CN112921099A - Kit and method for detecting human scientific gas potential genotype - Google Patents
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Abstract
The invention discloses a kit and a method for detecting human scientific gas potential genotypes. The invention adopts multiple PCR amplification and electrophoresis methods to analyze and identify the allelic polymorphism (SNP) of 5 genes: NRXN1, PCDH18, TCERG1L, COX10 and CADM2, comprising the steps of: a) collecting the cells dropped off from the oral cavity of a testee, storing the cells in a collection card or extracting DNA nucleic acid from a blood sample; b) adding a saliva acquisition card sample or an extracted DNA sample of a testee into a reaction system for PCR amplification; c) running an amplification program; d) and carrying out electrophoretic analysis on the amplification products, and judging according to a peak pattern. The invention can synchronously detect the SNP of a plurality of genes related to a testee, and realizes the simplicity, high efficiency and specificity of detection. The analysis provides reference for whether the testee has the potential of scientific quality.
Description
Technical Field
The invention relates to the field of gene detection, in particular to a kit and a method for detecting human scientific gas potential genotypes.
Background
Rockfililer has said that: the work is a stage which can perform own talents, the knowledge read by cold windows, the strain capacity, the decision capacity, the adaptability and the coordination capacity of the people are displayed on the stage. In addition to work, there is no activity that provides such a high degree of self-filling, self-expression, and such a strong sense of personal embarrassment and a reason for survival. The quality of work often determines the quality of life. ", a
Competence refers to the ability possessed in performing various activities, which ensures that people effectively recognize the world. The abilities include various abilities that an individual must possess in recognizing activities, such as perception (observation), memory, imagination, thinking ability, attention, and the like.
At present, research shows that the ability of people is the reaction and judgment of people or things shown by the mutual correlation, mutual alternation and mutual influence of various factors and the comprehensive experience, environment, demand and other factors. The influencing factors comprise various aspects such as family background, living environment, self experience, social administration and the like. On the basis of the influence factors, people consider to make judgment and decision after receiving social information and carrying out complex processes such as information feedback, decision making and the like. This judgment and decision making varies greatly from person to person. This difference, the physiological genetic genes of parents play a great role, just called "have their parents necessarily their children". Different persons, even if receiving the same information, grow in the same environment, and make different judgments. The baton for guiding self-behaviors and making judgment behavior rules is a gene with genetic characteristics, basic mechanisms and performances of supporters life in our body.
The gene has DNA segment with genetic information, and the segment can utilize Polymerase Chain Reaction (PCR) technology to know human biology and related human gene information along with the discovery of Taq enzyme and the gradual maturity and perfection of PCR. The human genome project was proposed as early as 1985, and was formally started in 1990, and through a concerted effort by many countries, it was declared that the work of drawing a draft of the human genome was completed in 2000.
The human genome sequence is decoded, human appearance and character characteristics such as character, physique, emotion, height and weight, and the like, which are related to the human body, and influence and related genome can be possibly found, and probably, the human can use biological technology to correct and adjust the bad genome in the human body, control and improve personal emotion and character, change the handling capacity and behavior attitude of one person and the like.
Scientific temperament is an expression which is expressed by integrating various characters and has the abilities of hunting heart, resisting pressure, keeping behavior, stabilizing heart, stronger logic, predicting the foreword and the like. But the heart hunting, the anti-stress capability and the behavior persistence play important roles in the field. These several abilities are expressed differently in different humans and may be related to differences in the 5 locus SNP sites in humans.
In the human genome, there is one SNP site every 100-300 bases, and various differences of human genetic genes are attributed to 90% of gene variations caused by SNPs. SNPs (single nucleotide polymorphisms) refer to changes in DNA sequence caused by changes in a single nucleotide-a, T, C or G, resulting in diversity of chromosomal genomes between species including humans and protein expression between individuals. The influence of gene mutation caused by SNP on human is not completely clear, but at present, research shows that the action of a small amount of SNP can be visually expressed, such as difference in behavior.
