CN105274204A - Method and kit for determination of human PLIN1 gene rs894160 site polymorphism - Google Patents

Abstract

A detection method and a kit for the polymorphism at the rs894160 site of the human PLIN1 gene. The method includes: providing human genomic DNA to be tested; providing an upstream primer and a downstream primer for amplifying the sequence near the rs894160 site of the human PLIN1 gene, wherein the downstream primer has a mismatched base G; using the human genomic DNA to be tested as a template , using upstream primers and downstream primers to carry out PCR amplification reactions to obtain amplification products containing ARGCTT fragments or RGCT fragments, wherein R is the undetermined base A or G on the rs894160 site of the human PLIN1 gene; provide restriction endonucleases; use Restriction endonuclease digests the obtained amplified product to obtain the corresponding digested product; and according to the obtained digested product, it is determined whether the undetermined base R on the rs894160 site of the human PLIN1 gene is A or G. The restriction endonuclease is a restriction endonuclease capable of cleaving either one of the AAGCTT fragment and the AGGCTT fragment, or one of the AGCT fragment and the GGCT fragment. The measuring means of the invention is not only fast and reliable, but also the measuring cost is greatly reduced.

Description

Human PLIN1 gene rs894160 polymorphism detection method and kit

technical field

The present invention relates to methods for determining single nucleotide polymorphisms.

Background technique

SNP (Single Nucleotide Polymorphism) refers to the DNA sequence variation caused by the change of a single nucleotide, including single base substitution, insertion and deletion. Gene polymorphism is an individual's innate genetic characteristics, does not change with the environment, and is not a specific or non-specific clinical manifestation of a certain disease, so its application does not have diagnostic and therapeutic functions.

Gene polymorphism detection is widely used in the field of genetics and medicine, examples are as follows: 1. To study genetic phenomena such as the origin and evolution of species. According to the genetic variation, the information of biological macromolecules can be obtained, the history of biological evolution can be inferred, and the evolution relationship of species can be clarified. It is used in archaeology to determine the genetic variation characteristics of ancient humans and the genetic relationship of different populations. 2. To study genetics such as polymorphism and population kinship. Polymorphism is also closely related to various human characteristics, including height, weight, skin color, facial features and so on. 3. In the field of preventive medicine, it can be used for disease prevention measures. Through the study of the tolerance of the human body to poisons, individuals who are prone to toxic effects on certain poisons are found, and the contact of the individuals with the poisons is avoided in time to protect the susceptible population. For example, conduct polymorphic detection on people who are going to be exposed to organic solvents such as benzene, screen out individuals who are prone to blood toxicity, neurotoxicity and other reactions, keep them away from organic solvents such as benzene, and protect such susceptible people from poisons . 4. It can be used in drug selection and dosage determination in pharmacology. Through polymorphic detection, it can be judged that the sick individual is sensitive to the toxic side effect of a certain drug, so as to avoid the use of this drug to avoid its toxic effect. Determine the drug dose by judging the individual's response to the drug effect, reducing the drug dosage and its side effects.

The PLIN1 gene is located on chromosome 15q26.1, and the encoded perilipin protein plays an important role in the process of fat metabolism. PLIN1 gene rs894160 polymorphism, there are two alleles A and G in the population, forming three genotypes of PLIN1 gene: AA type (the two allele bases of the rs894160 polymorphic site in the human genome are both A), AG type (the two allelic bases of the rs894160 polymorphic site in the human genome are A and G respectively) and GG type (the two allelic bases of the rs894160 polymorphic site in the human genome are both G) .

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technology is a fast, simple, accurate and low-cost classical method for determining SNP (single nucleotide polymorphism) genotype. In the method, primers are used to amplify the template to form a sufficient amount and stable amplification product, and the SNP is finally determined by digesting the amplified product and detecting the fragment length of the digested product. The rs894160 polymorphic site of PLIN1 gene is detected by PCR-RFLP technology, and the required endonuclease is MnlI endonuclease, which is expensive and requires the development of new detection technology. Similar methods can be found in, for example, the inventor's previous patent 102424849B (the entire content of which is incorporated herein by reference) and the like. However, there are still many defects in the existing determination methods, which need to be improved urgently.

Contents of the invention

The purpose of the present invention is to provide a reliable means for determining the rs894160 polymorphism of human PLIN1 gene for non-disease diagnosis or treatment purpose.

