CN105671186B - Gene and detection kit and detection method for evaluator mathematical ability - Google Patents
Gene and detection kit and detection method for evaluator mathematical ability Download PDFInfo
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Abstract
The present invention relates to field of biotechnology, in particular to for the gene of evaluator mathematical ability and detection kit and detection method.The present invention provides SPOCK1 gene in mankind's full-length genome, especially application of its SNP site rs1012694, rs11743006, rs17778739, rs17777541 in evaluator mathematical ability for the first time.The present invention has found 4 SNP sites for reaching whole-genome association notable level being located on SPOCK1 gene by the meta analysis of three independent crowd's samples, a kind of detection kit and detection method for assessing mathematical ability is provided based on this, experimental result is from Chinese population, with very strong Chinese population adaptability, detection means economy is convenient simultaneously, is conducive to promote on a large scale in crowd.
Description
Technical field
The present invention relates to field of biotechnology, more particularly to for the gene of evaluator mathematical ability and inspection
Test agent box and detection method.
Background technique
Mathematical ability has great importance for personal and entire society development.Mathematical ability is most important as the mankind
One of higher level cognitive capacity, be basic capacity necessary to individual correct understanding objective world, in addition to following work and
Life all has important influence.Therefore, fostering mathematical skill is educated always through the mankind.Research confirms that mathematical ability is by losing
It passes and environmental factor codetermines, inherent cause determines 20% to the 90% of mathematical ability.
Single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) is changing by single nucleotide acid
The change of DNA sequence dna caused by change causes the diversity of DNA sequence.SNP distribution is wide, density is high, it has also become after limit
Third generation genetic marker after property fragment length polymorphism (RFLP) mark and microsatellite, that is, Short tandem repeatSTR (STR) mark processed,
It plays an important role in the research of the complex inheritances disease such as tumour, diabetes, Dyslexia.Wherein, whole-genome association
(Genome Wide Association Study, GWAS) is the important means for studying this kind of complex inheritance character related gene.
Only there is a small amount of research in American-European crowd for the whole-genome association of mathematical ability, and does not find to show
Work property gene loci.At present in Chinese population, there are no the full-length genomes of the large-scale system carried out for mathematical ability
Association analysis research report, thus lack the genetic site for being used for mathematical ability assessment for Chinese population and association base
Because of detection kit.Therefore carry out the research of crowd's mathematical ability correlated inheritance site, have to China human mortality overall qualities are improved
Significance.
Summary of the invention
In view of this, the purpose of the present invention is to provide for evaluator mathematical ability gene and detection kit and
Detection method assesses mathematical ability by the whole-genome association to the gene, is suitable for Chinese population
For achieving the above object, the invention provides the following technical scheme:
Application of the SPOCK1 gene in evaluator mathematical ability in mankind's full-length genome.
Preferably, the SPOCK1 gene is SNP site rs1012694 thereon, SNP site rs11743006, SNP position
It is more than one or two of point rs17778739, SNP site rs17777541.
At present in Chinese population, there are no the associations point of the full-length genome of the large-scale system carried out for mathematical ability
Analysis research is reported, thus lacks detecting for the genetic site of mathematical ability assessment and associated gene for Chinese population
Kit.The present invention for the first time in Chinese population by GWAS analysis have found with the associated gene of Chinese population mathematical ability and
Its SNP site, and a kind of detection kit and detection method for assessing mathematical ability is provided on basis herein.
In terms of mathematical ability whole-genome association research, the present invention is for the first time research pair with Chinese general population
As having used structuring maths exam achievement as judgment criteria, minimum point of 0 point of best result 100 divides, and uses full-length genome later
Association analysis method, analyzes being associated between the mathematics achievement and full-length genome single nucleotide polymorphism of individual, and research obtains number
Study the association site for the mathematical ability that achievement is representative.
