CN108588215A - A kind of primer and its detection method for detecting the relevant SNP site of familial hypercholesterolemia neurological susceptibility - Google Patents
A kind of primer and its detection method for detecting the relevant SNP site of familial hypercholesterolemia neurological susceptibility Download PDFInfo
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Abstract
The invention discloses a kind of primers and its detection method for detecting the relevant SNP site of familial hypercholesterolemia neurological susceptibility.Primer includes the primer of the primer in the sites rs41291161, the primer in the sites rs12720772, the primer in the sites rs12084215 and the sites rs2922126, and primer sequence is shown in NO.1~8 SEQ ID；Detection method includes the following steps：(1) extraction patient gene's group DNA；(2) using gained DNA in step (1) as template, PCR amplification is carried out using primer described in claim 1；(3) amplified production is recycled, PCR amplification is then carried out, then purified, analysis determination is carried out to SNP partings finally by sequenator.The method of the present invention accuracy is high, and primer specificity is good, can be used for the risk assessment of familial hypercholesterolemia, and guidance is provided for the disease prevention of high cholesterol.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to one kind is susceptible for detecting familial hypercholesterolemia
The primer and its detection method of the relevant SNP site of property.
Background technology
Familial hypercholesterolemia is also known as familial hyperbetalipoproteinemia, and most common heredity is high in fat childhood being
One kind of most serious in mass formed by blood stasis and lipid-metabolism disease, can lead to going out for the angiocardiopathy complication of various threat to life
It is existing, it is one kind of coronary artery disease.The clinical manifestation of the disease be low-density lipoprotein cholesterol level increase, xanthoma, angle
Film bends and premature coronary heart disease.With the improvement of living standards, more and more people also extremely focus on own health.Familial
Its serious symptom of hypercholesterolemia, therefore very must to the detection of Familial HypercholesterolemicPatients Patients early gene and screening
It wants, passes through detection and the relevant SNP of familial high cholesterol neurological susceptibility, it will be able to high cholesterol Susceptible population be helped to understand its trouble
The risk of upper high cholesterol, carries out prevention work in advance, avoids the generation of familial hypercholesterolemia.Single nucleotide polymorphism
(SNP) the after restriction fragment length polymorphism (RFLP) mark and microsatellite tandem sequence repeats (STR) mark is had become at present
Three generations's genetic marker.It is more by mononucleotide in order to realize the early discovery, early prevention and early control of familial hypercholesterolemia
State property (SNP) detection high cholesterol neurological susceptibility have become research hotspot both domestic and external, have genome-wide association study show this 4
A sites (rs41291161, rs12720772, rs12084215, rs2922126) SNP and the close phase of high cholesterol neurological susceptibility
It closes, by detecting the polymorphism with the relevant SNP site of high cholesterol neurological susceptibility, it can be estimated that suffer from familial hypercholesterolemia
Susceptible risk, provide guidance for the disease prevention of high cholesterol.
Invention content
For above-mentioned deficiency in the prior art, the present invention provides a kind of for detecting familial hypercholesterolemia SNP
The primer and its detection method in site, primer specificity is good, and detection method accuracy is high, can be used for familial hypercholesterolemia
The risk assessment of disease provides guidance for the disease prevention of high cholesterol.
To achieve the above object, the technical solution adopted by the present invention to solve the technical problems is：
A kind of primer for detecting the relevant SNP site of familial hypercholesterolemia neurological susceptibility, primer include
The primer in the sites rs41291161, the primer in the sites rs12720772, the primer in the sites rs12084215 and the sites rs2922126
Primer, each primer particular sequence is as follows：
rs41291161-F：5’-CTTCACAAATACACACCTG-3’；(SEQ ID NO.1)
rs41291161-R：5’-TGTCCTATCTGAACCTTAGC-3’；(SEQ ID NO.2)
rs12720772-F：5’-AAGTACACACTTGGAAGGAA-3’；(SEQ ID NO.3)
rs12720772-R：5’-GTCCACACCTATCTCTAACTAT-3’；(SEQ ID NO.4)
rs12084215-F：5’-GACACAGATTAGTTCTGGAT-3’；(SEQ ID NO.5)
rs12084215-R：5’-AAGATGATGTGACCACTGT-3’；(SEQ ID NO.6)
rs2922126-F：5’-AGTCTCACGCAGTTATAGG-3’；(SEQ ID NO.7)
rs2922126-R：5’-CTTCTACTTCTCAACAGCAAC-3’.(SEQ ID NO.8)
A kind of detection method of the relevant SNP site polymorphism of familial hypercholesterolemia neurological susceptibility, including following step
Suddenly：
(1) extraction patient gene's group DNA；
(2) using gained DNA in step (1) as template, PCR amplification is carried out using primer described in claim 1；
(3) amplified production is recycled, and carries out concentration mensuration, calculation template amount carries out PCR amplification and to amplified production again
It is purified, analysis determination is carried out to SNP partings finally by sequenator.