According to the first aspect of the present invention, a method for determining the polymorphism at the rs894160 site of the human PLIN1 gene is provided, comprising:

Provide human genomic DNA to be tested;

Provide an upstream primer and a downstream primer for amplifying the sequence near the rs894160 site of the human PLIN1 gene, wherein the downstream primer has a mismatched base G;

Using the human genomic DNA to be tested as a template, carry out PCR amplification reaction using upstream primers and downstream primers to obtain amplification products containing ARGCTT fragments or ARGT fragments, wherein R is the undetermined base A or G;

Provide restriction enzymes;

Carry out enzyme digestion (reaction) to the obtained amplified product with a restriction endonuclease to obtain a corresponding enzyme digestion product; and

Determine whether the undetermined base R on the rs894160 site of the human PLIN1 gene is A or G according to the obtained enzyme digestion product,

Wherein, the restriction endonuclease is a restriction endonuclease capable of cutting one of the AAGCTT fragment and the AGGCTT fragment, or one of the AGCT fragment and the GGCT fragment.

According to an embodiment of the present invention, on the obtained amplification product, there is a base C between the mismatched base G of the downstream primer and the paired base of R. Surprisingly, setting mismatched bases in this way will make the enzyme digestion reaction go on extremely smoothly, and there will be no phenomenon such as insufficient enzyme digestion. Moreover, setting the downstream primers with mismatched bases in this way enables the PCR reaction to proceed smoothly and improves the sensitivity and specificity of PCR, which may be related to changes in the secondary structure of the amplified sequence after the mismatched bases.

In a specific embodiment of the present invention, on the obtained amplification product, the terminal base of the downstream primer is adjacent to the paired base of R. For example, the end of the downstream primer can be a structure such as ... GC.

In a preferred embodiment of the present invention, the restriction endonuclease can be HindIII or its isoschizase which can only cut the AAGCTT fragment. This kind of HindⅢ enzyme is cheap and can greatly reduce the cost of determination.

According to an embodiment of the present invention, the resulting enzyme digestion products include three types of fragments: uncut amplification products with the total fragment length, amplification products with longer fragments after cleavage, and (usually not effectively observable) cleaved amplification products with Amplification products of shorter fragments (often not effectively visualized). Determine whether the undetermined base at the rs894160 site of the human PLIN1 gene is A or G by observing (judging) the type of fragment contained in the digestion product. For example, in the case of using HindⅢ enzyme, judge according to the observation results of two fragments, the total fragment and the longer fragment after cleavage: if only one uncleaved amplification product with the total fragment length is observed, Then the undetermined base is G; if it is observed that there is only one cut product with a longer fragment (less than the total fragment length), the undetermined base is A; if the above two are observed, then the undetermined base includes both A includes G in turn.

In one embodiment of the present invention, judging (or observing) the types of fragments contained in the digested products is carried out by gel electrophoresis combined with ultraviolet light taking pictures and comparing with reference pictures. In this case, the preferred reference image is obtained after the amplification product is photographed by an ultraviolet light after the gel electrophoresis standard setting time and only has a first reference image position and a second reference image position, wherein the first reference image position corresponds to The first reference image position is for amplicons with untruncated total fragment lengths, while the second reference image position is for amplified products with longer fragments after cleavage. For example, the reference image used in the present invention is obtained by photographing the synthesized 150bp and 99bp two base fragments through gel electrophoresis at the same time and then taken by ultraviolet light. Compared with the composite reference image of the conventional DNAladder, since the reference image with only two specific reference image positions is used, the method of the present invention can intuitively and quickly observe or judge the type of fragment contained in the digestion product, while maximizing to avoid misjudgment. After the enzyme cleavage reaction, it is preferable to concentrate the enzyme cleavage product at a concentration of 1 to 2 times and then perform electrophoresis to increase the brightness of the image and improve the accuracy of judgment.