The first stage of whole-genome association, (sample size is respectively 494 Hes based on two independent crowds
504) SNP site that 28 be located on autosome meet the further validation criteria of GWAS, is obtained altogether.To this 28 SNP
Point is verified in the crowd that another sample size is 599, as a result such as table 1, on 1 result verification of table SPOCK1 gene
SNP site rs1012694, SNP site rs11743006, SNP site rs17778739, SNP site rs17777541 have aobvious
Write sex differernce.
Table 1
Hereafter meta analysis is carried out to the data result of three crowd's samples, shows that 4 of the present invention are located at SPOCK1
SNP site on gene reaches whole-genome association notable level (5 × 10-8), respectively rs1012694 (p=1.870
×10-9), (p=1.450 × 10 rs11743006-9), (p=2.170 × 10 rs17778739-9) and rs17777541 (p=
7.840×10-9), it the results are shown in Table 2.
Table 2
| SNP | Allele | β | SE | P |
| rs1012694 | T/C | -2.396 | 0.398 | 1.87E-09 |
| rs11743006 | A/C | -2.404 | 0.397 | 1.45E-09 |
| rs17778739 | G/C | -2.386 | 0.398 | 2.17E-09 |
| rs17777541 | C/G | -2.302 | 0.399 | 7.84E-09 |
Note, SNP: single nucleotide polymorphism；Allele: allele；β: addition model regression coefficient；SE: regression coefficient
Standard error；Wherein one risk allele of every increase is (corresponding to first etc. in the minorAllele and table 2 in table 1
Position gene), β unit of mathematics achievement mean change, therefore β can be used as score value to assess mathematical ability height.
Based on above-mentioned summary of the invention, the present invention provides a kind of detection kits of evaluator mathematical ability, comprising:
SNP site rs1012694, SNP site rs11743006, SNP site rs17778739, SNP site
The amplimer and its Single base extension primer of the above SNP site of one or both of rs17777541.
Preferably, the SNP site rs1012694, SNP site rs11743006, SNP site rs17778739, SNP
The amplimer and its Single base extension primer of site rs17777541 is as follows:
SNP site rs1012694 amplimer sequence is as shown in SEQ ID NO:1-2, and Single base extension primer sequence is such as
Shown in SEQ ID NO:3；
SNP site rs11743006 amplimer sequence is as shown in SEQ ID NO:4-5, and Single base extension primer sequence is such as
Shown in SEQ ID NO:6；
SNP site rs17778739 amplimer sequence is as shown in SEQ ID NO:7-8, and Single base extension primer sequence is such as
Shown in SEQ ID NO:9；
SNP site rs17777541 amplimer sequence is as shown in SEQ ID NO:10-11, Single base extension primer sequence
As shown in SEQ ID NO:12.
Preferably, the kit further include:
Taq enzyme, shrimp alkaline phosphotase, shrimp alkaline phosphotase buffer, without enzyme water and SNP site rs1012694, SNP
The respective standard DNA sample of site rs11743006, SNP site rs17778739, SNP site rs17777541.Wherein, standard
DNA is used primarily as control, and whether all reagents can work normally in test kit.It is advised according to the operation of default
Model will do it standard DNA detection in specifically test first time implementation process.
