CN104894250B - The detection method of rs492594 genotype and its application - Google Patents

Abstract

The invention discloses a genotype detection method of rs492594 and its application. The invention provides the application of a product for detecting the genotype of the rs492594 SNP site in the human genome or a product for detecting the genotype of the rs570876 SNP site in the human genome in the preparation of a product for predicting the hypoglycemic effect of pioglitazone on humans. The present invention proves that the genotype at rs492594 in diabetic patients is related to the hypoglycemic effect of pioglitazone, and the detection of rs492594 can be used to screen type 2 diabetic patients who are relatively sensitive to pioglitazone, and suitable hypoglycemic drugs can be selected according to the genotype at rs492594 in diabetic patients , Improve the level of individualized treatment. The present invention further discloses a restriction fragment length polymorphism (restriction fragment length polymorphism, RFLP) method of the genotype at rs492594. The method can identify the genotype at the rs492594 site reliably, conveniently, economically and rapidly.

Description

Genotype detection method of rs492594 and its application

technical field

The invention relates to a genotype detection method of rs492594 and its application, belonging to the field of biomedicine,

Background technique

Thiazolidinedione drugs, also known as insulin sensitizers, are commonly used oral hypoglycemic drugs in clinical practice. Thiazolidinedione drugs include pioglitazone, troglitazone, rosiglitazone, etc. These drugs have a similar chemical structure, that is, 2,4-thiazolidinedione structure, and the difference between different drugs is that they have different side chain substitutions base. However, the safety of these compound drugs is not the same, and only pioglitazone is still widely used clinically. Thiazolidinediones are peroxisome proliferator-activated receptor gamma (PPARγ) agonists. Structural changes occur after PPARγ is activated, and it can recruit a variety of co-activators to bind with it and play different biological roles, including increasing insulin sensitivity in peripheral tissues, reducing fat deposition in skeletal muscle and liver, and promoting skeletal muscle to circulation. Glucose utilization in the middle, inhibition of glycogen output. There are also studies suggesting that thiazolidinediones can protect pancreatic β-cell function.

Although there are a variety of oral hypoglycemic drugs clinically, on the one hand, it is difficult to achieve individualized treatment to maximize the efficacy of the drugs, and on the other hand, the occurrence of side effects limits the use of some hypoglycemic drugs. Differences in drug effects are mainly determined by differences in individual genetic backgrounds related to drug effects and metabolism. By studying the pharmacogenetics of oral hypoglycemic drugs, genetic information can be used to provide a theoretical basis for individualized medication. Pioglitazone is a thiazolidinedione drug still in use on the market. Studies have shown that in addition to lowering blood sugar and delaying the progression of prediabetes to diabetes, it also has anti-inflammatory and anti-atherosclerosis, and has potential cardiovascular protection. There are also adverse effects leading to edema, weight gain, increased fracture risk, and heart failure. The indications for pioglitazone are all type 2 diabetes, but due to the heterogeneity of the etiology and pathogenesis of type 2 diabetes, if it is not subdivided, there will inevitably be a small number of patients with adverse reactions or poor curative effect, resulting in increased medical expenses for patients. Physical and mental harm caused by adverse reactions. Therefore, finding out the special group of type 2 diabetes that is sensitive to this drug and using individualized drug dosage may bring more benefits to patients.

The G6PC2 gene is located at 2q24.3 and encodes the catalytic subunit 2 of glucose-6-phosphatase. This protein is specifically expressed in pancreatic β cells. Cell studies have not found that this protein has phosphohydrolase activity, but it is the main target protein of autoimmune diabetes mediated by cellular immunity. Knockout of the G6PC2 gene in animal experiments resulted in relative activation of glucokinase, which altered the state of glucose-stimulated insulin secretion. A number of studies in people with type 2 diabetes at home and abroad have found that the G6PC2 gene is associated with fasting blood sugar or the risk of type 2 diabetes. One of the SNP sites, rs492594, is located on exon 5 of the G6PC2 gene, which is a missense mutation (c.655G>C, Val219Leu). According to the PubMed Chinese typing data, the minor allele G frequency of rs492594 is 0.463.

Contents of the invention

A technical problem to be solved by the present invention is to predict the hypoglycemic effect of pioglitazone.

