CN102586404A - Kit for specifically detecting genotype of ApoE4 and application of kit - Google Patents

Abstract

The invention relates to a kit for specifically detecting the genotype of ApoE4 and application of the kit. The kit comprises sucrose water of 4 percent, a NaOH solution of 10mmol/L, a Tris-HCL (pH 7.5) solution of 1mol/L, DMSO (Dimethylsulfoxide), a reagent I, a reagent II, Hha I incision enzyme and 10*M Buffer. The application of the kit comprises the following steps of: preparing and preserving a sample; extracting genome DNA; carrying out PCR (Polymerase Chain Reaction) amplification; and identifying the genotype of ApoE4. Comparing with the prior art, the reagent provided by the kit can noninvasively obtain individual genome DNA; the ApoE4 gene can be efficiently amplified, the genotype of the ApoE4 can be quickly and accurately determined, and the genotype determination of a large quantity of individuals can be simultaneously carried out; and the kit has the advantages of short determination time and high accuracy and is suitable for large-scale screening of molecular epidemiology and preventive medicine.

Description

A kind of special genotypic test kit of detection ApoE4 and application thereof

Technical field

The invention belongs to molecular biological field, specifically be a kind of fast, the genotypic test kit of specific detection ApoE4 and application thereof.

Background technology

Human apo E (Apolipoprotein E, ApoE) coded by 19q11.2, its gene is common to have 3 kinds of phenotypes, and promptly 3 kinds of coded Apolipoprotein E2s, E3 and E4 of ε 2, ε 3 and ε 4 allelotrope constitute 6 kinds of different gene types.Comprise 3 kinds of homozygotes (ε 2/ ε 2, ε 3/ ε 3, ε 4/ ε 4) and 3 kinds of heterozygotes (ε 2/ ε 3, ε 3/ ε 4, ε 2/ ε 4).Allelic change causes ApoE4 bigger to ldl receptor avidity, i.e. ApoE4＞ApoE3＞ApoE2, thus influencing lipoprotein metabolism and lipid level, the ApoE4/4 genotype is as the cardiovascular and cerebrovascular disease independent risk factor.Because the ApoE gene is relevant with insulin resistant (IR), obesity etc., it is significant to the research of medical science Molecular Epidemic therefore to detect the ApoE genotype.

At present; It mainly is PCR-RFLP that the medical science Molecular Epidemic detects the genotypic method of ApoE4; At first from peripheral blood extracting genomic dna, select for use ApoE exon 4 flanking sequences design primer to carry out the amplification of ApoE4, Hha I carries out enzyme and cuts; Then enzyme is cut product and carry out vertical non-sex change PAGE and separate, to confirm its genotype.But this kind method sampling request is high, detects loaded down with trivial detailsly, is unfavorable for the use of epidemiology generaI investigation, and traumatic blood drawing to obtain genomic dna be not by each individual acceptance, the especially individuality of normal control group.ApoE exon 4 sequence GC content are very high, are difficult for amplification, and genotypic detected result is had very big influence, are necessary amplification condition is optimized.Therefore; Set up a kind of simple, AT extracting genomic dna; Efficient specific amplification ApoE 4 gene fragments, the genotypic method of rapid detection ApoE4 has important meaning to the relevant molecular epidemiology of ApoE4 genotype and the research of preventive medicine.

Summary of the invention

The object of the invention is exactly to provide a kind of judgement time short for the defective that overcomes above-mentioned prior art existence, genotypic test kit of the specific detection ApoE4 that accuracy is high and application thereof.

The object of the invention can be realized through following technical scheme:

The genotypic test kit of a kind of special detection ApoE4; It is characterized in that this test kit is made up of following reagent: Tutofusin tris (Tris)-HCl (pH 7.5) solution of the NaOH solution of 4wt% sucrose water, 10mmol/L, 1mol/L, DMSO 99.8MIN. (DMSO), reagent I, reagent II, restriction nuclease (Hha I) restriction endonuclease, 10 * M Buffer.

