Probe primer and kit for detecting insulin-like growth factor 1 gene polymorphism

Abstract

The invention discloses a probe primer and a kit for detecting gene polymorphism of an insulin-like growth factor 1 (IGF-1). In allusion to polymorphic sites rs972936 and rs5742632 in the IGF-1 gene, two groups of specific primers and corresponding fluorescent probe sequences are designed, and the two groups of specific primers and the corresponding fluorescent probe sequences are used for realizing typing detection of the polymorphism of the gene. According to the method, different genotypes can be accurately distinguished by combining melting curve analysis corresponding to different Tm values through a molecular beacon multiple fluorescent quantitative PCR technology. The kit is simple in composition and convenient to operate, is suitable for large-batch sample detection, has the advantages of high sensitivity, strong specificity, rapid detection, high throughput and the like, and is suitable for accurate detection and analysis of IGF-1 gene polymorphism in scientific research and clinical application.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and specifically to probe primers and kits for detecting the polymorphism of the insulin-like growth factor 1 (also known as "IGF-1") gene. Background Art

[0002] The insulin-like growth factor 1 (IGF-1) gene is located on human chromosome 12, with a full length of approximately 84,734 bases and contains 3,103 known single nucleotide polymorphism (SNP) sites. Depending on their different positions in the gene, these SNP sites may play different biological functions by regulating gene expression, influencing post-transcriptional regulation processes, or altering the amino acid sequence of proteins.

[0003] IGF-1 is a single-chain basic polypeptide with a structure homologous to insulin and plays an important role in promoting growth and development, regulating material metabolism, and immune function. Studies have found that IGF-1 can play a key role in insulin regulation by downregulating the level of endogenous growth hormone, enhancing insulin sensitivity, and maintaining the normal function of pancreatic islet β cells.

[0004] Existing studies have shown that a decrease in IGF-1 levels is closely related to an increase in insulin resistance, which may induce type 2 diabetes mellitus (T2DM) and its related complications. A number of studies have confirmed multiple risk genes related to the onset of T2DM, among which IGF-1 is one of the candidate genes with a relatively significant impact.

[0005] As an important tool for evaluating the functional status of IGF-1, gene polymorphism detection is of great significance in studying the relationship between it and the susceptibility to T2DM. Currently, the commonly used detection method is DNA direct sequencing, that is, sequence analysis of PCR amplification products to identify mutation sites. Although this method has high accuracy, it has the disadvantages of complex operation, long detection cycle, and high cost, and is difficult to apply to high-throughput detection of a large number of samples.

[0006] Therefore, there is an urgent need to develop a method for detecting IGF-1 gene polymorphism with high sensitivity, high specificity, and suitable for large-scale sample screening to meet the actual needs of clinical research and the development of personalized medicine. Summary of the Invention

[0007] The present invention provides a specific probe primer for detecting the polymorphism of insulin-like growth factor 1 (IGF-1) gene and its supporting kit. Combining with the molecular beacon multiplex fluorescence quantitative PCR detection method, by simultaneously performing specific amplification on multiple target sites (such as rs972936, rs5742632), high-throughput detection of the target SNP sites is achieved. This method can simultaneously identify and distinguish multiple target sequences in a single reaction, significantly improving the detection efficiency, reducing the usage of samples and reagents, thus effectively saving the detection cost, and is applicable to the rapid screening of clinical samples and individualized medication guidance.

[0008] A probe primer for detecting the polymorphism of insulin-like growth factor 1 gene according to the present invention, the artificially synthesized DNA primer sequence is:

[0009] The primer sequences for the rs972936 site of IGF-1 are as follows:

[0010] The A1 discrimination primer sequence is:

[0011] 5’-TCTATCTGGCCTGAACTTCTGCATTGCT-3’;

[0012] The A2 discrimination primer sequence is:

[0013] 5’-TTTCTATCTGGCCTGAACTTCTGCATTACC-3’;

[0014] The Pc universal primer sequence is:

[0015] 5’-CTGAATGTCAGAATAACTACACATACCACACTT-3’;

[0016] The primer sequences for the rs5742632 site of IGF-1 are as follows:

[0017] The A1 discrimination primer sequence is:

[0018] 5’-CTCCTAGTATGGAACAGATATTTGAGGATGAAGTT-3’;

[0019] The A2 discrimination primer sequence is:

[0020] 5’-TTCTCCTAGTATGGAACAGATATTTGAGGATGAAGTC-3’;

[0021] The Pc universal primer sequence is:

[0022] 5’-TGAGAAGATCAATGACACACATACAATAAGC-3’.