In order to solve the above technical problems, the present invention provides a product for detecting the genotype of the rs492594SNP site in the human genome or a product for detecting the genotype of the rs570876SNP site in the human genome in the preparation of products for predicting the hypoglycemic effect of pioglitazone on humans after intervention application.

The above applications are specifically reflected in the following:

rs492594 SNP site GG genotype in diabetic patients to be tested, after pioglitazone intervention, the degree of decrease in blood sugar, HbA1c and/or insulin resistance level is higher than or candidate higher than rs492594 SNP site CC genotype or rs492594 SNP site GC The genotype of the diabetes patients to be tested is the degree of blood sugar reduction, HbA1c (glycosylated hemoglobin) reduction and/or insulin resistance reduction after pioglitazone intervention; the GG genotype is homozygous for G at the rs492594SNP site, and the GC gene The CC genotype is a heterozygote for G and C at the rs492594 SNP site, and the CC genotype is a homozygote for C at the rs492594 SNP site;

rs570876 SNP site AA genotype in diabetic patients to be tested, after pioglitazone intervention, the degree of decrease in blood glucose, HbA1c and/or insulin resistance level is higher than or candidate higher than rs570876 SNP site TT genotype or rs570876 SNP site AT The genotype of the diabetes patient to be tested is the degree of blood sugar reduction, HbA1c (glycosylated hemoglobin) reduction and/or insulin resistance level reduction after pioglitazone intervention; the AA genotype is homozygous for A at the rs570876 SNP site, and the AT gene The genotype is a heterozygote of A and T at the rs570876 SNP site, and the TT genotype is a homozygote of T at the rs570876 SNP site;

The degree of decrease in blood sugar after the intervention of pioglitazone is [(difference between blood sugar before pioglitazone intervention and blood sugar after pioglitazone intervention)/blood sugar before pioglitazone intervention] * 100%;

The reduction degree of HbA1c after pioglitazone intervention is [(difference between HbA1c before pioglitazone intervention and HbA1c after pioglitazone intervention)/HbA1c before pioglitazone intervention]*100%.

The above blood glucose is the OGTT 2-hour blood glucose.

The pioglitazone intervention refers to taking a dose of 30 mg per day for one year.

The above-mentioned hypoglycemic effect is reflected in at least one of the following (1)-(3):

(1) The degree of decline in blood glucose concentration after pioglitazone intervention;

(2) The degree of decline in the concentration of glycosylated hemoglobin after pioglitazone intervention;

(3) The degree of decline in human insulin resistance after pioglitazone intervention;

Good hypoglycemic effect is reflected in at least one of the following A'-C':

A', after the pioglitazone intervention, the blood sugar concentration of people decreased to a high degree;

B', after the intervention of pioglitazone, the concentration of glycosylated hemoglobin decreased to a high degree;

C', after the intervention of pioglitazone, the level of insulin resistance decreased to a high degree.

The hypoglycemic effect of pioglitazone on people whose genotype of rs492594 locus is GG is better than that of pioglitazone on people whose genotype of rs492594 locus is CG or CC.

The hypoglycemic effect of pioglitazone on people whose genotype of rs570876 locus is AA is better than that of pioglitazone on people whose genotype of rs492594 locus is AT or TT.

Another technical problem to be solved by the present invention is to screen pre-diabetic patients who are sensitive to pioglitazone.

In order to solve the above technical problems, the present invention provides the product for detecting the genotype of the rs492594SNP site in the human genome or the application of the product for detecting the genotype of the rs570876SNP site in the human genome in the preparation of products for screening pioglitazone-sensitive prediabetic patients .

Prediabetic patients who are relatively sensitive to pioglitazone show a high degree of blood glucose reduction, a high degree of HbA1c reduction and/or a high degree of insulin resistance reduction after pioglitazone intervention.

In the above application, the product is the DNA molecule shown in Sequence 1 and Sequence 2.

The third technical problem to be solved by the present invention is to detect the genotype of the rs492594 SNP site in the human genome.

In order to solve the above technical problems, the present invention provides the application of the product for detecting the genotype of the rs570876 SNP site in the human genome in the preparation of the product for detecting the genotype of the rs492594 SNP site in the human genome.

The fourth technical problem to be solved by the present invention is to detect the genotype of the rs492594 SNP site in the human genome.