With respect to heart hunting, stress resistance and behavioral persistence, a 11000-person research project was developed in 2012 to explore the relationship of these three abilities to loci. After analyzing more than 120 million gene loci, the project researchers eventually find several gene loci that can affect knock-hearting, behavior persistence and high stress tolerance, such as NRXN1, PCDH18, TCERG1L, COX10 and CADM 2. By analyzing the relation between SNP sites and capabilities, researchers find that polymorphic G allele has more strange heart than A at site rs6754640 in gene NRXN 1; the rs987360 polymorphic T allele in the locus of the gene PCDH18 has more strange heart than C; the rs11018023 polymorphic A allele at the bit point in the gene TCERG1L has more fantastic hearts than G; polymorphic C allele in locus rs17608059 in gene COX10 has higher behavior persistence and high pressure tolerance than that of T gene; the polymorphic C allele in locus rs12494658 in gene CADM2 is more persistent and more resistant to stress than the T-type. Persons who have a combination of hunter hearts, behavioral persistence and high stress tolerance are able to generate a greater interest in novelty or problems encountered, and are able to continue to focus and explore in behavior without easily receding even if they encounter difficulties. The analysis method can provide reference for whether the testee has the potential of scientific quality.
Disclosure of Invention
The invention aims to provide a kit and a method for detecting the human scientific gas potential genotype, wherein the kit can detect the related genes of a testee and give analysis.
Specifically, the invention discloses a kit and a method for detecting the human scientific gas potential genotype, which comprises the following steps:
a) collecting the cells dropped off from the oral cavity of a testee, storing the cells in a collection card or extracting DNA nucleic acid from a blood sample;
b) adding a saliva acquisition card sample or an extracted DNA sample of a testee into a reaction system for PCR amplification;
c) running an amplification program;
d) carrying out electrophoretic analysis on the amplification product, and judging according to a peak pattern;
the kit comprises the following components: primer Mix, PCR reaction solution and DNA/RNA-free water.
Wherein the Primer Mix comprises a Primer group for amplifying 5 gene SNP polymorphic sites and a Primer group for 3 human genome DNA internal references and reaction internal references pcDNA, which are detailed in Table 1.
TABLE 1 SNP detection sites, primer sequences and PCR amplification fragment lengths of the kit
The kit can carry out synchronous multiplex PCR amplification on a detected sample, and can accurately detect the genotypes of detected genes NRXN1, PCDH18, TCERG1L, COX10 and CADM2 through the design of SNP primers: heterozygotes or homozygotes; the pcDNA is used for detecting a reaction system, monitoring whether the reaction system is effective or not and whether amplification is normal or not; 3 human genome DNA is internally referred to for detecting human samples, so that the human samples are guaranteed to be effective.
Wherein the PCR reaction solution comprises the following components: 2 XPCR buffer, DNA polymerase, dNTPs, potassium chloride, magnesium chloride, etc.
The PCR amplification reaction conditions of the kit are shown in Table 2.
TABLE 2 PCR amplification reaction conditions of the present invention
The amplification product of the kit is analyzed by electrophoresis; the preferred electrophoresis is capillary electrophoresis.
The kit can simultaneously amplify a plurality of loci, realizes the simplicity, high efficiency and specificity of detection, and provides reference for whether a testee has the potential of scientific quality by analyzing an amplification map, which is shown in Table 3.
TABLE 3 detection site-character reference information of the present invention
Drawings
FIG. 1 is a negative control DNA/RNA-free water amplification map of the kit. Only characteristic peaks of pcDNA appear.
FIG. 2 is an amplification profile of a sample of exfoliated cells from the oral cavity of subject 1. TCERG1L genotype is GA, PCDH18 is CT, CADM2 is CT, COX10 is CT, NRXN1 is GA, and pcDNA peak and characteristic peaks of human genome DNA (huDNA) ginseng-1, human genome DNA (huDNA) ginseng-3 and human genome DNA (huDNA) ginseng-8 appear.
Fig. 3 is an amplification map of a sample of exfoliated cells from the oral cavity of subject 2. TCERG1L genotype A, PCDH1 is CT, CADM2 is CT, COX10 is C, NRXN1 is G; the pcDNA peak and characteristic peaks of human genome DNA (huDNA) internal reference-1, human genome DNA (huDNA) internal reference-3 and human genome DNA (huDNA) internal-8 appear.
FIG. 4 is an amplification map of a DNA sample extracted from blood of subject 2, as shown in FIG. 3.
