CN112980931A - Kit and method for detecting human music epigenotype - Google Patents
Kit and method for detecting human music epigenotype Download PDFInfo
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Abstract
The invention discloses a kit and a method for detecting human music epigenotype. The invention adopts a multiplex PCR combined fragment length/quality analysis method to synchronously and rapidly qualitatively detect 5 Single Nucleotide Polymorphism (SNP) sites on 5 genes, namely UGT8, FAM76B, GATA2, NDFIP1 and PCDH7, which are related to the capability of distinguishing sound and the capability of synthesizing music. The method 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) 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. Through the analysis, reference is provided for whether the testee possesses the music talent.
Description
Technical Field
The invention relates to the field of gene detection, in particular to a kit and a method for detecting human music endowment genotypes.
Background
Since darwinian first proposed ringing, it was recognized that ringing is an alternating signal. Through evolution, humans have evolved the perception, generation, and processing of crews into musical art. The music art can be used for mastering sentiments, expressing the sentiments and being used as a survival skill; learning music can exercise intelligence, imagination and memory of people, and can also arouse inspiration and temperament.
The music talent is a performance which integrates various abilities and has the capabilities of strong voice distinguishing ability, accurate pronunciation, high absolute pitch and the like. These several abilities are expressed differently in different humans and may be related to differences in the 5 locus SNP sites in humans.
More than 80% of gene polymorphisms in the human genome are Single Nucleotide Polymorphisms (SNPs). SNPs refer to DNA sequence polymorphisms at the genomic level caused by variations of a single nucleotide, including base transitions, transversions, deletions, and insertions, 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.
Musical ability is a complex cognitive skill in non-language, with a potential biological basis. The newborn can distinguish the frequency and humming tune without receiving formal music training. It has long been agreed that these musics can be genetically determined. Research shows that brain structures influence the music ability to some extent, the sound sensation and the sound production ability are established in brain activity areas related to auditory pathways and neurocognitive processes, and the genotype with strong correlation with the brain activity areas is a strong influence factor of individual music ability expression.
Based on the research of measuring the musical ability of the subjects by adopting a pitch precision test method in the Finnish family and the Mongolia family, the research shows that one non-synonymous SNP in the UGT8 gene is highly related to the musical ability through family linkage and association analysis, exome sequencing and genome hybridization analysis comparison (rs4148254, p is 8.0 multiplied by 10-17). The GATA binding protein 2 gene regulates the development of cochlear hair cells and the hypothalamus, influences the audio localization function of human, and has strong correlation with the auditory pathway function because the chromosome 3q21.3(rs9854612) is positioned at the upstream of the gene. The PCDH7 gene is expressed in amygdala of both newborn and adult animals, and related to emotional interpretation of music, the linkage probability of distinguishing the 4q14 (rs13146789) site next to the PCDH7 gene is highest. Rs2186739 on FAM76B gene and rs2961694 on NDFIP1 gene have also been shown to be involved in embryonic development, chondrogenesis, and neurodegeneration. By detecting SNP sites related to the development of the inner ear and the auditory pathway, the inherent ability of distinguishing sound and the comprehensive music ability of the detected person can be judged, music learning suggestions are provided for the detected person, the learning interest is improved, and the advantages of the person are enhanced.
At present, in the market, there are three major SNP typing detection techniques: sequencing method, fluorescent quantitative PCR method, and gene chip method.
Fluorescent quantitative PCR
The fluorescent quantitative PCR adopts fluorescent quenching and double-end labeling technology, and specific probes are designed aiming at SNP sites, so that the fluorescent quantitative PCR has the advantages of high sensitivity, high accuracy, high specificity and the like. However, the fluorescent quantitative PCR flux is low, and if a plurality of SNP sites are to be detected simultaneously, tube-by-tube detection is required, the operation is complex, the sample dosage is large, and the clinical requirement is difficult to adapt. In addition, the fluorescent quantitative PCR is difficult to set internal reference quality control, and false positive and false negative cannot be avoided.
The gene chip technology comprises the following steps:
the gene chip is a two-dimensional DNA probe array formed by regularly arranging and fixing tens of thousands of DNA fragments (gene probes) with specific sequences on supports such as a silicon chip, a glass slide and the like by a micromachining technology, and can carry out rapid qualitative and quantitative analysis on biological information of a gene expression profile of a sample by hybridizing the chip with a marked biological sample. Its advantages are high flux and simple operation; the defects are high detection cost, poor repeatability and lower sensitivity. The variety of the chip is large, and the difficulty in establishing a uniform quality control standard also limits the popularization of the gene chip technology.