In a preferred embodiment of the present invention, the total fragment length of the amplified product is between 100 and 300 bp by designing the length of the upstream primer and the downstream primer, and the fragment length of the upstream primer is at least 35 bp, preferably 40 to 60 bp ( In this preferred range, the amplification reaction is particularly smooth and sufficient), so that the length of some fragments excised by enzyme digestion accounts for more than 20% of the total fragment length. The design of the present invention can make the positions of the uncut amplification product with the total fragment length and the electrophoretic ultraviolet photo of the amplified product with longer fragments significantly different after cutting. However, if the total fragment is too long, the electrophoretic shift will not be obvious; if it is too short, the image will become blurred and cannot be accurately distinguished. The fragment length range of the upstream primer ensures that the PCR amplification reaction can proceed smoothly, and avoids insufficient amplification that occurs when there is a base mismatch.

In a preferred embodiment of the present invention, by controlling the scope of the annealing temperature in the PCR amplification program between 55～70°C, the preferred temperature is 60～69°C (this temperature can improve the specificity of the amplification reaction), so that the designed The PCR product has a high purity. The design of the present invention can make the bands of the PCR amplification products clear, free from miscellaneous bands, and easy to identify by electrophoresis. However, if the temperature is too low, there will be few specific bands and too many miscellaneous bands, making it difficult to distinguish and judge after electrophoresis; if the temperature is too high, it will affect the efficiency of PCR amplification, so that there are too few specific amplification products, which will affect the observation effect.

In a preferred embodiment of the present invention, by controlling the scope of the extension time in the PCR amplification program between 10-60s, preferably 15-30s (this time can improve the efficiency of the amplification reaction and the specificity of the amplified product) , so that the designed PCR reaction time is shorter and the purity of the amplified product is higher. The design of the present invention can make the strips of the PCR amplification products clear, free of miscellaneous bands, easy to identify by electrophoresis, and the PCR time is short. However, if the time is too short, the specific bands cannot be amplified completely, resulting in fewer amplified products, and it is difficult to distinguish and judge after electrophoresis; if the time is too long, the probability of non-specific bands will increase, and the appearance of mixed bands will affect the observation effect .

In the present invention, the PCR reaction components may include DNA template, upstream and downstream primers, TaqDNApolymerase (DNA polymerase or also called "Taq enzyme"), buffer, Mg ²⁺ and so on. In a preferred embodiment of the present invention, TaqDNApolymerase (DNA polymerase) and its TaqAntibody can be used in the PCR amplification reaction. TaqAntibody is a monoclonal antibody of TaqDNApolymerase. It binds to TaqDNApolymerase and inhibits the activity of DNA polymerase. The affinity between the two is very high. Even at 65°C, it can still block the activity of TaqDNApolymerase, so it can effectively inhibit the non-specific annealing and annealing of primers. The non-specific amplification caused by primer dimer has extremely high amplification sensitivity and specificity. ChampagneTaqAntibody only needs to be heated at 95°C for 30s to completely inactivate and release TaqDNApolymerase activity, ensuring the smooth progress of subsequent PCR amplification reactions.

Tris-HCl buffer with a pH value of 8.1-8.7 can be added to the PCR reaction, and the pH value of the PCR solution should be adjusted between 6.8-7.8 during the extension reaction at 72°C, so that the Taq enzyme can perform better in an alkaline environment. active. In addition, gelatin (0.01%) can also be added to the PCR solution to reduce the adsorption of the PCR tube to the Taq enzyme, stabilize the enzyme activity and protection, and promote the PCR reaction.

In addition, when the concentration of Mg ²⁺ in the PCR solution is between 1.5 and 2.0 mmol/L, the yield and specificity of the PCR reaction can be well controlled.

The final concentration of PCR primers is generally about 0.1-1 μM. If the concentration is too high, it will lead to non-specific amplification, and if it is too low, the amplified product will be too little.

In addition, cosolvents dimethyl sulfoxide (DMSO) and ammonium sulfate can also be added to the PCR reaction to reduce the level of base mismatch and improve the amplification efficiency of GC-rich templates. This may be completely related to eliminating the secondary structure of primers and templates, lowering the melting temperature of DNA and denaturing DNA.

According to another aspect of the present invention, there is provided a kit for determining the polymorphism at the rs894160 site of the human PLIN1 gene, which comprises:

Upstream primers and downstream primers for PCR amplification of sequences near the rs894160 site of the human PLIN1 gene, wherein the downstream primers have mismatched base G to obtain amplification products containing ARGCTT fragments or RGCT fragments, where R is the rs894160 site of the human PLIN1 gene The pending base A or G on ; and

A restriction enzyme that recognizes one of the AAGCTT fragment and the AGGCTT fragment, or a restriction enzyme that recognizes one of the AGCT fragment and the GGCT fragment.

