CN102796823A

CN102796823A - Fluorescence detection kit and detection method for motor function related gene CKMM

Info

Publication number: CN102796823A
Application number: CN2012103170908A
Authority: CN
Inventors: 步迅; 夏子芳
Original assignee: Shandong Bio-Focus Gene Science & Technology Co Ltd
Current assignee: Shandong Bio-Focus Gene Science & Technology Co Ltd
Priority date: 2012-08-30
Filing date: 2012-08-30
Publication date: 2012-11-28

Abstract

The invention discloses a fluorescence detection kit and a detection method for a motor function related gene CKMM and belongs to the technical field of biological detection. The kit comprises a reagent before amplification and a reagent after amplification, wherein the reagent before amplification comprises a PCR buffer solution, a reaction mixture of MgCl2 and dNTPs, Taq enzyme, ultrapure water and a primer mixture of high-specificity amplification CKMM gene hotspot mutation; and the reagent after amplification comprises a genotype standard substance and an internal label. Genotyping can be performed by using the gene as a detection object through PCR amplification, capillary electrophoresis detection and comparison with the genotype standard substance, so that the individual with the specific genotype can be screened out and provides reference for sports person selection. In the aspect of sports person selection, the fluorescent-labeling technology and the capillary electrophoresis technology are adopted for the first time to detect the high sensitivity and specificity of the motor function related gene, so that manpower, materials and time are saved.

Description

Motion function genes involved CKMM fluorescence detection reagent kit and detection method

Technical field

The present invention relates to a kind of motion function genes involved CKMM fluorescence detection reagent kit and detection method, belong to technical field of biological.

Background technology

Along with the completion of the Human Genome Project, being in full swing of post genome project paid close attention in the application of sports problem domain with the biotechnology that genetically engineered is taken as the leading factor widely.The gene genetic practical applications is historical inevitable, trend of the times in selection of athletes, and it will fundamentally cause the revolution of motion selection theory and method.Use genetic theory and method to carry out the motion selection preliminary progress has been arranged.

Tn (CK) is the key enzyme of Cr+MgATP+H-PCr+MgADP reaction.CK and phosphocreatine are formed creatine one phosphocreatine shuttle system, in intracellular effect: the one,, when high energy demand, keep the ratio of ATP/ADP as " temporary transient energy snubber system "; The 2nd,, as energy transportation organization, energy is transported to and utilizes the position from producing the position.Flesh type Tn is a kind of tissue specificity enzyme, mainly in Skelettmuskel, expresses, and a small amount of expression is also arranged in the cardiac muscle.CKMM mainly concentrates near the M line in the muscle; Its major function is the ATP that generates high density at the myosin head, and for Muscle contraction and motion provide energy, CKMM also exists near the sarcoplasmic reticulum Ca ATP enzyme; Possibly influence reassociating of Ca, thereby influence the work capacity of muscle.Research shows that the polymorphum of people's CKMM gene is relevant with endurance and endurance training effect.

Three kinds of genotype of CKMM gene pleiomorphism are respectively: AA type, GG type and heterozygote AG type.Result of study shows: with compare before the endurance training, when communicating inferior US motion after the training, people's motor capacity of AA, AG type improves significantly, but the various indexs changes in GG type training back are not obvious.Therefore GG type gene is least responsive in the endurance training, carries the allelic AA type of A, AG type people is significantly higher than the type with GG to the susceptibility of endurance training.

Current research is illustrated in rhabdomyolysis symptoms such as the myalgia, the Tn that occur behind the high-intensity exercise increase, and with ACE and CKMM gene confidential relation is arranged.The motion back more is prone to the high reaction of Tn with the individuality of D allelotrope (ACE gene) and G allelotrope (CKMM gene).And the people who has DD genotype (ACE gene) and GG genotype (CKMM gene) simultaneously, the back Tn that can occur moving raises unusually, and the D+G genotype has summation action.

Gene tester commonly used at present has: (direct sequencing directly checks order; DS), ligase enzyme detection reaction (ligase detection reaction; LDR), restriction fragment length polymorphism analysis (restriction fragment length polymorphism; RFLP), dhplc analysis (denaturing high performance liquid chromotography, DHPLC), quantitative PCR.All there is different defects in these methods, and for example complex operation, result are difficult for problems such as interpretation, poor repeatability, false negative false positive be many.