Sequencing method:
the Sanger sequencing method is an SNP typing gold standard, and can detect known SNPs and discover unknown SNPs. However, the Sanger sequencing method is complex in operation and high in cost. When the number of sequencing sites is large, sequencing needs to be carried out one by one, the time consumption is long, and the accumulated price is relatively expensive. The second-generation sequencing technology realizes sequencing while synthesis, and has the advantages of high flux and high efficiency. However, the second generation sequencing platform is expensive and low in popularity, and is not mature enough to be used as an SNP detection technology in clinic.
In conclusion, the application of the technology to music talent inheritance guide multiple gene detection has obvious limitations, and at present, no report about a music talent inheritance guide multiple gene detection scheme based on multiple PCR and CE separation technologies exists in China.
Disclosure of Invention
The invention aims to provide a kit and a method for detecting human music endowment genotypes, wherein the kit can detect related genes of a testee and give analysis.
Specifically, the invention discloses a kit and a method for detecting human music epigenotype, 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, PCR buffer solution, vitamin Bt 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.
The kit can carry out synchronous multiplex PCR amplification on a detected sample, and can accurately detect the genotypes of detected genes UGT8, FAM76B, GATA2, NDFIP1 and PCDH7 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 amplify a plurality of loci simultaneously, realizes the simplicity, high efficiency and specificity of detection, and provides reference for whether a testee has the potential of music talent through analysis of amplification maps, which is shown in Table 3.
TABLE 3 detection site-character reference information of the present invention
Drawings
FIG. 1 is an amplification profile of a sample of a subject's mouth. GATA2 genotype is GA, FAM76B is CC, PCDH7 is GT, NDFIP1 is AA, UGT8 is CC, and characteristic peaks of pcDNA peak, human genome DNA (huDNA) ginseng-1, human genome DNA (huDNA) ginseng-4 and human genome DNA (huDNA) ginseng-8 appear.
FIG. 2 is an amplification map of a DNA sample extracted from blood of a subject. GATA2 genotype is GA, FAM76B is CT, PCDH7 is GT, NDFIP1 is AA, UGT8 is CT; the pcDNA peak and characteristic peaks of human genome DNA (huDNA) internal reference-1, human genome DNA (huDNA) internal reference-4 and human genome DNA (huDNA) internal-8 appear.
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 kit of music talent from birth or not, including box body and the reagent of depositing in the box body, including 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 PrimerMix comprises a primer group for amplifying 5 SNP polymorphic sites, 3 human genome references and pcDNA reaction references, and is detailed in Table 1.
Wherein the PCR reaction solution comprises the following components: 2 XPCR buffer solution, DNA polymerase, dNTPs, vitamin Bt, potassium chloride, magnesium chloride and the like.
In the embodiment, the sample is an oral cavity wall exfoliative cell collection card of a testee 1, and a blood DNA extraction sample of a testee 2.
In the implementation case, the electrophoresis is to carry out 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 sound distinguishing capability and the comprehensive music capability of the testee.
Example one
(1) The selected samples were: a sample of cells exfoliated from the oral wall of subject 1.
(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 results are shown in FIG. 1.
(7) Analyzing data
The experiment uses a sample of the testee, determines the genotype of the testee according to the positions of the target peaks appearing in the map, guides and judges whether the testee has the ability of distinguishing sound and the ability of synthesizing music, and further analyzes whether the testee has the potential of music talent.
Fig. 1 is an analysis pattern of a saliva sample of a subject 1, and fig. 2 is an analysis pattern of a blood DNA extraction sample of a subject 2.
The analysis according to FIG. 1 is as follows:
the electrophoresis pattern of the amplification product of the testee 1 shows the following characteristic peaks: GATA2 genotype is GA, FAM76B site is TC, PCDH7 site is GT, NDFIP1 site is AA, UGT8 site is CT, pcDNA peak and characteristic peaks of ginseng-1, ginseng-4 and genomic DNA (huDNA) of human, and ginseng-8.
