CN110734969A - PCR-RFLP method for detecting single nucleotide polymorphism of gene CREB1 - Google Patents

Abstract

The invention discloses PCR-RFLP methods for detecting single nucleotide polymorphism of gene CREB1, firstly, according to the sequencing result of a DNA pool, the detected gene polymorphism comprises single nucleotide polymorphism of a CREB1 gene promoter region from a transcription start point to-1354 bit T or G and single nucleotide polymorphism of-1343 bit T or A, taking the genome DNA of a sample to be detected as a template and a primer pair P as primers, carrying out PCR amplification on a CREB1 gene sequence containing a polymorphic site under the condition that Taq DNA polymerase, Buffer (Buffer environment), Mg + +, dNTPs exist, then dividing a PCR product into two parts, digesting the two parts by Hha I and Xsp I restriction enzymes respectively, and carrying out agarose gel electrophoresis typing on the enzyme digestion products.

Description

PCR-RFLP method for detecting single nucleotide polymorphism of gene CREB1

Technical Field

The invention belongs to the technical field of molecular biological detection, and particularly relates to PCR-RFLP methods for detecting single nucleotide polymorphism of gene CREB 1.

Background

Diabetes is considered to be the fifth most serious disease in the world, and 90% of the diseases are non-insulin-dependent diabetes mellitus, also called type II diabetes mellitus. The disease belongs to a complex metabolic disorder, the pathogenesis of which is complex and the result of genetic and environmental interactions, wherein genetic polymorphisms may be the major factors influencing disease susceptibility among individuals. Early clinical symptoms of type II diabetes are not obvious, often delaying the optimal treatment time for the patient. Therefore, the search and identification of genetic markers in the human genome associated with type II diabetes would facilitate early screening and diagnosis of the disease, and control the progression of the disease in a timely manner.

The genetic markers are mainly divided into two types, wherein the type I is an apparent marker which indirectly reflects the genetic variation at the DNA level, such as a morphological marker, a cell marker and a biochemical marker, and the type II is a molecular marker which directly reflects the genetic variation at the DNA level, the molecular markers are important components of the genetic markers and can reflect various genetic variations at the DNA level, including point mutation, deletion, insertion, translocation, inversion, rearrangement or repeated polymorphism with length and rearrangement less than .

The diagnosis technology developed based on the molecular marker can accurately detect the mutation sites related to the human complex diseases, and is an effective method for early screening and diagnosis of the diseases. The application of molecular marker diagnosis firstly detects genetic markers closely related to disease clinical detection indexes on a DNA level, secondly establishes a rapid detection method of the genetic polymorphism, and finally realizes the application of the molecular marker detection method in early disease diagnosis and susceptible population screening.

The Single Nucleotide Polymorphism (SNP) is an important component of a molecular marker and is widely distributed in a genome, the restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP) is effective techniques for detecting the SNP, if no natural restriction enzyme cutting site exists near the SNP site, a specific restriction enzyme cutting site is introduced through the PCR and is cut, and then agarose or polypropylene gel electrophoresis analysis is carried out, so that different genotypes of the SNP site can be accurately identified.

The CREB response element binding protein 1(CREB1) is important transcription factor containing leucine zipper structure domain, the increase of cAMP level in cells can activate CREB1, and further regulate the downstream gene containing CREs element, the research shows that CREB1 can also regulate the expression of the downstream gene by recruiting costimulatory molecule (such as PGC-1) to participate in the metabolic process of blood sugar and glucagon, the research of Herzig S and the like shows that the mice knocking out CREB1 gene show fatty liver phenotype and reduce the sensitivity of insulin (Herzig S, Hedrick S, Motterte I, Koo S-ranH, Galimi F, MontminM (2003) B controlled lipid metabolism nuclear hormone receptor PPAR- [ gamma ] 426. 193: 190. 193. the CREB1 gene is closely related to the generation of diabetes.

At present, the research on CREB1 gene at home and abroad mainly focuses on the aspects of function and regulation mechanism, and the genetic variation of CREB1 gene and the relevance of the genetic variation and the type II diabetes are not reported. Therefore, the research on the polymorphism of the type II diabetes susceptibility gene CREB1 is necessary, and the identification of the molecular marker related to type II diabetes in the CREB1 gene sequence has great practical significance for early screening and diagnosis of type II diabetes.

Disclosure of Invention

The invention aims to provide PCR-RFLP methods for detecting single nucleotide polymorphism of gene CREB1, and the polymorphic sites are taken as molecular markers.