In order to solve the above technical problems, the present invention provides a method for detecting the genotype of the rs492594SNP site in the human genome, 1) direct sequencing;

2) Detecting the genotype of the rs570876 SNP site in the human genome to be tested, and determining the genotype of the rs492594 SNP site in the human genome to be tested according to the genotype of the rs570876 SNP site in the human genome to be tested.

Above-mentioned 2) shown method, comprises the steps:

1) performing PCR amplification on the human genomic DNA to be tested with primers for amplifying the DNA fragment containing the rs570876 SNP site to obtain a PCR amplification product;

2) Digesting the PCR product with ApoI to obtain a digested product, judging the genotype of the rs570876SNP site in the human genome to be tested according to the size of the digested product, and then determining the genotype of the rs570876SNP site according to the genotype of the rs570876SNP site. The genotype of the rs492594 SNP locus.

In the above method, the primer pair for amplifying the DNA fragment containing the rs492594SNP site is composed of the DNA molecule shown in sequence 1 and the DNA molecule shown in sequence 2;

The genotype of the rs570876SNP site in the human genome to be tested is determined according to the size of the digestion product, and then the genotype of the rs492594SNP site is determined according to the genotype of the rs570876SNP site as follows: if the enzyme If the cut product only contains target fragments of 307bp and 396bp in size, the genotype of the rs570876SNP site in the human genome is AA, and the genotype of the rs492594SNP site in the human genome is GG; if the digested product contains 307bp, 396bp and 703bp DNA fragments, the genotype of the rs570876SNP site in the human genome is AT, and the genotype of the rs492594SNP site in the human genome is GC; if the enzyme digestion product only contains a 703bp DNA fragment, then The genotype of the rs570876 SNP site in the human genome is TT, and the genotype of the rs492594 SNP site in the human genome is CC.

The nucleotide of the rs570876 SNP site is the 308th nucleotide of sequence 3 or sequence 4;

The nucleotide of the rs492594 SNP site is the 501st nucleotide of sequence 7 or the 11427th deoxyribonucleotide of the GenBank Accession Number NCBI Reference Sequence: NG_011682.1 (May 4, 2014) of the G6PC2 gene.

The methods are non-diagnostic and non-therapeutic methods.

The nucleotide sequence of the 307bp target fragment is sequence 5;

The nucleotide sequence of the 396bp target fragment is sequence 6;

The nucleotide sequence of the 703bp target fragment is the DNA molecule shown in Sequence 4.

The fifth technical problem to be solved by the present invention is to prepare products for predicting the hypoglycemic effect of pioglitazone on humans or to prepare products for screening diabetics who are sensitive to pioglitazone.

In order to solve the above technical problems, the present invention provides the application of the genotype of the rs570876 site and/or the genotype of the rs492594 site in the human genome to prepare products for predicting the hypoglycemic effect of pioglitazone on humans;

or,

Application of the genotype of the rs570876 site and/or the genotype of the rs492594 site in the human genome in the preparation of products for screening diabetic patients sensitive to pioglitazone.

The sixth technical problem to be solved by the present invention is a product for detecting the genotype of the rs492594 site in the human genome and/or detecting the genotype of the rs570876 site in the human genome.

In order to solve the above technical problems, the present invention provides a product for detecting the genotype of the rs492594 site in the human genome and/or detecting the genotype of the rs570876 site in the human genome, which comprises amplifying the product comprising the rs570876 site and the site immediately adjacent to the site PCR primers for human genomic DNA fragments 5 nucleotides downstream of 3';

The PCR primers are specifically a primer pair composed of the DNA molecule shown in Sequence 1 and the DNA molecule shown in Sequence 2.

In the above application, method or product, said human is a diabetic patient.

The diabetic patient is specifically a prediabetic patient.

There is linkage disequilibrium between the rs570876 and the rs492594 (r ² =1), and the A allele of rs570876 (rs570876-A) and the G allele of rs492594 (rs492594-C) constitute a haplotype.