Adopt fluorescent mark technology, capillary electrophoresis technique; Through with the primer of different fluorescent markers to the specific amplification of motion function genes involved CKMM; And after capillary electrophoresis post analysis PCR product goes out the peak situation; Can realize the detection to this motion function genes involved site, highly sensitive, interpretation is clear accurately, sampling is convenient.

Summary of the invention

The purpose of this invention is to provide a kind of motion function genes involved CKMM fluorescence detection reagent kit and detection method.

Gene type after detection motion function genes involved CKMM fluorescence detection reagent kit of the present invention, this test kit comprise amplifing reagent and increase is used reagent;

Reagent comprises before the said amplification: PCR damping fluid, MgCl ₂, dNTPs mixture, Taq enzyme, primer mixture and ultrapure water;

Said amplification back reagent comprises: interior mark and be used for the gene type standard substance of gene type;

Use described test kit to carry out the condition of pcr amplification reaction: the pH value of pcr amplification system is 8.0-9.0; Magnesium ion concentration is 1.5-3.5mM; The final concentration of 4 kinds of dNTP respectively is 200-300 μ M, and the consumption of Taq enzyme is 0.1-0.4U/ μ L, and the primer final concentration is 0.2-0.4 μ M.

The primer that is used for the CKMM gene test is:

SEQ?NO.1：5’-AGG?GTG?TCA?GGT?GTG?AAA?GAA?TCA-3’；

SEQ?NO.2：5’-GTATCCT?TAC?TTT?CTC?AAG?AAC?CTG?CCG-3’；

SEQ?NO.3：5’-CCT?TAC?TTT?CTC?AAG?AAC?CTG?CCA-3’。

5 ' of SEQ NO.1 end carries out fluorochrome label in the described primer, and the used optical dye of interior mark is different resorcinolphthalein.

Said amplification adopts the polymerase chain reaction to realize, adopts multiple tracks or single track capillary electrophoresis to detect.

Adopting said test kit to be applied to the method that the motion function genes involved detects, is through fluorescent primer PCR and capillary electrophoresis specific detection motion function genes involved CKMM; The primer that is used for the CKMM detection is: SEQ NO.1, SEQ NO.2, SEQ NO.3, the amplification of said gene adopts the polymerase chain reaction to realize, adopts multiple tracks or single track capillary electrophoresis to detect.

The human gene group DNA who is wherein detected is: use Chelex method, magnetic bead extraction method or phenol/chloroform extraction method that the source sample is handled the DNA that obtains; Described source sample is: derive from human: filter paper blood cake/buccal swab sample, FTA card blood cake/buccal swab sample, buccal swab sample, blood.

When using described test kit to carry out pcr amplification, the amplification elementary reaction carries out amplification program on the PCR of any model appearance: 94-98 ℃ of 1-5min; 26-32 round-robin 94-98 ℃ 15-60s, 55-65 ℃ of 15-60s, 72 ℃ of 15-60s; 60 ℃ of 30-60min.

Beneficial effect of the present invention is following:

The present invention is a detected object with the CKMM gene, and through pcr amplification, capillary electrophoresis detects, and with the contrast of gene type standard substance, can carry out gene type, filters out the individuality with specific gene type, for the physical culture selection provides reference.Having adopted fluorescent mark technology, capillary electrophoresis technique to realize highly sensitive specific detection aspect the physical culture selection first, manpower and materials and time have been practiced thrift to the motion function genes involved.

Description of drawings

Fig. 1 is a test kit of the present invention to the figure as a result of the somatotype after the individual DNA cloning of 71-2 sample CKMM gene;

Fig. 2 is a test kit of the present invention to the figure as a result of the somatotype after the individual DNA cloning of 74-2 sample CKMM gene;

Fig. 3 is a test kit of the present invention to the figure as a result of the somatotype after No. 87 individual DNA cloning of sample CKMM gene.

Embodiment

Below in conjunction with embodiment the present invention is done and to further describe.

Embodiment 1

Test kit of the present invention detects the dna sample of 40 Different Individual.Be used for the primer employing blue fluorescent dyes mark that the motion function genes involved detects, interior mark adopts the red fluorescence dyestuff mark.

