CN101654710A

CN101654710A - Fluorescent quantitative PCR detection kit and method for gene type of Hu sheep estrogen receptor

Info

Publication number: CN101654710A
Application number: CN200910152668A
Authority: CN
Inventors: 王争光; 俞颂东; 董文艳
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2009-09-17
Filing date: 2009-09-17
Publication date: 2010-02-24

Abstract

The invention provides fluorescent quantitative PCR detection kit and method for a gene type of a Hu sheep estrogen receptor. The fit mainly comprises a specific amplimer, a PCR buffer solution, a deoxynucleotide triphosphate mixture, a DNA dye and DNA polymerase. The fluorescent quantitative PCR detection kit is characterized in that the specific amplimer comprises an upstream primer 5'-GCACCAGATCCAAGCCAACGAG-3' and a downstream primer 5'-GTACCTGTA GAAGGCGGGAGGG-3'. The invention has the advantages of step reduction, sensitivity improvement, high reaction speed, efficiency increase, environmentally friendly operation and personnel security.

Description

The fluorescent quantificationally PCR detecting kit of gene type of Hu sheep estrogen receptor and method

(1) technical field

The present invention relates to a kind of fluorescent quantificationally PCR detecting kit and detection method of gene type of Hu sheep estrogen receptor.

(2) background technology

Sheep belongs to short broadtail sheep, be rare white lambskin sheep variety, mainly being distributed in the some areas in Zhejiang Province, Jiangsu Province and Shanghai City, having that early sexual maturity, the four seasons are oestrused, polyembryony high yield and excellent economic characters such as fast of growing, is the rare and precious polyembryony variety source in the world.At present, because of the adjustment of market product structure and traditional mode of selecting and remain, make sheep be faced with the problem of endogamy, germplasm degeneration, lambing percentage decline, and directly influenced sheep Industrial economic benefit and sustainable development.By research to polyembryony mark and the polyembryony functional gene of sheep, can be the molecular breeding technology of using, directive breeding sheep polyembryony system provides theoretical foundation and technical foundation.

(estrogen receptor, ESR) gene is a major gene of control sheep fecundity trait to estrogen receptor.Sheep ESR gene exists wild homozygous genotype (AA), sudden change heterozygous genes type (AB) and three kinds of genotype of sudden change homozygous genotype (BB).B allelotrope and its prolificacy of sheep ESR gene have dependency, and three kinds of genotypic sheep litter sizes are followed successively by BB, AB, AA from high to low.Therefore, from colony, filter out the genotypic sheep of BB, set up core breeding group, cultivation sheep polyembryony strain, raising sheep Industrial economic benefit are had crucial meaning.The genotypic main method of detection sheep ESR gene of document record be the single-strand conformation polymorphism method (single strand conformation polymorphism, PCR-SSCP).The PCR-SSCP method uses a pair of primer to carry out the regular-PCR amplification, then with special pcr amplification product sex change, snapback then, make it to become single strand dna with certain space structure, an amount of single stranded DNA is carried out native polyacrylamide gel electrophoresis, dye by radioactive automatic developing, silver at last or ethidium bromide develops the color and judges genotype.This method operation steps is many, process is complicated, consuming time longer; When the fragment that is detected was too big, recall rate was lower; When the point mutation of some position when inoperative or effect is very little to the change of single strand dna three-dimensional conformation, easily cause polyacrylamide gel electrophoresis to differentiate and cause omission; In addition, this method need be made gel, makes operator contact poisonous and harmful reagent, and electrophoretic strength of current and time are difficult to control, and easily electrophoresis failure causes band to see clearly and influences genotypic judgement.

(3) summary of the invention

The object of the invention provides a kind of genotypic fluorescent quantificationally PCR detecting kit of sheep ESR gene and detection method, and the genotypic method accuracy rate of detection sheep ESR gene of the prior art is low, operation steps is many, process is complicated, the time is long, the deleterious technical problem of detection reagent to solve.