The above kit may also include other components, such as PCR reaction Mix (including 4 kinds of dNTP mixture, Mg ²⁺ , TaqDNApolymerase, TaqAntibody, buffer, DMSO and ammonium sulfate) and instructions for performing the assay, etc.

Those skilled in the art can understand that, unless there is an obvious conflict, the relevant features of the first aspect and the second aspect of the present invention can be combined with each other.

The measuring means of the invention is not only fast and reliable, but also the measuring cost is greatly reduced.

Description of drawings

Fig. 1 has described the electrophoresis ultraviolet photo of the digestion product obtained according to the method of the present invention. Where Marker is: standard reference position; lane 1: AA genotype; lane 2: AG genotype; lane 3: GG genotype.

detailed description

The present invention is described in detail below. It should be understood by those skilled in the art that the following detailed description is only for illustrating rather than limiting the present invention.

(1) Acquisition of DNA to be tested

Peripheral blood was collected from different populations, and the genomic DNA was extracted to determine the polymorphism of the rs894160 site of the PLIN1 gene as follows.

For the selection of DNA to be tested, the present invention is not particularly limited. It can be an in vitro sample obtained from body fluid or tissue of the human body, or a pre-degraded genome. For ease of practice, it is generally preferred to extract ex vivo samples from blood. The tissues mentioned therein include tissues containing all or at least the PLIN1 gene in the human body, regardless of whether the PLIN1 gene is expressed or not. Methods for extracting DNA from body fluids and/or tissues are well known to those skilled in the art, and can be performed with reference to commonly used molecular biology manuals, such as "Molecular Cloning" 2nd edition.

(2) Primer design and synthesis

Search the Gene database and the SNP database of the NCBI website to obtain the complete sequence of the PLIN1 gene and the information of the rs894160 polymorphic site, respectively. The nucleotide sequence before and after the polymorphic site R of rs894160 (the nucleotide sequence is represented by 5'→3', with the same uppercase and lowercase meanings) is as follows (SEQ ID NO: 1):

Upstream primers and downstream primers are designed according to the gene sequence, wherein the downstream primers introduce mismatched bases. On the final amplified product, there is a base C between the mismatched base G of the downstream primer and the paired base of R.

In the present invention, the downstream primer has mismatched base G and is 35-60 bp in length. In one embodiment, the primers are as follows, the upstream primer: 5'TGCAGTGGGCAGAACCTCACGT3' (SEQ ID NO: 2), the downstream primer: 5'AGAAATTGACTGAGCAAGGGGGCTGGTATCTCCTGAGGCACATTCTAA G C3' (SEQ ID NO: 3), wherein the underlined bases of the downstream primers are mismatched bases.

(3) Preparation of PCR amplification products

According to the characteristics of upstream primers and downstream primers and corresponding PCR reagents, PCR amplification procedures and conditions are formulated to prepare PCR amplification products. In one of the examples, take genomic DNA (50-80ng), upstream and downstream primers (0.1-1 μM final concentration), 2×PCRMix 7.5 μL (including 4 kinds of dNTP mixture, Mg ²⁺ , TaqDNApolymerase, TaqAntibody, buffer, DMSO and ammonium sulfate) and double distilled water to form a 15 μL reaction system, and adjust the pH of the solution to about 7.3. The PCR reaction conditions were: pre-denaturation at 95°C for 30s, denaturation at 95°C for 30s→annealing at 65°C for 30s→extension at 72°C for 20s, a total of 30 cycles, and extension at 72°C for 7min. A stable amplification product was obtained after the PCR reaction.

(4) enzyme digestion reaction

The present invention can adopt HindⅢ endonuclease or AluI endonuclease, and provides multiple selectable endonucleases for the polymorphism identification of PLIN1 gene rs894160 site, and can select suitable endonucleases according to the market price during experiments. At present, the price of these endonucleases is relatively low compared with the endonuclease Mnl that can be used when there is no base mismatch near the polymorphic site, as shown in Table 1.