Described test kit is made up of the reagent of the said proportioning of table 1,

Table 1

Sequence number	Reagent name	Concentration/purity/explanation	Volume (μ L)
				1	Sucrose water	4wt％	10000
2	NaOH solution	10mmol/L	2000
				3	Tris-HCl (pH 7.5) solution	1mol/L	500
4	DMSO	Analytical pure	12.5
				5	Reagent I	See table 2 for details	192.5
6	Reagent II	See table 3 for details	20
				7	Hha I restriction endonuclease (TaKaRa company)	4～12U/μL	10
8	10×M?Buffer	See table 4 for details	10

The composition of described reagent I is as shown in table 2,

Table 2

Sequence number	Moity	TV (μ L)
			1	2.0Taq premix (TaKaRa company)	125
2	Aqua sterilisa	67.5
			Add up to		192.5

The composition of described reagent II is as shown in table 3,

Table 3

The composition of described 10 * M Buffer is as shown in table 4,

Table 4

Sequence number	Moity	Concentration (mM)
			1	Tris-HCl，pH7.5	100
2	MgCl 2	100
			3	Dithizone	10
4	NaCl	500

The application of the genotypic test kit of a kind of special detection ApoE4 is characterized in that the step of the application of test kit is following:

(1) preparation of sample and storage: the 10mL 4% sucrose water centrifugal 5min of 3000r/min that gargles behind the 10s collects and contains the epithelial deposition of buccal mucosa; The sample that obtains is if detect in the short period of time and can be stored in-20 ℃, if can be stored in-80 ℃ or the liquid nitrogen subsequent use in long-time detection;

(2) carry out the pcr amplification of ApoE4 gene;

(3) the ApoE4PCR product is after Hha I enzyme is cut, and enzyme is cut product and carried out the 15%PAGE electrophoresis;

(4) promptly can simplify judgement ApoE4 genotype with reference to the Richard method.

Described ApoE4 gene is utilizing pcr amplification, adopts the DMSO 99.8MIN. of 5wt% that the PCR response procedures is optimized, and the annealing temperature of amplification is 70 ℃.

Compared with prior art; The present invention adopts non-invasive methods to pass through simple alkaline lysis and obtains the oral mucosa genomic dna quickly and easily; Utilize 5%DMSO to optimize the PCR program ApoE4 gene order is efficiently increased, the genotype of rapidly obtain ApoE4, the judgement time is short; Accuracy is high, for the molecular epidemiology of ApoE4 genotype relative disease and the research of preventive medicine provide high efficiency method.

Description of drawings

The 9 routine ApoE4 genotype electrophorograms of Fig. 1 for using present method to obtain.

Wherein: swimming lane 1 is a dna molecular amount standard, and that use in this instance is 20bp DNA Ladder Maker; The ApoE4 genotype of swimming lane 2,3,4,5,8 is ε 3/ ε 3 types, totally 5 routine Different Individual; The ApoE4 genotype of swimming lane 6 is ε 4/ ε 4 types, totally 1 example; The ApoE4 genotype of swimming lane 7 is ε 2/ ε 2 types, totally 1 example; The ApoE4 genotype of swimming lane 9 is ε 3/ ε 4 types, totally 1 example; The ApoE4 genotype of swimming lane 10 is ε 2/ ε 3 types, totally 1 example.

Embodiment

Below in conjunction with accompanying drawing and specific embodiment the present invention is elaborated.

Embodiment

One, the reagent and the explanation that comprise of test kit

Test kit of the present invention comprises 8 kinds of reagent, is respectively that 4% sucrose water, 10mmol/L NaOH dissolve, 1mol/L Tris-HCl (pH 7.5) solution, DMSO, reagent I, reagent II, Hha I restriction endonuclease, Buffer.Wherein, 4% sucrose water is used for the epithelial collection of buccal mucosa; 10mmol/L NaOH dissolves, 1mol/L Tris-HCl (pH 7.5) solution is the extraction reagent of genomic dna, and reagent I is the PCR of a TaKaRa company operation reagent, and DMSO is the optimization reagent of PCR program; Reagent II is the PCR primer of Gene A poE4, and Hha I restriction endonuclease, Buffer are used for the enzyme of Gene A poE4PCR amplified production and test conscientiously.

Specifically as table 1 ' shown in, the concrete component of reagent I, reagent II and Buffer '-4 ' that see table 2 wherein.

