CN110607364B - Method for determining genotype of allele by detecting SNP locus - Google Patents
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Abstract
The invention provides a method for determining the genotype of an allele by detecting SNP loci, which detects one or more SNP loci of the alleles to be detected by an AS-PCR method and then determines the genotype of each allele to be detected according to the detection result of each SNP locus. The method of the invention does not need expensive detection instruments, can be carried out in small laboratories and hospital laboratories, and meets the requirements of high sensitivity, convenience and rapidness of clinical examination.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a method for determining the genotype of an allele by detecting SNP loci.
Background
Alleles refer to genes that are located at the same position on a pair of homologous chromosomes and control different morphologies of the same trait, and the english text is "allele". Different alleles produce changes in genetic characteristics such as color development or blood type. Alleles control the apparent-recessive relationship and genetic effects of relative traits, and can be distinguished into different categories. In an individual, a certain form (dominant) of an allele may be expressed much more than other forms (recessive). When an organism carries a pair of identical alleles, then the organism is homozygous for the gene (homozygous) or can be called true-breeding; conversely, if a pair of alleles are not identical, the organism is heterozygous (hybrid) or known as hybrid. Alleles each encode a protein product, determine a trait, and may be disabled by mutation. There is an interaction between alleles. A recessive relationship occurs when one allele determines a biological trait more strongly than another allele and causes an organism to exhibit only its own trait. The effect is dominant and hidden, but the effect is hidden from being expressed.
A single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) is a genetic marker located on the human genome. With the completion of the human genome project, it was found that human DNA sequences were not completely identical, including certain variations. These variations lead to the development of a variety of genetic diseases. As a third generation genetic marker, SNP sites are closely related to the generation of genetic diseases. Therefore, the research and detection of SNP loci provide a new way for finding out high-risk groups of genetic diseases, detecting related genes of diseases, basic research of biomedicine and the like. Existing methods for detecting SNPs can be broadly divided into two main categories: one general class is the traditional classical detection methods based on gel electrophoresis, represented by Single Strand Conformation Polymorphism (SSCP), allele-specific PCR (AS-PCR), and the like. Another broad class is the high throughput, high degree of automation detection methods represented by direct sequencing, mass spectrometry detection techniques, and the like. Among the various detection methods, the direct sequencing method has the highest accuracy and is often used as a gold standard for SNP locus detection. However, the method requires high instrument cost, takes long time for sequencing, and is difficult to popularize in general hospitals or laboratories.
Disclosure of Invention
In order to solve the above technical problems, the present invention provides a method for determining the genotype of an allele by detecting SNP sites, which detects one or more SNP sites of alleles to be detected by an AS-PCR method and then determines the genotype of each allele to be detected according to the detection result of each SNP site.
In one embodiment, the method comprises the steps of: step 1: designing three amplification primers aiming at each SNP locus of the allele to be detected, wherein one amplification primer is an upstream primer or a downstream amplification primer, and the other two amplification primers are respectively a downstream amplification primer or an upstream amplification primer; the bases at the 3' -end of the two amplification primers are the same as and complementary to the bases of the SNP locus respectively, and the bases at other positions of the two amplification primers are the same; step 2: constructing a positive plasmid control group which accords with all possible genotypes of the SNP loci, and obtaining genotype interpretation intervals of each SNP locus to be detected according to the QPCR result of the positive control group; and, step 3: and (3) carrying out QPCR (quantitative polymerase chain reaction) on the sample to be detected by using the specific amplification primer designed in the step (1), and judging the genotype of the SNP locus of the sample to be detected according to the obtained result according to the different genotype interpretation intervals of the SNP locus determined in the step (2).