The specific implementation method comprises the following steps:
for a better understanding of the present invention, reference is made to the following detailed description and accompanying drawings. It is to be understood that these examples are for further illustration of the invention and are not intended to limit the scope of the invention. Moreover, it should be understood that the invention is not limited to the above-described embodiments, but is capable of various modifications and changes within the scope of the invention.
Detect whether to possess the detection kit of scientific quality of smell potentiality originally, including box body and the reagent of depositing in the box body, including the following step:
a) collecting the cells dropped off from the oral cavity of a testee, storing the cells in a collection card or extracting DNA nucleic acid from a blood sample;
b) adding a saliva acquisition card sample or an extracted DNA sample of a testee into a reaction system for PCR amplification;
c) running an amplification program;
d) carrying out electrophoretic analysis on the amplification product, and judging according to a peak pattern;
the kit comprises the following components: primer Mix including Primer set, PCR reaction solution and DNA/RNA-free water.
The PCR reaction system Primer Mix comprises a Primer group for amplifying 5 SNP polymorphic sites, 3 personal genome references and pcDNA reaction references, and the details are shown in Table 1.
Wherein the PCR reaction solution comprises the following components: 2 XPCR buffer, DNA polymerase, dNTPs, potassium chloride, magnesium chloride, etc.
In the embodiment, the samples are DNA/RNA-free water, an oral cavity wall cast-off cell collecting card of the testee 1, an oral cavity wall cast-off cell collecting card of the testee 2 and a blood DNA extracting sample of the testee 2.
In the embodiment, the electrophoresis is to perform electrophoresis on the amplification product through capillary electrophoresis, and an amplification map is given and analyzed by combining analysis software, so as to judge the cheerful heart, behavior persistence and high pressure tolerance of the testee.
Example one
(1) The selected samples were: DNA/RNA-free water.
(2) Architecture configuration
According to the specification, preparing a reaction system by the Primer Mix and the PCR reaction solution on ice according to the proportion of the specification, carrying out vortex mixing, centrifuging by a centrifuge, mixing uniformly by a gun head, and subpackaging.
(3) Adding the sample
According to the instructions, using a pipette to take a corresponding volume of DNA/RNA-free water, and adding into the reaction system which is divided.
(4) Amplification procedure
The amplification procedure on the PCR instrument is as in table 2.
(5) Detection of amplification product on 3500DX genetic analyzer
A sample mixture consisting of deionized formamide and an internal molecular weight standard in the system (Size-500) { (1. mu.L Size-500+ 12. mu.L deionized formamide) × (number of samples) }. Mix 9. mu.L of the sample mixture with 1. mu.L of the amplification product to avoid the formation of bubbles and to perform electrophoresis as soon as possible. Detection and analysis are carried out by an ABI 3500 genetic analyzer (purchased from ABI company of America), and specific analysis parameters are sample injection voltage: 1.2kv, sample injection time: 15s, electrophoresis time 1210-. The results of the detection are shown in FIG. 1.
(6) Analyzing data
The experiment uses DNA/RNA-free water, and the electrophoresis pattern should not have target peaks of each gene locus, and only has characteristic peaks of the pcDNA gene, as follows: the peak appearance corresponding to the pcDNA site is: singlet, showing pcDNA in the map;
as shown in FIG. 1, the electrophoretically analyzed DNA/RNA-free water-amplified product showed only the peak of the internal reference pcDNA of the reaction.
The experiment can effectively verify the amplification effectiveness of the reaction reagent and has no external pollution source.
Example two
(1) The selected samples were: samples of cells exfoliated from the oral wall of subjects 1 and 2.
(2) Sample collection
The collection type is as follows: cells are shed from the oral wall.
The acquisition method comprises the following steps: the saliva collecting rod is adopted to wipe the inner wall of the oral cavity back and forth for 4 times, the reverse side of the saliva collecting rod is used to wipe the inner wall of the oral cavity back and forth for 4 times, the saliva collecting rod is taken out, the saliva collecting rod is repeatedly pressed on a saliva sample collecting card, cells on the inner wall of the saliva are transferred to the saliva sample collecting card, and the collected saliva sample collecting card is dried in a pollution-free area.
Valid samples: the area of the saliva sample acquisition card with the pink area changed into light pink or white is the effective saliva sample area.