In any of the applications or products described above, the product can be a reagent or a test kit, and can also be a combination product of a reagent or a kit and an instrument, such as a combination product consisting of primers and a DNA sequencer, consisting of PCR reagents and Combination product consisting of DNA sequencing reagents and DNA sequencer;

The rs492594 is a biallelic polymorphic SNP site on human chromosome 2, and the variation is to switch G/C (then being C/G on its complementary chain); the genotype of the rs492594 site is CC, CG or GG; the CC is a homozygous type whose rs492594 site is C, the GG is a homozygous type whose rs492594 site is G, and the CG is a heterozygous type whose rs492594 site is G and C, and the The detection of the genotype of the rs492594 site in the human genome can specifically be the detection of the nucleotide type of the rs492594 site;

The rs570876 is a biallelic polymorphic SNP site on human chromosome 2, and the variation is conversion A/T (then being T/A on its complementary chain); the genotype of the rs570876 site is AA, TT or AT; the AA is a homozygous type whose rs570876 site is A, the TT is a homozygous type whose rs570876 site is T, and the AT is a heterozygous type whose rs570876 site is A and T. The genotype of the rs570876 site in the genome can specifically be the nucleotide type of the rs570876 site;

The present invention proves that the degree of reduction of blood sugar and HbA1c (glycosylated hemoglobin) in prediabetic patients whose genotype is GG at rs492594 is significantly greater than that of prediabetic patients whose genotype is CG or CC at rs492594 after using pioglitazone Genotype is related to the hypoglycemic effect of pioglitazone. The detection of rs492594 genotype can be used to screen prediabetic patients who are relatively sensitive to pioglitazone. Appropriate hypoglycemic drugs can be selected according to the genotype at rs492594 of prediabetic patients to improve the level of individualized treatment. The present invention further provides a restriction fragment length polymorphism (restriction fragment length polymorphism, RFLP) analysis method of the genotype at rs492594, which can identify the genotype at the rs492594 site reliably, conveniently, economically and rapidly.

Description of drawings

Fig. 1 is the flow chart of pharmacogenetic research of pioglitazone.

Fig. 2 is a comparison between the typing results of the chip and the RFLP typing results of the present invention.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The patients in the following examples are all pre-diabetic patients.

Embodiment 1, the study of pioglitazone on the curative effect of different genotype diabetes patients

Ethics statement

This study was approved by the Ethics Committee (IRB) of Peking University Health Science Center, and all patients with prediabetes signed an informed consent.

1. Collection of clinical data and biological samples

Carry out clinical trials of pioglitazone drugs, establish research cohorts, and collect clinical data and biological samples at various stages.

The study is a randomized, drug-blind, parallel, placebo-controlled prospective multicenter clinical trial. According to the WHO1999 diagnostic criteria, 1903 pre-diabetic patients were included, of which 953 were treated with pioglitazone (the dose of pioglitazone was 30mg/d, and all subjects completed one-year follow-up. The baseline and follow-up data of the subjects were collected, including the height of the patients. , body weight, waist circumference, hip circumference, fasting blood glucose, HbA1c (glycosylated hemoglobin), fasting C-peptide, fasting insulin, 2-hour postprandial blood glucose, blood lipids, urinary microalbumin, etc. Dropping off during the first year of follow-up and incomplete baseline data The final number of selected cases was 761 patients with prediabetes using pioglitazone.

2. Microarray design and genotyping

In this study, Illumina's Infinium HD iSelect custom chip (article number WG-401-1004) and MassARRAY flight mass spectrometer were used for SNP site typing. The iSelect custom chip includes pharmacokinetic and diabetes candidate gene-related loci, covering almost all gene loci related to drug metabolism, and taking into account the characteristics of the genetic background of the Chinese Han population. In the phase 1 iSelect chip study, 477 of the 761 prediabetic patients obtained in step 1 were selected for genotyping experiments. 12 related loci with statistical difference were obtained. In the second stage, it was verified in the remaining 285 prediabetic patients in step 1. The 12 sites were repeatedly typed by flight mass spectrometry, and finally the results of the two types were combined for analysis, and the position with the highest correlation with the hypoglycemic effect was obtained. point.

The criterion for judging the effectiveness of pioglitazone intervention is: the patients return to the state of normal glucose tolerance after 1 year of follow-up. According to the WHO 1999 standard, in the oral glucose tolerance test, FPG<6.1mmol/L and 2hPG<7.8mmol/L are considered normal glucose tolerance.

The experimental process is shown in Figure 1.

