CN1078616C - Method for testing or predicting senile dementia and kit - Google Patents

Abstract

The present invention provides a gene diagnosis method for diagnosing and predicting senile dementia and a kit thereof. More specifically, the present invention provides an improved method for detecting and predicting senile dementia by detecting whether apolipoprotein E epsilon 4 allele ((APOE<*>4 gene) exists and a related kit thereof. The present invention has the advantages of low cost, simple process, less time consumption, etc.

Description

The method and the test kit that whether have the senile dementia ospc gene in a kind of test sample

The present invention relates to technical field of biological and disease gene diagnostic field.More specifically, the present invention relates to a kind ofly whether exist apo E ε 4 allelotrope (APOE*4 gene) to detect or predict the method for senile dementia by detecting, and the test kit that is used for this method.

To the discovering of AlzheimerShi disease (being a kind of of dementia), in these patients' brain, there are a large amount of unusual senile plaques and have a liking for Congo red vascular lesion in recent years.Though the biochemical component to these abnormal structure's structures has carried out broad research, their roles in causing senile dementia are still not quite clear.Sophisticated senile plaque is a kind of structure of complexity, is made up of the centronucleus of amyloid fiber silk, and the amyloid fiber silk is surrounded by underfed aixs cylinder, AT and dendron, nervelet erosion cell plastid and star inoglia cell.Senile plaque amyloid centronucleus is a kind of polypeptide that contains 40-43 amino-acid residue that is called amyloid-beta (A β), is surrounded by blood vessel on every side, has a liking for Congo red vascular lesion thereby produce.Find that by experiment in the body amyloid-beta polypeptide has neurotoxicity [LaFerla et al.Nat.Genet.1995.9:21-29], can find in AlzheimerShi disease, Down's syndrome, Dutch type hereditary cerebral hemorrhage and the elderly's brain.The amyloid-beta polypeptide is to be produced by cracking by normal amyloid precursor protein (Amyloid precursor protein abbreviates " APP " as).

Apo E (ApoE) is a kind of very important albumen that plays a part in vivo, and for example: it is the moiety of plasma lipoprotein, participates in cholesterol metabolic, still is one of composition of liver synthetic vldl simultaneously.ApoE albumen mainly contains three kinds of shaped bodies, is respectively: ApoE2, ApoE3, ApoE4.These three kinds of shaped bodies are any two allelic expression products in three allelotrope on gene locus of No. 19 karyomit(e) of people.Have three kinds of homozygous genotypes, be respectively: APOE2/2, APOE3/3, APOE4/4, and three kinds of heterozygous genes types are respectively: APOE2/3, APOE2/4, APOE3/4.APOE3/3 is modal genotype.Three kinds of proteic amino acid of ApoE are formed only fine distinction: for the 112nd and the 158th amino acids residue, ApoE3 albumen is respectively halfcystine and arginine; ApoE4 albumen is respectively arginine and arginine; ApoE2 albumen is respectively halfcystine and halfcystine.

Discover, there are dependency in APOE gene and senile dementia, this is a very marvelous achievement in research, wherein mainly contains the evidence of three aspects: (1) linkage analysis assignment of genes gene mapping method finds that No. 19 chromosomal APOE gene region is with the senile dementia tight association; (2) ApoE albumen is fixed together with the amyloid-beta polypeptide in the cerebrospinal fluid; (3) in senile dementia people cerebral nerve mat-ups and senile plaque, found the albumen of anti-ApoE.The strong evidence of this three aspect just, making the APOE gene is the approval that this judgement of risk factor of senile dementia obtains nearly all in the world related experiment chamber.Outside the Pass having with senile dementia, also there are dependency in APOE gene and senile dementia [Altstiel et al.1997.Lancet 349:1451], coronary heart disease [Moore et al.1997.Clin.Genet.51:22-5], gallbladdergallstonecholetithiasis [Bertomeu et al.1996.Gastroenterology 111:1603-10], diabetes and atherosclerosis [Semenkovich 1997.Diabetes 46:327-34], large bowel cancer [Kervinen et al.Gastroenterology 1996.110:1785-90] etc.

Studies show that further senile dementia philtrum APOE*4 gene frequency is significantly higher than normal the elderly.According to this principle, whether contain the APOE*4 gene by detecting the experimenter, but just the auxiliary diagnosis patient may suffer from senile dementia or tell the positive experimenter should note preventing senile dementia.

Detect whether to be the method for APOE*4 gene pure or heterozygote have two classes, they directly or on protein level are detecting on the nucleic acid level respectively indirectly, thereby are called direct method and indirect method.Direct method mainly be to use based on polymerase chain reaction (polymerase chain reaction, PCR) Kuo Zeng restriction enzyme enzyme process is directly done gene type; Indirect method is by separating ApoE albumen with isoelectric focusing method, identify with the proteic antiserum(antisera) of anti-ApoE4, thereby infers the experimenter's genotype indirectly.