In one embodiment, the step 2 includes the steps of: a. designing at least three different gradient DNA positive template experiment groups for all genotypes and primer combinations which can occur in the SNP loci, designing three amplification primers for each genotype according to the method of the step 1, designing six different genotypes and primer combinations for each SNP locus, and performing QPCR (quantitative polymerase chain reaction) by using the combinations; b. the primer and the corresponding homozygous genotype plasmid template are a specific group, the primer and the non-corresponding homozygous genotype plasmid template are a non-specific group, and the primer and the mixed genotype plasmid template are a mixed group; c. converting QPCR results of the specific group, the non-specific group and the mixed group into gene copy numbers, and comparing the gene copy numbers according to homozygous genotype/homozygous genotype and heterozygous genotype/heterozygous genotype to obtain ratio of multiplying power of comparison of specific combination and non-specific combination and mixed group combination under each concentration gradient of each genotype; d. and expressing the multiplying power ratio of the obtained genotypes in a logarithmic form, and drawing a quantitative typing area chart of the SNP locus genotypes to obtain the genotype interpretation interval of each SNP locus to be detected.
In one embodiment, the test gene is an collectin gene.
In one embodiment, the SNP sites are rs11136000, rs2279590 and/or rs9331888.
In one embodiment, the rs11136000 locus is the three amplification primers in step 1: the upstream primer SEQ No.3:5'-TAAAGAATCCACTCATCCTTCAAGA-3', downstream primer SEQ No.4:5'-GCAAGGGCCCGTTAGAGAA-3' and SEQ No.5:5'-GCAAGGGCCCGTTAGAGAG-3'.
In one embodiment, the rs2279590 locus is the three amplification primers in step 1: the upstream primer SEQ No.8:5'-GGAAGTCCTCCTGCTT-3', upstream primer SEQ No.9:5'-GGAAGTCCTCCTGCTC-3' and downstream primer SEQ No.10:5'-TGGAAACCACTTTTATTC-3'.
In one embodiment, the rs9331888 locus is the three amplification primers in step 1: the upstream primer SEQ No.13:5'-GGAAGTCCTCCTGCTT-3', upstream primer SEQ No.14:5'-GGAAGTCCTCCTGCTC-3' and downstream primer SEQ No.15:5'-TGGAAACCACTTTTATTC-3'.
Single Nucleotide Polymorphism (SNP) refers to a polymorphism of a DNA sequence at the genomic level caused by variation of a single nucleotide, including transitions, transversions, deletions and insertions. Since each SNP locus usually contains only two alleles, genotyping can be performed by a simple "+/-" analysis without analysis of fragment length. Existing methods for detecting SNPs can be broadly divided into two main categories: one general class is the traditional classical detection methods based on gel electrophoresis, represented by Single Strand Conformation Polymorphism (SSCP), allele-specific PCR (AS-PCR), and the like. Another broad class is the high throughput, high degree of automation detection methods represented by direct sequencing, mass spectrometry detection techniques, and the like. Among the various detection methods, the direct sequencing method has the highest accuracy and is often used as a gold standard for SNP locus detection. However, the method requires high instrument cost, takes long time for sequencing, and is difficult to popularize in general hospitals or laboratories. The AS-PCR method adopted by the invention does not need expensive detection instruments or redundant experimental processes, and can realize high-throughput sequencing of SNP loci of genes to be detected by only designing specific primers and combining QPCR technology.
The detection result of the method is compared with the result of the direct sequencing method, and the result shows that the detection result and the result completely accord with each other, which shows that the detection sensitivity of the method to SNP locus genotypes is the same as the gold standard. Because the method does not need expensive detection instruments, the method can be carried out in small laboratories and test rooms of hospitals, and meets the requirements of high sensitivity, convenience and rapidness of clinical tests. Therefore, the method can be used for detecting the genotype of the CLU gene and the related SNP locus of the AD in clinical hospital examination rooms, and provides a simple and convenient detection method for clinically searching the susceptibility gene of the AD.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of genotyping at locus rs11136000 (A), locus rs2279590 (B) and locus rs9331888 (C);
FIG. 2 is a graph showing the results of detecting the rs11136000 genotype by the gene sequencing method and the QPCR quantitative typing method of the present invention, (1) and (2) are the results of detecting the rs11136000 genotype by the gene sequencing method, and the genotypes are CC and CT respectively; (3) the quantitative typing method is the result of the QPCR quantitative typing method;
FIG. 3 is a graph showing the results of detecting the rs2279590 genotype by a gene sequencing method and a QPCR quantitative typing method, wherein (1) and (2) are the results of detecting the rs2279590 genotype by the gene sequencing method, and the genotypes are CC and CT respectively; (3) the quantitative typing method is the result of the QPCR quantitative typing method; and
FIG. 4 is a graph showing the results of detecting the rs9331888 genotype by the gene sequencing method and the QPCR quantitative typing method of the present invention, wherein (1), (2) and (3) are the results of detecting the rs9331888 genotype by the gene sequencing method, and the genotypes are CC, CG and GG respectively; (4) the result of the QPCR quantitative typing method is shown.