Table 1 Recognition sequences and prices of several endonucleases from NEB Company

Note: N is A or T or C or G

In one example, take 10 μL of PCR product, add 5U of endonuclease HindIII or endonuclease AluI, 2 μL of 10× digestion buffer, and 7.5 μL of double distilled water to form a 20 μL reaction system, and digest in a water bath at 37°C for 4 to 12 hours. The digested product was obtained, and the digested product was concentrated by 1 times and observed by electrophoresis.

(5) Electrophoresis test

In one of the examples, the PCR product is still 150 bp if it cannot be cut after HindⅢ enzyme digestion, and 99 bp and 51 bp if it is cut (because HindⅢ enzyme digestion produces a cohesive end that makes the number of bases in the complementary chain different, so the cut The length of the fragment after opening is calculated based on the number of single-strand bases on the gene sequence), and the 51bp fragment is difficult to distinguish in the electropherogram.

The digested product was subjected to electrophoresis for 30-80 min in 2-4% agarose gel under the condition of 3-8 V/cm, and photographed under ultraviolet light for determination. Enzyme electrophoresis is shown in Figure 1. The genotype is judged based on the presence or absence of 150bp and 99bp fragments: GG type has a 150bp fragment, AG type has 150bp and 99bp fragments, and AA type has a 99bp fragment.

The following are several examples of implementing the present invention.

Example 1 Extraction of Human Peripheral Blood Leukocyte DNA Determination of rs894160 Polymorphism

1 Materials and methods

1.1 Main reagents and instruments

Reagent: 2×PCRMix (including 4 kinds of dNTP mixture, Mg ²⁺ , TaqDNApolymerase, TaqAntibody, buffer, DMSO and ammonium sulfate) (Vazyme Company), restriction endonuclease HindIII (NEB Company), agarose (Biowest Company) , primers were synthesized by Shanghai Sangon Company.

Instruments: Model 9600 PCR instrument (PE Company), mini electrophoresis tank (PharmaciaBiotech, EPS1000), GelDoc2000 gel imager (Bio-RAD Company).

1.2 Extract DNA from peripheral blood leukocytes as the template of genomic DNA to be tested

1mL of human peripheral blood was collected in EDTA-K ₂ anticoagulant tubes, leukocytes were separated by referring to the leukocyte separation method in "Practical Flow Cytometry Color Atlas", and leukocyte genomic DNA was extracted by referring to the sodium chloride salting out method in "Molecular Cloning", and As the human genome DNA template to be tested.

1.3 Sequence search and primer design

Search the PLIN1 gene sequence and rs894160 polymorphic site information on the NCBI website to design primers, as follows:

Upstream primer: 5'TGCAGTGGGCAGAACCTCACGT3' (SEQ ID NO: 2);

Downstream primer: 5' AGAAATTGACTGAGCAAGGGGGCTGGTATCTCCTGAGGCACATTCTAA G C3' (SEQ ID NO: 3)

1.4PCR amplification

Take genomic DNA (50-80ng), upstream and downstream primers (final concentration: 0.1-1 μM), 2×PCRMix 7.5 μL (including 4 kinds of dNTP mixture, Mg ²⁺ , TaqDNApolymerase, TaqAntibody, buffer, DMSO and ammonium sulfate), Make up 15 μl of the reaction system with sterilized double distilled water, and adjust the pH of the solution to about 7.3.

The PCR reaction conditions were: pre-denaturation at 95°C for 30s, denaturation at 95°C for 30s→ annealing at 65°C for 30s→ extension at 72°C for 20s, a total of 30 cycles, and extension at 72°C for 7min. A stable amplification product was obtained after the PCR reaction.

1.5 Enzyme digestion identification

After PCR amplification, take 10 μl of the PCR product, add 5U endonuclease HindIII, 2 μl 10× digestion buffer and sterilized double distilled water to form a 20 μl reaction system, and digest in a water bath at 37°C for 4 hours to obtain the digested product.

After the above digested product was concentrated by 1 times, it was electrophoresed for 40 min under the condition of 3% agarose gel 5V/cm, and the polymorphic type was identified by taking pictures under ultraviolet light.

2 results

2.1PCR amplification results

The amplified sequence (which is located at 402-551 of the base sequence of SEQ ID NO: 1, 150 bp in total), the obtained amplification product sequence is as follows (SEQ ID NO: 4):

The underlined parts in the amplified product sequence are the sequences corresponding to the upstream and downstream primers respectively, and R represents the A/G polymorphism, that is, the SNP site rs894160.