Carrying out the APOE gene type indirectly with isoelectrofocusing partition method and antibodies color reaction is than method early.It is iso-electric point (pI) difference of protein molecule that isoelectrofocusing separates ratio juris.The proteic iso-electric point of ApoE2, ApoE3 and ApoE4 is respectively: 5.9,6.0 and 6.1, thus the electrophoretic migration speed difference of the ApoE albumen of different iso-electric points in the gel of continuous pH gradient, thereby can effectively be separated.Isolate after the protein, by half-dried electrotransfer method with protein transduction to the solid supporting film, then by the Western blotting of standard, carry out combination mensuration and make evaluation with special anti-ApoE protein antibodies respectively.Isoelectrofocusing-antibody act is the method for early using since complicated operation, step many, cost an arm and a leg and day by day eliminated by PCR-restriction enzyme enzyme process (PCR-RFLP) (being also referred to as " the restriction enzyme enzyme process that PCR-based increases ").

In directly detecting experimenter's body in the genotypic direct method of APOE, have other several different methods to can be used for substituting the restriction enzyme enzyme process of PCR-based amplification, comprising for example: labeled oligonucleotide probe hybrid method that can be detected, ligase chain reaction, chain replace TRAP, reverse transcription amplification method, independently keep sequence replicating method, amplification of nucleic acid sequences method, duplicate chain reaction method etc.Such as, in " labeled oligonucleotide probe hybrid method that can be detected " detection technique of aforesaid APOE gene, available specific combination detects to one of the APOE*4 gene, a pair of or two pairs of probes whether to contain the APOE*4 gene (be that genotype is APOE2/4, APOE3/4 and APOE4/4), and this probe under identical condition not can with APOE*2, APOE*3 gene (being that genotype is APOE2/2, APOE3/3 and APOE2/3) combination.Similarly, available specific combination detects whether contain the APOE*2 gene to the probe of APOE*2 gene; Detect whether contain the APOE*3 gene with specific combination to the probe of APOE*3 gene.Comprehensively use these probes, just can determine the genotype [Wenham et al.Clin.Chem.1991.37:241-244] of APOE in object under inspection or the sample.Yet the labeled oligonucleotide probe hybrid method has following shortcoming: cost height, operational condition are difficult to control.

At present, the restriction enzyme enzyme process of PCR-based amplification is that comparatively ideal carries out the detection method of APOE gene type.Pcr amplification reaction can carry out according to a conventional method.Typical PCR reaction comprises three steps: (1) elevated temperature heat sex change, the template DNA two strands in the detected sample is separated into strand; (2) annealing reaction allows a pair of special primer combine closely by the base complementrity pair principle with template DNA5 ' and 3 ' end respectively; (3) extension under the katalysis of thermostable high-temperature polysaccharase, is pressed synthetic two the complementary double-stranded DNAs of base complementrity pair principle with four kinds of free bases (A, T, G and C) according to the sequence of template respectively.General through 30-35 circulation, can be with the DNA of long 2kb from the original lpg 0.5-1 μ g that increases, [J. Sa nurse Brooker, E.F. is the Ritchie not, T. Manny A Disi." molecular cloning experiment guide " 1992pp672-681 of second edition Science Press].Usually, this content is enough done further molecular biology experiment.

After increasing by the various APOE genotype of PCR reaction pair, can do gene type with special probe, digestion with restriction enzyme, denaturing gradient gel or existing other possible technique.

Wenham etc. have delivered by the digestion with restriction enzyme technology at The Lancet 1991.337:1158-1159 and have carried out the APOE methods of genotyping.In the method, the PCR product that amplifies has covered the pleomorphism site of coding the 112nd and 158 amino acids residues in the APOE gene, PCR product length is 227 base pairs, amplified production is cut with restriction enzyme CfoI enzyme in the amplification back, separate endonuclease bamhi with 20% native polyacrylamide gel electrophoresis then, because 6 kinds of APOE genotype enzymes are cut product length difference, so can carry out gene type accurately.

(J.Lipid Res.1990.31:545-48) is very nearly the same with people's such as Wenham method for people's such as Hixon method, and difference only is to cause because of used primer is different the PCR product length difference that amplifies, and people's such as Hixon length is 244 base pairs.

Though people's such as people such as Hixon and Wenham detection method can be carried out the APOE gene type exactly, still has some distinct disadvantage, as the cost height, length consuming time.

Therefore, the purpose of this invention is to provide a kind of method of improved detection APOE*4 gene, the cost of this method is lower and/or consuming time shorter, detects simultaneously that tolerance range remains unchanged or higher.

