CN102796822A

CN102796822A - Motion function gene ACTN3 (alpha-Actinin) fluorescent detection kit and detection method

Info

Publication number: CN102796822A
Application number: CN2012103168363A
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 fluorescent detection kit for detecting motion function related gene ACTN3 (alpha-Actinin), belonging 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 reaction mixture of PCR (Polymerase Chain Reaction) buffer solution, MgCl2 and NTPs (Nucleoside Triphosphate), Taq enzyme, super pure water and primer mixture for high-specificity amplification of ACTN3 gene hotspot mutation; and the reagent after amplification comprises genetic typing standard substance and an internal standard. According to the fluorescent detection kit, the gene is the detection object; and compared with the genetic typing standard substance, genetic typing can be performed through PCR amplification and capillary electrophoresis detection to screen out individuals with specific genotype and reference is provided for material selection in sports. In the aspect of material selection in sports, high-sensitivity specific detection of the motion function related gene is realized by using a fluorescence labeling technology and a capillary electrophoresis technology for the first time, and the manpower and material resources and the time are greatly saved.

Description

Motion function Gene A CTN3 fluorescence detection reagent kit and detection method

Technical field

The present invention relates to a kind of motion function Gene A CTN3 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.

Discover that the ACTN3 gene is really relevant with muscle explosive force, can to athletic explosive power development prediction accurately be arranged in early days.The ACTN3 gene engineering is applied in the selection of athletes field, not only can improves the accuracy rate of selection, can also significantly reduce elite's cultivation cost, the more important thing is and realize the early exercise item location, can improve lumber recovery effectively.

ACTN (α-actinine) is the conjugated protein of Actin muscle, mainly is distributed near myofiber Z line (similar DB) and helps location sarcostyle actinmicrofilament.ACTN has ACTN1,2,3,4 kind of existence form, and the mankind have only ACTN2 and ACTN3.ACTN2 is present in Skelettmuskel and the cardiac muscle, and ACTN3 exists only in the Skelettmuskel fast muscle fiber.Confirm that 1747 Nucleotide of No. 16 exons of ACTN3 gene C → T sudden change take place and produce terminator codon, replace the l-arginine of 577R amino-acid residue; Form 577X, making influences muscle stretch speed by the ACTN3 protein delation; Reduce explosive power, about 16% people exists this polymorphic.

ACTN3T allelotrope stops this proteinic manufacturing fully.Have two allelic people of T can not in their quick muscle fiber, make alpha Actinin 3, thereby aspect explosive power, be in a disadvantageous position, those CT genotype are because there is a functional gene, thereby can also make alpha Actinin 3.The CC genotype then has other people incomparable gene condition owing to having two ACTN3 genes.ACTN3 gene pleiomorphism and explosive power quality have dependency, and the homozygous speed quality of CC is superior to CT, TT type, and the investigator thinks that 577R can be used as the athletic gene selection of explosive power relevant item index.

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 ACTN3; 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 Gene A CTN3 fluorescence detection reagent kit and detection method.

Detection motion function genes involved ACTN3 fluorescence detection reagent kit of the present invention, the gene type after comprising amplifing reagent and increasing 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 ACTN3 gene test is:

SEQ?NO.1：5’-TTT?CTG?TTG?CCT?GTG?GTAAGT?GG-3’；

SEQ?NO.2：5’-GTTAGAT?GGC?ACC?TCG?CTC?TCA-3’

SEQ?NO.3：5’-GAT?GGC?ACC?TCG?CTC?TCG-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 ACTN3; The primer that is used for the ACTN3 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 ACTN3 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 greatly 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 ACTN3 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 73-4 sample ACTN3 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 ACTN3 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 ACTN3 was carried out order-checking and detects.The somatotype result of wherein, 71-2 number, 73-4 number, No. 87 samples is followed successively by: C/T, T/T, C/C.

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 bimodal of the about 1200H of 127bp peak value, the about 1450H of 131bp peak value appears in 71-2 sample ACTN3 gene type position, examines the heterozygosis into C/T, and be consistent with sequencing result.

Fig. 2: the unimodal of the about 2100H of 131bp peak value appears in 73-4 sample ACTN3 gene type position, examines to T/T isozygotys, and be consistent with sequencing result.

Fig. 3: No. 87 the unimodal of the about 2600H of 127bp peak value appears in sample ACTN3 gene type position, examines to C/C isozygotys, and be consistent with sequencing result, points out this individuality to have explosive power 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. a motion function Gene A CTN3 fluorescence detection reagent kit is characterized in that comprising archaeal dna polymerase and buffered soln thereof, the primer of amplification gene, 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 ACTN3 gene test is:

SEQ?NO.1：5’-TTT?CTG?TTG?CCT?GTG?GTAAGT?GG-3’

SEQ?NO.2：5’-GTTAGAT?GGC?ACC?TCG?CTC?TCA-3’

SEQ?NO.3：5’-GAT?GGC?ACC?TCG?CTC?TCG-3’。

3. test kit according to claim 1 and 2 is characterized in that: the primer of the described ACTN3 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 ACTN3 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 ACTN3; The primer that is used for the ACTN3 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.