Further, PCR amplification system is in step (2)：100~150ng of DNA profiling, a concentration of 5pmol/ μ L are just
To 3.0 μ L of primer, the 3.0 μ L of reverse primer of a concentration of 5pmol/ μ L, 25.0 μ L of Prime STAR MAX, ddH is finally used2O is mended
Sufficient volume is to 50 μ L.
Further, PCR reaction conditions are in step (2)：95 DEG C of pre-degeneration 5min, 95 DEG C of denaturation 30s, 55 DEG C are annealed
15s, 72 DEG C of extension 15s, 30 recycle, in 4 DEG C of preservations after last 72 DEG C of extensions 5min.
Further, PCR amplification system is in step (3)：2.5 μ L of DTCS Master Mix, a concentration of 5pmol/ μ L
1.0 μ L of reverse primer, DNA profiling 20ng, finally use ddH2O supplies volume to 10 μ L.
Further, PCR reaction conditions are in step (3)：94 DEG C of pre-degeneration 30s, 94 DEG C of denaturation 25s, 55 DEG C are annealed
25s, 60 DEG C of extension 3min, 30 cycles, last 60 DEG C of extensions 20min.
Beneficial effects of the present invention are：
The present invention provides respectively detection familial hypercholesterolemia related gene rs41291161,
The primer and detection method of rs12720772, rs12084215, rs2922126 site-specific single nucleotide polymorphism, primer specific
Property it is good, detection method accuracy is high, can be used for the risk assessment of familial hypercholesterolemia, is the disease prevention of high cholesterol
Guidance is provided.
Description of the drawings
Fig. 1 is pcr amplification product agarose gel electrophoresis figure；
Fig. 2, Fig. 3, Fig. 4 are the sequencing result figure of rs41291161 loci polymorphisms detection；
Fig. 5, Fig. 6, Fig. 7 are the sequencing result figure of rs12720772 loci polymorphisms detection；
Fig. 8, Fig. 9, Figure 10 are the sequencing result figure of rs12084215 loci polymorphisms detection；
Figure 11, Figure 12, Figure 13 are the sequencing result figure of rs2922126 loci polymorphisms detection；
Figure 14, Figure 15, Figure 16 are the fluorescence proof diagram of rs41291161 loci polymorphism testing results；
Figure 17, Figure 18, Figure 19 are the fluorescence proof diagram of rs12720772 loci polymorphism testing results；
Figure 20, Figure 21, Figure 22 are the fluorescence proof diagram of rs12084215 loci polymorphism testing results；
Figure 23, Figure 24, Figure 25 are the fluorescence proof diagram of rs2922126 loci polymorphism testing results.
Specific implementation mode
The specific implementation mode of the present invention is described below, in order to facilitate understanding by those skilled in the art this hair
It is bright, it should be apparent that the present invention is not limited to the ranges of specific implementation mode, for those skilled in the art,
As long as various change is in the spirit and scope of the present invention that the attached claims limit and determine, these variations are aobvious and easy
See, all are using the innovation and creation of present inventive concept in the row of protection.
Embodiment
1, it determines and the relevant SNP site of familial hypercholesterolemia neurological susceptibility
The present invention filters out 4 and high cholesterol neurological susceptibility associated SNP positions, specially：rs41291161、
Rs12720772, rs12084215 and rs2922126 are located at the rs41291161 of APOB genes on human chromosomal 2p24.1
Susceptibility loci is A, and rs12720772 susceptibility locis are A, are located at PCSK9 genes on the 1p24.1 of human chromosomal
Rs12084215 susceptibility locis are A, are located at the rs2922126 susceptibility locis of GHSR genes on human chromosomal 3q26.31
For A.