Another object of the present invention provides a kind of test kit that is used to detect the APOE*4 gene.

In one aspect of the invention, provide the method that whether has the APOE*4 gene in a kind of test sample, it comprises step:

(a) with specimen preparation PCR reaction template;

(b) with template in Auele Specific Primer that detects the APOE*4 gene and the step (a), under the specific amplification condition, carry out pcr amplification;

(c) pcr amplification product of step (b) being carried out enzyme cuts;

(d) the PCR enzyme of step (c) is cut product and carry out electrophoresis;

(e) existence of judging the APOE*4 gene according to the electrophoresis result of step (d) whether, wherein,

In the step (a) of preparation template, when sample was tissue sample, the consumption of proteolytic enzyme was 30-75 μ g/ml; When sample was anticoagulation or clot sample, the consumption of proteolytic enzyme was 0-50 μ g/ml.

In another aspect of this invention, a kind of test kit of the APOE*4 of detection gene is provided, this test kit contains the required reagent of preparation template, carry out the required reagent of pcr amplification, carry out enzyme cuts and handles required reagent, wherein, in the required reagent of preparation template, when sample was tissue sample, the consumption that is used to prepare the proteolytic enzyme (as Proteinase K) of 1 duplicate samples was 30-75 μ g/ml; When sample was anticoagulation or clot sample, the consumption that is used to prepare the proteolytic enzyme (as Proteinase K) of 1 duplicate samples was 0-50 μ g/ml.In a preferred example, the content that is used to prepare the proteolytic enzyme (as Proteinase K) of 1 duplicate samples is 0 μ g/ml.

For the object of the invention, employed in this application science title and term are as this area scientific and technical literature is defined, perhaps as those skilled in the art are known usually.

" APOE*4 gene " refers to contain gene or the assortment of genes form of APOE ε 4, and it has comprised that the APOE4 gene is to isozygoty or the form of heterozygosis, and particularly it has comprised genotype forms such as APOE2/4, APOE3/4 and APOE4/4.

" (can be used for) detects APOE*4 " refers to this method or test kit can be used in the detection testing sample whether have the APOE*4 gene.Should understand, though claim this method or test kit " to detect APOE*4 ", clearly this method or test kit also can be used for detecting whether have APOE*2 or APOE*3 gene.Therefore, method of the present invention or test kit also are used for detecting testing sample and whether have APOE*2 or APOE*3 gene, and/or are used for detecting the genotype (can carry out the APOE gene type) of testing sample APOE.

" Auele Specific Primer of APOE*4 " refers to such primer, it can utilize the pleomorphism site of coding the 112nd and 158 amino acids in the APOE gene, thereby makes the different amplified productions that obtain when increasing the APOE*4 gene be different from resulting amplified production when increasing APOE*2 gene and/or APOE*3 gene.These APOE*4 Auele Specific Primers are well-known to those skilled in the art, comprising (but being not limited to): Wenham primer, Hixon primer etc.In addition, also can be according to the encoding sequence of known APOE gene, in conjunction with nucleotide sequence, design various primers (as long as promptly making the primer designed cover nucleotide polymorphisms site) corresponding to the 112nd and 158 amino acids with known ordinary method corresponding to the 112nd and 158 amino acids.Designing this class primer is that those skilled in the art can be unlabored.

" Hixon primer " refers to disclosed primer, i.e. primer 1:5 '-TAA GCT TGG CAC GGC TGT CCA AGG A-3 ' in the method (J.Lipid Res.1990.31:545-48) people such as Hixon; Primer 2: 5 '-ACA GAA TTC GCC CCG GCC TGG TAC AC-3 '

" Wenham primer " refers to disclosed primer, i.e. primer 1:5 '-TCC AAG GAG CTG CAG GCG GCG CA-3 ' in the method (The Lancet 1991.337:1158-1159) people such as Wenham; Primer 2: 5 '-ACA GAA TTC GCC CCG GCC TGG TAC ACT GCC A-3 '.

" preparation template required reagent " refers to detect the used reagent of extracting genome DNA purge process in the sample, comprising for example: PBS, lysis buffer, proteolytic enzyme (as Proteinase K) etc.

" carry out the required reagent of pcr amplification " and refer to the denier APOE gene in the template is carried out the used reagent of pcr amplification reaction process, comprising for example: dNTP, high temperature-resisting DNA polymerase, Mg ²⁺, DMSO (methyl-sulphoxide), template DNA, Auele Specific Primer, 10 * damping fluid and sterilization ultrapure water etc.

" carry out enzyme cut handle required reagent " refers to that the APOE gene that pcr amplification is gone out carries out the used reagent of restriction enzyme reaction, comprising for example: restriction enzyme CfoI or HhaI and enzyme cutting buffering liquid etc.

