CN102676639A - Molecular marker kit for formulating individual weight reduction scheme - Google Patents

Abstract

The invention relates to a molecular marker kit for formulating an individual weight reduction scheme. The kit comprises nucleotide shown in SEQ ID NO:1-10 and can comprise restriction endonucleases Hha I, Msp I and Bbv I. The kit is used for analyzing SNPs (single nucleotide polymorphisms) genotypes of a subject and issuing a comprehensive genetic predisposition analysis report, and nutritionists are invited to recommend a diet style (low fat diet, low carbohydrate diet and balanced diet) suitable to weight reduction to the subject, analyze intake calorie of the subject required every day according to the practical situation of the subject and individually arrange the proportion of three nutrient elements (fat, protein and carbohydrate) in three meals a day, so that healthy, scientific and effective weight reduction is achieved.

Description

A kind of molecule marker test kit that is used to formulate individuation loss of weight scheme

Technical field

The present invention relates to a kind of test kit, relate in particular to a kind of molecule marker test kit that is used to formulate individuation loss of weight scheme.

Background technology

In February, 2007, the report of survey of the World Health Organization's fat ratio in various countries of issue.Report shows that in current 6,500,000,000 populations, the whole world has 1,700,000,000 people overweight approximately, and wherein 300,000,000 is fat.Show according to statistics: the present overweight person of China is up to 200,000,000, and wherein the obese person is also above 9,000 ten thousand.Scholarly forecast, China's obese people in following 10 years will surpass 200,000,000.

In the past few decades, gradually reduce with food by people's physical activity level and to become too abundant, cause people's mode of life to change, thereby caused fat spreading worldwide.Yet in fact, although people are in the identical environment, whether everyone does not become fat, and this shows that inherited genetic factors also can produce certain influence to the mankind's changes of weight.That is to say when heredity can determine different individualities in being in identical environment, whether to have fat susceptibility, simultaneously, also determined the responsiveness that different individualities is intervened different diet and motion.Therefore, realization individuation Weight management is not only the developing direction of trophology, and has crucial social effect and economic worth.

Following with the gene that the fat degree of correlation is bigger:

The FTO gene: the FTO gene is the enzyme of nearest found a kind of modifying DNA of can encoding.Another research finds that also in obese people, the level of FTO gene in fatty tissue increases.But can't recognize accurately still this gene is how to influence BMI's at present.As if the SNP site of this gene region is all relevant with BMI, is directly or indirectly but can't understand these SNP sites to the influence of BMI.

The SNP site of FTO gene is nearly all only relevant with fatty tissue.This just means that the risk allelotrope of this gene is that the approach of human body weight increase is that fatty tissue is increased, rather than increases the volume or the skeleton density of muscle.

Data shows that people FTO genovariation may cause the rise or the imbalance of FTO genetic expression, possibly cause obesity through excessive ingesting and carry risk allelotrope person under the situation that does not change energy expenditure.Cecil etc. have studied possibly getting in touch of FTO genovariation and obesity, energy expenditure and food intake; But caused fat susceptibility but not demonstrate its direct regulation and control energy expenditure though find the variation of FTO gene; But possibly in control ingestion of food and food selection, play a role, showing the phenotype of FTO gene and bulimia nervosa or liking high-energy food has relation.

If the FTO gene has only a copy variation, with the physiognomy ratio that does not have the variation copy, fat probability also will exceed 30%.It is very general in obese individuals that small variation takes place.Carry the people of two variation copies, mean body weight is than three kilograms of the individual weights that has normal copy.

And another is about the discovering of FTO gene, carries the FTO gene but the people that actively participates in physical activity, and their body weight is the same with non-ob gene carrier, this means, carries the crowd of FTO, and physical activity will be best fat-reducing means.

The FABP2 gene: FABP2 genes encoding small intestine fatty acid binding protein plays an important role in the process of lipid acid absorption and transhipment in small intestinal lumen.This albumen in intestinal epithelial cell, come to light (absorption to fat of intestinal epithelial cell has intense influence).And the sudden change of this gene can cause the ability of the fatty acid binding protein conjugated fatty acid of its coding to increase greatly, and in other words, this sudden change has caused the increase greatly of the ability of absorption of human body fat.

Had a lot of clinical studyes to prove, carried the allelic individuality of risk, the ability of its intestinal absorption dietary fatty acid has obtained reinforcement.And the rising of risk allelotrope and BMI, body fat, increase, obesity and the leptin secretion level of stomach fat are relevant.Many clinical meals intervention studies show that the concentration level of intravital after the meal triglyceride level of risk allelotrope carrier and 14-18 carbon longer chain fatty acid substantially exceeds undesired individuality.Therefore, risk allelotrope carrier needs the fatty intake of strict control.

The PPARG gene:Peroxisome proliferation-activated receptors (PPARs) is nuclear hormone receptor (NHR) superfamily member, they be the part activating transcription factor and and lipid metabolism, glycaemic homeostasis and hyperplasia are relevant with multiple paths such as secretions.Peroxisome proliferation-activated receptors G (PPARG) high expression level and in the formation of fat, play important effect in adipocyte; Multiple participation lipid acid transhipment and metabolic gene are regulated and control by PPARG at transcriptional level; Like adipocyte fatty acid binding protein (AFABP), fatty acid transport protein (FATP), lipoprotein lipase (LPL) etc.; Can increase the expression of fatty acid transport protein and lipid acid transhipment enzyme, irritation cell is to the picked-up of lipid acid with to the conversion of acyl CoA.

Up to the present, nearly more than 30 high-level scientific research papers point out, the PPARG gene exists close relatedly with fat, and wherein, the Pro12Ala polymorphic site of this gene also becomes the most representative discovery with the cognation of obesity.People such as Memisoglu discover, Pro allelotrope carries individuality, and are more responsive to the total amount of fat in the food.Take in the less Pro/Pro homozygote allelotrope of dietary fat than those and carry individuality, take in the more individuality of dietary fat, its average BMI significantly increases, and this situation does not then take place in Ala allelotrope carrier colony.In the large-scale research about mellitus of Finland, scientists has obtained the strong evidence that genotype influences effects of dietary intervention.In this motion meals intervention experiment in 3 years by a definite date, (8.3Kg), weight loss effect is superior to Pro/Ala allelotrope carrier (4.0Kg) to Ala/Ala allelotrope carrier; And the latter is superior to Pro/Pro allelotrope carrier (3.4Kg).These researchs point out that Ala allelotrope carrier has better metabolism handiness aspect the storage and transfer of fat.And have easily usually get fat, diet and motion intervene its loss of weight is more effective, insulin sensitivity is better and type ii diabetes hangs down onset risk.Therefore, Pro allelotrope is risk allelotrope.