Detailed Description
In order that those skilled in the art will better understand the technical solutions of the present application, the present invention will be further described with reference to examples, and it is apparent that the described examples are only some of the examples of the present application, not all the examples. All other embodiments, which can be made by one of ordinary skill in the art based on the embodiments herein without making any inventive effort, shall fall within the scope of the present application.
Examples
1. Material
1.1. Experimental samples 13 blood samples were from outpatient or inpatient clinic in the Dongzhi gate hospital of the university of Beijing traditional Chinese medicine in 2019. Wherein, after 8 men and 5 women, all study subjects signed written informed consent, 2ml of peripheral blood was collected and stored in a-20 ℃ refrigerator. The normal group, 3, was healthy and had no family genetic history of AD, with the remaining 10 blood samples from AD patients.
1.2. Reagent and instrument whole blood genomic DNA extraction kit (QIAamp DNA Blood Mini Kit) (beijing orchid bordetella commercial limited); taKaRa TaqTM HotStar DNA polymerase and other PCR reagents (Beijing orchid Boli commercial Co., ltd.); taKaRa DL2000DNA marker (Beijing orchid Boli commercial Co., ltd.); quantiNova (TM)
Green PCR Kit (Beijing orchid Bode commercial Co., ltd.); ultra-micro spectrophotometers (America Valley instruments (Shanghai) Inc.); t100 TM Type PCR instrument (Bio RAD); DYY-6C electrophoresis apparatus (Beijing six biotechnology Co., ltd.); quantum studio TM 6Flex fluorescent quantitative PCR apparatus (Thermo Fisher); the primer is synthesized by Shenzhen Hua big gene company; sequencing was done by Shanghai bioengineering company.
2. Method of
2.1. Extraction of genomic DNA extraction was performed with reference to QIAamp DNA Blood Mini Kit instructions. The extracted nucleic acid is used for measuring the content of DNA by using an ultra-micro spectrophotometry, detecting the integrity of the DNA by using agarose gel electrophoresis and preserving at-20 ℃ for standby.
2.2. Design of specific primers complete sequence of CLU and information of SNP sites to be detected were downloaded from Genbank, and specific primers were designed for three SNP sites using primer Premier 5.0 software. 3 pairs of specific primers are designed aiming at SNP locus rs11136000, the outer primers are used for conventional PCR amplification and sequencing, and 2 pairs of inner primers are used for identifying 3 genotypes (CC type, CT type and TT type) of rs11136000 genes. 3 pairs of specific primers are designed aiming at SNP locus rs2279590, the outer primers are used for conventional PCR amplification and sequencing, and 2 pairs of inner primers are used for identifying 3 genotypes (CC type, CT type and TT type) of rs2279590 genes. 3 pairs of specific primer outer primers are designed for conventional PCR amplification and sequencing aiming at SNP locus rs9331888, and 2 pairs of inner primers are designed for identifying 3 genotypes (CC type, CG type and GG type) of rs9331888 genes. The primer sequences are shown in Table 1 below.