2, design primer
Sequence with the above SNP site is template, using (the Molecular Biology of primer-design software Oligo 7
Insights, USA), by comparing the several key factors for influencing primer, the primer sequence filtered out, particular sequence is such as
Under：
rs41291161-F：5’-CTTCACAAATACACACCTG-3’；(SEQ ID NO.1)
rs41291161-R：5’-TGTCCTATCTGAACCTTAGC-3’；(SEQ ID NO.2)
rs12720772-F：5’-AAGTACACACTTGGAAGGAA-3’；(SEQ ID NO.3)
rs12720772-R：5’-GTCCACACCTATCTCTAACTAT-3’；(SEQ ID NO.4)
rs12084215-F：5’-GACACAGATTAGTTCTGGAT-3’；(SEQ ID NO.5)
rs12084215-R：5’-AAGATGATGTGACCACTGT-3’；(SEQ ID NO.6)
rs2922126-F：5’-AGTCTCACGCAGTTATAGG-3’；(SEQ ID NO.7)
rs2922126-R：5’-CTTCTACTTCTCAACAGCAAC-3’.(SEQ ID NO.8)
Above-mentioned primer sequence is synthesized by Chengdu Qin Ke biologies Co., Ltd, and it is standby that the primer dry powder of synthesis is diluted to 5pmol/ μ L
With, then by PCR amplification, (Fig. 1) is verified through agarose gel electrophoresis, it is found that purpose band is clear, no miscellaneous band, and segment is big
It is small in the same size with design, show that the primer specificity of design is good.
3, with the detection of the relevant SNP site polymorphism of high cholesterol neurological susceptibility
(1) extraction of DNA and Purity
Using people's anticoagulated whole blood as sample, using Full automatic instrument for extracting nucleic acid, genomic DNA is extracted, detailed process is as follows：
After 300 μ L whole bloods and 60 μ L (1mol/L) DTT are added to 804 nucleic acid extraction Mini reagents of Lab-Aid, open
Open Full automatic instrument for extracting nucleic acid, run about 45min, take out the DNA that extraction obtains, then by the DNA of extraction be added to it is micro from
In heart pipe, the measurement of DNA Purities and concentration is carried out using ultramicron nucleic acid-protein concentration analyzer.
(2) PCR amplification and recycling
To extract obtained DNA as template, PCR amplification is carried out to it respectively using above-mentioned primer, reaction system is shown in Table
1, amplification program is shown in Table 2.
Table 1PCR reaction systems
Primer mix：It is added to system by primer, primer dry powder need to be diluted to 100 μm of ol/mL, then with 1:1:
Sense primer, downstream primer and sterilizing ultra-pure water are mixed into mix primer (Primers mix) by 8 ratio.
Table 2PCR amplification programs
Electrophoresis, glue recycling：Using 1% agarose gel electrophoresis, electrophoretic parameters are voltage 120V, time 30min；Electrophoresis is complete
The gel containing target fragment is cut under gel electrophoresis images analyzer after, gel piece is fitted into EP pipes, glue recycling
The specification of method reference TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 kits,
Concentration mensuration finally is carried out to the product of glue recycling, 4 DEG C save backup.
(3) it marks and purifies
Label：A clean 0.2ml PCR pipe is taken, is sequentially added：DTCS Master Mix, sequencing primer, sterilizing are super
Pure water, template DNA, the dosage (X μ L) of template DNA is determined according to the nucleic acid concentration of template DNA in marking reaction system, reaction
System (10 μ L) is shown in Table 3, table 4 respectively with amplification program.