The technical solution used in the present invention is:

A kind of fluorescent quantificationally PCR detecting kit of gene type of Hu sheep estrogen receptor, mainly comprise specificity amplification primer, PCR damping fluid, deoxidation nucleoside triphosphate mixture, DNA dyestuff and archaeal dna polymerase, it is characterized in that: described specificity amplification primer is as follows: upstream primer: 5 '-GCACCAGATCCAAGCCAACGAG-3 ', downstream primer: 5 '-GTACCTGTAGAAGGCGGGAGGG-3 '.The sheep ESR gene that described sheep ESR gene specific primer is delivered according to GenBank (accession number: first exon dna sequence dna design X98010).

Concrete, described DNA dyestuff is SYBR Green I or the EvaGreen that similar spectral response curve is arranged with it ^TMSYBR Green I is a kind of DNA dyestuff of double-stranded specific, have only with could emitting fluorescence after double-stranded DNA combines.The fluorescence that produces during by denaturation temperature when double-stranded DNA decay fast form special melting curve, if the fluorescence that in the pcr amplification working cycle SYBR Green I is sent continues to monitor, utilize this feature to observe so to the sex change of different products.The melting curve of product depends on GC content, sequence length.If there is the mutant sequence in the amplified production, so in denaturation process between it and the wild-type sequence melting temperature(Tm) be that Tm will produce difference.Tm differs more than 2 ℃, just can it be separated by they melting curves separately.Two peak values can appear in the sudden change heterozygous after carrying out the melting curve analysis, and a peak value only appears in the homozygous and wild-type of suddenling change, but the varying in size of peak value.

The working concentration of SYBR Green I is to influence Success in Experiment whether key factor, if the unsaturated meeting of concentration of SYBR Green I makes the variation that fluorescent signal can not the reaction product amount.Excessive concentrations then can suppress the PCR reaction, reduces the PCR reaction efficiency.SYBR Green I is the patent of Molecular Probes under the Invitrogen house flag, usually test kit has more ready-made scheme, generally should optimize working concentration according to practical situation when using SYBR Green I to be: the final concentration in the reaction is 1 * to 0.2 * between.EvaGreen ^TMBe Biotium company product, stability is extremely strong, can be not destroyed in normal storage, operation and PCR process, and what the dyestuff in buffered soln can safety is stored in room temperature or the refrigerator, also can multigelation.

Key of the present invention is the design of Auele Specific Primer, and other components in the test kit such as archaeal dna polymerase, PCR damping fluid, deoxidation nucleoside triphosphate mixture etc., all can be formed with reference to the conventional test kit in this area.

The invention still further relates to a kind of fluorescent quantitative PCR detection method of gene type of Hu sheep estrogen receptor, described method comprises:

(1) extracts testing sample DNA;

(2) get specificity amplification primer, PCR damping fluid, deoxidation nucleoside triphosphate mixture, DNA dyestuff and archaeal dna polymerase, add testing sample DNA and be made into the PCR reaction solution, carry out pcr amplification reaction, when each loop ends, measure light absorption value, amplification cycles finishes the back PCR product is done 70 ℃～100 ℃ (0.5 ℃/s) melting curve analysis is adopted the analysis software of real-time fluorescence PCR instrument to obtain the melting curve of testing sample DNA;

Described specificity amplification primer is as follows:

Upstream primer: 5 '-GCACCAGATCCAAGCCAACGAG-3 '

Downstream primer: 5 '-GTACCTGTA GAAGGCGGGAGGG-3 ';

(3) according to the testing sample melting curve, judge testing sample female hormone receptor gene type: if two peak values appear in testing sample DNA melting curve, then testing sample is the sudden change heterozygous; If a peak value only appears in testing sample DNA melting curve, then testing sample is the homozygous or wild-type of sudden change, and the peak value of melting curve is then homozygous for sudden change between 85～90 ℃, and the peak value of melting curve then is wild-type between 90～95 ℃.