3. Research results

In the first stage, 3089 SNP sites were typed using iSelect custom chips, and the sites with a detection rate ≥ 90% and a minor allele frequency (MAF) ≥ 1% were included in the analysis, a total of 782 SNP sites. All the results were statistically performed using the genetics statistical software PLINK version1.07.

After 1 year of follow-up, the reversal of prediabetes to normal glucose tolerance was effective as pioglitazone intervention. After adjusting confounding factors such as gender, age, baseline body mass index (BMI) and lifestyle intervention, logistic regression analysis was performed, and the results indicated that there were 39 Sites P<0.05 were arranged in ascending order of p value, and the top 19 SNP sites with the highest correlation were selected for the second phase of MassARRAY flight mass spectrometry typing verification. Validated loci include: rs2236135, rs10849577, rs683369, rs492594, rs8133052, rs17036104, rs3788007, rs2276707, rs11807, rs4715332, rs1800822, rs7294. In the verification study, it was confirmed that rs492594 was still associated with the reversal of prediabetes, which was consistent with the results of the first phase study (OR (95% CI) = 1.634 (1.126-2.357), p = 9.7×10 ^-3 ).

After merging the data from the first phase and the second phase, the minor allele G of rs492594 and the curative effect of pioglitazone intervention were analyzed in additive, recessive, and dominant inheritance modes, respectively, after adjusting sex, age, and baseline BMI. Logistic regression analysis. It was found that the minor allele G of rs492594 is beneficial to the reversal of normal glucose tolerance after pioglitazone intervention (OR (95%CI)=1.499 (1.214-1.852), p=1.6×10 ^-4 in the additive mode), And the p-value drops further. The research results are shown in Table 1.

Table 1 Correlation between rs492594 locus and effective pioglitazone intervention ^*

*: In addition mode, after merging the data of the first stage and the second stage

Of the 761 prediabetic patients, 749 were successfully typed, 145 samples were GG genotype at rs492594 locus, 366 samples were CC genotype at rs492594 locus, and 238 samples were GC genotype at rs492594 locus.

The blood glucose changes of patients carrying different genotypes of rs492594 were compared, and the results are shown in Table 2. It can be found that from CC genotype, CG genotype to GG genotype, the degree of decline in oral glucose tolerance test (OGTT) 2-hour blood glucose and glycosylated hemoglobin (HbA1c) before and after pioglitazone intervention is gradually increasing, and the prediabetic patients with GG genotype The decrease degree of blood glucose and HbA1c at 2 hours of OGTT before and after pioglitazone intervention was the most significant, followed by CG genotype and CC genotype. However, baseline clinical characteristics were not different among the three genotypes, indicating that the decrease in blood glucose and HbA1c at 2 hours of OGTT before and after pioglitazone intervention was not related to the blood glucose level at baseline. The above results prove that the blood glucose and HbA1c are more significantly reduced and the curative effect is better after the prediabetic patients carrying the G gene use pioglitazone. It was also found that the improvement of insulin resistance (HOMA-IR) was the most obvious in prediabetic patients with GG genotype after intervention with pioglitazone.

Table 2 Comparison of related indicators of clinical characteristics among different genotypes of rs492594

*: HOMA-IR is non-normally distributed and expressed as median (interquartile range). The P value is obtained by ANOVA analysis after logarithmic transformation of the above parameters.

It can be seen from the above results that the rs492594 genotype of the diabetic patient to be tested can be detected to assist in the detection of blood glucose and/or HbA1c traits of the diabetic patient to be tested before and after pioglitazone intervention, as follows:

Detect the genotype of the rs492594 SNP site of the diabetic patient before and after the intervention of pioglitazone, and determine the blood glucose and/or HbA1c traits before and after the intervention of pioglitazone according to the genotype of the diabetic patient to be tested: After the intervention, the blood glucose and/or HbA1c decline degree is higher than or the candidate is higher than the rs492594 SNP site CC genotype or the rs492594 SNP site GC genotype in the diabetic patients to be tested after the pioglitazone intervention blood sugar and/or HbA1c decline degree; the GG The genotype is a homozygote for G at the rs492594SNP site, the GC genotype is a heterozygote for G and C at the rs492594SNP site, and the CC genotype is a homozygote for C at the rs492594SNP site;

The nucleotide of the above-mentioned rs492594SNP site is the 501st nucleotide of sequence 7 or the 11427th deoxyribonucleotide of the GenBank Accession Number NCBI Reference Sequence: NG_011682.1 (May 4, 2014) of the G6PC2 gene.