The technical scheme adopted by the invention for solving the technical problems is as follows: the PCR-RFLP method for detecting the mononucleotide polymorphism of the type II diabetes susceptibility gene CREB1 is characterized in that blood genome DNA of a diabetic patient and a normal person is taken as a template, PCR amplification is carried out on P by utilizing a polymerase chain reaction primer under the condition that Taq DNA polymerase, a buffer environment, Mg + +, dNTPs exist, then restriction enzyme is utilized to carry out enzyme digestion on the P, and the mononucleotide polymorphism of a sample to be detected can be accurately identified through electrophoresis detection;

the polymerase chain reaction primer pair P is as follows:

an upstream primer P-F: 5'-CCTGGAGTACCAGGAAGGACAGC-3' 23nt

The downstream primer P-R: 5'-TTACACGTATGAGCCACC-3' 18nt

The underlined bases in the primer P-F are mismatched bases, so that the restriction enzyme cutting site of Hha I is introduced;

after PCR amplification, products are divided into two parts, parts of PCR products are digested by restriction enzyme Hha I, the fragments after digestion are subjected to agarose gel electrophoresis, the base polymorphism of the CREB1 gene at the-1354 th site is identified according to the agarose gel electrophoresis result, the other parts of PCR products are digested by restriction enzyme Xsp I, the fragments after digestion are subjected to agarose gel electrophoresis, and the base polymorphism of the CREB1 gene at the-1343 th site is identified according to the agarose gel electrophoresis result.

The PCR amplification conditions are as follows: 20 μ L reaction system, including 0.625U Taq DNA polymerase, 2 XBuffer 10 μ L (including Mg + +, dNTPs, etc.), 0.45 μ L genomic DNA, 10pmol/μ L upstream and downstream primers each 0.5 μ L and sterilized ultrapure water 8.3 μ L;

the PCR reaction program is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 54.5 ℃ for 30s, extension at 72 ℃ for 35s, 35 cycles; extension at 72 ℃ for 10 min.

The concentrations of the agarose gels were all 3.0%.

The polymorphism of the human CREB1 gene promoter region is detected as follows: a T > G mutation at position-1354 of the genome; a T > a mutation at position-1343 of the genome.

The base polymorphism of CREB1 gene at the-1354 th site is judged by electrophoresis as follows: the TT genotype is expressed as a 161bp band; TG genotypes appear as 161, 139 and 22bp bands; the GG genotypes showed 139 and 22bp bands.

The base polymorphism of the CREB1 gene at the-1343 th site is judged by electrophoresis as follows: the AA genotype is expressed as a 161bp band; AT genotypes appear as 161, 125 and 36bp bands; the TT genotypes appeared as 125 and 36bp bands.

Compared with the prior art, the DNA pool sequencing screening SNP and the PCR-RFLP are combined, so that the complexity and the instability of SSCP are solved, and genetic markers which are simple, rapid, low in cost, high in accuracy and convenient to screen and detect the genetic markers related to the type II diabetes susceptibility on the DNA level are provided, wherein the genetic markers are applied to .

The 2 detected SNP sites of the CREB1 gene are positioned in a promoter region, and the promoter is an important cis-acting element at the 5' end of a gene sequence and can be combined with a plurality of trans-acting factors to further regulate the expression level of the gene. When the promoter sequence is mutated, the original transcription factor binding site can be eliminated, and a new transcription factor binding site can be generated, and finally the promoter activity and the expression level of CREB1 protein are influenced.

The detection method provided by the invention lays a foundation for establishing the relationship between the CREB1 gene polymorphism and the type II diabetes.

Drawings

FIG. 1 is a diagram showing the sequencing result of SNP polymorphism of CREB1 gene-1354T > G mutation screened by DNA pool sequencing in the invention.

FIG. 2 is a diagram showing the sequencing result of SNP polymorphism of CREB1 gene-1343T > A mutation screened by DNA pool sequencing.

FIG. 3 shows the distribution of polymorphic sites of CREB1 genes-1354 and-1343 in type II diabetic patients and normal persons.

Detailed Description

The invention is described in further detail at , which is illustrative and not limiting, in accordance with routine experimentation or with the manufacturer's instructions, unless otherwise specified.