TABLE 1 SNP locus conventional PCR and QPCR primer sequences
2.3. Gene sequencing typing PCR amplification was performed using conventional PCR primers designed and TaqTM HotStar DNA polymerase. The PCR reaction system consists of the following components: 10x PCR Buffer 2.5ul,dNTP Mixture (2.5 mM) 2ul, taKaRa Taq enzyme (5U/ul) 0.125ul, upstream and downstream primers (10 uM) 1ul each, DNA template 1ul, sterile water 17.375ul, and the reaction overall system 25ul. Amplification reaction conditions: 95 ℃ for 3min,95 ℃ for 30s,58 ℃ for 30s and 72 ℃ for 30s, and 35 cycles are total; the final extension was carried out at 72℃for 5min and stored at 4 ℃. The PCR products were analyzed by agarose gel electrophoresis, DL2000DNA markers were used as a reference, and the desired DNA bands were recovered and purified and sent to Shanghai Bioengineering to complete sequencing.
2.4. The construction of the positive control group plasmid template uses blood of 3 healthy persons collected by Tongzhengmen Hospital in Beijing as a sample, extracts genomic DNA from the blood, carries out PCR amplification according to the method, recovers the required DNA band and sends the DNA band to be tested, and the PCR product is used as a template to construct a plasmid vector. Because the sites of s11136000, rs2279590 and rs9331888 respectively correspond to 2 bases, three genotypes. And performing point mutation experiments by taking the constructed plasmid vector as a template according to the sequencing result to obtain another base corresponding to each SNP locus, and constructing a new plasmid vector. And mixing the two homozygote plasmid vectors in equal proportion to obtain a heterozygote plasmid vector, thereby constructing a positive control group plasmid template conforming to the genotypes of the SNP loci. Construction of the vector the PCR product was used as template and pMDTM18-TVector (TaKaRa) as vector, steps were described in TaKaRa pMDTM18-T Vector Cloning Kit. The point mutation experiment adopts a sequenced plasmid vector as a template, a point mutation primer is designed by itself, and a PCR reaction reagent in QuikChangeLightning Multi Site-Directed Mutagenesis Kit is selected, wherein the PCR reaction system comprises the following components: 10x reaction Buffer 5ul,dNTP Mixture 1ul, each 1ul,QuickSolutionreagent 1.5ul of upstream and downstream mutation primer (10 uM), 1ul of plasmid DNA template, 14.5ul of sterile water, followed by Quickchange lightening Enzyme ul of reaction total 26ul. Amplification reaction conditions: 2min at 95 ℃, 20s at 95 ℃, 10s at 60 ℃ and 30s at 68 ℃ for 18 cycles; the final extension was performed at 68℃for 5min and 4 ℃. The PCR products were used to construct plasmid vectors according to the instructions.
QPCR quantitative typing the QPCR detection was performed by selecting SYBR Green qPCR Master using designed QPCR primers. The QPCR reaction system consists of the following components: 2X SYBR Green Master Mix ul, 1ul of upstream and downstream primer (10 uM), 1ul of DNA template, 7ul of sterilized water, 25ul of reaction system. The amplification reaction conditions of rs11136000 and rs9331888 are 95 ℃ 2min,95 ℃ 5s and 64 ℃ 20s, and 40 cycles are total. The rs2279590 amplification reaction conditions are 95 ℃ 2min,95 ℃ 5s and 55 ℃ 20s, and 40 cycles are total. And comparing the results obtained by the experimental group and the positive control group, and carrying out SNP typing on the clinical samples of each group.