[0023] The present invention also provides a kit for detecting the rs972936 locus and the rs5742632 locus of IGF-1. This kit is used to detect the rs972936 locus and the rs5742632 locus of the IGF-1 gene. The kit contains the primer-probe group as described in claim 1, and also contains Taq enzyme, dNTP and / or Mg2+;

[0024] The Taq enzyme in the kit is used to catalyze the synthesis of DNA strands;

[0025] dNTP serves as the four deoxynucleotide substrates required for DNA synthesis and is recognized and utilized by the polymerase;

[0026] Mg2+ serves as the auxiliary ion required for the polymerase reaction, can provide a suitable catalytic environment for Taq enzyme, and assist it to achieve efficient and specific DNA amplification;

[0027] The above three components together constitute the basic components of the PCR reaction system in gene polymorphism detection.

[0028] The present invention also discloses a method for detecting the polymorphism of the insulin-like growth factor 1 gene using the kit, including the following steps:

[0029] (1) Extract the genomic DNA of the sample to be tested;

[0030] (2) Perform fluorescence quantitative PCR reaction using the above probe primers;

[0031] Perform the reaction on a fluorescence quantitative PCR instrument, and the PCR reaction conditions are:

[0032] Pre-denaturation at 92 - 97°C for 5 - 15 minutes;

[0033] Denaturation at 92 - 97°C for 10 - 30 seconds;

[0034] Annealing at 57 - 65°C for 10 - 30 seconds;

[0035] Extension at 70 - 75°C for 10 - 30 seconds;

[0036] 40 - 50 cycles;

[0037] Extension at 72°C for 10 minutes;

[0038] Denaturation at 92 - 97°C for 1 minute, renaturation at 40°C for 1 minute, and monitor the fluorescence signal in real time during the melting temperature process of 45 - 80°C, and record 5 times for each 1°C increase in temperature;

[0039] (3) Analyze the data obtained from fluorescence quantitative PCR amplification, set appropriate baselines and thresholds, and based on the melting curve (Melting Curve) formed by the amplification curve, judge the genotype of the target site according to the differences in hybridization peaks corresponding to different Tm values;

[0040] The methods of fluorescence quantitative PCR amplification and Tm value detection are well-known conventional techniques to those skilled in the art, so they will not be elaborated here.

[0041] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0042] 1. The present invention uses a fluorescent probe that can specifically recognize the target nucleic acid sequence. By hybridizing with the target sequence, a conformational change occurs, releasing the fluorophore from the quenched state and then emitting a fluorescent signal. According to the differences in the fluorescence melting temperatures (Tm values) corresponding to different genotypes, different characteristic peaks are formed on the melting curve, thereby realizing the accurate determination of genotypes. In the case of target DNA deletion, a stable structure can be formed between the fluorophore and the quencher, and no fluorescent signal is generated, ensuring a low detection background and strong signal specificity.

[0043] 2. When the target DNA exists, the fluorescent probe is completely complementary to it and binds, causing the probe structure to be damaged, separating the fluorophore from the quencher, and releasing a detectable fluorescent signal, thereby realizing the real-time monitoring and specific recognition of the target sequence.

[0044] 3. Compared with traditional genotyping techniques, the method of the present invention is simple to operate and has a short cycle. Usually, the detection of multiple samples can be completed within 2 - 3 hours, with significant advantages such as high sensitivity, good specificity, fast detection speed, and high throughput. The fluorescent probes used can be labeled with different fluorophores at different sites, thereby realizing the simultaneous detection of multiple targets. This method monitors the fluorescence change in real time throughout the fluorescence quantitative PCR reaction process, without the need for post-PCR processing or electrophoresis and other steps, and can achieve true closed-tube operation, effectively avoiding the risk of contamination, and is suitable for clinical applications and large-scale screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is the melting curve diagram in Example 3;

[0046] Figure 1 A is the melting curve diagram of three genotypes (TT, CC, TC) at the rs972936 site of the IGF-1 gene;

[0047] Figure 1 B is the melting curve diagram of three genotypes (GG, AA, AG) at the rs5742632 site of the IGF-1 gene. DETAILED DESCRIPTION OF THE INVENTION

[0048] The present invention will be described in detail below in conjunction with embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several adjustments and improvements can still be made, and these all fall within the protection scope of the present invention.