Referring to table 3, the test subject 1 had 1 genotype with strong comprehensive musical ability, with homozygote at UGT8 site, homozygote at GATA2 site, and heterozygote at PCDH7 site; the FAM76B locus is homozygote, the NDFIP1 locus is homozygote, and the kit has 1 genotype with weak sound distinguishing capability and 1 genotype with strong sound distinguishing capability, so that the kit may have strong comprehensive music distinguishing capability and medium sound distinguishing capability.
Example two
(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. 2.
(8) Analyzing data
Fig. 2 is a blood-extracted DNA sample analysis map of subject 2, analyzed according to fig. 2 as follows:
the electrophoresis pattern of the amplification product of the testee 2 shows the following characteristic peaks: GATA2 genotype is GA, FAM76B site is TC, PCDH7 site is GT, NDFIP1 site is AA, UGT8 site is CT, pcDNA peak and characteristic peaks of ginseng-1, ginseng-4 and genomic DNA (huDNA) of human, and ginseng-8.
Referring to table 3, the UGT8 site, GATA2 site, and PCDH7 site of test subject No. 2 were all heterozygotes, and possessed 3 genotypes with moderate comprehensive musical ability; the FAM76B site is heterozygote, the NDFIP1 site is homozygote, and the 1 genotype with strong sound distinguishing capability is possessed, so that the medium comprehensive music capability and the strong sound distinguishing capability can be possessed.
Claims (7)
1. A kit and a method for detecting human music epigenotypes 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) and 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 human music epigenotypes according to 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 human music epigenotypes according to claim 1, wherein the blood sample is a blood sample of a subject or a blood sample collected from a blood storage card, and the extracted DNA can be directly used for PCR amplification.
4. The kit and the method for detecting human music epigenotypes according to claim 1, wherein the reaction system comprises Primer Mix including Primer set, PCR reaction solution and DNA/RNA-free water.
5. The kit and method for detecting human music Tianzhu genotype of claim 1, wherein the Primer Mix in the reaction system comprises primers for detecting all genotypes of SNPs of UGT8, FAM76B, GATA2, NDFIP1 and PCDH7, and comprises pcDNA and 3 human genomic DNA internal reference primers.
6. The kit and method for detecting human music epigenotypes according to claim 1, wherein the PCR reaction solution in the reaction system comprises 2 XPCR buffer, DNA polymerase, dNTPs, vitamin Bt, potassium chloride, magnesium chloride, etc.
7. The kit and method for detecting human musical epigenotypes according to claim 1, wherein the amplification products are analyzed by electrophoresis; preferably the electrophoresis is capillary electrophoresis.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113774168A (en) * | 2021-09-18 | 2021-12-10 | 北京艾瑞克阳医疗科技有限公司 | 2019 novel coronavirus, Deltay and lambda variant strain typing nucleic acid detection kit and detection method thereof |
Citations (2)
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| JP2011182780A (en) * | 2010-02-12 | 2011-09-22 | Hubit Genomix Inc | Polymorphism of efficacy and side effect expression of il-6 inhibitor treatment and use thereof |
| CN109207579A (en) * | 2018-09-06 | 2019-01-15 | 宁波海尔施基因科技有限公司 | A kind of Multiple detection kit and application thereof detecting malignant fever tumor susceptibility gene |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011182780A (en) * | 2010-02-12 | 2011-09-22 | Hubit Genomix Inc | Polymorphism of efficacy and side effect expression of il-6 inhibitor treatment and use thereof |
| CN109207579A (en) * | 2018-09-06 | 2019-01-15 | 宁波海尔施基因科技有限公司 | A kind of Multiple detection kit and application thereof detecting malignant fever tumor susceptibility gene |
Non-Patent Citations (3)
| Title |
|---|
| "rs2186739", 《UCSC》 * |
| HANSOO PARK等: "Comprehensive genomic analyses associate UGT8 variants with musical ability in a Mongolian population", 《J MED GENET》, vol. 49 * |
| J OIKKONEN等: "A genome-wide linkage and association study of musical aptitude identifies loci containing genes related to inner ear development and neurocognitive functions", 《MOLECULAR PSYCHIATRY》, vol. 20, pages 6 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113774168A (en) * | 2021-09-18 | 2021-12-10 | 北京艾瑞克阳医疗科技有限公司 | 2019 novel coronavirus, Deltay and lambda variant strain typing nucleic acid detection kit and detection method thereof |
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Application publication date: 20210618 |