2.6. DNA templates of 4 different gradients were designed for each SNP site before genotyping QPCR experiments (10 9 Copy number, 10 8 Copy number, 10 7 Copy number, 10 6 Copy number) positive control group. Since one SNP site has three genotypes and two pairs of specific primers, each SNP site has 6 different genotypes and primer combinations. The primers and the corresponding homozygous genotype plasmid templates are positive control groups, the primers and the non-corresponding homozygous genotype plasmid templates are non-specific groups, and the primers and the mixed genotype plasmid templates are mixed groups. After the reaction, a quantitative standard curve (correlation coefficient R) is drawn by taking the ct value as the abscissa and the logarithm of the initial copy number concentration of the specific primer and the corresponding homozygous genotype group as the ordinate 2 >0.99). And converting the mixed group and the nonspecific group ct values into copy numbers through quantitative standard curves in equal proportion, comparing the conversion results according to homozygous genotypes/homozygous genotypes, reflecting the obtained results according to multiplying power forms, converting the multiplying power according to logistic logarithmic forms, and analyzing the results. Obtaining the required SNP locus base according to the QPCR result of the positive control groupThe cause type interpretation interval is: for the purpose of rs11136000, when the result falls to [1, ++ infinity]The genotype is CC; [ -1,1]Genotype is CT; [ -1, - ≡]Genotype is TT. For the purpose of rs2279590, when the result falls to [1, ++ infinity]The genotype is CC; [ -1,1]Genotype is CT; [ -1, - ≡]Genotype is TT. For the purpose of rs9331888, when the result falls to [1, ++ infinity]The genotype is CC; [ -1,1]Genotype is CG; [ -1, - ≡]The genotype is GG. The genotype interpretation interval of each SNP site is shown in FIG. 1 below. The construction process of the interpretation interval of QPCR quantitative typing is described by taking CLU R1rs11136000 as a specific example.
The following table shows QPCR results for six different genome and primer combinations for CLU R1 (rs 11136000) positive plasmids. Wherein the combination of the CLU-P1-FOR/P3C-REV primer and the CC homozygous plasmid template of the CLU R1, and the combination of the CLU-P1-FOR/P3T-REV primer and the TT homozygous plasmid template of the CLU R1 are a specific group; the CLU-P1-FOR/P3C-REV primer is combined with the TT homozygous plasmid template of the CLU R1, and the CLU-P1-FOR/P3T-REV primer is combined with the CC homozygous plasmid template of the CLU R1 into a nonspecific group; the remaining two groups are mixed groups. According to the above experimental method, four gradients were set with the specific group as positive standard (10 9 Copy number, 10 8 Copy number, 10 7 Copy number, 10 6 Copy number), QPCR result constructs quantitative standard curve (correlation coefficient R 2 >0.99 The mixed and nonspecific group ct values were scaled to copy number by a quantitative standard curve to obtain the following table.
TABLE 2 results of plasmid template QPCR with CLU R1 (rs 11136000) specific primers
The lower graph shows that after conversion of copy numbers of QPCR results of six combinations (specific group, non-specific group and mixed group), the ratio of multiplying power of comparison of specific combinations with non-specific combinations and mixed group combinations under each gradient of CC, TT and CT genotypes is obtained respectively according to comparison copy numbers of homozygous genotype/homozygous genotype and heterozygous genotype/heterozygous genotype.
TABLE 3 quantitative typing multiplying power ratio of QPCR for each genotype of CLU R1 (rs 11136000)
And finally, the multiplying power ratio of the three genotypes is expressed in a logarithmic form, and a genotyping quantitative typing area diagram is drawn. As shown in fig. 1A.
3. Results
3.1. Extraction of genomic DNA the genomic DNA was extracted from peripheral blood of 10 AD patients using a DNA extraction kit, and absorbance (A), A was detected by an ultra-micro spectrophotometer 260nm /A 280nm The ratio is 1.8-2.0, which shows that the purity of the extracted DNA meets the requirement of the subsequent experiment.
3.2. Results of the gene sequencing method and the QPCR quantitative typing method are compared, and the results of the gene sequencing and the QPCR quantitative typing of the rs11136000, the rs2279590 and the rs9331888 3 SNP loci of 10 groups of samples are shown in fig. 2, fig. 3 and fig. 4. The coincidence rate of the genotypes of the loci rs11136000, rs2279590 and rs9331888 of 10 samples detected by the two methods is 100%, and the results are shown in tables 4-6.
Table 4 two methods detect rs11136000 genotype distribution (n=10)
Table 5 two methods detect rs2279590 genotype distribution (n=10)
Table 6 two methods detect rs9331888 genotype distribution (n=10)
It is to be understood that this invention is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also encompassed by the appended claims.