The PCR-RFLP method for detecting the single nucleotide polymorphism of the gene CREB1 is characterized in that: taking blood genome DNA of a diabetic patient and a normal person as a template, taking a primer pair P (F, R) as a primer, carrying out PCR amplification on a CREB1 gene, carrying out enzyme digestion on the CREB1 gene by using restriction endonuclease, and accurately identifying the single nucleotide polymorphism of a sample to be detected through electrophoresis detection; carrying out PCR amplification on P by using a polymerase chain reaction primer under the conditions of existence of Taq DNA polymerase, a buffer environment, Mg + +, and dNTPs, then carrying out enzyme digestion on P by using restriction enzyme, and accurately identifying the single nucleotide polymorphism of a sample to be detected by electrophoresis detection;

the polymerase chain reaction primer pair P is as follows:

an upstream primer P-F: 5'-CCTGGAGTACCAGGAAGGACAGC-3' 23nt

The downstream primer P-R: 5'-TTACACGTATGAGCCACC-3' 18nt

The underlined bases in the primer P-F are mismatched bases, so that the restriction enzyme cutting site of Hha I is introduced;

after PCR amplification, products are divided into two parts, parts of PCR products are digested by restriction enzyme Hha I, the fragments after digestion are subjected to agarose gel electrophoresis, the base polymorphism of the CREB1 gene at the-1354 th site is identified according to the agarose gel electrophoresis result, the other parts of PCR products are digested by restriction enzyme Xsp I, the fragments after digestion are subjected to agarose gel electrophoresis, and the base polymorphism of the CREB1 gene at the-1343 th site is identified according to the agarose gel electrophoresis result.

Wherein, the PCR amplification conditions are as follows: 20U L reaction system, including 0.625U Taq DNA polymerase, 2 x Buffer 10U L (including Mg + +, dNTPs, etc.), 0.45U L genome DNA, 10 pmol/U L upstream, downstream primer each 0.5U L and sterilized ultrapure water 8.3U L;

the PCR reaction program is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 54.5 ℃ for 30s, extension at 72 ℃ for 35s, 35 cycles; extension at 72 ℃ for 10 min.

Wherein, the concentration of the agarose gel is 3.0%.

And , detecting polymorphisms of a promoter region of the human CREB1 gene, wherein T AT the-1354 th site of the genome is greater than G mutation, T AT the-1343 th site of the genome is greater than A mutation, and the base polymorphisms of the CREB1 gene AT the-1354 th site are judged by electrophoresis, wherein TT genotypes are represented by 161bp bands, TG genotypes are represented by 161, 139 and 22bp bands, GG genotypes are represented by 139 and 22bp bands, the base polymorphisms of the CREB1 gene AT the-1343 th site are judged by electrophoresis, AA genotypes are represented by 161bp bands, AT genotypes are represented by 161, 125 and 36bp bands, and TT genotypes are represented by 125 and 36bp bands.

The invention utilizes a PCR-RFLP method to detect the mononucleotide polymorphism sites of the susceptibility gene CREB1 of the type II diabetes.

1. Blood sample collection

The invention specifically takes II type diabetes mellitus patients and normal individuals as detection objects, blood samples of 126 II type diabetes mellitus patients are collected from the department of endocrinology of Suzhou-shi hospital of Wuhan university, and blood samples of 100 normal people are collected from the department of physical examination of the school hospital of Wuhan university. The experimental contents have been approved by the ministry of medical ethics committee of the ministry of medical science of hospitals of the Wuhan university of science and technology.

2. Separation, extraction and purification of genome DNA

Reference is made to the Sambrock et al (2002) method.

3. Construction of DNA pools

The OD values of the DNA samples at 260nm and 280nm were measured by an ultraviolet spectrophotometer. The DNA content and the ratio OD260/OD280 were calculated. If the ratio of OD260/OD280 is less than 1.6, indicating that the sample contains more protein or phenol, then purification is carried out; if the ratio is greater than 1.8, RNA removal should be considered.

DNA concentration (ng) ═ 50 XOD 260 value Xdilution multiple

After the DNA detection is finished, fixed amount is taken out and diluted to 50 ng/. mu.L, then 30 individuals are randomly selected from type II diabetes patients and normal people respectively, and 10. mu.L of DNA sample of each individual is taken and mixed to construct a DNA pool.

4. CREB1 gene polymorphic site screening

① PCR amplification of CREB1 Gene promoter region

Sequence information of CREB1 gene (NC-000002.12) was obtained from NCBI database (http:// www.ncbi.nlm.nih.gov /), and primers for amplifying CREB1 gene promoter region were designed using Primer 5.0 and the Primer information was as follows:

an upstream primer 1-F: 5'-TGGGAGGTGGGACATGAAAG-3' 20nt

Downstream primer 1-R: 5'-TCAGAGGTCTCTCAGGACGG-3' 20nt

The constructed DNA pool is used as a template, the designed primers are used for PCR amplification, a mixed sample adding method is adopted for a PCR reaction system, namely the total amount of various reaction components is calculated according to the quantity of various components required by every reaction systems and the quantity of PCR reactions required by 1 reaction, the total amount is added into 1 1.5mL centrifuge tubes, the mixture is subjected to instantaneous centrifugation after being fully mixed, the mixture is then subpackaged into 0.2mL Eppendorf PCR tubes, template DNA is added, and the PCR amplification is carried out after the instantaneous centrifugation.