Table 3 marks reaction system
Table 4 marks amplification program
Purifying：A clean PCR pipe is taken, by a concentration of 3mol/L, sodium-acetate buffer, 0.1mol/ of the pH value for 5.2
L, the Na that pH value is 8.02Edta buffer liquid and Glycogen are with volume ratio for 2:2:1 is formulated as terminate liquid；
It takes 5 μ L of above-mentioned terminate liquid to be added in the EP pipes of label reaction, mixes well, brief centrifugation；It will be then by liquid
It is transferred in a new 1.5ml EP pipe, and the absolute ethyl alcohol of 50 μ L precoolings is added, mix well, in -20 DEG C of freezings
10min；Then 5min is centrifuged in 12000rpm, abandons supernatant；70% ethyl alcohol that 150 μ lL are pre-chilled is added to EP pipes again, is put into high speed
In centrifuge, 2min is centrifuged in 12000rpm, abandons supernatant；It slightly centrifuges, remaining liquid in EP pipes is blotted with pipettor；Open EP
Pipe lid, 37 DEG C are dried to white precipitate bleach；Then 25 μ L SLS (Sample Loading are added into EP pipes
Solution) dissolving DNA stands 8min；Brief centrifugation.
(4) it is sequenced
The dissolved DNA liquid of previous step is all added in one piece of 96 clean hole sample plate hole, in the process of addition
In cannot generate bubble, add half drop mineral oil；One piece of 96 new orifice plate is taken, 10 drop separation buffers are added in corresponding aperture
Liquid；Ultra-pure water is added to liquid close to index line to glue groove；96 hole sample panels, 96 hole buffer solution plates and glue groove are packed into and are sequenced
Instrument selects corresponding program to be sequenced.
(5) sequencing result is analyzed
By sequence analysis software (GenomeLabTM GeXP (Genetic Analysis System)), will be sequenced
Row are compared with standard sequence, find SNP site, pass through the type of base at SNP site, so that it may to obtain SNP site
Genotype, and preserve analysis collection of illustrative plates and original testing result.
Wherein, Fig. 2, Fig. 3, Fig. 4 are the sequencing result figure of rs41291161 loci polymorphisms detection, and genotype is followed successively by
AA、AT、TT；Wherein, AA and AT is the susceptible genotype in the sites rs41291161, and TT is the non-easy sensillary base in the sites rs41291161
Because of type.
Fig. 5, Fig. 6, Fig. 7 be rs12720772 loci polymorphisms detection sequencing result figure, genotype be followed successively by GG, AG,
AA；Wherein, AA and AG is the susceptible genotype in the sites rs12720772, and GG is the non-susceptible genotype in the sites rs12720772.
Fig. 8, Fig. 9, Figure 10 be rs12084215 loci polymorphisms detection sequencing result figure, genotype be followed successively by CC, AC,
AA；Wherein, AA and AC is the susceptible genotype in the sites rs12084215, and CC is the non-susceptible genotype in the sites rs12084215.
Figure 11, Figure 12, Figure 13 be rs2922126 loci polymorphisms detection sequencing result figure, genotype be followed successively by AA,
AT、TT；Wherein, AA and AT is the susceptible genotype in the sites rs2922126, and TT is the non-susceptible genotype in the sites rs2922126.
Above-mentioned sequencing result figure without set peak, background peaks, miscellaneous peak, float peak phenomena such as, show to detect SNP through the invention
The method of point polymorphism, specificity is good, accuracy is high, therefore, testing agency can according to the polymorphism of above-mentioned SNP site,
Assessment subject suffers from the risk of high cholesterol, and guidance is provided for the disease prevention of high cholesterol.
4, with the verification of the relevant SNP site polymorphic detection result of high cholesterol neurological susceptibility
Above-mentioned testing result is verified using quantitative fluorescent PCR sonde method, wherein Figure 14, Figure 15, Tu16Wei
The fluorescence proof diagram of rs41291161 loci polymorphism testing results, genotype are followed successively by AA, AT, TT；Wherein, AA and AT are
The susceptible genotype in the sites rs41291161, TT are the non-susceptible genotype in the sites rs41291161.
Figure 17, Figure 18, Figure 19 are the fluorescence proof diagram of rs12720772 loci polymorphism testing results, and genotype is followed successively by
GG、AG、AA；Wherein, AA and AG is the susceptible genotype in the sites rs12720772, and GG is the non-easy sensillary base in the sites rs12720772
Because of type.
Figure 20, Figure 21, Figure 22 are the fluorescence proof diagram of rs12084215 loci polymorphism testing results, and genotype is followed successively by
CC、AC、AA；Wherein, AA and AC is the susceptible genotype in the sites rs12084215, and CC is the non-easy sensillary base in the sites rs12084215
Because of type.