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Claims (1)
1. A method for determining the genotype of an allele by detecting SNP loci, characterized in that the method detects one or more SNP loci of the alleles to be detected by an AS-PCR method, and then determines the genotype of each allele to be detected according to the detection result of each SNP locus;
the method comprises the following steps:
step 1: designing three amplification primers aiming at each SNP locus of the allele to be detected, wherein one amplification primer is an upstream primer or a downstream amplification primer, and the other two amplification primers are respectively a downstream amplification primer or an upstream amplification primer; the bases at the 3' -end of the two amplification primers are the same as and complementary to the bases of the SNP locus respectively, and the bases at other positions of the two amplification primers are the same;
step 2: constructing a positive plasmid control group which accords with all possible genotypes of the SNP loci, and obtaining genotype interpretation intervals of each SNP locus to be detected according to the QPCR result of the positive control group;
step 3: carrying out QPCR experiments on the sample to be detected by using the specific amplification primers designed in the step 1, and judging genotypes of SNP loci of the sample to be detected according to different genotype interpretation intervals of the SNP loci determined in the step 2 by the obtained results;
the step 2 comprises the following steps:
a. designing at least three different gradient DNA positive template experiment groups for all genotypes and primer combinations which can occur in the SNP loci, designing three amplification primers for each genotype according to the method of the step 1, designing six different genotypes and primer combinations for each SNP locus, and performing QPCR experiments by using the combinations;
b. the primer and the corresponding homozygous genotype plasmid template are a specific group, the primer and the non-corresponding homozygous genotype plasmid template are a non-specific group, and the primer and the mixed genotype plasmid template are a mixed group;
c, after the QPCR reaction is finished, drawing a quantitative standard curve by taking a ct value as an abscissa and taking the logarithm of the initial copy number concentration of the specific primer and the corresponding homozygous genotype group as an ordinate; converting QPCR results of the non-specific group and the mixed group into gene copy numbers, and comparing the gene copy numbers according to homozygous genotype/heterozygous genotype to obtain ratio of specific combination to non-specific combination and mixed group comparison under each concentration gradient of each genotype;
d. the multiplying power ratio of the obtained genotypes is expressed in a logarithmic form, and a quantitative typing area diagram of the SNP locus genotypes is drawn to obtain the interpretation interval of each SNP locus genotype to be detected;
the alleles to be detected are collectin genes, and the SNP loci are rs11136000, rs2279590 and/or rs9331888;
the rs11136000 locus is respectively formed by the following three amplification primers in the step 1: the upstream primer SEQ No.3:5'-TAAAGAATCCACTCATCCTTCAAGA-3', downstream primer SEQ No.4:5'-GCAAGGGCCCGTTAGAGAA-3' and SEQ No.5:5'-GCAAGGGCCCGTTAGAGAG-3';
the rs2279590 locus is respectively formed by the following three amplification primers in the step 1: the upstream primer SEQ No.8:5'-GGAAGTCCTCCTGCTT-3', upstream primer SEQ No.9:5'-GGAAGTCCTCCTGCTC-3' and downstream primer SEQ No.10:5'-TGGAAACCACTTTTATTC-3';
the rs9331888 locus is respectively formed by the following three amplification primers in the step 1: the upstream primer SEQ No.13:5'-GGAAGTCCTCCTGCTT-3', upstream primer SEQ No.14:5'-GGAAGTCCTCCTGCTC-3' and downstream primer SEQ No.15:5'-TGGAAACCACTTTTATTC-3'.
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| CN102471800A (en) * | 2009-07-03 | 2012-05-23 | 加的夫大学学院咨询有限公司 | Diagnosis and treatment of alzheimer's disease |
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| CN101054601A (en) * | 2006-04-13 | 2007-10-17 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Oligonucleotide for detecting cytochrome P450 enzyme series mutation site and gene chip |
| AU2013203473A1 (en) * | 2008-04-30 | 2013-05-02 | Integrated Dna Technologies, Inc. | RNase-H-Based Assays Utilizing Modified RNA Monomers |
| CN102471800A (en) * | 2009-07-03 | 2012-05-23 | 加的夫大学学院咨询有限公司 | Diagnosis and treatment of alzheimer's disease |
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