The ADRB2 gene:The albumen adrenergic receptor albumen of ADRB2 genes encoding is distributed widely in adipocyte.It combines back activated adenyl cyclase (AC) that cyclic monophosphate (cAMP) generation is increased with catecholamine, further activated protein kinase A acts on multiple lipid metabolism involved enzyme class, ionic channel and transcription factor, thereby promotes steatolysis.A plurality of mutational sites that cause amino acid to change of this gene are identified.

The clinical study of some longer cycles shows, the allelic carrier of ADRB2 no matter be the body weight gain from the Childhood to the Adulthood, or the body weight gain of Adulthood is all more.A clinical study shows; High carbohydrate meals can cause the allelic female carrier's of SNP site 27Glu of ADRB2 gene risk of obesity obviously to increase; Simultaneously, this diet style is irrelevant with the risk of obesity that does not carry this allelic women, this expression; 27Glu allelotrope has increased the risk of individual obesity, takes the factor mode of Hi CHO for a long time when this individuality.

The ADRB3 gene:The ADRB3 acceptor is distributed in the brown of people and tissues such as white adipose tissue, the interior retinulae of abdomen, gall-bladder; Because these tissues contain abundant sympathetic nerve; They mainly are through the usually exciting ADRB3 acceptor of release noradrenaline, thereby the coupling protein on the activation of mitochondria stimulates Fatty Acid Oxidation and phosphorylation and the interior excess fat of consumer; It is relevant with fat pathologic, physiologic that this receptor function descends. and β 3 adrenergic receptor genes are polymorphum in the crowd expresses; Having found at present has 4 SNPs on this receptor gene, one of them is functional variation, and this possibly be that ADRB3 genovariation realizes that through binding the ADRB3 receptor protein function performance causes active decline; Reduce interior fat decomposition and energy and produce, cause the accumulation of fat at internal organ and belly.

Beta-3 adrenergic receptor albumen mainly is expressed in adipocyte, especially in visceral adipocytes, participates in regulating the steatolysis and the exothermal process of intraperitoneal fatty tissue.T on this gene order → C sudden change causes the tryptophane on the amino acid polypeptide sequence to be replaced by l-arginine, because this sudden change just in time is positioned at first endocellular function district of acceptor, can cause fatty resolving power decline in the body, and thermogenesis weakens.

In the research, 76 Japanese women have participated in the research of a prevention of obesity, comprise the change (reducing the energy absorption) and the physical exertion (walking) of diet, and personalized monitoring and the treatment of supportive group.The result shows, takes two allelic women of A, and it loses weight and reduces that energy is taken in and it is directly related to increase the walking amount.Yet, there is the allelic women of one or two risk equipotential not have significantly and loses weight, take in although obviously reduced their energy, increased their walking amount.

Therefore, need set up a cover is the fat-reducing scheme of the personalization of reference with individuality to the genetic predisposition of obesity, to improve fat-reducing and to keep the effect of body weight.And to understand the difference of individuality to the reaction of the genetic predisposition of obesity, different diet and mode of motion, and just need set up a cover detection method, differentiate the gene genotype relevant with human energy metabolism.Through detecting these gene locuss, can understand the difference of individuality, and personalized Weight management scheme is provided to the tested person to the reaction of the genetic predisposition of obesity, different diet and mode of motion.

At present, the methods of genotyping of commercial usefulness mainly includes: 1) though the Microarray chip can high-throughput, detect somatotype comparatively exactly, cost is higher, and instrument costs an arm and a leg; 2) genome sequencing, though the fine somatotype of ability, machine is expensive, cost is high, simultaneously, is used for the obviously too waste of special SNP site somatotype.3) though the more preceding two kinds of methods of fluorescent PCR system are more more economical, the detection scale is limited, is unfavorable for commercialization.

Comparatively speaking, enzyme is cut and PCR-SSCP SNP site typing is more suitable for the gene type that commercial undertaking carries out To Template: the cost that 1) needs is lower; 2) sensitivity is higher relatively; 3) high specificity; 4) can carry out the detection of a large amount of samples simultaneously, help mass-producing and detect, be suitable for commercial applications.

Enzyme is cut typing PCR-RFLP:

Restriction enzyme can be incorporated within one section dna sequence dna that is called as the Restriction Enzyme recognition sequence or near the specific site it specifically, and cutting double-stranded DNA.

5′…G↓AATTC…3′

3′…CTTAA↑G…5′

If SNP produces or eliminated certain restriction endonuclease sites, then can detect behind the electrophoresis through PCR product enzyme is cut.This method can utilize the different bases in the special identification SNP site of specific limited property restriction endonuclease, thereby Accurate Analysis go out the genotype in this SNP site to the SNP site of known dna sequence.Binding sequence is analyzed; Can accomplish that not only cost is low, and level of automation is high to " full scan " analysis of the target DNA sheet degree of batch samples; Therefore; Carrying out gene diagnosis and polymorphism analysis, comprising in the detection of mononucleotide polymorphic (SNP), PCR-RFLP technology a kind of accurate, quick, easy, economic technique means of can yet be regarded as.

The PCR-SSCP typing:

Dna single chain conformation polymorphum (Single Strand Conformation Polymorphism, SSCP)

SSCP is that the single stranded DNA of isodactylism produces conformation variation, the difference that shows as electrophoretic mobility in non-sex change Vestolen PP 7052 phthalein amine because of the difference of examining the former times acid sequence.The single stranded DNA conformational analysis is very responsive to the change of dna sequence dna, and usually a base difference can both show.In sscp analysis; Utilize a certain purpose fragment in the round pcr fixed point amplifying genom DNA, amplified production is carried out denaturing treatment, double-stranded DNA is separated into strand; Separate with non-sex change Vestolen PP 7052 phthalein amine gel electrophoresis again, judge whether there is sudden change in the purpose fragment according to the band change in location.