The degree of decrease in blood sugar after the intervention of pioglitazone is [(difference between blood sugar before pioglitazone intervention and blood sugar after pioglitazone intervention)/blood sugar before pioglitazone intervention] * 100%;

The reduction degree of HbA1c after pioglitazone intervention is [(difference between HbA1c before pioglitazone intervention and HbA1c after pioglitazone intervention)/HbA1c before pioglitazone intervention]*100%.

The above blood glucose is the OGTT 2-hour blood glucose.

The pioglitazone intervention refers to taking a dose of 30 mg per day for one year.

Therefore, the detection of rs492594 genotype can be used to screen prediabetic patients who are relatively sensitive to pioglitazone and improve the level of individualized treatment.

rs492594 is a single nucleotide polymorphism site on human chromosome 2, and the site variation is to switch G/C (on its complementary strand, it is C/G). The genotype of rs492594 locus is CC, CG or GG. The CC is homozygous for C at the rs492594 site, the GG is homozygous for G at the rs492594 site, and the CG is heterozygous for G and C at the rs492594 site. The detection of the polymorphism (ie allele) or genotype of rs492594 in the human genome can specifically be the detection of the nucleotide type of rs492594.

Example 2, Genotype detection of rs492594 site

1. Primer design for genotype detection of rs492594 locus

1. Detection principle

The rs492594 site is usually typed by chip detection and flight mass spectrometry. This method is complex and suitable for multi-site and high-throughput research, but for studies with fewer typing sites and fewer cases, the experimental The conditions and costs are relatively high, which is not conducive to the development of clinical testing. Based on the results of this study, it is necessary to design a convenient and practical kit.

Restriction fragment length polymorphism (RFLP), as the first generation of molecular biology markers, is widely used in biological research, with low cost and simple operation, and can be operated by general laboratories. The experimental principle is: DNA restriction endonuclease has the activity of recognizing a specific DNA sequence and cutting the DNA double strand at a specific position, and the DNA molecule is changed (or formed) due to mutation (nucleotide substitution, insertion or deletion) The recognition sequence of the restriction endonuclease enables the DNA restriction endonuclease to cut off the target DNA fragment or not, and the length of the DNA fragment can be detected by electrophoresis to determine the nucleotide type at a specific position.

2. rs492594 does not have a restriction endonuclease recognition site

Search found linkage disequilibrium between rs570876 and rs492594 (r ² =1), rs570876 site A allele (rs570876-A) and rs492594 site G allele (rs492594-C) constitute the haplotype, ie rs570876-A and rs492594-G can replace each other.

rs570876 is a single nucleotide polymorphism site on human chromosome 2, and the mutation is switching A/T (T/A on its complementary strand). The genotype of rs570876 locus is AA, TT or AT. The AA is a homozygous type whose rs570876 site is A, the TT is a homozygous type whose rs570876 site is T, and the AT is a heterozygous type whose rs570876 site is A and T. The detection of the polymorphism (ie allele) or genotype of rs570876 in the human genome can specifically be the detection of the nucleotide type of rs570876.

It can be concluded that the genotype AA of the rs570876 site corresponds to the genotype GG of the rs492594 site, the genotype TT of the rs570876 site corresponds to the genotype CC of the rs492594 site, and the genotype AT of the rs570876 site corresponds to the genotype of the rs492594 site The genotype GC of the point, so the genotype of the rs492594 locus can be determined by detecting the genotype of the rs570876 locus.

The rs570876 site is located in the recognition sequence of the restriction endonuclease ApoI, so PCR primers and restriction endonuclease types were designed for the rs570876 site.