In order successfully to carry out pcr amplification reaction, it has been generally acknowledged that and to digest sample with proteolytic enzyme, this mainly because DNA be and numerous protein (especially histone combines), so be necessary these protein digestioies are fallen, thereby discharge DNA.In preparation during dna profiling, the main effect that adds proteolytic enzyme be with DNA bonded protein digestion, for example histone etc. is removed, thereby makes the easier purification of template.The concentration of (when for example being template with anticoagulation) used proteolytic enzyme (Proteinase K) was that (J. Sa nurse Brooker, E.F. is the Ritchie not, T. Manny A Disi at least 100 μ g/ml when template prepared usually." (the molecular cloning experiment guide " 1992 pp465 of second edition Science Press).Yet, the inventor of the present invention is surprised to find that, when the preparation pcr template, reduce the consumption of proteolytic enzyme, perhaps even do not carrying out under the situation of protease treatment, still can obtain identical or the better detecting result, simplify trace routine, and reduced the detection cost, the cost when especially preparing template.The present invention finishes on this unexpected basis of finding just.

In addition, the inventor also is surprised to find that, even for the tissue sample that cerebral tissue contains a large amount of tissue proteins like this, also only needs to use small amounts of protease just can prepare dna profiling.For example use the Proteinase K of 30-75 μ g/ml, and successfully use the gene type that tissue sample (as cerebral tissue) has been carried out APOE.

In the present invention, operable proteolytic enzyme comprises the enzyme of any digestible protein, and they comprise (but being not limited to): Proteinase K, papoid and trypsinase etc.Even more preferably Proteinase K and papoid, most preferably Proteinase K.

In detection method of the present invention, the consumption of proteolytic enzyme is generally 0-50 μ g/ml sample, preferably is 0-30 μ g/ml, more preferably is 0-20 μ g/ml.

The treatment time of proteolytic enzyme is not particularly limited, as long as sample is by homogeneous phaseization (homogenize).The usually conduct time is 50 ℃ hatched at least about 4 hours, preferably was 50 ℃ and hatched 6 hours, and complete digestion can 50 ℃ of overnight incubation if detect sample.

Be applicable to that sample of the present invention can be any tissue, body fluid or the blood that contains gene.Normally used sample is blood or tissue (as cerebral tissue), but also can use sludged blood (like this, just can reduce and treat the detected object misery that repeatedly blood-sample withdrawal brought) remaining when doing conventional or other detections.

In the step (a) with specimen preparation PCR reaction template, except different aspect handling with proteolytic enzyme (for example Proteinase K), all the other aspects are identical with ordinary method.

After the preparation template, use the template that makes in APOE*4 Auele Specific Primer and the step (a) exactly, carry out pcr amplification.The condition of carrying out pcr amplification is exactly conventional specific amplification condition.The specific amplification condition can and/or add an amount of denaturing agent (as methane amide etc.) by higher annealing temperature usually and realize.Various specific amplification conditions all are well known by persons skilled in the art, and on the books in various documents, as people such as Sambrook, and molecular cloning: in the laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989).

In one embodiment of the invention, used pcr amplification condition is: first 92-97 ℃ (preferably about 96 ℃) pre-sex change 5 minutes is 3-8 (preferably about 5) partial circulating then: each circulation is made up of three steps respectively: (1) 66-69 ℃ of (preferably about 68 ℃) 25-40 second (preferably about 30 seconds); (2) 71-73 ℃ (preferably about 72 ℃) 1.5-2.5 minute (preferably about 2 minutes); (3) 92-97 ℃ (preferably about 96 ℃) 25-40 second (preferably about 30 seconds); Carry out 28-35 (preferably about 32) major cycle again: each circulation is made up of three steps respectively: (1) 58-64 ℃ of (preferably 61 ℃) 25-40 second (preferably about 30 seconds); (2) 71-73 ℃ (preferably about 72 ℃) 1.5-2.5 minute (preferably about 2 minutes); (3) 92-97 ℃ (preferably about 96 ℃) 25-40 second (preferably about 30 seconds).Last 71-73 ℃ (preferably about 72 ℃) extended 5-10 minute.

After PCR reaction amplification, the adding restriction enzyme carries out enzyme and cuts, operational enzyme can be any somatotype enzyme commonly used in the PCR-restriction enzyme enzyme process (PCR-RFLP) (being also referred to as " the restriction enzyme enzyme process of PCR-based amplification "), as long as this restriction endonuclease can be discerned APOE gene specific site, comprising (but being not limited in) CfoI, HhaI etc.