The sample selecting method comprises the following steps: manual punch sampling was performed using a dabber plastic punch (1.0 mm).
(3) Architecture configuration
According to the specification, preparing a reaction system by the Primer Mix and the PCR reaction solution on ice according to the proportion of the specification, carrying out vortex mixing, centrifuging by a centrifuge, mixing uniformly by a gun head, and subpackaging.
(4) Adding a sample: and taking 1-2 effective samples in the saliva card by using a puncher, and adding the effective samples into the prepared reaction system.
(5) Amplification procedure
The amplification procedure on the PCR instrument is as in table 2.
(6) Detection of amplification product on 3500DX genetic analyzer
A sample mixture consisting of deionized formamide and an internal molecular weight standard in the system (Size-500) { (1. mu.L Size-500+ 12. mu.L deionized formamide) × (number of samples) }. Mix 9. mu.L of the sample mixture with 1. mu.L of the amplification product to avoid the formation of bubbles and to perform electrophoresis as soon as possible. Detection and analysis are carried out by an ABI 3500 genetic analyzer (purchased from ABI company of America), and specific analysis parameters are sample injection voltage: 1.2kv, sample injection time: 15s, electrophoresis time 1210-. The detection results are shown in fig. 2 and fig. 3, respectively.
(7) Analyzing data
The experiment uses a sample of a testee, determines the genotype of the testee according to the positions of all target peaks appearing in the map, guides and judges whether the testee has the wonderful heart, behavior persistence and high pressure tolerance, and further analyzes whether the testee has the potential of scientific quality.
Fig. 2 is an analysis pattern of a saliva sample of the subject 1, and fig. 3 is an analysis pattern of a saliva sample of the subject 2.
The analysis according to FIG. 2 is as follows:
the electrophoresis pattern of the amplification product of the testee 1 shows the following characteristic peaks: the TCERG1L genotype is GA, PCDH18 site is TC, CADM2 site is CT, COX10 site is CT, NRXN1 site is GA, pcDNA peak and characteristic peaks of ginseng-1, ginseng-3 and human genome DNA (huDNA) and ginseng-8.
Wherein locus G of the TCERG1L gene is shown as TCERG1L G, and locus A is shown as TCERG1L A; the PCDH18 gene, site C, is shown as PCDH18C, and site T is shown as PCDH 18T; the CADM2 gene locus T is shown as CADM 21T, and locus C is shown as CADM 21C; the COX10 gene site C is shown as COX 10C, and the SNP site T is shown as COX 10T; NRXN1 gene site G is shown as NRXN 1G and site a is shown as NRXN1 a; the pcDNA locus is unimodal; human genomic DNA (huDNA) reference-1 is a single peak, and B1 is shown in the map; human genomic DNA (huDNA) reference-3 is a single peak, and B3 is shown in the map; the human genome DNA (huDNA) has a single peak at the reference-8 locus, and B8 is shown in the map.
Referring to table 3, subject No. 1 had a heterozygous at TCERG1L site, PCDH18 site, CADM2 site, COX10 site, NRXN1 site, and thus had 5 genotypes with scientific potential for temperament, and thus might have strong knock-hearts, behavioral persistence, and high stress tolerance.
Fig. 3 is an analytical profile of a saliva sample from subject 2.
The electrophoresis pattern of the amplification product of the testee 2 shows the following characteristic peaks: TCERG1L genotype A, PCDH1 is CT, CADM2 is CT, COX10 is C, NRXN1 is G; pcDNA peak, human genome DNA (huDNA) reference-1, human genome DNA (huDNA) reference-3, and human genome DNA (huDNA) reference-8 characteristic peak.
Wherein TCERG1L gene locus A is shown as TCERG1L A; the PCDH18 gene, site C, is shown as PCDH18C, and site T is shown as PCDH 18T; the CADM2 gene locus T is shown as CADM 21T, and locus C is shown as CADM 21C; COX10 gene site C is shown as COX 10C; NRXN1 gene site G is shown as NRXN 1G; the pcDNA locus is unimodal; human genomic DNA (huDNA) reference-1 is a single peak, and B1 is shown in the map; human genomic DNA (huDNA) reference-3 is a single peak, and B3 is shown in the map; the human genome DNA (huDNA) has a single peak at the reference-8 locus, and B8 is shown in the map.