3. PCR primers for rs570876

Forward primer: 5'-CCTGCATGTATGTGTGGGGT-3'; (SEQ ID NO: 1)

Reverse primer: 5'-TCTTCTTAAGGCCAGGCTGC-3'. (Sequence 2)

2. Genotype detection of rs570876 locus to determine the genotype of rs492594 locus

1. PCR amplification

The genomic DNA of the detection object is used as a template, and the forward primer and reverse primer in step 1 are used as primers to carry out PCR amplification to obtain PCR amplification products. The PCR amplification products are composed of DNA molecules shown in sequence 3 and sequence 4. At least one composition, there are three situations as follows:

A. PCR amplification products are all DNA molecules shown in sequence 3;

B, PCR amplification product is the DNA molecule shown in sequence 4;

C. The PCR amplification product is the DNA molecule shown in sequence 3 and the DNA molecule shown in sequence 4.

The 308th position of the PCR amplification product from the 5' end is a SNP site, which has two nucleotide types, A or T. If the SNP site is A, ApoI can cut the sequence containing this site in the PCR amplification product (i.e. the sequence shown in sequence 3), if the SNP site is T, ApoI can not cut The sequence containing this site (ie the sequence shown in sequence 4) in the PCR amplification product was cut.

2. ApoI digestion

Each PCR amplified product is digested with ApoI to obtain each digested product, and each digested product is carried out to 2% agarose gel (the ratio of agarose and electrophoresis buffer is 2g: 100ml) electrophoresis, and it appears as follows (1 )-(3) cases:

(1) The digestion product is two bands and the bands are located at about 307bp and 396bp;

(2) The digestion product is a band and the band is located at about 700bp;

(3) The digested products are three bands, and the positions of the bands are about 307bp, 350bp and 700bp respectively.

The bands of each digested product were sequenced, and the results were as follows:

The enzyme digestion products corresponding to the isolated blood samples of patients with GG genotype at the rs492594 site in prediabetic patients are two different DNA molecules of 307bp and 396bp bands, which are the DNA shown in sequence 5 (307bp) and sequence 6 (396bp) molecule, the PCR amplification product corresponding to enzyme digestion is the situation shown in A in step 1, that is, the PCR amplification product is the DNA molecule shown in sequence 3, indicating that the genotype of the rs570876 locus of the prediabetic patient is AA;

The enzyme digestion product corresponding to the in vitro blood sample of the CC genotype patient at the rs492594 site in patients with prediabetes is a band of about 700 bp, which is the DNA molecule shown in sequence 4, and the PCR amplification product corresponding to the enzyme digestion is the band in step 1 In the situation shown, that is, the PCR amplification products are all DNA molecules shown in sequence 4, indicating that the genotype of the rs570876 locus in the prediabetic patient is TT;

The enzyme digestion products corresponding to the isolated blood samples of patients with rs492594 locus GC genotype in patients with prediabetes were 307bp, 396bp and 700bp bands, which were three different DNA molecules, sequence 5 (307bp) and sequence 6 (396bp ), the DNA molecule shown in sequence 4 (700bp), the corresponding PCR amplification product is the situation shown in C in step 1, that is, the PCR amplification product is the DNA molecule shown in sequence 3 and sequence 4 The DNA molecules shown indicate that the genotype of the rs570876 locus in the prediabetic patient is AT.

Therefore, the genotype of the rs570876SNP site in the genomic DNA of the prediabetic patient to be tested can be determined to determine the genotype of the rs492594SNP site in the genomic DNA of the prediabetic patient to be tested. The genotype of the rs492594 SNP site of the prediabetic patient to be tested is GG, if the genotype of the rs570876 SNP site in the genomic DNA of the prediabetic patient to be tested is TT, the genotype of the rs492594 SNP site of the prediabetic patient to be tested is CC, and if the genotype of the rs492594 SNP site of the patient to be tested is CC. If the genotype of the rs570876 SNP site in the patient's genomic DNA is AT, then the genotype of the rs492594 SNP site of the prediabetic patient to be tested is GC.

The method for the genotype of the rs492594 SNP site of the prediabetic patient to be tested is as follows: the genomic DNA of the prediabetic patient to be tested is used as a template, and the DNA molecules shown in sequence 1 and sequence 2 are used as primers to perform PCR amplification to obtain PCR amplification products , and then digest the PCR amplification product with ApoI to obtain the digested product;

Detect the size of the digested product,

If the digestion products are two DNA molecules shown in sequence 5 (307bp) and sequence 6 (396bp), then the PCR amplification products are all DNA molecules shown in sequence 3, and the rs570897 genotype of the detected patient is AA, The genotype of rs492594 is GG; if the digested product is the DNA molecule shown in sequence 4 (703bp), the PCR amplification products are all DNA molecules shown in sequence 4, and the genotype of rs570897 of the detected patient is TT, and the genotype of rs492594 is CC; if the digested products are the three DNA molecules shown in sequence 5 (307bp), sequence 6 (396bp), and sequence 4 (703bp), then the PCR amplification product is the DNA molecule shown in sequence 3 and sequence 4. The DNA molecule shown in the figure shows that the rs570897 genotype of the detected patient is AT, and the rs492594 genotype is CG. This detection method is named RFLP detection method of rs492594 genotype.