1, sample to be tested is 40 parts, and that hereinafter is enumerated is the result of individual 3 parts of different genotype.Correlated samples all uses the technological method of " DNA extraction-pcr amplification-order-checking " that CKMM was carried out order-checking and detects.The somatotype result of wherein, 71-2 number, 74-2 number, No. 87 samples is followed successively by: G/G, A/A, A/G.

2, the extracting genome DNA of sample

Chelex extraction method: cut 1～3mm blood cake and place the 1.5mL centrifuge tube, add sdH ₂O 1mL, it is centrifugal to vibrate, and abandons supernatant, and repeating step twice is abandoned supernatant, and draw 200 μ L with the rifle head of cutting fast after the 5%Chelex-100 concussion is suspended and add in the centrifuge tube, the vibration several seconds.Behind 56 ℃ of water bath heat preservation 30min, the vibration several seconds.95 ℃ of boiling water bath 10min slightly vibrate the several seconds.The centrifugal 5min of 2000rpm, the DNA for extracting in the supernatant.

3, the check and analysis of amplification and amplified production

3.1PCR amplification system:

3.2PCR amplification program:

4, amplified production fluoroscopic examination on genetic analyzer

Form appearance mixture ((mark in the 0.2 μ L molecular weight) * (sample introduction number)+(9.8 μ L deionized formamide) * (sample introduction number)) by mark in deionized formamide and the molecular weight.Appearance mixture on the 10 μ L is mixed with 1 μ L amplified production or allelotrope analytical standard, avoid producing bubble.95 ℃ of sex change 5min, ice bath 5min, and electrophoresis as early as possible.Use the genetic analyzer check and analysis.

5, conclusion

Somatotype of the present invention is complete clear, and the result is shown in Fig. 1-3:

Fig. 1: the unimodal of the about 3500H of 166bp peak value appears in 71-2 sample CKMM gene type position, examines to G/G isozygotys, and be consistent with sequencing result.

Fig. 2: the unimodal of the about 2800H of 162bp peak value appears in 74-2 sample CKMM gene type position, examines to A/A isozygotys, and be consistent with sequencing result, points out this individuality to have and train susceptibility preferably.

Fig. 3: No. 87 the bimodal of the about 2650H of 162bp peak value, the about 2200H of 166bp peak value appears in sample CKMM gene type position, examines the heterozygosis into A/G, and be consistent with sequencing result, points out this individuality to have and train susceptibility preferably.

2.5 * PCR damping fluid of used different pH among the above embodiment, with the Tris-HCl damping fluid preparation of different pH values, the Tris-HCl concentration in 1 * PCR damping fluid is 10mM, KCl concentration is 50mM; Used Taq polysaccharase and other reagent and material are the commercially available prod among the present invention.

Claims

1. motion function genes involved CKMM fluorescence detection reagent kit is characterized in that comprising archaeal dna polymerase and buffered soln thereof, the primer of each gene that increases, interior mark and gene type standard substance.

2. test kit according to claim 1 is characterized in that:

The primer that is used for the CKMM gene test is:

SEQ?NO.1：5’-AGG?GTG?TCA?GGT?GTG?AAA?GAA?TCA-3’；

SEQ?NO.2：5’-GTATCCT?TAC?TTT?CTC?AAG?AAC?CTG?CCG-3’；

SEQ?NO.3：5’-CCT?TAC?TTT?CTC?AAG?AAC?CTG?CCA-3’。

3. test kit according to claim 1 and 2 is characterized in that: the primer of the described CKMM of being used for gene amplification, and wherein, the 5 ' end of primer SEQ NO.1 carries out fluorochrome label, and interior mark is selected for use and is different from above-mentioned fluorochrome label.

4. test kit according to claim 1 and 2 is characterized in that: the amplification of said CKMM gene adopts the polymerase chain reaction to realize, adopts multiple tracks or single track capillary electrophoresis to detect.

5. one kind is adopted the said test kit of claim 1 to be applied to the method that the motion function genes involved detects, and it is characterized in that through fluorescent primer PCR and capillary electrophoresis specific detection motion function genes involved CKMM; The primer that is used for the CKMM detection is: SEQ NO.1, SEQ NO.2, SEQ NO.3, the amplification of said gene adopts the polymerase chain reaction to realize, adopts multiple tracks or single track capillary electrophoresis to detect.