2.2 Digestion results

The product sequences after digestion with endonuclease HindIII are as follows:

When the polymorphic site contains an A allele, the PCR product can be digested to form a cut fragment of 99 bp (the downstream sequence of the fragment is increased by 4 bases compared with the upstream sequence due to the formation of sticky ends by enzyme digestion) sequence (SEQ ID NO: 5):

When the polymorphic site contains an A allele, the PCR product can be digested to form a cut fragment of 51 bp (the downstream sequence of the fragment is reduced by 4 bases compared with the upstream sequence due to the formation of sticky ends by enzyme digestion) sequence (SEQ ID NO: 6):

agcttagaatgtgcctcaggagataccagcccccttgctcagtcaatttct51

After electrophoresis, the digested products were photographed and identified under ultraviolet light, and the results are shown in Figure 1. Among them, a 150bp fragment appeared after the rs894160 site was amplified. Three fragments of 150bp, 99bp and 51bp will appear in the electrophoresis after digestion with HindIII (51bp of which is not easy to distinguish in the electropherogram).

Genotype judgment was performed by electrophoresis after HindIII digestion: as shown in Figure 1, the GG type has a fragment of 150 bp, the AG type has two fragments of 150 bp and 99 bp, and the AA type has a fragment of 99 bp.

2.3 PLIN1 gene rs894160 polymorphism results

A total of 88 cases were detected, and the results found that 56 cases of GG type, 22 cases of AG type and 10 cases of AA type were found at the rs894160 locus.

Example 2 Determination of rs894160 polymorphism in human peripheral blood whole blood samples

The main reagents and instruments are the same as in Example 1. Refer to the phenol-chloroform extraction method in "Molecular Cloning Technology" to extract peripheral blood genomic DNA and use it as a human genomic DNA template to be tested.

The sequence search was the same as in Example 1, and the primer sequences were designed as follows:

Upstream primer: 5'CTGCAGTGGGCAGAACCTCACGTG3' (SEQ ID NO: 7);

Downstream primer: 5'AAGAAATTGACTGAGCAAGGGGGCTGGTATCTCCTGAGGCACATTCTAA G C3' (SEQ ID NO: 8)

Take genomic DNA (50-80ng), upstream and downstream primers (final concentration: 0.1-1 μM), 2×PCRMix 10 μL (including 4 kinds of dNTP mixture, Mg ²⁺ , TaqDNApolymerase, TaqAntibody, buffer, DMSO and ammonium sulfate), and sterilize Make up 20 μl of the reaction system with double distilled water, and adjust the pH of the solution to about 7.3.

The PCR reaction conditions were: pre-denaturation at 95°C for 30 s, denaturation at 95°C for 30 s → annealing at 65°C for 30 s → extension at 72°C for 20 s, a total of 30 cycles, and extension at 72°C for 10 min.

Enzyme digestion identification: Take 10 μl of the PCR product, add 5U endonuclease HindIII, 2 μl 10× digestion buffer and sterile double distilled water to form a 20 μl reaction system, digest in a 37°C water bath for 4 hours, and obtain the enzyme digestion product. After 1-fold concentration of the digested product, electrophoresis was carried out on 2.5% agarose gel at 5V/cm for 40min, and photographed under ultraviolet light for identification.

result:

The amplified sequence (which is located at 401-552 of the base sequence of SEQ ID NO: 1, 152 bp in total), the sequence of the amplified product obtained is as follows (SEQ ID NO: 9):

The underlined part in the amplified product sequence is the sequence corresponding to the upstream and downstream primers, and R represents the A/G polymorphism, that is, the SNP site rs894160.

The product sequences after digestion with endonuclease HindIII are as follows:

When the polymorphic site contains an A allele, the PCR product can be digested to form a cut fragment of 100 bp (the downstream sequence of the fragment is increased by 4 bases compared with the upstream sequence due to the formation of sticky ends by enzyme digestion) sequence (SEQ ID NO: 10):

When the polymorphic site contains an A allele, the PCR product can be digested to form a cut fragment of 52 bp (the downstream sequence of the fragment is reduced by 4 bases compared with the upstream sequence due to the formation of sticky ends by enzyme digestion) sequence (SEQ ID NO: 11):

agcttagaatgtgcctcaggagataccagcccccttgctcagtcaatttctt52

After electrophoresis, the digested product was photographed and identified under ultraviolet light, and a 152bp fragment appeared after the rs894160 site was amplified. Three fragments of 152bp, 100bp and 52bp will appear in the electrophoresis after digestion with HindIII (52bp is not easy to distinguish in the electrophoresis map)

The genotype was judged by electrophoresis after digestion with HindIII: GG type has a fragment of 152bp, AG type has two fragments of 152bp and 100bp, and AA type has a fragment of 100bp.