Meanwhile the present invention also provides a kind of detection methods of evaluator mathematical ability, comprising:
Step 1, with SNP site rs1012694, SNP site rs11743006, SNP site rs17778739, SNP site
The amplimer of the above SNP site of one or both of rs17777541 distinguishes PCR amplification human gene group DNA target fragment；
After PCR amplification described in step 2, step 1, PCR product is handled with shrimp alkaline phosphotase, is removed free
dNTPs；
After the completion of step 3, step 2, corresponding Single base extension primer is added into PCR product and carries out PCR respectively
Single base extension；
Step 4, by the PCR product purifying resin after Single base extension, chip point sample, mass spectral analysis, testing result uses
Software is to SNP site rs1012694, SNP site rs11743006, SNP site rs17778739, SNP site rs17777541
Carry out allelic gene typing；
Step 5, according to following criterion evaluation mathematical ability:
SNP site rs1012694, carrying the crowd that allelic gene typing is C/C is reference, allelic gene typing T/C
Crowd is low 2.396 points average compared with C/C parting crowd's mathematical ability, and allelic gene typing is T/T crowd compared with C/C parting crowd's mathematics
Ability is 4.792 points low；
SNP site rs11743006, carrying the crowd that allelic gene typing is C/C is reference, allelic gene typing A/C
Crowd is 2.404 points low compared with C/C parting crowd's mathematical ability, and allelic gene typing is A/A crowd compared with C/C parting crowd's mathematical ability
Low 4.808 points；
SNP site rs17778739, carrying the crowd that allelic gene typing is C/C is reference, allelic gene typing G/C
Crowd indicates 2.386 points low compared with C/C parting crowd's mathematical ability, and allelic gene typing is that G/G crowd indicates compared with C/C parting crowd
Mathematical ability is 4.772 points low；
SNP site rs17777541, carrying the crowd that allelic gene typing is G/G is reference, allelic gene typing C/G
Crowd is 2.302 points low compared with G/G parting crowd's mathematical ability, and allelic gene typing is C/C crowd compared with G/G parting crowd's mathematical ability
Low 4.604 points；
Mathematical ability is assessed according to score value size.
Preferably, step 1 are as follows:
With SNP site rs1012694, SNP site rs11743006, SNP site rs17778739, SNP site
The amplimer of the above SNP site of one or both of rs17777541 distinguishes PCR amplification human peripheral genomic DNA purpose piece
Section；
PCR amplification program are as follows: 94 DEG C of 4min；94 DEG C of 20s, 56 DEG C of 30s, 72 DEG C of 1min, totally 45 recycle；72℃5min；4
DEG C keep；
PCR amplification system are as follows: system totally 5 μ l, including 2 μ l Taq enzymes, 1.5 μ l are without enzyme water, upstream and downstream amplimer
Each 0.25 μ l, 1 μ l human peripheral genomic DNA sample solution, concentration are 50ng/ μ l.
Preferably, step 2 are as follows:
After PCR amplification described in step 1, prepares shrimp alkaline phosphotase processing reaction solution and reacted with PCR product, remove trip
From dNTPs；
Reaction condition is 37 DEG C of 40min, 85 DEG C of 5min, 4 DEG C of holdings；
Shrimp alkaline phosphotase handles reaction solution: totally 2 μ l, including 1.53 μ l without enzyme water, 0.17 μ l shrimp alkaline phosphotase
Buffer, 0.3 μ l shrimp alkaline phosphotase.
Preferably, step 3 are as follows:
After the completion of step 2, corresponding Single base extension primer is added into PCR product and carries out PCR single base respectively
Extend；
PCR reaction system: 0.2 μ l of reaction buffer, 0.94 0.2 μ l of μ l, ddNTP of Single base extension primer, Taq enzyme
0.041 μ l, no 0.619 μ l of enzyme water；
PCR response procedures: I.94 DEG C 30s；II.94℃5s；III.52℃5s；IV.80℃5s；V. III is returned to, 4 are followed
Ring；VI. II, 39 circulations are returned；VII.72 DEG C of holding 3min, VIII4 DEG C of holding.
Reagent is commercial reagents used in the process of PCR in the present invention, can be bought by commercially available approach, such as various buffers
Deng.
Preferably, software described in step 4 is TYPER4.0 software (Sequenom).
From the above technical scheme, the present invention has found that 4 are located at by the meta analysis of three independent crowd's samples
The SNP site for reaching whole-genome association notable level on SPOCK1 gene provides a kind of assessment mathematics based on this
The detection kit and detection method of ability, experimental result have very strong Chinese population adaptability from Chinese population,
Detection means economy is convenient simultaneously, is conducive to promote on a large scale in crowd.