Table 1 ': the reagent that test kit of the present invention comprised

Sequence number	Reagent name	Concentration/purity/explanation	Volume (μ L)
				1	4% sucrose water	Self-control	10000
2	NaOH solution	10mmol/L	2000
				3	Tris-HCl (pH 7.5) solution	1mol/L	500
4	DMSO	Analytical pure	12.5
				5	Reagent I	See table 2 for details	205
6	Reagent II	See table 3 for details	20
				7	Hha I restriction endonuclease (TaKaRa company)	4～12U/μL	10
8	Buffer	See table 4 for details	10

Annotate: this shows listed amount of reagent is 10 pattern detection

Table 2 ': the component of reagent I

Sequence number	Moity	TV (μ L)
			1	2.0Taq premix (TaKaRa company)	125

2	Aqua sterilisa	67.5
			Add up to		192.5

Table 3 ': the component of reagent II

The said gene primer can be synthetic with synthetic through existing gene by one of skill in the art.

Table 4 ': the component of Buffer

Sequence number	Moity	Concentration (mM)
			1	Tris-HCl，pH7.5	100
2	MgCl 2	100
			3	Dithiothreitol	10
4	NaCl	500

One, operation steps

This step comprises preparation and storage, extracting genomic dna, the pcr amplification of ApoE4 gene, the genotypic evaluation of ApoE4 of sample.

(1) preparation of sample and storage

Sample derives from individual sucrose collutory, and promptly the 10mL 4% sucrose water centrifugal 5min of 3000r/min that gargles behind the 10s collects and contains the epithelial deposition of buccal mucosa.The sample that obtains is if detect in the short period of time and can be stored in-20 ℃, if detection can be stored in-80 ℃ or the liquid nitrogen subsequent use after three days.

(2) extracting genomic dna

Present method is applicable to the processing of a sample, and a plurality of samples are parallel running simultaneously.

1. the epithelial deposition of buccal mucosa that also has that will collect moves in the EP pipe of 1.5mL.

2. in deposition, add 200 μ L 10mmol/L NaOH solution, 100 ℃ of alkaline lysis 5min behind the concussion 15s.The centrifugal 5min of 3000r/min under the room temperature.

3. supernatant is transferred in the EP pipe of a clean 1.5mL, adds 50 μ L 1mol/L Tris-HCl (pH7.5) neutralization ,-20 ℃ frozen subsequent use.

(3) pcr amplification of ApoE4 gene

The supernatant 2.5 μ L that get above-mentioned acquisition are used for the pcr amplification of ApoE4 gene, and the PCR system is configured according to component shown in the table 5.

Table 5:PCR reaction system component

Reagent name	Volume (μ L)
		Reagent I	19.25
Reagent II	2
		DMSO	1.25
Genomic dna	2.5
		Add up to	25

After pressing table 5 preparation PCR reaction system, mixing carries out the PCR reaction simultaneously according to condition shown in the table 6 gently, carries out the reaction of HhaI endonuclease digestion after reaction finishes.

The PCR reaction conditions of table 6:ApoE4 gene

(4) endonuclease reaction of ApoE4 gene PCR product

Get above-mentioned PCR product 8 μ L, according to the configuration of component shown in the table 7 endonuclease reaction system.

Table 7: endonuclease reaction system

Reagent name	Volume (μ L)
		The PCR product	8
Hha I restriction endonuclease (TaKaRa)	1
		Buffer	1

Mixing gently after reaction system prepares, 37 ℃ of reaction 1h, product carries out 15% native polyacrylamide gel electrophoresis.

(5) 15%PAGE electrophoresis

1, reagent preparation and preparation

The native polyacrylamide gel electrophoresis damping fluid of conventional configuration 15% etc., specific as follows:

(1) configuration 5 * TBE solution: dissolving 54g Tris alkali in 0.6L zero(ppm) water, add 27.5ml glacial acetic acid and 20m L 0.5moL/L EDTA solution (p H8.0), adding distil water is settled to 1L again, room temperature preservation.

(2) preparation of electrophoretic buffer (TBE).Get above-mentioned 5 * TBE, the adding distil water dilution is that 1 * TBE is as electrophoretic buffer.

The preparation of (3) 15% non-denaturing polyacrylamide gels.Prepare according to ratio shown in the table 8.

Table 8: prepare 15% polyacrylamide gel agents useful for same volume ratio

With the abundant mixing of the coagulant liquid for preparing, pour in the Bio-Rad vertical gel electrophoresis appearance that has assembled, insert the suitably comb of size, it is subsequent use to carry out electrophoresis after at room temperature placing about 30min.

(4) preparation of sample loading buffer (Loading Buffer): conventional configuration 6 * Loading Buffer.