Referring to table 3, subject No. 2, lacking the TCERG1L G, COX 10T and NRXN1 a genotypes, represented that subject 2 was likely to have weaker hunter hearts, behavioral persistence, and high stress tolerance than subject No. 1, and thus subject No. 2 was likely to be inferior to subject No. 1 in terms of scientific vapour potential.
EXAMPLE III
(1) The selected samples were: blood sample of subject 2.
(2) Sample type: a blood sample.
(3) And (3) extracting DNA: and (3) carrying out DNA extraction on the blood sample by using a nucleic acid extractor.
(4) Architecture configuration
According to the specification, preparing a reaction system by the Primer Mix and the PCR reaction solution on ice according to the proportion of the specification, carrying out vortex mixing, centrifuging by a centrifuge, mixing uniformly by a gun head, and subpackaging.
(5) Adding a sample: according to the instruction, a certain amount of the extracted DNA sample is taken by a pipette and added to the reaction system.
(6) Amplification procedure
The amplification procedure on the PCR instrument is as in table 2.
(7) Detection of amplification product on 3500DX genetic analyzer
A sample mixture consisting of deionized formamide and an internal molecular weight standard in the system (Size-500) { (1. mu.L Size-500+ 12. mu.L deionized formamide) × (number of samples) }. Mix 9. mu.L of the sample mixture with 1. mu.L of the amplification product to avoid the formation of bubbles and to perform electrophoresis as soon as possible. Detection and analysis are carried out by an ABI 3500 genetic analyzer (purchased from ABI company of America), and specific analysis parameters are sample injection voltage: 1.2kv, sample injection time: 15s, electrophoresis time 1210-. The results of the measurements are shown in FIG. 4.
(8) Analyzing data
FIG. 4 is an analysis chart of a blood extracted DNA sample of a subject 2, which lacks a TCERG1L G peak, a COX 10T peak and a NRXN 1A peak, and the other peaks are all present, and is consistent with FIG. 3, which shows that the detection kit is also applicable to the blood extracted DNA sample.
Claims (7)
1. A kit and a method for detecting the genotype of the potential of human scientific gas quality comprise the following steps:
a) collecting the cells dropped off from the oral cavity of a testee, storing the cells in a collection card or extracting DNA nucleic acid from a blood sample;
b) adding a saliva acquisition card sample or an extracted DNA sample of a testee into a reaction system for PCR amplification;
c) running an amplification program;
d) carrying out electrophoretic fragment analysis on the amplified product, and judging according to a peak pattern;
the PCR reaction system comprises primer groups for amplifying 5 gene SNP polymorphic sites, 3 human genome DNA internal references and one PCR reaction internal reference, and the primer groups are shown in table 1, and the following primer sequence directions are all from 5 'end to 3' end.
TABLE 1 SNP detection sites, primer sequences and PCR amplification fragment lengths of the kit
2. The kit and method for detecting the human scientific gas potential genotype of claim 1, wherein the exfoliated cells in the mouth are exfoliated cells on the inner wall of the mouth, and are preserved on a cell collection card and can be directly used for PCR amplification.
3. The kit and method for detecting the human scientific temperament potential genotype of claim 1, wherein the blood sample is a blood sample of a subject or a blood sample collected on a blood storage card, and the extracted DNA can be directly used for PCR amplification.
4. The kit and the method for detecting the human scientific gas potential genotype of claim 1, wherein the reaction system comprises a Primer Mix including a Primer group, a PCR reaction solution and DNA/RNA-free water.
5. The kit and method for testing human scientific gas potential genotypes as claimed in claim 1, wherein the Primer Mix in the reaction system comprises primers for testing all genotypes of SNPs of NRXN1, PCDH18, TCERG1L, COX10 and CADM2, and comprises pcDNA and 3 human genomic DNA internal reference primers.
6. The kit and the method for detecting the human scientific gas potential genotype of claim 1, wherein the PCR reaction solution in the reaction system comprises 2 XPCR buffer solution, DNA polymerase, dNTPs, potassium chloride, magnesium chloride and the like.
7. The kit and method for detecting the human scientific gas potential genotype of claim 1, wherein the amplification products are analyzed by electrophoresis; preferably the electrophoresis is capillary electrophoresis.
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Application publication date: 20210608 |