The PCR-SSCP technology is to detect dna mutation one of experimental technique means the most intuitively and accurately; The sensitivity that the DNA sheet degree sudden change between 100-300bp detects to length of this method can reach 100%, and the binding sequence analysis can be accomplished " full scan " analysis to the target DNA sheet degree of batch samples; Not only cost is low; And level of automation is high, therefore, is carrying out gene diagnosis and polymorphism analysis; Comprise in the detection of mononucleotide polymorphic (SNP) PCR-SSCP technology a kind of accurate, quick, easy, economic technique means of can yet be regarded as.

Summary of the invention

The object of the present invention is to provide a kind of molecule marker test kit that is used to prepare individuation loss of weight scheme, it comprises the Nucleotide shown in the SEQ ID NO:1-10.

Said test kit also comprises restriction enzyme Hha I, Msp I and BbvI.

Said test kit also comprises DNA extraction liquid.Said DNA extraction liquid comprises: damping fluid GA, damping fluid GB, damping fluid GD, rinsing liquid PW, elution buffer TB and Proteinase K.

Said test kit can also comprise adsorption column, collection tube and centrifuge tube.

Particularly, the Nucleotide shown in the SEQ ID NO:1-10 can be used for preparing the molecule marker test kit of individuation loss of weight scheme, and it can be used as primer.Wherein, the primer SEQ ID NO:1-2 FTO gene that is used to increase; The primer SEQ ID NO:3-4 FABP2 gene that is used to increase; The primer SEQ ID NO:5-6 ADRB3 gene that is used to increase; The primer SEQ ID NO:7-8 PPARG gene that is used to increase; The primer SEQ ID NO:9-10 ADRB2 gene that is used to increase.

Through test kit provided by the invention; To the genotypic analysis of tester SNPs; Provide complex inheritance PAA report; Recommend to be fit to the diet style (low fat diet, low carbohydrate diet, balanced diet) of its loss of weight by the nutritionist to the measured, and, analyze the absorption heat that need its every day according to measured's practical situation; The ratio of 3 big nutritive elements (fat, protein, glucide) in the breakfast, lunch and dinner is arranged on individuation ground, thereby reaches the purpose of health, science, effective loss of weight.

And, the test kit that the present invention adopts, it can be cut and PCR-SSCP SNP site typing is more suitable for the gene type that commercial undertaking carries out To Template through enzyme: the cost that 1) needs is lower; 2) sensitivity is higher relatively; 3) can carry out the detection of a large amount of samples simultaneously, help mass-producing and detect, be suitable for commercial applications.

For let above and other objects of the present invention, feature and advantage can be more obviously understandable, hereinafter is special lifts preferred embodiment, and conjunction with figs., elaborates as follows.

Description of drawings

Fig. 1 is an experiment process synoptic diagram of the present invention;

Fig. 2 is the pcr amplification result, M wherein, and 600bp DNA marker, swimming lane 1 are the sheet degree of primer SEQ IDNO:1-2 to FTO gene amplification; Swimming lane 2 is the sheet degree of primer SEQ ID NO:3-4 to FABP2 gene amplification; Swimming lane 3 is the sheet degree of primer SEQ ID NO:5-6 to ADRB3 gene amplification; Swimming lane 4 is the sheet degree of primer SEQID NO:7-8 to PPARG gene amplification; Swimming lane 5 is the sheet degree of primer SEQ ID NO:9-10 to ADRB2 gene amplification;

Fig. 3 is that the rs1799883 site enzyme of FABP2 gene is cut and declared type figure, M wherein, 600bp DNAmarker; Swimming lane 1-3 is the allelic homozygote of G; Swimming lane 4-6 is the allelic homozygote of A; Swimming lane 7-9 is the allelic heterozygote of G/A;

Fig. 4 is that the enzyme in ADRB3rs4994 site is cut and declared type figure, wherein, M, 600bp DNAmarker swimming lane 1-2 is the allelic homozygote of T; Swimming lane 3 is the allelic heterozygote of T/C;

Fig. 5 is that the enzyme in ADRB2rs1042714 site is cut and declared type figure, and wherein, the M swimming lane is DNAMarkerDL 1000, and swimming lane 1-2 is that CC is homozygous; Swimming lane 3-4 is the GC heterozygous; Swimming lane 5 is that GG is homozygous;

Fig. 6 is the polyacrylamide gel electrophoresis figure as a result in FTO rs9939609 site, swimming lane 2,5, A/A allelotrope homozygote; Swimming lane 1,3,6,7, A/T allelotrope heterozygote; Swimming lane 4, T/T allelotrope homozygote ( swimming lane 2,4 is a reference substance);

Fig. 7 is the allelic homozygote sequencer map of the A/A of FTO rs9939609;

Fig. 8 is the allelic homozygote sequencer map of the T/T of FTO rs9939609;

Fig. 9 is the polyacrylamide gel electrophoresis figure as a result in PPARG rs1801282 site, wherein, and swimming lane 1,3,7, C/C allelotrope homozygote; Swimming lane 2,5,6, C/G allelotrope heterozygote; Swimming lane 4, G/G allelotrope homozygote ( swimming lane 1,4 is a reference substance);

Figure 10 is the allelic homozygote sequencer map of the C/C of PPARG rs1801282;

Figure 11 is the allelic homozygote sequencer map of the G/G of PPARG rs1801282.

Embodiment

Please refer to Fig. 1, it is an experiment process synoptic diagram of the present invention, and its fundamental test flow process is:

1, extracts dna profiling;

2, pcr amplification contains the fragment in specific SNP site;

3, utilize enzyme cutting method respectively to the rs1799883 site of FABP2 gene, the rs4994 site of ADRB3 gene, and somatotype is carried out in the rs1042714 site of ADRB2 gene; Utilize the rs9939609 site of PCR-SSCP typing to the FTO gene, somatotype is carried out in the rs1801282 site of PPARG gene;

According to the somatotype result experimenter is provided rational fat-reducing scheme at last.