3. Verification of the RFLP detection method for the genotype of rs492594

(1) From the genomic DNA of 761 prediabetic patients in step 1, DNA samples from 96 patients with existing microarray results were randomly selected. The genotype detection results of the rs492594 site of each patient were obtained by detecting and typing at the rs492594 site.

(2) Using the detected 96 cases of genomic DNA as templates and using the DNA molecules shown in Sequence 1 and Sequence 2 as primers for PCR amplification to obtain PCR amplification products, the PCR amplification products were digested with ApoI to obtain Enzyme digestion products; and perform sequencing analysis on the enzyme digestion products, and judge the genotype of the detected patients at rs492594 according to the following criteria:

If the digested product is the DNA molecule shown in sequence 5 (307bp) and sequence 6 (396bp), the corresponding PCR amplification products are all DNA molecules shown in sequence 3, and the genotype of rs570897 of the detected patient is AA, rs492594 The genotype is GG;

If the digestion product is the DNA molecule shown in sequence 4 (703bp), the corresponding PCR amplification products are all DNA molecules shown in sequence 4, and the rs570897 genotype of the detected patient is TT, and the rs492594 genotype is CC;

If the digestion product is shown in sequence 5 (307bp), sequence 6 (396bp), sequence 4 (703bp), the corresponding PCR amplification product is the DNA molecule shown in sequence 3 and the DNA molecule shown in sequence 4, the detected The patient's rs570897 genotype was AT and rs492594 genotype was CG.

The result is shown in Figure 2:

Figure 2A is the sequencing result of the rs570897 genotype of the patient whose rs492594 genotype is CC detected by the iSelect chip;

Figure 2B is the results of rs570897 genotype enzyme digestion electrophoresis in patients with rs492594 genotype CC detected by iSelect chip;

Figure 2C is the rs570897 genotype sequencing result of the patient whose rs492594 genotype is GG detected by the iSelect chip;

Figure 2D is the result of rs570897 genotype enzyme digestion electrophoresis in patients with rs492594 genotype GG detected by iSelect chip;

Figure 2E is the rs570897 genotype sequencing results of GC patients detected by iSelect chip as rs492594 genotype;

Figure 2F is the result of rs570897 genotype enzyme digestion electrophoresis of GC patients with rs492594 genotype detected by iSelect chip;

Figure 2G is a schematic diagram of enzyme digestion electrophoresis;

Statistics show that the genotype at rs570897 of 11 of the 96 patients detected is AA, and their genotype at rs492594 is GG, which is 100% consistent with the genotype at rs492594 detected by the iSelect chip;

The genotype at rs570897 of 45 of the 96 patients detected was AT, and their genotype at rs492594 was GC, which was 100% consistent with the genotype at rs492594 detected by the iSelect chip;

Among the 96 patients detected, the genotype at rs570897 of 40 patients was TT, and their genotype at rs492594 was CC, which was 100% consistent with the genotype at rs492594 detected by the iSelect chip.

Experiments have proved that the above-mentioned RFLP detection method for the genotype of rs492594 can detect the genotype of the rs492594 locus reliably, simply, economically and rapidly.

Claims (3)

1. The application of the product for detecting the genotype of the rs492594SNP site in the human genome or the product for detecting the genotype of the rs570876SNP site in the human genome in the preparation of products for predicting the hypoglycemic effect of pioglitazone on humans after intervention. 2. The application of the product for detecting the genotype of the rs492594 SNP site in the human genome or the product for detecting the genotype of the rs570876 SNP site in the human genome in the preparation of products for screening pre-diabetic patients sensitive to pioglitazone. 3. The application according to claim 1 or 2, characterized in that: the product is the DNA molecule shown in Sequence 1 and Sequence 2.