Usually, described deoxidation nucleoside triphosphate mixture (dNTP) is that the ratio of dATP, dTTP, dCTP, dGTP amount of substance is 1: 1: 1: 1 mixture.

Concrete, described PCR reaction solution final concentration is composed as follows:

PCR damping fluid final concentration is 1 *

Each 0.1～0.3 μ M of specificity amplification primer

dNTPs 0.1～0.3mM

Testing sample DNA 10～100ng/ μ L

Taq archaeal dna polymerase 0.05～0.5U/ μ L

SYBR Green I final concentration is 1 *

Solvent is ddH ₂O.

SYBR Green I is available from U.S. hero's life technology company limited (Invitrogen), concentration is 10000 *, be diluted to the working fluid of 20 * concentration during use, add during pcr amplification in per 20 μ LPCR reaction solutions and to add 1 μ L, 20 * SYBR Green I working fluid, thus in the PCR reaction solution final concentration of SYBR Green I be 1 *.

PCR damping fluid final concentration is 1 *, be meant that the final concentration of each component in reaction solution is identical with 1 * PCR buffer in the PCR damping fluid, with 10 times of 10 * PCR buffer dilutions, its concentration of component is promptly identical with 1 * PCR buffer.10 * PCR buffer consists of: 100mmol/l Tris-HClpH8.3,15mmol/L KCl, 15mmol/L MgCl ₂, 0.01% (W/V) gelatin, solvent is a DEPC water.

Described pcr amplification reaction condition is as follows: 95 ℃ of pre-sex change 5min at first, then 94 ℃ of sex change 1min, 59 ℃ of renaturation 1min, 72 ℃ extend 1min, totally 39 circulations, 72 ℃ are extended 10min again.

Beneficial effect of the present invention is mainly reflected in:

1. step reduces, and sensitivity improves

The present invention only needs sample DNA is carried out fluorescent quantitative PCR, both can obtain detected result, reduced electrophoresis, silver and step such as dyed, avoided gel electrophoresis can't differentiate the defective of the point mutation that does not change the single strand dna three-dimensional conformation, and avoided of the detrimentally affect of factors such as electrophoretic current instability detecting.Owing in the system of sealing, finish amplification and The real time measure, greatly reduce contamination of heavy, also reduced the probability of result error.

2. reaction is quick, and efficient increases

The present invention adopts fluorescent quantitative PCR detection method, will increase and detects synchronously and finish, the level of automation height, there is not aftertreatment, need not hybridization, electrophoresis, take pictures, shortened the reaction times, can obtain detected result in 3 hours, than adopting the PCR-SSCP method can save 5 hours at least.

3. operation environmental protection, personal security

The present invention adopts fluorescent quantitative PCR detection method, does not need steps such as gel electrophoresis, silver dye, and need not to use carcinogenic reagent such as polyacrylamide, ethidium bromide, has avoided in the process of the test the healthy hidden danger of experiment operator has been got rid of in the pollution of environment.

(4) description of drawings

Fig. 1 is the melting curve figure of embodiment 1 detected a kind of wild homozygous sheep ESR gene;

Fig. 2 is the melting curve figure of embodiment 1 detected a kind of homozygous sheep ESR gene that suddenlys change;

Fig. 3 is the melting curve figure of embodiment 1 detected a kind of heterozygous sheep ESR gene that suddenlys change.

(5) embodiment

The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:

Embodiment 1:

56 sheep blood samples are gathered by magnificent animal husbandry company limited (national sheep seed farm) from Zhejiang at random, with-20 ℃ of frozen laboratories of taking back after the ACD anti-freezing, and the experimental applications under the extraction genomic dna is provided with:

1. design of primers

(accession number: first exon dna sequence dna design primer X98010), product length is 335bp to the sheep ESR gene of delivering according to GenBank.Primer sequence is:

Upstream primer: 5 '-GCACCAGATCCAAGCCAACGAG-3 '

Downstream primer: 5 '-GTACCTGTAGAAGGCGGGAGGG-3 '

2. fluorescence quantitative PCR detection

Sample DNA and sheep ESR gene specific primer are carried out fluorescent quantitative PCR in the PCR reaction system that contains DNA dyestuff (SYBR GreenI).