Polymorphic results: A total of 96 cases were tested, and the results found that 61 cases of GG type, 21 cases of AG type and 14 cases of AA type were found at the rs894160 locus.

Example 3 Determination of rs894160 polymorphism in human peripheral blood clot

Refer to the phenol-chloroform extraction method in "Molecular Cloning Technology" to extract genomic DNA from blood clots.

The upstream primer used in the PCR reaction is SEQNO:12, and the downstream primer is SEQNO:13.

Upstream primer: 5'CCGCCTGCAGTGGGCAGAACCTCACGT3' (SEQ ID NO: 12);

Downstream primer: 5'AAATTGACTGAGCAAGGGGGCTGGTATCTCCTGAGGCACATTCTAA G C3' (SEQ ID NO: 13)

Except that the annealing temperature of PCR amplification is 66° C., other reaction conditions are the same as in Example 1; enzyme digestion identification is the same as in Example 1.

result:

The amplified sequence (located at 397-549 of the base sequence of SEQ ID NO: 1, 153 bp in total), the sequence of the amplified product obtained is as follows (SEQ ID NO: 14):

The underlined parts in the amplified product sequence are the sequences corresponding to the upstream and downstream primers respectively, and R represents the A/G polymorphism, that is, the SNP site rs894160.

The product sequences after HindIII digestion are as follows:

When the polymorphic site contains an A allele, the PCR product can be digested to form a cut fragment of 104bp (the downstream sequence of the fragment is increased by 4 bases compared with the upstream sequence due to the formation of sticky ends due to enzyme digestion) sequence (SEQ ID NO: 15):

When the polymorphic site contains an A allele, the PCR product can be digested to form a cut fragment of 49 bp (the downstream sequence of the fragment is reduced by 4 bases compared with the upstream sequence due to the formation of sticky ends by enzyme digestion) sequence (SEQ ID NO: 16):

agcttagaatgtgcctcaggagataccagcccccttgctcagtcaattt49

After electrophoresis, the digested product was photographed and identified under ultraviolet light, and a 153bp fragment appeared after the rs894160 site was amplified. Three fragments of 153bp, 104bp and 49bp will appear in the electrophoresis after digestion with HindIII (49bp is not easy to distinguish in the electrophoresis map)

The genotype was judged by electrophoresis after digestion with HindIII: the GG type has a fragment of 153bp, the AG type has two fragments of 153bp and 104bp, and the AA type has a fragment of 104bp.

Polymorphic results: A total of 80 cases were tested, and the results found that 54 cases of GG type, 24 cases of AG type and 12 cases of AA type were found at the rs894160 locus.

Example 4 Determination of rs894160 polymorphism in human oral mucosa cells

The main reagents are the same as in Example 1 except that the endonuclease is AluI; the apparatus is the same as in Example 1. Genomic DNA was extracted from oral mucosal cells using a micro DNA extraction kit.

The upstream primer used in the PCR reaction is SEQNO:17, and the downstream primer is SEQNO:18.

Upstream primer: 5'GCCTGCAGTGGGCAGAACCTCACGT3' (SEQ ID NO: 17);

Downstream primer: 5'GAAATTGACTGAGCAAGGGGGCTGGTATCTCCTGAGGCACATTCTAA G C3' (SEQ ID NO: 18)

For PCR amplification, except that the annealing temperature is 68° C., other reaction conditions are the same as in Example 1; for enzyme digestion identification, except that AluI enzyme is used as the endonuclease, other conditions are the same as in Example 1.

result:

The amplified sequence (which is located at the 399-550 position of the base sequence of SEQ ID NO: 1, 152 bp in total), the obtained amplification product sequence is as follows (SEQ ID NO: 19):

The underlined part in the amplified product sequence is the sequence corresponding to the upstream and downstream primers, and R represents the A/G polymorphism, that is, the SNP site rs894160.