The PCR reaction system is as follows: a20. mu.L reaction system comprising 0.625U of Taq DNA polymerase, 10. mu.L 2 XBuffer (containing Mg + +, dNTPs, etc.), 0.45. mu.L genomic DNA, 0.5. mu.L each of 10 pmol/. mu.L upstream and downstream primers, and 8.3. mu.L sterilized ultrapure water.

The PCR reaction program is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 54.5 ℃ for 30s, extension at 72 ℃ for 35s, 35 cycles; extension at 72 ℃ for 10 min.

② DNA pool sequencing analysis

The PCR amplification product is sent to Wuhan Pongziaceae biotechnology limited company for gel cutting purification and sequencing, and a sequencing peak map is analyzed, wherein two different peaks at the same site have single nucleotide mutation, T, G detection results appear at the 3 rd site from the left of the figure 1, and T, A detection results appear at the 3 rd site from the left of the figure 2, namely the invention screens 2 SNP sites of CREB1 gene, wherein the nucleotide polymorphism of the CREB1 gene with T or G at the-1354 site can be expressed as-1354T > G, and the nucleotide polymorphism of the CREB1 gene with T or A at the-1343 site can be expressed as-1343T > A.

5. PCR-RFLP detection of CREB1 gene polymorphic site

① polymorphic site analysis

When the CREB1 gene site-1354 generates T > G mutation, other original sequences near T base can not form recognition sequence of endonuclease, at this time, mismatch is introduced by primer, so that primer 3 ' end and mutant type ' G ' at this point form recognition sequence GCGC of Hha I restriction endonuclease after PCR amplification, thus when the site-1354 generates T > G mutation, namely T mutation is G, the GCTG sequence correspondingly becomes recognition sequence GCGC of endonuclease, thus forming recognition site of Hha I restriction endonuclease, the mutation can be detected by Hha I-PCR-RFLP method, when the CREB1 gene site-1343 generates T > A mutation, the original sequence CTAG of Xsp I restriction endonuclease is correspondingly changed into GAAG, which belongs to natural restriction enzyme cutting site, and can be directly detected by Xsp I-PCR-RFLP method.

② PCR-RFLP primer design

Aiming at the T > G mutation at the-1354 th site of the CREB1 gene, designing a mismatch primer pair P by utilizing a technology of introducing an enzyme cutting site, wherein an upstream primer sequence is as follows:

P-F:5’-CCTGGAGTACCAGGAAGGACAGC-3’23nt

g is introduced with mismatched base, and forms a recognition sequence of Hha I restriction enzyme with mutant type 'G';

in addition, the original Xsp I restriction endonuclease recognition site can disappear due to the mutation of the T & gtA at the position-1343 of the CREB1 gene, the restriction endonuclease recognition site belongs to a natural enzyme digestion site, and the distance between the site and the mutation site of the T & gtG at the position-1354 is only 12bp apart on a genome, so that the method innovatively detects 2 mutation sites simultaneously by using pairs of primers (P), and has the advantages of simplicity, rapidness, cost saving and the like compared with the traditional method.

The PCR reaction system of the primer pair P is as follows: a20. mu.L reaction system comprising 0.625U of Taq DNA polymerase, 10. mu.L 2 XBuffer (containing Mg + +, dNTPs, etc.), 0.45. mu.L genomic DNA, 0.5. mu.L each of 10 pmol/. mu.L upstream and downstream primers, and 8.3. mu.L sterilized ultrapure water.

The PCR reaction program of the primer pair P is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 54.5 ℃ for 30s, extension at 72 ℃ for 35s, 35 cycles; extension at 72 ℃ for 10 min.

③ PCR product enzyme digestion and RFLP detection

20 mu L of the PCR product was divided into two portions, cleaved with Hha I and Xsp I, respectively, and then the SNP polymorphism was determined based on the results of electrophoresis.

1) The enzyme cutting system is 20 mu L and comprises: mu.L of PCR product, 1. mu.L (10U/. mu.L) of restriction enzyme, 2. mu.L of digestion Buffer, and 7. mu.L of sterilized distilled water.

2) Digestion conditions of enzyme digestion: digesting for 5-10 h in a constant temperature incubator at 37 ℃.