Figure 23, Figure 24, Figure 25 are the fluorescence proof diagram of rs2922126 loci polymorphism testing results, and genotype is followed successively by
AA、AT、TT；Wherein, AA and AT is the susceptible genotype in the sites rs2922126, and TT is the non-tumor susceptibility gene in the sites rs2922126
Type.
Above-mentioned fluorescence probe method testing result is consistent with PCR sequencing PCR testing result 100%, further demonstrates using sequencing
Method detects the specificity and accuracy of SNP site polymorphism.
Sequence table
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Claims (6)
1. a kind of primer for detecting the relevant SNP site of familial hypercholesterolemia neurological susceptibility, which is characterized in that described
Primer include the primer in the sites rs41291161, the primer in the sites rs12720772, the sites rs12084215 primer and
The primer in the sites rs2922126, each primer particular sequence are as follows：
rs41291161-F：5’-CTTCACAAATACACACCTG-3’；
rs41291161-R：5’-TGTCCTATCTGAACCTTAGC-3’；
rs12720772-F：5’-AAGTACACACTTGGAAGGAA-3’；
rs12720772-R：5’-GTCCACACCTATCTCTAACTAT-3’；
rs12084215-F：5’-GACACAGATTAGTTCTGGAT-3’；
rs12084215-R：5’-AAGATGATGTGACCACTGT-3’；
rs2922126-F：5’-AGTCTCACGCAGTTATAGG-3’；
rs2922126-R：5’-CTTCTACTTCTCAACAGCAAC-3’.
2. a kind of detection method of the relevant SNP site polymorphism of familial hypercholesterolemia neurological susceptibility, which is characterized in that packet
Include following steps：
(1) extraction patient gene's group DNA；
(2) using gained DNA in step (1) as template, using primer pair described in claim 1, it carries out PCR amplification；
(3) amplified production is recycled, and carries out concentration mensuration, calculation template amount carries out PCR amplification and carried out to amplified production again
Purifying, analysis determination is carried out finally by sequenator to SNP partings.
3. the detection side of the relevant SNP site polymorphism of familial hypercholesterolemia neurological susceptibility according to claim 2
Method, which is characterized in that PCR amplification system is described in step (2)：100~150ng of DNA profiling, a concentration of 5pmol/ μ L are just
To 3.0 μ L of primer, the 3.0 μ L of reverse primer of a concentration of 5pmol/ μ L, 25.0 μ L of Prime STAR MAX, ddH is finally used2O is mended
Sufficient volume is to 50 μ L.
4. the detection side of the relevant SNP site polymorphism of familial hypercholesterolemia neurological susceptibility according to claim 2
Method, which is characterized in that PCR reaction conditions are described in step (2)：95 DEG C of pre-degeneration 5min, 95 DEG C of denaturation 30s, 55 DEG C are annealed
15s, 72 DEG C of extension 15s, 30 recycle, in 4 DEG C of preservations after last 72 DEG C of extensions 5min.
5. the detection side of the relevant SNP site polymorphism of familial hypercholesterolemia neurological susceptibility according to claim 2
Method, which is characterized in that PCR amplification system is described in step (3)：2.5 μ L of DTCS Master Mix, a concentration of 5pmol/ μ L
1.0 μ L of reverse primer, DNA profiling 20ng, finally use ddH2O supplies volume to 10 μ L.
6. the detection side of the relevant SNP site polymorphism of familial hypercholesterolemia neurological susceptibility according to claim 2
Method, which is characterized in that PCR reaction conditions are described in step (3)：94 DEG C of pre-degeneration 30s, 94 DEG C of denaturation 25s, 55 DEG C are annealed
25s, 60 DEG C of extension 3min, 30 cycles, last 60 DEG C of extensions 20min.
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| CN114410769A (en) * | 2021-12-14 | 2022-04-29 | 上海大格生物科技有限公司 | SNP marker related to hypercholesterolemia based on SOD3 gene, kit and application |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114410769A (en) * | 2021-12-14 | 2022-04-29 | 上海大格生物科技有限公司 | SNP marker related to hypercholesterolemia based on SOD3 gene, kit and application |
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