After enzyme is cut, exactly enzyme is cut product and carry out the electrophoresis gene type.This can adopt the conventional used deposition condition of detection APOE*4 gene.People's such as Wenham, Hixon native polyacrylamide gel electrophoresis separation condition (document is the same) for example.

In a preferred example of the present invention, the deposition condition that uses is the non-denaturing polyacrylamide gel of deposition condition as 15-18%, voltage 150-200 volt, and the time is 1.5-2.5 hour.

Compare with people's such as Wenham, Hixon native polyacrylamide gel electrophoresis separation condition, the advantage of this deposition condition is the time short (only needing 1.5-2.5 hour), and available homemade 8-15 centimetre (preferably 10-14 centimetre) gel electrophoresis apparatus (equipment cost is low) can effectively separate.By contrast, humans such as Wenham 20% non-denaturing polyacrylamide gel, 100 volts of electrophoresis spend the night, and the time is oversize; Humans such as Hixon 8% native polyacrylamide gel electrophoresis separates under constant current (45 milliamperes) condition with 43 centimetres of electrophoresis apparatuss, and equipment requirements height, electrophoresis time are also grown (at least 3 hours).

In another aspect of this invention, a kind of test kit of the APOE*4 of detection gene is provided, this test kit contains the required reagent of preparation template, carry out the required reagent of pcr amplification, carry out enzyme cuts and handles required reagent, wherein, in the required reagent of preparation template, when sample was tissue sample, the consumption that is used to prepare the proteolytic enzyme (as Proteinase K) of 1 duplicate samples was 30-75 μ g/ml; When sample was anticoagulation or clot sample, the consumption that is used to prepare the proteolytic enzyme (as Proteinase K) of 1 duplicate samples was 0-50 μ g/ml.In a preferred example, the content that is used to prepare the proteolytic enzyme (as Proteinase K) of 1 duplicate samples is 0 μ g/ml.

Test kit of the present invention can be used to detect or predicts whether contain the excessive risk gene of suffering from senile dementia, its principle is: senile dementia philtrum APOE*4 gene frequency is significantly higher than normal the elderly, so around this principle, contain the APOE*4 gene if detect the experimenter, but just the auxiliary diagnosis patient may suffer from senile dementia or tell the experimenter should note preventing senile dementia.

Test kit of the present invention can be used for detecting whether contain or do not contain APOE*4 gene (comprising homozygote or heterozygote) in experimenter's peripheral blood DNA.If the experimenter contains APOE*4 gene (comprising homozygote or heterozygote), then the experimenter is contained the excessive risk gene of suffering from senile dementia; If the experimenter contains APOE*4 gene pure, then he suffers from or easily to suffer from the possibility of senile dementia higher than the experimenter who contains APOE*4 genetic heterozygosis; If the experimenter does not contain the APOE*4 gene, then he suffer from or easily suffer from the possibility of senile dementia will be lower.

For the senile dementia patient that can not definitely diagnose clinically, the elderly of gradual hypomnesis is arranged and the people of familial senile dementia medical history is arranged, relatively be fit to do the detection of senile dementia tumor susceptibility gene.For example: with studies show that the inventive method and test kit are done, in the Chinese population of area, Shanghai, contain the genotypic experimenter of APOE4/4 and will suffer from high 9.4 times (table 1) of senile dementia risk, moreover age of onset early reaches average survival time weak point than the people who does not contain the APOE*4 gene.

Evidence suggests: if patient contain one or two APOE4 allelotrope add existing clinical data than clinical data is only arranged diagnosis senile dementia accurate many [Mayeux R.et al.N.Engl J Med.1998,338:506-11].So, in order to improve the clinical diagnosis level, be service for patients, be necessary to do the detection that has or not the APOE*4 gene.

Use optimization method of the present invention and test kit, domestic common laboratory or clinical biochemical testing laboratory all can be made APOE gene type accurately easily.This test kit is compared with the used reagent in domestic and international other laboratories, has the following advantages:

1. the template preparation is easier, purity is higher, and used experimenter's blood sample is (only 100 μ l anticoagulations get final product) still less, and cost is low.

2. can be used for there is not anticoagulant occasion, also enough carry out the APOE gene type, be fit to fairly large generaI investigation, moreover do the APOE gene type, almost do not see the domestic and foreign literature report with sludged blood with residue sludged bloods such as clinical liver function tests.