Specific embodiment
The invention discloses the gene and detection kit and detection method for evaluator mathematical ability, this field skills
Art personnel can use for reference present disclosure, be suitably modified realization of process parameters.In particular, it should be pointed out that all similar replacements and
Change apparent to those skilled in the art, they are considered as being included in the present invention.It is of the present invention application,
Product and detection method are described by preferred embodiment, related personnel obviously can not depart from the content of present invention,
To method described herein and application is modified or appropriate changes and combinations in spirit and scope, carry out the implementation and application present invention
Technology.
In terms of mathematical ability whole-genome association research, the present invention is ground using Chinese general population as research object
Study carefully the association site for the mathematical ability for showing that mathematics achievement is representative.The first stage of whole-genome association, with two
Based on independent crowd (sample size is respectively 494 and 504), it is further that total 28 obtained on autosome meet GWAS
The SNP site of validation criteria.This 28 SNP sites are verified in the crowd that another sample size is 599.At three
Find that 4 reach whole-genome association notable level (5 on SPOCK1 gene altogether in the meta analysis of crowd's sample
×10-8) SNP site, respectively (p=1.870 × 10 rs1012694-9), (p=1.450 × 10 rs11743006-9)、
(p=2.170 × 10 rs17778739-9) and (p=7.840 × 10 rs17777541-9)。
On the basis of newfound herein, the present invention further developed the gene detecting kit of mathematical ability assessment, packet
Containing the PCR primer pair and specific detection above-mentioned 4 loci gene type for expanding above-mentioned 4 SNP site genetic fragments
Sequenom MassARRAY Single base extension primer.Sequence such as table 3 of the PCR primer to sequence and Single base extension primer
It is shown.
Table 3
The kit further includes Taq enzyme, SAP (shrimp alkaline phosphatase, shrimp alkaline phosphotase),
The standard in SAP Buffer, no enzyme water and the site rs1012694, rs11743006, rs17778739 and rs17777541
DNA sample.
PCR primer containing specific amplification target fragment in kit of the invention is to, specific detection genes of individuals
The Sequenom MassARRAY Single base extension primer (specificity of primer ensure that the accuracy of subsequent detection) of type, and
General components, reagent, operating procedure etc. for PCR amplification and progress Sequenom MassARRAY parting detecting reagent.
Detection method of the invention is used to measure the genomic DNA from people, and clinic materials samples sources are extensive, such as body
Liquid, histocyte etc. can prepare genomic DNA by extracting and purifying these sample standard deviations.
Reagent used in the present invention is commercial product, is purchased from Tiangeng biochemical technology.
According to the operating procedure that product description provides, using OSR-M102-T1 poba gene group DNA extraction kit and
Its matched OSE-M48 instrument for extracting nucleic acid (Tiangeng biochemical technology) prepares people DNA sample of the human peripheral genomic DNA as detection
This.
The present invention detects above-mentioned 4 SNP sites using Sequenom MassARRAY, and detection method is as follows:
1,4 amplimers shown in table 1 are to amplifying target genes segment respectively.PCR amplification: 94 DEG C of 4min；94℃
20s, 56 DEG C of 30s, 72 DEG C of 1min, totally 45 recycle；72℃5min；4 DEG C of holdings.Each reaction volume is 5 μ l, including 2 μ lTaq
Enzyme, 1.0 μ l are without enzyme water, PCR upstream and downstream primer each 0.25 μ l, 0.5 μ ldNTP (10mMeach), 1 μ lDNA sample solution
(concentration is 50ng/ μ l).
2, after PCR, by PCR product respectively with alkaline phosphatase (SAP, shrimp alkaline
Phosphatase) processing reaction solution reaction, with remove system middle reaches from dNTPs.Alkaline phosphatase treatment reaction solution (SAP
Mix, totally 2 μ l, including 1.53 μ l without enzyme water, 0.17 μ l SAP Buffer, 0.3 μ l SAP Enzyme) reaction condition: 37
DEG C 40min, 85 DEG C of 5min, 4 DEG C of holdings, starting PCR instrument carry out alkaline phosphatase treatment.