[0049] Example 1: Probe and Primer Design

[0050] The present invention designs fluorescence probes and primer sequences for the SNP sites rs972936 and rs5742632 of the IGF-1 gene. The principle is that after the fluorescence probe specifically hybridizes with the target sequence, the conformation of the probe structure changes, separating the fluorescent group from the quenching group and generating a fluorescent signal; the genotype result is judged according to the melting temperature (Tm) and the curve peak shape of different hybridization products.

[0051] In the absence of target DNA, the fluorescence probe maintains its original conformation and the fluorescence signal is not released; once it hybridizes with the target DNA, the structure breaks and releases fluorescence, and its signal change is monitored in real time by fluorescence quantitative PCR and the melting curve is plotted.

[0052] The probes and primers for detecting the polymorphism of the insulin-like growth factor 1 (IGF-1) gene of the present invention adopt the following DNA oligonucleotide sequences:

[0053] Locus rs972936:

[0054] A1 discrimination primer: 5’-TCTATCTGGCCTGAACTTCTGCATTGCT-3’;

[0055] A2 discrimination primer: 5’-TTTCTATCTGGCCTGAACTTCTGCATTACC-3’;

[0056] Pc universal primer: 5’-CTGAATGTCAGAATAACTACACATACCACACTT-3’;

[0057] Locus rs5742632:

[0058] A1 discrimination primer: 5’-CTCCTAGTATGGAACAGATATTTGAGGATGAAGTT-3’;

[0059] A2 discrimination primer: 5’-TTCTCCTAGTATGGAACAGATATTTGAGGATGAAGTC-3’;

[0060] Pc universal primer: 5’-TGAGAAGATCAATGACACACATACAATAAGC-3’。

[0061] The above probes and primers were independently designed by the applicant according to the target SNP site sequence information, and optimized in combination with the melting temperature, specific region and fluorescence labeling strategy to ensure good recognition ability and signal-to-noise ratio at polymorphic sites, and improve the detection sensitivity and accuracy. At present, no identical design has been disclosed in the public literature or patents, and it has high innovation and industrial practical value.

[0062] Example 2:

[0063] A kit for detecting the polymorphism of insulin-like growth factor 1 gene, which is used to detect the rs972936 site and rs5742632 site of the IGF-1 gene. The kit contains the primer-probe group as described in claim 1, and also contains Taq enzyme, dNTP and / or Mg2+;

[0064] The Taq enzyme in the kit is used to catalyze the synthesis of DNA strands;

[0065] dNTP serves as the four deoxynucleotide substrates required for DNA synthesis, which are recognized and utilized by the polymerase;

[0066] Mg2+ serves as an auxiliary ion required for the polymerase reaction, which can provide a suitable catalytic environment for Taq enzyme and assist it to achieve efficient and specific DNA amplification;

[0067] The above three components together constitute the basic components of the PCR reaction system in gene polymorphism detection.

[0068] Example 3: Detection of different genotype standards using the kit of Example 2

[0069] 1. Standard construction: The applicant constructed wild-type (TT / AA) and mutant (CC / GG) standard plasmids containing the rs972936 and rs5742632 sites of the IGF-1 gene, which were synthesized by Shanghai Tianhao Biotechnology Co., Ltd. and verified for sequence accuracy by Sanger sequencing. The obtained standard DNA was uniformly diluted to 10 ng / μL and used for verification of the fluorescence quantitative PCR detection system.

[0070] 2. PCR detection system: Fluorescence quantitative PCR detection was performed using the probes and primers described in Example 1;

[0071] The total volume of the reaction system was 15 μL, and the specific composition was as follows:

[0072] PCR Mix: 7.5 μL (2× concentration);

[0073] Concentration of each set of primers (forward and reverse): 0.5 μM;

[0074] Probe concentration in each group: 0.1 μM;

[0075] Add sample DNA (wild type, mutant type, mixed type): 2 μL (10 ng / μL);

[0076] Add sterile distilled water to make up to 15 μL.

[0077] The PCR program was set up as follows:

[0078] Pre-denaturation: 95°C, 5 minutes;

[0079] Amplification cycles (45 in total):

[0080] Denaturation: 95°C, 30 seconds;

[0081] Annealing: 60°C, 30 seconds;

[0082] Extension: 72°C, 30 seconds;

[0083] Final extension: 72°C, 10 min.

[0084] Melting curve analysis:

[0085] Denature at 95°C for 1 minute;

[0086] Renature at 40°C for 1 minute;

[0087] Melting range: 45℃ to 80℃, and record 5 fluorescence signals for every 1℃ increase.

