 gene according to claim 2, characterized in that: The concentration of the genomic DNA is above 50 ng / μL.

6. The method for analyzing DNA methylation level in the promoter region of MYH3 gene according to claim 2, characterized in that: The OD of the genomic DNA 260 / OD 280 The ratio is 1.8~2.

0.

7. The method for analyzing DNA methylation level in the promoter region of MYH3 gene according to claim 2, characterized in that: The method for analyzing the DNA methylation level in the promoter region of the MYH3 gene also includes: step (c) sequencing the product obtained from the second PCR amplification reaction, and calculating the DNA methylation level in the promoter region of the MYH3 gene according to the sequencing result.

8. The method for analyzing DNA methylation level in the promoter region of MYH3 gene according to claim 7, characterized in that: The products obtained from the second PCR amplification reaction are recovered from agarose gel, connected, transformed, screened for positive clones, identified by PCR, and then sequenced.

9. A kit for detecting the DNA methylation level in the promoter region of the MYH3 gene, characterized in that: Comprising the primer set of claim 1.

10. A method for analyzing animal meat quality, characterized in that: The method comprises the method for analyzing DNA methylation level in the promoter region of the MYH3 gene according to any one of claims 2 to 8.

Images