The product sequences after AluI digestion are as follows:

When the polymorphic site contains the A allele, the PCR product can be digested to form a cut fragment 104bp sequence (SEQ ID NO: 20):

When the polymorphic site contains the A allele, the PCR product can be digested to form a cut fragment 48bp sequence (SEQ ID NO: 21):

ccttagaatgtgcctcaggagataccagcccccttgctcagtcaatttc48

After electrophoresis, the digested product was photographed and identified under ultraviolet light, and a 152bp fragment appeared after the rs894160 site was amplified. Three fragments of 152bp, 104bp and 48bp will appear in the electrophoresis after digestion with AluI (48bp of which is not easy to distinguish in the electropherogram).

The genotype was judged by electrophoresis after digestion with AluI: GG type has a fragment of 152bp, AG type has two fragments of 152bp and 104bp, and AA type has a fragment of 104bp.

Polymorphic results: A total of 122 cases were tested, and the results found that 65 cases of GG type, 22 cases of AG type and 29 cases of AA type were found at the rs894160 locus.

Claims (10)

1. measure a method for people PLIN1 gene rs894160 loci polymorphism, comprising:

Human gene group DNA to be measured is provided;

There is provided upstream primer and the downstream primer of amplification people PLIN1 gene rs894160 location proximate sequence, wherein downstream primer has base mismatch G;

With described human gene group DNA to be measured for template, utilize upstream primer and downstream primer to carry out pcr amplification reaction to obtain the amplified production containing ARGCTT fragment or RGCT fragment, wherein R is base A undetermined on people PLIN1 gene rs894160 site or G;

Restriction enzyme is provided;

Utilize restriction enzyme to carry out enzyme to gained amplified production to cut to obtain corresponding digestion products;

And determine that the base R undetermined on people PLIN1 gene rs894160 site is A or G according to gained digestion products,

Wherein, restriction enzyme is for can cut one of AAGCTT fragment and AGGCTT fragment, or the restriction enzyme of one of AGCT fragment and GGCT fragment.

2. method according to claim 1, wherein on gained amplified production, a base C of being separated by between the pairing base of base mismatch G and the R of downstream primer.

3. method according to claim 2, wherein on gained amplified production, the pairing base of downstream primer terminal bases and R is adjacent.

4. method according to claim 1, wherein restriction enzyme is Hind III or its isoschizomers that can cut AAGCTT fragment.

5. method according to claim 1, wherein restriction enzyme is AluI or its isoschizomers that can cut AGCT fragment.

6. method according to claim 1, wherein gained digestion products comprises three kinds of clip types: have total fragment length do not cut off amplified production, cut off after have compared with the amplified production of long segment and after cutting off, there is amplified production compared with short-movie section, by judging that the clip types that digestion products comprises determines that the base undetermined on people PLIN1 gene locus rs894160 is A or G.

7. method according to claim 6, wherein judge clip types that digestion products comprises taken pictures by gel electrophoresis associating ultraviolet lamp after with reference to scheming to contrast to carry out.

8. method according to claim 7, wherein reference figure is that amplified production obtains and only has first with reference to picture position and second with reference to picture position after the gel electrophoresis standard setting time after ultraviolet lamp is taken pictures, wherein first with reference to picture position for be the amplified production of the total fragment length do not cut off, second with reference to picture position then for be cut off after there is amplified production compared with long segment.

9. method according to claim 1, wherein make total fragment length of amplified production between 100 to 300bp by designing the length of upstream primer and downstream primer, and the fragment length of downstream primer is at least 35bp, makes enzyme fall Partial Fragment earnestly and account for the per-cent of the length of total fragment more than 20%.

10., for measuring a test kit for people PLIN1 gene rs894160 loci polymorphism, it comprises:

For upstream primer and the downstream primer of pcr amplification people PLIN1 gene rs894160 location proximate sequence, wherein downstream primer has base mismatch G to obtain the amplified production containing ARGCTT fragment or RGCT fragment, and wherein R is base A undetermined on people PLIN1 gene rs894160 site or G; And

The restriction enzyme of one of AAGCTT fragment and AGGCTT fragment can be identified, or identify the restriction enzyme of one of AGCT fragment and GGCT fragment.