Embodiment 1: extract the oral mucosa cell DNA

Gather the intraoral mucous membrane cell of person under inspection with the oral mucosa swab, utilize the buccal swab genome DNA extracting reagent kit of sky, Shanghai root biological production to extract person under inspection's genomic dna.Step is following:

1. processing material:

Swab transposition that will wiping in cheek is cut the cotton swab part with scissors in the 2ml centrifuge tube from its bar, add 400 μ l damping fluid GA.

2. add 20 μ l Proteinase K solution, 10 seconds mixings of vortex were placed 60 minutes for 56 ℃, and per therebetween 15 minutes vortex mixings for several times.

3. add 400 μ l damping fluid GB, fully put upside down mixing, placed 10 minutes for 70 ℃, this moment, the solution strain was limpid, and is brief centrifugal to remove the drop of cap wall.

4. add 200 μ l absolute ethyl alcohols, fully put upside down mixing, brief centrifugal to remove the drop of cap wall.

5. will go up step gained solution and a flocks and all add (adsorption column CR puts into collection tube) among the adsorption column CR, 12,000rpm (～13,400 * g) centrifugal 30 seconds, outwell the waste liquid in the collection tube, CR puts back in the collection tube with adsorption column.

6. in adsorption column CR, add 500 μ l damping fluid GD (please checking to have added absolute ethyl alcohol whether earlier before using), 12,000rpm (～13,400 * g) centrifugal 30 seconds, outwell the waste liquid in the collection tube, CR puts back in the collection tube with adsorption column.

7. in adsorption column CR, add 700 μ l rinsing liquid PW (please checking to have added absolute ethyl alcohol whether earlier before using), 12,000rpm (～13,400 * g) centrifugal 30 seconds, outwell the waste liquid in the collection tube, CR puts back to collection tube with adsorption column.

8. in adsorption column CR, add 500 μ l rinsing liquid PW, 12,000rpm (～13,400 * g) centrifugal 30 seconds, outwell the waste liquid in the collection tube.

9. adsorption column CR is put back in the collection tube, 12,000rpm (～13,400 * g) centrifugal 2 minutes, outwell waste liquid.Adsorption column CR room temperature was placed several minutes, thoroughly to dry rinsing liquid remaining in the sorbing material.

10. adsorption column CR is changed in the clean centrifuge tube, to the unsettled dropping in adsorption film mid-way 20-50 μ l elution buffer TB, room temperature was placed 2-5 minute, and 12,000rpm (～13,400 * g) centrifugal 2 minutes.

11. just obtained also having the elutriant of genomic dna after centrifugal, can directly use as dna profiling.

Embodiment 2: pcr amplification contains the fragment in specific SNP site

1. the preparation of primer: be followed successively by SEQ ID NO:1-10

Below No. 1 to No. 5 primer synthetic by the big gene of Shenzhen China:

Table 1

Need ultrapure water,, add water the upstream and downstream primer is diluted to the concentration of 10 μ molL-1,4 ℃ of preservations according to the quality of primer.

More than the gene orders announced according to NCBI of the five pairs of primers, the sequence number of gene is respectively FTOrs9939609, FABP2 rs1799883, ADRB3 rs4994, PPARG rs1801282, ADRB2rs1042714.Wherein, SEQ NO:1-2,7-10 utilize software oligo 6.0 designs; SEQ NO:3-4 is from document: Peng Xian pretty young woman opens that tinkling of pieces of jade king is blue and green to wait the research related with non-alcoholic fatty liver disease of .FABP2 Ala54 Thr gene pleiomorphism. health research .2009,4:401-404; SEQ NO:5-6 is from document: Elisabeth Wid é n; Markku Lehto; Timo Kanninen, et.al.Association of aPolymorphism in the β 3-Adrenergic-Receptor Gene with Features of the InsulinResistance Syndrome in Finns.N Engl J Med.1995; 333:348-352.

2.PCR amplification:

The pcr amplification reaction system is 25 μ l, comprising: each 1 μ l of 10 μ molL-1 upstream and downstream primers; GreenMaster Mix 12.5 μ l; Dna profiling 2 μ l; , ultrapure water 8.5 μ l.

Operation steps is:

1. in the little centrifuge tube of a 200ul specification; Add 10 μ molL-1 upstream and downstream primers (preparing in the last step), Green Master Mix 12.5 μ l (U.S. Promega company respectively with liquid-transfering gun; GOTaqGreen Master Mix), dna profiling 2 μ l (extract among the embodiment 1 and obtain), ultrapure water 8.5 μ l; Use the vibrator mixing, brief centrifugal to remove the drop of cap wall.

The centrifuge tube that 2. will contain mixture is put into the pcr amplification appearance and is continued pcr amplification.

The pcr amplification program is:

Table 2

Its amplified fragments is followed successively by: SEQ ID NO:11-15.

3.PCR the detection of amplified production

After the EP of pcr amplification appearance, the mixture in original 200ul centrifuge tube is exactly a pcr amplification product.Utilizing concentration is that 1.5% agarose gel electrophoresis detects.Detected result is seen Fig. 2.Swimming lane 1 is the sheet degree of primer SEQ ID NO:1-2 to FTO gene amplification; Swimming lane 2 is the sheet degree of primer SEQ ID NO:3-4 to FABP2 gene amplification; Swimming lane 3 is the sheet degree of primer SEQ ID NO:5-6 to ADRB3 gene amplification; Swimming lane 4 is the sheet degree of primer SEQ ID NO:7-8 to PPARG gene amplification; Swimming lane 5 is the sheet degree of primer SEQ ID NO:9-10 to ADRB2 gene amplification.

Embodiment 3: the SNP somatotype of goal gene

The purpose fragment SEQ ID NO:11-15 of above-mentioned 5 genes that obtain is carried out snp analysis with diverse ways respectively; Utilize enzyme cutting method respectively to the rs1799883 site of FABP2 gene; The rs4994 site of ADRB3 gene, and somatotype is carried out in the rs1042714 site of ADRB2 gene; Utilize the PCR-SSCP method respectively somatotype to be carried out in the rs9939609 site of FTO gene, the rs1801282 site of PPARG gene.