3. shorten than human native polyacrylamide gel electrophoresis isolation identification times such as Wenham, Hixon,

4. favorable reproducibility.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, such as people such as Sambrook, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

Detection APOE*4 gene through experimenter's peripheral blood cells sample that anti-freezing is handled from-20 ℃ of preservations

(a) use the specimen preparation pcr template: draw respectively through 100 μ l peripheral blood samples of-20 ℃ of preservations of glucose-citric acid-sodium citrate (ACD) anti-freezing and put into the 1.5ml conical centrifuge tube, add 500 μ l PBS and (in 800ml distilled water, dissolve 8gNaCl, 0.2gKCl, 1.44gNa ₂HPO ₄, and 0.24gKH ₂PO ₄PH value to 7.4 with the HCl regulator solution, add water and be settled to 1L, the 0.1034MPa autoclaving was stored in room temperature after 20 minutes), vortex oscillation device thermal agitation 30 seconds, abandon supernatant liquor after centrifugal 4 minutes, add 300 μ l lysis buffers (10mmol/L Tris-HCl pH8.0,0.5%SDS) and 1 μ l Proteinase K (20mg/ml) for 12,000 rev/mins, thermal agitation, 50 ℃ of water bath heat preservations at least 3 hours (generally can spend the night).Add 400 μ l chloroforms, thermal agitation, is drawn onto another conical centrifuge tube with supernatant liquid by 12,000 rev/mins after centrifugal 5 minutes; Add 1: 1 phenol-chloroform 400 μ l, thermal agitation, is drawn onto another conical centrifuge tube with supernatant liquid by 12,000 rev/mins after centrifugal 5 minutes; Add 400 μ l chloroforms, thermal agitation, is drawn onto another conical centrifuge tube with supernatant liquid by 12,000 rev/mins after centrifugal 5 minutes; The dehydrated alcohol 600 μ l that add-20 ℃ of precoolings are inverted gently and shake, and visible cotton-shaped DNA precipitation is separated out; 12,000 rev/mins centrifugal 10 minutes, topple over and supernatant liquor, drain, room temperature was placed 20 minutes or 40 ℃ of oven dry 10 minutes; Add 150 μ l aqua sterilisas, 50 ℃ of water-baths were dissolved 3 hours at least, promptly can be used as the template of PCR reaction.

(b) pcr amplification reaction: in the conical centrifuge tube of 0.5ml, add 13 μ l aqua sterilisas, each 0.2 μ l (8pmol/L) Hixon primer (primer 1:5 '-TAA GCT TGG CAC GGC TGT CCA AGG A-3 '; 5 '-ACA GAA TTC GCC CCG GCC TGG TAC AC-3 '), 2.5 μ l, 10 * damping fluid, 2.5 μ l dimethyl sulfoxide (DMSO) (DMSO), 2.5 μ l Mg primer 2: ²⁺(25mmol/L), 1 μ l dNTP (200 μ mol/L), 0.2 μ l (5 units/μ l) the high temperature resistant polymerase of DNA and 3 μ l dna profilings, add 15 μ l paraffin oils behind the mixing again through sterilization, put into the amplification of DNA cloning instrument subsequently.Pcr amplification reaction 96 ℃ of pre-sex change earlier 5 minutes, the PCR circulating reaction is made up of two programs subsequently: 1. 5 partial circulatings: annealed 30 seconds for 68 ℃; 72 ℃ were extended 2 minutes; 96 ℃ of sex change 30 seconds; 2. 32 major cycles: annealed 30 seconds for 61 ℃; 72 ℃ were extended 2 minutes; 96 ℃ of sex change 30 seconds.Last 72 ℃ of extensions 10 minutes.The pcr amplification reaction product generally only has a band, 4 ℃ of preservations of product.

(c) digestion with restriction enzyme reaction: get above-mentioned PCR product 12 μ l and put into another conical centrifuge tube, add 5 CfoI of unit restriction endonucleases, 37 ℃ of enzymes are cut and are spent the night.

(d) cut adding 2.5 μ l gel loading buffers (6 * damping fluid) in the product at enzyme, go up sample behind the mixing, use the 15-18% native polyacrylamide gel electrophoresis, [electrophoretic buffer is formed (every liter): 10.8g Tris alkali, 5.5g boric acid, 4ml 0.5mol/L EDTA (pH8.0)], voltage 150-200 volt, electrophoresis 1.5 to 2 hours, then gel is taken out, putting into the container that contains 0.5 μ g/ml ethidium bromide dyeed 8 minutes, be put into observation and Taking Pictures recording under the 254nm ultraviolet lamp again, as permanent preservation, make the APOE gene type according to endonuclease bamhi length difference.

As a result, as coming to the same thing of drawing in the Hixon document, can carry out somatotype to the APOE gene exactly with the method for the embodiment of the invention 1.

Embodiment 2

Basic embodiment 1 program of pressing is carried out the APOE*4 gene test to the tester's peripheral blood cells sample handled through anti-freezing from-20 ℃ of preservations, and difference is: the consumption of Proteinase K changes 50 μ g/ml into.

As a result, can carry out somatotype to the APOE gene exactly.