3, after the completion of step 2, into PCR product, corresponding Single base extension primer carries out PCR respectively in addition table 3
Single base extension；
PCR reaction system: 0.2 μ l of reaction buffer, 0.94 0.2 μ l of μ l, ddNTP of Single base extension primer, Taq enzyme
0.041 μ l, no 0.619 μ l of enzyme water；
PCR response procedures: I.94 DEG C 30s；II.94℃5s；III.52℃5s；IV.80℃5s；V. III is returned to, 4 are followed
Ring；VI. II, 39 circulations are returned；VII.72 DEG C of holding 3min, VIII4 DEG C of holding.
4, purifying resin, chip point sample, mass spectral analysis, testing result TYPER4.0 software (Sequenom) parting are simultaneously defeated
Result out.
5, according to following criterion evaluation mathematical ability:
SNP site rs1012694, carrying the crowd that allelic gene typing is C/C is reference, allelic gene typing T/C
Crowd is low 2.396 points average compared with C/C parting crowd's mathematical ability, and allelic gene typing is T/T crowd compared with C/C parting crowd's mathematics
Ability is 4.792 points low；
SNP site rs11743006, carrying the crowd that allelic gene typing is C/C is reference, allelic gene typing A/
C crowd is 2.404 points low compared with C/C parting crowd's mathematical ability, and allelic gene typing is A/A crowd compared with C/C parting crowd's mathematics energy
Power is 4.808 points low；
SNP site rs17778739, carrying the crowd that allelic gene typing is C/C is reference, allelic gene typing G/C
Crowd indicates 2.386 points low compared with C/C parting crowd's mathematical ability, and allelic gene typing is that G/G crowd indicates compared with C/C parting crowd
Mathematical ability is 4.772 points low；
SNP site rs17777541, carrying the crowd that allelic gene typing is G/G is reference, allelic gene typing C/G
Crowd is 2.302 points low compared with G/G parting crowd's mathematical ability, and allelic gene typing is C/C crowd compared with G/G parting crowd's mathematical ability
Low 4.604 points；
Mathematical ability is assessed according to score value size.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (8)
1. in mankind's full-length genomeSPOCK1SNP site rs1012694, the SNP site rs11743006, SNP site of gene
The above application in evaluator mathematical ability of one or two of rs17778739, SNP site rs17777541.
2. a kind of detection kit of evaluator mathematical ability characterized by comprising
SNP site rs1012694, SNP site rs11743006, SNP site rs17778739, in SNP site rs17777541
The amplimer and its Single base extension primer of one or more SNP site.
3. kit according to claim 2, which is characterized in that the SNP site rs1012694, SNP site
Rs11743006, SNP site rs17778739, the amplimer of SNP site rs17777541 and its Single base extension primer are such as
Under:
SNP site rs1012694 amplimer sequence is as shown in SEQ ID NO:1-2, Single base extension primer sequence such as SEQ
Shown in ID NO:3；
SNP site rs11743006 amplimer sequence is as shown in SEQ ID NO:4-5, Single base extension primer sequence such as SEQ
Shown in ID NO:6；
SNP site rs17778739 amplimer sequence is as shown in SEQ ID NO:7-8, Single base extension primer sequence such as SEQ
Shown in ID NO:9；
SNP site rs17777541 amplimer sequence is as shown in SEQ ID NO:10-11, and Single base extension primer sequence is such as
Shown in SEQ ID NO:12.
4. kit according to claim 2, which is characterized in that further include:
Taq enzyme, shrimp alkaline phosphotase, shrimp alkaline phosphotase buffer, without enzyme water and SNP site rs1012694, SNP site
The respective standard DNA sample of rs11743006, SNP site rs17778739, SNP site rs17777541.