1, the rs1799883 site somatotype of FABP2 gene

The pcr amplification sheet degree of FABP2 gene is following: SEQ ID NO:12

1 ACAGGTGTTA?ATATAGTGAA?AAGGAAGCTT?GCAGCTCATG?ACAATTTGAA?GCTGACAATT

61 ACACAAGAAG?GAAATAAATT?CACAGTCAAA?GAATCAAGC N?CTTTTCGAAA?CATTGAAGTT

121 GTTTTTGAAC?TTGGTGTCAC?CTTTAATTAC?AATCTAGCAG?ACGGAACTGA?ACTCAGGGTA

181 AGAATTTTTT?TTTTTATGAG?CAATGCATTC?TTGATTTTTC?TACCCAATAT?TAAAATGATT

241 TCTGCTCTAT?TTCATTGGAT?GGTTTAATTA?ATGCAGGTCT?CCTTCACTAA?CTGAAGAAGC

301 CAAT

Wherein " N " is the position in representative SNP site.HhaI can specific recognition ... GCG ↓ C ... This sequence, and its incision become " ... GCG and C ... "

The step of the rs1799883 site enzyme cutting type of FABP2 gene is:

1. with 1 μ l restriction enzyme Hha I (Takara company); 2.5 μ l 10 * enzyme cutting buffering liquid (Buffer M, takara company), PCR product 7ul; Ultrapure water 9.5ul together adds in 1 200ul centrifuge tube and uses the vibrator mixing, and is brief centrifugal to remove the drop of cap wall.

The centrifuge tube that 2. above mixture will be housed is put into 37 ℃ water-bath water-bath 4 hours, and the mixture in original 200ul centrifuge tube was exactly that enzyme is cut product.

3. 5 μ L enzymes are cut product and add appearance buffering (Loading buffer, Beijing ancient cooking vessel state) liquid mixing on the 1 μ L, pick up, add in the glue hole of sepharose with liquid-transfering gun.

4. pick up DNA Marker (Marker I, sky, Shanghai root) 4 μ L with liquid-transfering gun, add in the glue hole of sepharose.

The 150V constant voltage, electrophoresis 15min; Take out gel, put into the gel imaging appearance and take pictures, record.

Its result asks for an interview the rs1799883 site somatotype cleavage map of the FABP2 gene of Fig. 3.According to the data that NCBI announces, the rs1799883 site is the sudden change of a G → A, and (recognition sequence does HhaI ... GCG ↓ C ...) can be special the G allelotrope in this site of identification, cut through a dna sequence dna, be divided into size two for 100bp and 204bp; When G sported A, this restriction enzyme site disappeared.Therefore, according to this result, the allelotrope that can distinguish on the rs1799883 site is G or A.Fig. 3 cuts for the enzyme in FABP2rs1799883 site and declares type figure.Wherein, M600bp DNA marker, swimming lane 1-3 are the allelic homozygote of G; Swimming lane 4-6 is the allelic homozygote of A; Swimming lane 7-9 is the allelic heterozygote of G/A.

2, the rs4994 site somatotype of ADRB3 gene

The pcr amplification sheet degree of ADRB3 gene is following: SEQ ID NO:13

1 CGCCCAATAC?CGCCAACACC?AGTGGGCTGC?CAGGGGTTCC?GTGGGAGGCG?GCCCTAG CCG

61 GGGCCCTGCT?GGCGCTGGCG?GTGCTGGCCA?CCGTGGGAGG?CAACCTGCTG?GTCATCGTGG

121 CCATCGCC YG?GACTCCGAGA?CTCCAGACCA?TGACCAACGT?GTTCGTGACT?TCGCTGGCCG

181 CAGCCGACCT?GGTGATGGGA?CTCCTGGTGG

Because in the fragment of primer amplification, contain the CCGG sequence, therefore, MSP I also can discern this sequence, and at the 58th the C base degree of hacking of ingressing." Y " represents the position in this SNP site, and when Y was T, the sequence at Y place became " CCTG ", and MSP I can't discern this section sequence, therefore, can extension increasing sequence be cut 2 sections sequences for the 58+152bp size; When Y is that C is, the sequence at Y place becomes " CCGG ", and MSPI can discern this section sequence.Therefore can this extension increasing sequence be cut be 58,70,3 bands of 82bp size.The different bases in this SNP site of identification that therefore, MSPI can be special.

Because it is more approaching that the ADRB3 gene enzyme is cut the segmental size of product, the present invention has adopted the higher polyacrylamide gel electrophoresis of resolving power.

The step of the rs4994 site enzyme cutting type of ADRB3 gene is:

1. with 1 μ l restriction enzyme Msp I (NEB company); 2.5 μ l 10 * enzyme cutting buffering liquid (Buffer2, NEB company), PCR product 7ul; Ultrapure water 9.5ul together adds in 1 200ul centrifuge tube and uses the vibrator mixing, and is brief centrifugal to remove the drop of cap wall.

2. 37 ℃ of water-baths are 4 hours, just can obtain enzyme and cut product.

3. 2 μ L enzymes are cut product and add 1 μ L sample-loading buffer (Loading buffer, Beijing ancient cooking vessel state) mixing, pick up, accurately click and enter in the glue hole of polyacrylamide gel (39: 1,12%, see after one section in face of oneself preparation, refinement explanation) with liquid-transfering gun.(polyacrylamide gel is with in electrophoresis chamber, installing in advance).

4. pick up DNA Marker (Marker I, sky, Shanghai root) 4 μ L with liquid-transfering gun, add in the glue hole of polyacrylamide gel., leave standstill about 10min, sample and DNAMarker are all sunk.

5. energized then, electrophoresis 5～10min under the 300V constant voltage, 120V constant voltage then, electrophoresis 6h, after surge was accomplished, silver dyed (the refinement explanation is seen after) and shows band, just obtains restriction enzyme mapping.