2, step

(1) prepares 15% the polyacrylamide gel mixed solution of 10mL in proportion, pour in the glass guide channel of vertical gel electrophoresis appearance, insert the suitably comb of size; After treating that gel polymerisation fully; Hold comb and gel with 1 * TBE wetted paper handkerchief, preservative film sealing monoblock gel, 4 ℃ of preservations; Until using (can preserve 1-2 days, and also can buy commercial gel).

When (2) preparing electrophoresis, gel is put into gel vertical electrophoresis appearance, assembled is prepared electrophoretic buffer, and washes well with electrophoretic buffer.

(3) 20bp DNA Ladder Maker 4 μ L add in the hand-hole as reference.Enzyme is cut product and is mixed with an amount of 6 * Loading Buffer, carries out the electrophoresis operation in the adding in the appearance hole.

(4) electrode is linked to each other with power supply, opening power is carried out electrophoresis.Electrophoresis to standard reference fuel migration stops electrophoresis to desired location.

(5) with gel separation, ethidium bromide staining, uv lamp is observed and is taken pictures, and the judgement of ApoE genotype is simplified with reference to methods such as Richard.

Two, method evaluation

Test the said method of sampling with this respectively and collect the individual buccal mucosa epithelial cell of 9 examples; The peripheral blood sample 200 μ L of the same individuality of parallel collection simultaneously; Draw the drawer genomic dna that uses the same method; Pcr amplification ApoE4 gene, Hha I enzyme is cut, and 15% native polyacrylamide gel electrophoresis is analyzed the genotype of individual ApoE4.The result shows that the result of determination in two kinds of sample sources is in full accord, always distinguishes that coincidence rate is 100%, distinguishes that alternately rate of load condensate is 100%, and sensitivity is 100%, and specific degree is 100%, result such as Fig. 1.

Claims (4)

1. genotypic test kit of special detection ApoE4; It is characterized in that this test kit is made up of following reagent: Tutofusin tris (Tris)-HCl (pH 7.5) solution of the NaOH solution of 4wt% sucrose water, 10mmol/L, 1mol/L, analytical pure DMSO 99.8MIN. (DMSO), reagent I, reagent II, restriction nuclease (Hha I) restriction endonuclease, 10 * M Buffer.

2. the genotypic test kit of a kind of special detection ApoE4 according to claim 1 is characterized in that described test kit is made up of the reagent of the said proportioning of table 1,

Table 1

Sequence number Reagent name Concentration/purity/explanation Volume (μ L) 1 Sucrose water 4wt％ 10000 2 NaOH solution 10mmol/L 2000 3 Tris-HCl (pH 7.5) solution 1mol/L 500 4 DMSO Analytical pure 12.5 5 Reagent I See table 2 for details 192.5 6 Reagent II See table 3 for details 20 7 Hha I restriction endonuclease (TaKaRa company) 4～12U/μL 10 8 10×M?Buffer See table 4 for details 10

The composition of described reagent I is as shown in table 2,

Table 2

Sequence number Moity TV (μ L) 1 2.0Taq premix (TaKaRa company) 125 2 Aqua sterilisa 67.5 Add up to 192.5

The composition of described reagent II is as shown in table 3,

Table 3

The composition of described 10 * M Buffer is as shown in table 4,

Table 4

Sequence number Moity Concentration (mM) 1 Tris-HCl，pH7.5 100 2 MgCl ₂ 100 3 Dithizone 10 4 NaCl 500

3. the application of the genotypic test kit of special detection ApoE4 as claimed in claim 1 is characterized in that the step of the application of test kit is following:

(1) preparation of sample and storage: the 10mL 4% sucrose water centrifugal 5min of 3000r/min that gargles behind the 10s collects and contains the epithelial deposition of buccal mucosa; The sample that obtains is if detect in the short period of time and can be stored in-20 ℃, if can be stored in-80 ℃ or the liquid nitrogen subsequent use in long-time detection;

(2) carry out the pcr amplification of ApoE4 gene;

(3) the ApoE4PCR product is after Hha I enzyme is cut, and enzyme is cut product and carried out the 15%PAGE electrophoresis;

(4) promptly can simplify judgement ApoE4 genotype with reference to the Richard method.

4. the application of the genotypic test kit of a kind of special detection ApoE4 according to claim 3; It is characterized in that; Described ApoE4 gene is utilizing pcr amplification, adopts the DMSO 99.8MIN. of 5wt% that the PCR response procedures is optimized, and the annealing temperature of amplification is 70 ℃.

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