[0088] 3. Melting curve analysis and graph interpretation, such as Figure 1 As shown:

[0089] like Figure 1 As shown in A, it is the melting curve diagram of three genotypes (TT, CC, TC) of the rs972936 site of the IGF-1 gene:

[0090] TT-type samples showed a single high Tm peak;

[0091] The CC type samples showed low Tm peaks;

[0092] The TC type sample showed double peaks, corresponding to the two Tm values ​​of TT and CC, indicating a heterozygous type.

[0093] like Figure 1 As shown in B, it is the melting curve of the three genotypes of GG, AA, and AG at the rs5742632 site:

[0094] GG type is a low Tm peak;

[0095] Type AA has a single high Tm peak;

[0096] The AG type shows two Tm peaks, which are typical features of the heterozygous type;

[0097] The above melting curve results were analyzed by SLAN software, and the genotypes were judged by combining the differences in different Tm values, so as to achieve accurate genotyping of the polymorphic sites of the IGF-1 gene.

[0098] The method for detecting the polymorphism of the insulin-like growth factor 1 gene using the kit in the present invention, the embodiment gives a specific detection method, and when actually using the dosage of the reaction system and the like, the raw materials can be added according to the multiple of the dosage in the embodiment.

[0099] The present invention provides an SNP detection system with high specificity, high sensitivity and suitable for batch detection, which can be widely used in the fields of gene polymorphism research, genetic disease screening, individualized drug use and population genetics investigation, etc.

Claims

1. Probe primers for detecting polymorphisms of the insulin-like growth factor 1 gene, characterized in that, The synthetic DNA primer sequences are as follows: The primer sequences for the rs972936 locus of IGF-1 are as follows: The A1 discrimination primer sequence is: 5’-TCTATCTGGCCTGAACTTCTGCATTGCT-3’; The A2 discrimination primer sequence is: 5’-TTTCTATCTGGCCTGAACTTCTGCATTACC-3’; The Pc universal primer sequence is: 5’-CTGAATGTCAGAATAACTACACATACCACACTT-3’; The primer sequences for the rs5742632 locus of IGF-1 are as follows: The A1 discrimination primer sequence is: 5’-CTCCTAGTATGGAACAGATATTTGAGGATGAAGTT-3’; The A2 discrimination primer sequence is: 5’-TTCTCCTAGTATGGAACAGATATTTGAGGATGAAGTC-3’; The Pc universal primer sequence is: 5’-TGAGAAGATCAATGACACACATACAATAAGC-3’.

2. Kit for detecting insulin-like growth factor 1 gene polymorphism, characterized in that, The kit is used for detecting the rs972936 locus and rs5742632 locus of the IGF-1 gene. The kit contains the primer-probe group as described in claim 1, and also contains Taq enzyme, dNTP and / or Mg2+; The Taq enzyme in the kit is used to catalyze the synthesis of DNA strands; dNTP serves as the four deoxynucleotide substrates required for DNA synthesis, which are recognized and utilized by the polymerase; Mg2+ serves as the auxiliary ion required for the polymerase reaction, which can provide a suitable catalytic environment for Taq enzyme and assist it to achieve efficient and specific DNA amplification; The above three components together constitute the basic components of the PCR reaction system in gene polymorphism detection.

3. A method for detecting the polymorphism of insulin-like growth factor 1 gene using the kit of claim 2, characterized in that, It includes the following steps: (1) Extract the genomic DNA of the sample to be tested; (2) Perform fluorescence quantitative PCR reaction using the primer-probe group as described in claim 1; (3) Analyze the data obtained from the fluorescence quantitative PCR amplification, set appropriate baselines and thresholds, and judge the genotype of the target locus according to the melting curve formed by the amplification curve and the hybridization peak differences corresponding to different Tm values.

4. The method for detecting the polymorphism of insulin-like growth factor 1 gene using the kit according to claim 2, characterized in that The fluorescence quantitative PCR reaction conditions in step (2) are: Pre-denaturation at 92 - 97°C for 5 - 15 minutes; Denaturation at 92 - 97°C for 10 - 30 seconds; Annealing at 57 - 65°C for 10 - 30 seconds; Extension at 70 - 75°C for 10 - 30 seconds; 40 - 50 cycles; Extension at 72°C for 10 minutes; Denaturation at 92 - 97°C for 1 minute, renaturation at 40°C for 1 minute, and monitor the fluorescence signal in real time during the melting temperature range of 45 - 80°C, and record 5 times for each 1°C increase in temperature.

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