Wherein, above-mentioned 39: 1, the preparation method of 12% polyacrylamide gel was following:

39: 1,12% polyacrylamide gel was by 39: 1, and 40% polyacrylamide gel stoste obtains through dilution.At first, disposed 39: 1 40% polyacrylamide gel stoste:

A polyacrylamide gel (40%, acrylic amide: N, N '-methylene diacrylamide=39: 1) collocation method: in 800mL water, add acrylic amide 390g, N, N '-methylene diacrylamide 10g is settled to 1L.

B is measured polyacrylamide gel solution 9.0mL, 10 * tbe buffer liquid 3.0mL, 10% ammonium persulphate 0.21mL, TEMED 10.5 μ L, the water 17.79mL of 40% (39: 1); Be total to the 30mL mixing; Can obtain 12%39: 1 polyacrylamide gel liquid; Through the operation of glue device, can obtain 12%39: 1 polyacrylamide gel.

10% Ammonium Persulfate 98.5: be dissolved in whole amount to 1g Ammonium Persulfate 98.5 (Beijing ancient cooking vessel state) in the aqueous solution of 10ml, this solution can be preserved several weeks at 4 ℃.

TEMED: i.e. N, N, N ', N '-Tetramethylethylenediamine, Chinese N by name, N, N ', N '-tetramethyl-diethylamine.Molecular formula is (CH3) 2NCH2CH2N (CH3) 2, and molecular weight is 116.20.(Beijing ancient cooking vessel state)

Method that silver dyes and step are explained as follows:

Electrophoresis unloads gel after finishing from electrophoresis chamber, carefully gel is taken out from the glass offset plate, cuts Yi Xiaojiao and serves as a mark, and places the pallet (pallet is placed on the shaking table) that deionized water is housed soaks, and opens shaking table, prepares silver and dyes.

Step is following:

A. wash glue: in pallet, add the 500mL rinsed with deionized water, deionized water is outwelled in 60r/min vibration 30 seconds after the completion.

B. fixing: in pallet, add the ethanol of 500mL 20%, the 60r/min 15min that vibrates outwells ethanolic soln after the completion, and repeating step a is promptly with outwelling behind the water rinse gel 30s.

C. oxidation: in pallet, add 500mL 1%HNO3 solution, the 60r/min 3min that vibrates outwells solution after the completion.

D. dyeing: in pallet, add the AgNO3 solution of 500mL 0.1%, the 60r/min 30min that vibrates outwells solution after the completion, and repeating step a is promptly with outwelling behind the water rinse gel 30s

E. colour developing: in pallet, add the yellow soda ash colour developing liquid (adding 80 μ L/100mL formaldehyde) of 300mL 2%, brown band appears in the about 10min of 60r/min, outwells solution after the completion, and repeating step a is promptly with outwelling behind the water rinse gel 30s.

F. stop to show: in pallet, add the acetic acid soln of 500mL 4%, 60r/min 3min uses water rinse 30s.

G. keep band, level cuts up and down redundance, and gel is packed in the valve bag, takes pictures and analyzes the resolution genotype.

The silver transfection reagent:

Shaking table, deionized water

0.1% silver nitrate solution: 0.5 gram Silver Nitrate is dissolved in the 500ml distilled water, places brown bottle, room temperature preservation.

Colour developing liquid: add soda ash light 6g, 240 μ L formaldehyde in the 300mL water, mixing, the time spent joins at present

Acetate (4%): get 4mL acetate and add water and be settled to 100mL

Data according to the NCBI announcement; The rs4994 site is the sudden change of a T → C; (recognition sequence does Msp I ... the C allelotrope in this site of identification of C ↓ CGG...) can be special, is that this restriction enzyme site occurs when T sports C, can amplification sheet degree be divided into that size is 58,70,82bp three bands.Therefore, according to this result, the allelotrope that can distinguish on the rs4994 site is T or C.Fig. 4 cuts for the enzyme in ADRB3rs4994 site and declares type figure.Wherein, M, 600bp DNAmarker 1-2 are the allelic homozygote of T; 3 is the allelic heterozygote of T/C.In all samples that detect, temporarily do not find C/C gene pure.

3, the rs1042714 site somatotype of ADRB2 gene

The pcr amplification sheet degree of ADRB2 gene is following: SEQ ID NO:15

1 AAGCCATGCG?CCGGACCACG?ACGTCACGCA?G SAAAGGGAC?GAGGTGTGGG?TGGTGGGCAT

61 GGGCATCGTC?ATGTCTCTCA?TCGTCCTGGC?CATCGTGTTT?GGCAATGTGC?TGGTCATCAC

121 AGCCATTGCC?AAGTTCGAGC?GTCTGCAGAC?GGTCACCAAC?TACTTCATCA?CTTCACTGGC

181 CTGTGCTGAT?CTGGTCATGG?GCCT

Wherein " S " is the position in representative SNP site.BbvI can specific recognition ... GCAGC (N) ₈↓ ... This sequence.

The step of the rs1042714 site enzyme cutting type of ADRB2 gene is:

1. with 1 μ l restriction enzyme BbvI (Fermentas company); 2.5 μ l 10 * enzyme cutting buffering liquid (10 * FastDigest Green Buffer; Fermentas company); PCR product 7ul, ultrapure water 9.5ul together add in 1 200ul centrifuge tube and use the vibrator mixing, and be brief centrifugal to remove the drop of cap wall.

The centrifuge tube that 2. above mixture will be housed is put into 65 ℃ water-bath water-bath 4 hours, and the mixture in original 200ul centrifuge tube was exactly that enzyme is cut product.

3. 5 μ L enzymes are cut product and add appearance buffering (Loading buffer, Beijing ancient cooking vessel state) liquid mixing on the 1 μ L, pick up, add in the glue hole of sepharose with liquid-transfering gun.

4. pick up DNA Marker (Marker I, sky, Shanghai root) 4 μ L with liquid-transfering gun, add in the glue hole of sepharose.

The 150V constant voltage, electrophoresis 15min; Take out gel, put into the gel imaging appearance and take pictures, record.