Embodiment 3

Basic embodiment 1 program of pressing is carried out the APOE*4 gene test to the tester's peripheral blood cells sample handled through anti-freezing from-20 ℃ of preservations, and difference is: the consumption of Proteinase K changes 0 μ g/ml into.

As a result, can carry out somatotype to the APOE gene exactly.

Embodiment 4

Basic embodiment 1 program of pressing is carried out the APOE*4 gene test to the tester's peripheral blood cells sample handled through anti-freezing from-20 ℃ of preservations, and difference is: primer changes the Wenham primer into.

As a result, as coming to the same thing of drawing in the Wenham document, can carry out somatotype to the APOE gene exactly.

Embodiment 5

Basic embodiment 1 program of pressing, tester's peripheral blood cells sample of handling through anti-freezing from-20 ℃ of preservations is carried out the APOE*4 gene test, and difference is: it is HhaI that the deposition condition of the deposition condition employing Hixon in the step (d) and enzyme are cut the restriction endonuclease that uses in the step (c).

As a result, can carry out somatotype to the APOE gene exactly.

To Shanghai area Chinese population, it is as shown in table 1 below to carry out the result that check and analysis drew according to embodiment 1-5.The result shows that the APOE*4 gene is highly enriched in the senile dementia people, is the excessive risk factor of suffering from senile dementia.In other words, whether the existence of APOE*4 gene has dependency with senile dementia (AD) disease.

The APOE gene frequency distribution table of table 1 Shanghai area senile dementia (AD) patient and normal control

Patient's AD control group (n=200)

(n＝163)

Sex (woman/man) 85,/78 105/95

Age (year) 73.5 5.6 72.7 ± 44.9

ε2/2 4 2

ε2/3 12 20

ε2/4 5 1

ε3/3 93 149

ε3/4 37 26

ε4/4 12 2

ε2 25(7.7％) 25(6.3％)

ε3 235(72.1％) 344(86.0％)

ε4 66(20.2％) ^a 31(7.7％)

^a: the ratio of patient's AD group and control group A POE*4 frequency: p=2.4 * 10 ^-6, df=2, x ²=25.9; The n=number. the statistical software of use: Epinfo.5.01 Chinese edition

In addition, press the method for embodiment 1-5, APO4*4 gene test and somatotype that Shanghai area Chinese population is carried out, and studied the vascular dementia dependency, the result who is drawn is as shown in table 2 below.The result shows first whether the existence of APOE*4 gene also has dependency with senile dementia (vascular dementia) disease.The APOE gene frequency distribution table of table 2 Shanghai area senile dementia (vascular dementia) patient and normal control

Vascular dementia patient's control group (n=200)

(n=60) sex, (woman/man) 8,/52 105/95 age, (year) 68.3 ± 6.2 72.7 ± 4.9 ε, 2/2 12 ε, 2/3 5 20 ε, 2/4 21 ε, 3/3 38 149 ε, 3/4 12 26 ε, 4/4 22 ε 29, (7.5%) 25, (6.3%) ε 3 93, (77.5%) 344, (86.0%) ε 4 18, (15.0%)^a31 (7.7%)

^a: the ratio of vascular dementia patient's group and control group A POE*4 frequency: P=0.046, df=2, x ²=6.2; The n=number. the statistical software of use: Epinfo.5.01 Chinese edition

Embodiment 6

Through the fresh peripheral blood cells that anti-freezing is handled, detect the APOE*4 gene from the senile dementia philtrum

The APOE*4 methods of genotyping is identical with embodiment 1, and is only slightly different in the template preparation process: fresh anticoagulation cirumferential blood cell can destroy erythrocyte membrane through-20 ℃ of freeze thawing half an hour.

As a result, can carry out somatotype to the APOE gene exactly.

Embodiment 7

Through the fresh peripheral blood cells that anti-freezing is handled, detect the APOE*4 gene from the senile dementia philtrum

Basic embodiment 1 program of pressing, tester's peripheral blood cells sample of handling through anti-freezing carries out the APOE*4 gene test, and difference is: fresh anticoagulation cleans without PBS, directly extracts template DNA.

As a result, can carry out somatotype to the APOE gene exactly.

Embodiment 8

From tester's peripheral blood cells (clot) of handling without anti-freezing, detect the APOE*4 gene

Basically press the program of embodiment 1, difference is: the 100mg sludged blood is shredded with scissors or make homogenate with homogenizer; The Proteinase K consumption is 40 μ g/ml.

As a result, can carry out somatotype to the APOE gene exactly.

Embodiment 9

From tester's peripheral blood cells (clot) of handling without anti-freezing, detect the APOE*4 gene

Basically press the program of embodiment 8, difference is: the Proteinase K consumption is 0 μ g/ml.