3) After digestion, the sample is put in water bath at 65 ℃ for 5min to terminate the digestion reaction, agarose Gel electrophoresis is carried out at 120V, the digestion result is detected by EB staining, the BIO-RAD Gel Doc 2000 Gel imaging analysis system is used for photographic analysis, and the genotype is judged and recorded.

The base polymorphism at the-1354 th site of the CREB1 gene is as follows: the TT genotype is expressed as a 161bp band; TG genotypes appear as 161, 139 and 22bp bands; the GG genotypes showed 139 and 22bp bands.

The base polymorphism at the position-1343 of the CREB1 gene is as follows: the AA genotype is expressed as a 161bp band; AT genotypes appear as 161, 125 and 36bp bands; the TT genotypes appeared as 125 and 36bp bands.

6. Distribution difference of CREB1 gene polymorphic site in population

The genotype distribution of CREB1 gene polymorphic sites in type II diabetes patients (T2D, n ═ 126) and normal individuals (Control, n ═ 100) is shown in figure 3, and for the-1354 mutation site, GG genotype belongs to the dominant genotype in diabetes patients and has the lowest frequency in normal people; for the-1343 mutation site, the AA genotype is the dominant genotype in diabetic patients, while the dominant genotype in normal persons is TA. It can be seen that the GG genotype at position-1354 and the AA genotype at position-1343 may be related to susceptibility to type II diabetes.

Sequence listing

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Claims (7)

1, PCR-RFLP method for detecting single nucleotide polymorphism of gene CREB1, which is characterized in that blood genome DNA of diabetics and normal people is taken as a template, PCR amplification is carried out on P by using polymerase chain reaction primers under the condition that TaqDNA polymerase, buffer environment, Mg + +, dNTPs exist, then restriction enzyme is used for carrying out enzyme digestion on P, and the single nucleotide polymorphism of a sample to be detected can be accurately identified through electrophoresis detection.

2. The PCR-RFLP method for detecting the mononucleotide polymorphism of gene CREB1 as claimed in claim 1, wherein the primer pair P of polymerase chain reaction is:

an upstream primer P-F: 5'-CCTGGAGTACCAGGAAGGACAGC-3' 23nt

The downstream primer P-R: 5'-TTACACGTATGAGCCACC-3' 18nt

The underlined bases in the primer P-F are mismatched bases, so that the restriction enzyme cutting site of Hha I is introduced;

after PCR amplification, products are divided into two parts, parts of PCR products are digested by restriction enzyme HhaI, then the fragments after digestion are subjected to agarose gel electrophoresis, the base polymorphism of the CREB1 gene at the-1354 th site is identified according to the agarose gel electrophoresis result, the other parts of PCR products are digested by restriction enzyme Xsp I, then the fragments after digestion are subjected to agarose gel electrophoresis, and the base polymorphism of the CREB1 gene at the-1343 th site is identified according to the agarose gel electrophoresis result.

3, PCR-RFLP method for detecting mononucleotide polymorphism of gene CREB1 as claimed in claim 1, wherein the PCR amplification condition is 20 μ L reaction system including 0.625U Taq DNA polymerase, 2 XBuffer 10 μ L (including Mg + +, dNTPs, etc.), 0.45 μ L genome DNA, 10pmol/μ L upstream and downstream primers 0.5 μ L each and 8.3 μ L sterilized ultrapure water;

the PCR reaction program is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 54.5 ℃ for 30s, extension at 72 ℃ for 35s, 35 cycles; extension at 72 ℃ for 10 min.

4. The PCR-RFLP method for detecting the mononucleotide polymorphism of gene CREB1 as claimed in claim 1, wherein the concentration of agarose gel is 3.0%.

5. The PCR-RFLP method for detecting single nucleotide polymorphism of gene CREB1 according to claim 1, wherein the polymorphism of the detected human CREB1 gene promoter region is as follows: a T > G mutation at position-1354 of the genome; a T > a mutation at position-1343 of the genome.

6. The PCR-RFLP method for detecting single nucleotide polymorphism of gene CREB1 according to claim 1, characterized in that the base polymorphism at the No. 1354 of CREB1 gene is judged to be: the TT genotype is expressed as a 161bp band; TG genotypes appear as 161, 139 and 22bp bands; the GG genotypes showed 139 and 22bp bands.

7. The PCR-RFLP method for detecting single nucleotide polymorphism of gene CREB1 according to claim 1, characterized in that the base polymorphism of the CREB1 gene at the-1343 th site is judged by electrophoresis as follows: the AA genotype is expressed as a 161bp band; AT genotypes appear as 161, 125 and 36bp bands; the TT genotypes appeared as 125 and 36bp bands.

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