According to the data that NCBI announces, there is the variation of C → G base in ADRB2 gene rs1042714 site, and the restriction enzyme site of variation back restriction endonuclease BbvI disappears, so CC is individual, and it is 40bp and 164bp that its enzyme is cut product; GC is individual, and it is 204bp, 164bp, 40bp that its enzyme is cut product; And GG is individual, because restriction enzyme site disappears, still is 204bp.The enzyme that the result asks for an interview the ADRB2 rs1042714 site of Fig. 5 is cut and is declared type figure, and wherein the M swimming lane is DNAMarker DL 1000, and the residue swimming lane is the PCR-RFLP product.Confirm genotype through observing banding pattern, swimming lane 1,2 has 40bp and 2 fragments of 164bp, for CC homozygous; It is the GC heterozygous that swimming lane 3,4 has 204bp, 164bp, a 40bp3 fragment; Swimming lane 5 has only 1 fragment of 204bp, for GG homozygous.

Above-mentioned 3 kinds of banding patterns be can find through agarose gel electrophoresis, CC, GC, three kinds of genotype of GG represented respectively.The variation that has this site is described.

4, the rs9939609 site somatotype of FTO gene

The pcr amplification sheet degree of FTO gene is following: SEQ ID NO:11

1 GAATGAAATA?GGATTCAGAA?GAGATGATCT?CAAATCTACT?TTATGAGATA?ATGTCCTTTT

61 TAAAAATAAA?CACTAACATC?AGTTATGCAT?TTAGAATGTC?TGAATTATTA?TTCTAGGTTC

121 CTTGCGACTG?CTGTGAATTT?WGTGATGCAC?TTGGATAGTC?TCTGTTACTC?TAAAGTTTTA

181 ATAGGTAACA?GTCAGAAATG?GAGTGGGAG

Wherein W represents the position of SNP, and this SNP site is the sudden change of " T → A ".

The PCR-SSCP somatotype step in the rs9939609 site of FTO gene is:

1. prepare polyacrylamide gel (39: 1,12%) with the ADRB3 gene.

2. polyacrylamide gel electrophoresis---need be to PCR product and previously prepd FTOrs9939609 site A/A allelotrope, rs9939609 site T/T allelotrope homozygote as with reference to doing denaturing treatment; Make the dna double chain separately, thereby utilize the specificity of banding pattern to judge.

Concrete grammar is: the pcr amplification product of FTO gene and object of reference 1.5 μ L and 6.0 μ L denaturing agents (98% methane amide, 0.025% tetrabromophenol sulfonphthalein, 0.025% YLENE cyanogen, 10mmol/L EDTA (pH8.0), 10% glycerine) are together added mixing in the model, and (adding model is 96 holes; Coincide with the hole of PCR appearance; In different holes, add different object of reference, product; Mark is good, and sex change just can be sequentially added into electrophoresis in the gel after accomplishing), add model well with rubber belt sealing; 98 ℃ of sex change 10min, ice bath 7min then.PCR product and object of reference pick up with the liquid-transfering gun of 10ul respectively and click and enter in the glue hole of gel after the sex change, electrophoresis 5～10min under the 300V constant voltage, and 120V constant voltage then, electrophoresis 24h, surge is accomplished back silver and is dyed to show and be with.

3. silver dyes (with the ADRB3 gene).

The reagent of using

Sex change sample loading buffer: in 9.8mL deionized formamide solution, add bromjophenol blue 2.5mg, YLENE cyanogen FF 2.5mg, glycerine 200 μ L, 0.01mol/L EDTA200 μ L, mixing packing, 4 ℃ of preservations.

The sex change sample loading buffer is a denaturing agent, and each constituent concentration is: 98% methane amide, 0.025% tetrabromophenol sulfonphthalein, 0.025% YLENE cyanogen, 10mmol/L EDTA (pH8.0), 10% glycerine.

Concrete outcome is asked for an interview the polyacrylamide gel electrophoresis figure as a result in the FTO rs9939609 site of Fig. 6, swimming lane 2,5, A/A allelotrope homozygote; Swimming lane 1,3,6,7, A/T allelotrope heterozygote; Swimming lane 4, T/T allelotrope homozygote.

According to the data that NCBI announces, there is the variation of T → A base in FTO gene rs9939609 site.Utilize the PCR-SSCP technology, obtained the result of Fig. 6.Wherein, swimming lane 2,5, A/A allelotrope homozygote; Swimming lane 1,3,6,7, A/T allelotrope heterozygote; Swimming lane 4, T/T allelotrope homozygote.

Wherein the allelic homozygote of A/A of swimming lane 2,4 and the allelic homozygote of TT have carried out the cloning and sequencing checking, and collection of illustrative plates can accurately analyze the genotype in this site.Sequencer map is asked for an interview Fig. 7 and Fig. 8, and the order-checking collection of illustrative plates is accomplished by the big gene of Shenzhen China.

Test sample the time, as reference, just can accurately distinguish the genotype in this site with FTO A/A allelotrope homozygote, T/T allelotrope homozygote according to these banding patterns.

5, the rs1801282 site somatotype of PPARG gene

The pcr amplification sheet degree of PPARG gene is following: SEQ ID NO:14

1 CAGTGTGAAT?TACAGCAAAC?CCCTATTCCA?TGCTGTTATG?GGTGAAACTC?TGGGAGATTC

61 TCCTATTGAC?SCAGAAAGCG?ATTCCTTCAC?TGATACACTG?TCTGCAAACA?TATCACAAGG

121 TAAAGTTCCT?TCCA

Wherein S represents the position of SNP, and this SNP site is the sudden change of " G → C ".

The PCR-SSCP somatotype step in the rs1801282 site of PPARG gene is:

1. prepare polyacrylamide gel (39: 1,12%) with the ADRB3 gene

2. (this part is different with the ADRB3 gene for polyacrylamide gel electrophoresis; Need do denaturing treatment to rs1801282 site C/C allelotrope, the G/G allelotrope homozygote object of reference of PCR product and previously prepd PPARG gene; Make the dna double chain separately, thereby utilize the specificity of banding pattern to judge.