As a result, can carry out somatotype to the APOE gene exactly.

Embodiment 10

From cerebral tissue, detect the APOE*4 gene

Basically press the program of embodiment 8, difference is that used sample is the cerebral tissue piece, and the Proteinase K consumption is 75 μ g/ml.

As a result, can carry out somatotype to the APOE gene exactly.

Embodiment 11

From cerebral tissue, detect the APOE*4 gene

Basically press the program of embodiment 10, difference is that used sample is the cerebral tissue piece, and the Proteinase K consumption is 40 μ g/ml.

As a result, can carry out somatotype to the APOE gene exactly.

Claims (10)

1. whether have the method for senile dementia ospc gene in the test sample, described senile dementia ospc gene is the APOE*4 gene, and it comprises step:

(a) with specimen preparation polymerase chain reaction reaction template, wherein said sample is selected from down group: tissue sample, anticoagulation and clot;

(b) with template in Auele Specific Primer that detects the APOE*4 gene and the step (a), under the specific amplification condition, carry out PCR amplification;

(c) amplified product of polymerase chain reaction of step (b) being carried out enzyme cuts;

(d) the polymerase chain reaction enzyme of step (c) is cut product and carry out electrophoresis;

(e) whether the existence of judging the APOE*4 gene according to the electrophoresis result of step (d) is characterized in that,

In the step (a) of preparation template, when sample was tissue sample, the consumption of proteolytic enzyme was 30-75 μ g/ml; When sample was anticoagulation or clot sample, the consumption of proteolytic enzyme was 0-50 μ g/ml.

2. the method for claim 1 is characterized in that, in step (d), deposition condition is the non-denaturing polyacrylamide gel of 15-18% (weight), voltage 150-200 volt, and the time is 1.5-2.5 hour.

3. method as claimed in claim 2 is characterized in that, the length of this non-denaturing polyacrylamide gel is 8-15 centimetre.

4. the method for claim 1 is characterized in that, when sample was anticoagulation or clot sample, the consumption of proteolytic enzyme was 0 μ g/ml.

5. the method for claim 1 is characterized in that, this proteolytic enzyme is selected from: Proteinase K or papoid.

6. the method for claim 1 is characterized in that, in step (b), the Auele Specific Primer of used APOE*4 gene is selected from:

Hixon primer, i.e. primer 1:5 '-TAA GCT TGG CAC GGC TGT CCA AGG A-3 '; Primer 2: 5 '-ACA GAA TTC GCC CCG GCC TGG TAC AC-3 '; With

Wenham primer, i.e. primer 1:5 '-TCC AAG GAG CTG CAG GCG GCG CA-3 '; Primer 2: 5 '-ACA GAA TTC GCC CCG GCC TGG TAC ACT GCC A-3 '.

7. the method for claim 1 is characterized in that, in step (b), the polymerase chain reaction condition is:

(a) 92-97 ℃ of pre-sex change is 5 minutes;

(b) be 3-8 partial circulating: each circulation is made up of three steps respectively: (1) 66-69 ℃ of 25-40 second; (2) 71-73 ℃ 1.5-2.5 minute; (3) 92-97 ℃ of 25-40 second;

(c) 28-35 major cycle: each circulation is made up of three steps respectively: (1) 58-64 ℃ of 25-40 second; (2) 71-73 ℃ 1.5-2.5 minute; (3) first 92-97 ℃ of 25-40 second;

(d) 71-73 ℃ was extended 5-10 minute.

8. the test kit that whether has the senile dementia ospc gene in the test sample, described senile dementia ospc gene is the APOE*4 gene, described test kit contains the required reagent of useful specimen preparation polymerase chain reaction template, carry out the required reagent of PCR amplification, carry out enzyme cuts and handles required reagent, it is characterized in that, sample wherein to be tested is selected from down group: tissue sample, anticoagulation and clot, and when sample was tissue sample, the consumption of proteolytic enzyme was 30-75 μ g/ml; When sample was anticoagulation or clot sample, the consumption of proteolytic enzyme was 0-50 μ g/ml.

9. as test kit as described in the claim 8, it is characterized in that this test kit does not contain proteolytic enzyme.

10. test kit as claimed in claim 8 is characterized in that, in carrying out the required reagent of PCR amplification, contained primer is selected from:

Hixon primer, i.e. primer 1:5 '-TAA GCT TGG CAC GGC TGT CCA AGG A-3 '; Primer 2: 5 '-ACA GAA TTC GCC CCG GCC TGG TAC AC-3 '; With

Wenham primer, i.e. primer 1:5 '-TCC AAG GAG CTG CAG GCG GCG CA-3 '; Primer 2: 5 '-ACA GAA TTC GCC CCG GCC TGG TAC ACT GCC A-3 '.