5. a kind of detection method of evaluator mathematical ability characterized by comprising
Step 1, with SNP site rs1012694, SNP site rs11743006, SNP site rs17778739, SNP site
The amplimer of the above SNP site of one or both of rs17777541 distinguishes PCR amplification human gene group DNA target fragment；
After PCR amplification described in step 2, step 1, PCR product is handled with shrimp alkaline phosphotase, removes free dNTPs；
After the completion of step 3, step 2, corresponding Single base extension primer is added into PCR product and carries out the mono- alkali of PCR respectively
Base extends；
Step 4, by the PCR product purifying resin after Single base extension, chip point sample, mass spectral analysis, testing result uses software
SNP site rs1012694, SNP site rs11743006, SNP site rs17778739, SNP site rs17777541 are carried out
Allelic gene typing；
Step 5, according to following criterion evaluation mathematical ability:
SNP site rs1012694, carrying the crowd that allelic gene typing is C/C is reference, and allelic gene typing is T/C crowd
Low 2.396 points average compared with C/C parting crowd's mathematical ability, allelic gene typing is T/T crowd compared with C/C parting crowd's mathematical ability
Low 4.792 points；
SNP site rs11743006, carrying the crowd that allelic gene typing is C/C is reference, and allelic gene typing is A/C crowd
2.404 points low compared with C/C parting crowd's mathematical ability, allelic gene typing is that A/A crowd is low compared with C/C parting crowd's mathematical ability
4.808 point；
SNP site rs17778739, carrying the crowd that allelic gene typing is C/C is reference, and allelic gene typing is G/C crowd
Indicate 2.386 points low compared with C/C parting crowd's mathematical ability, allelic gene typing is that G/G crowd indicates compared with C/C parting crowd's mathematics
Ability is 4.772 points low；
SNP site rs17777541, carrying the crowd that allelic gene typing is G/G is reference, and allelic gene typing is C/G crowd
2.302 points low compared with G/G parting crowd's mathematical ability, allelic gene typing is that C/C crowd is low compared with G/G parting crowd's mathematical ability
4.604 point；
Mathematical ability is assessed according to score value size.
6. detection method according to claim 5, which is characterized in that step 1 are as follows:
With SNP site rs1012694, SNP site rs11743006, SNP site rs17778739, SNP site rs17777541
One or both of the above SNP site amplimer distinguish PCR amplification human gene group DNA target fragment；
PCR amplification program are as follows: 94 DEG C of 4 min；94 DEG C of 20 s, 56 DEG C of 30 s, 72 DEG C of 1 min, totally 45 recycle；72℃ 5
min；4 DEG C of holdings；
PCR amplification system are as follows: system totally 5 μ l, including 2 μ l Taq enzymes, 1.0 μ l are without enzyme water, 0.5 μ l, 10 mM dNTP, on
Trip and each 0.25 μ l of downstream amplification primer, 1 μ l human gene group DNA's sample solution, concentration are 50 ng/ μ l.
7. detection method according to claim 5, which is characterized in that step 2 are as follows:
After PCR amplification described in step 1, prepares shrimp alkaline phosphotase processing reaction solution and reacted with PCR product, removed free
dNTPs；
Reaction condition is 37 DEG C of 40 min, 85 DEG C of 5 min, 4 DEG C of holdings；
Shrimp alkaline phosphotase handles reaction solution: totally 2 μ l, and including 1.53 μ l without enzyme water, 0.17 μ l shrimp alkaline phosphotase is slow
Fliud flushing, 0.3 μ l shrimp alkaline phosphotase.
8. detection method according to claim 5, which is characterized in that step 3 are as follows:
After the completion of step 2, corresponding Single base extension primer is added into PCR product and carries out PCR Single base extension respectively；
PCR reaction system: 0.2 μ l of reaction buffer, 0.94 0.2 μ l of μ l, ddNTP of Single base extension primer, Taq enzyme
0.041 μ l, no 0.619 μ l of enzyme water；
PCR response procedures: 94 DEG C of 30 s of I.；II. 94℃ 5 s；III. 52℃ 5 s；IV. 80℃ 5 s；V. it returns
III, 4 circulations；VI. II, 39 circulations are returned；VII.72 DEG C of holding 3min, VIII 4 DEG C of holding.
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