Concrete grammar is: the pcr amplification product of PPARG gene and object of reference 1.5 μ L and 6.0 μ L denaturing agents (98% methane amide, 0.025% tetrabromophenol sulfonphthalein, 0.025% YLENE cyanogen, 10mmol/L EDTA (pH8.0), 10% glycerine) are together added mixing in the model, and (adding model is 96 holes; Coincide with the hole of PCR appearance; In different holes, add different object of reference, product; Mark is good, and sex change just can be sequentially added into electrophoresis in the gel after accomplishing), add model well with rubber belt sealing; 98 ℃ of sex change 10min, ice bath 7min then.PCR product and object of reference pick up with the liquid-transfering gun of 10ul respectively and click and enter in the glue hole of gel after the sex change, electrophoresis 5～10min under the 300V constant voltage, and 120V constant voltage then, electrophoresis 24h, surge is accomplished back silver and is dyed to show and be with.

3. silver dyes (with the ADRB3 gene)

The reagent of using (identical) with FTO

According to the data that NCBI announces, there is the variation of C → G base in the rs1801282 site of PPARG gene.Utilize the PCR-SSCP technology, obtained the result of Fig. 9.Wherein, swimming lane 1,3,7, C/C allelotrope homozygote; Swimming lane 2,5,6, C/G allelotrope heterozygote; Swimming lane 4, G/G allelotrope homozygote.

Wherein swimming lane 1 and 4 the allelic homozygote of C/C and the allelic homozygote of G/G have carried out the cloning and sequencing checking, find that collection of illustrative plates can accurately analyze the genotype in this site.Sequencer map is asked for an interview Fig. 7 and Fig. 8, and the order-checking collection of illustrative plates is accomplished by the big gene of Shenzhen China.

Therefore, test sample the time, as reference, only need just can accurately distinguish the genotype in this site with the allelic homozygote of C/C and the allelic homozygote of G/G according to these banding patterns.

Embodiment 4: 5 adult females are carried out the Weight management service based on gene test

Step 1:DNA extracts

Instruct the examinee to use oral cavity sampling swab to carry out the mouth epithelial cells sampling by the surveyor, utilize the buccal swab genome DNA extracting reagent kit of sky, Shanghai root biological production to extract person under inspection's genomic dna.

Step 2: genotype tests

Use test kit provided by the invention,, FABP2 gene, ADRB3 gene, ADRB2 gene, FTO gene, the PPARG gene of detected person's genomic dna detected, confirm the genotype in these SNP sites according to the foregoing description 1-3.

Step 3: Weight management service

Through to the genotypic analysis of tester SNPs; Provide complex inheritance PAA report; Recommend to be fit to the diet style (low fat diet, low carbohydrate diet, balanced diet) of its loss of weight by the nutritionist to the measured, and, analyze the absorption heat that need its every day according to measured's practical situation; The ratio of 3 big nutritive elements (fat, protein, glucide) in the breakfast, lunch and dinner is arranged on individuation ground, thereby reaches the purpose of health, science, effective loss of weight.

The following genotype combination of table 3 is used to analyze the recipe classification

5 adult females of table 4 are according to the diet of gene test result adjustment

Numbering

FTO

FABP2

PPARG

ADRB2

ADRB3

1#

TT

Thr/Ala

Pro/Pro

Gln/Gln

Trp/Trp

2#

AT

Thr/Thr

Pro/Pro

Gln/Gln

Trp/Trp

3#

AT

Ala/Ala

Pro/Ala

Gln/Gln

Trp/Trp

4#

TT

Ala/Ala

Pro/Pro

Gln/Glu

Trp/Arg

5#

TT

Ala/Ala

Pro/Pro

Gln/Gln

Trp/Trp

1# advises low fat diet, and the activity of medium tenacity carries out Weight management.

2# advises low fat diet, and the activity of medium tenacity carries out Weight management.

3# advises low carbohydrate diet, and high activity carries out Weight management.

4# advises low carbohydrate diet, and high activity is measured and carried out Weight management.

5# advises balanced diet, and the activity of medium tenacity carries out Weight management.

Low carbohydrate diet refers to that the heat ratio that glucide provides must not be higher than 49% in breakfast, lunch and dinner; Low fat diet refers to that the absorption total amount of fat in the meals and the intake of sfas must not surpass 50g;

Balanced diet refers to that the ratio of balance three big nutritive elements should be controlled at:

Glucide: 55%-65%

Fat: 20%-30%

Protein: 10-15%

The activity of medium tenacity is meant: design the motion of a series of suitable individualities by the nutritionist of specialty, the activity that makes its every day is between 1.4～1.6 times of its health basal metabolic rate(BMR) (BMR).

High-intensity activity is meant: design the motion of a series of suitable individualities by the nutritionist of specialty, the activity that makes its every day is between 1.6～1.8 times of its health basal metabolic rate(BMR) (BMR).

Though the present invention discloses as above with preferred embodiment; Right its is not in order to limit the present invention; Any person of ordinary skill in the field; In spirit that does not break away from the present invention and scope, when can doing a little change and improvement, so the present invention's protection domain is as the criterion when looking the claim person of defining.

Claims (8)

1. a molecule marker test kit that is used to formulate individuation loss of weight scheme is characterized in that, comprises the Nucleotide shown in the SEQID NO:1-10.

2. test kit according to claim 1 is characterized in that, said test kit also comprises restriction enzyme Hha I, Msp I and BbvI.

3. test kit according to claim 1 and 2 is characterized in that said test kit also comprises DNA extraction liquid.

4. test kit according to claim 3 is characterized in that, said DNA extraction liquid comprises: damping fluid GA, damping fluid GB, damping fluid GD, rinsing liquid PW, elution buffer TB and Proteinase K.

5. test kit according to claim 4 is characterized in that said test kit also comprises adsorption column, collection tube and centrifuge tube.

6.SEQ the application of the Nucleotide shown in the ID NO:1-10 in the molecule marker test kit of preparation individuation loss of weight scheme.

7. application according to claim 6 is characterized in that, said Nucleotide is as primer.

8. application according to claim 7 is characterized in that, the primer SEQ ID NO:1-2 FTO gene that is used to increase; The primer SEQ ID NO:3-4 FABP2 gene that is used to increase; The primer SEQ ID NO:5-6 ADRB3 gene that is used to increase; The primer SEQ ID NO:7-8 PPARG gene that is used to increase; The primer SEQID NO:9-10 ADRB2 gene that is used to increase.

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