The amplification reaction system final concentration consists of:

Taq archaeal dna polymerase (triumphant outstanding biotechnology (Shanghai) Co., Ltd.) 0.1U/ μ l

Each 0.2 μ M of upstream and downstream primer

dNTP 0.2mM

(dATP, dTTP, each 0.05mM of dCTP, dGTP, Takala company)

Sample DNA 50ng/ μ l

20 * SYBR Green I dyestuff working fluid (Invitrogen company), 1 μ l

10 * PCR damping fluid (triumphant outstanding biotechnology (Shanghai) Co., Ltd.), 2 μ l

Add ddH ₂O to 20 μ l.

Amplification reaction condition is:

95 ℃ of pre-sex change 5min at first, then 94 ℃ of sex change 1min, 59 ℃ of renaturation 1min, 72 ℃ extend 1min, totally 39 circulations, 72 ℃ are extended 10min again.

When each loop ends, measure every hole SYBR Green light absorption value, after amplification cycles finishes, the PCR product done 70 ℃ to 100 ℃ melting curve analysis with 0.5 ℃/s.According to Ct value and the solubility curve that the analysis software of real-time fluorescence PCR instrument obtains, analytical results, the genotype of judgement sample DNA.

3. detected result

In 56 sheep, wild homozygous individuality is that 13 (melting curve is referring to Fig. 1), sudden change heterozygous individuality are that 24 (melting curve is referring to Fig. 3), the homozygous individuality of sudden change are 19 (melting curve is referring to Fig. 2).

SEQUENCE?LISTING

＜110〉Zhejiang University

＜120〉fluorescent quantificationally PCR detecting kit of gene type of Hu sheep estrogen receptor and method

<130>

<160>2

<170>PatentIn?version?3.4

<210>1

<211>22

<212>DNA

<213>Unknown

<220>

＜223〉artificial sequence

<400>1

gcaccagatc?caagccaacg?ag 22

<210>2

<211>22

<212>DNA

<213>Unknown

<220>

＜223〉artificial sequence

<400>2

gtacctgtag?aaggcgggag?gg 22

Claims

1. the fluorescent quantificationally PCR detecting kit of a gene type of Hu sheep estrogen receptor, mainly comprise specificity amplification primer, PCR damping fluid, deoxidation nucleoside triphosphate mixture, DNA dyestuff and archaeal dna polymerase, it is characterized in that: described specificity amplification primer is as follows: upstream primer: 5 '-GCACCAGATCCAAGCCAACGAG-3 ', downstream primer: 5 '-GTACCTGTAGAAGGCGGGAGGG-3 '.

2. test kit as claimed in claim 1 is characterized in that described DNA dyestuff is SYBR GreenI or EvaGreen ^TM

3. the fluorescent quantitative PCR detection method of a gene type of Hu sheep estrogen receptor, described method comprises:

(1) extracts testing sample DNA;

(2) get specificity amplification primer, PCR damping fluid, deoxidation nucleoside triphosphate mixture, DNA dyestuff and archaeal dna polymerase, add testing sample DNA and be made into the PCR reaction solution, carry out pcr amplification reaction, when each loop ends, measure light absorption value, amplification cycles finishes the back PCR product is done 70 ℃～100 ℃ melting curve analysis, obtains the melting curve of testing sample DNA;

Described specificity amplification primer is as follows:

Upstream primer: 5 '-GCACCAGATCCAAGCCAACGAG-3 '

Downstream primer: 5 '-GTACCTGTA GAAGGCGGGAGGG-3 ';

4. method as claimed in claim 3 is characterized in that the ratio that described deoxidation nucleoside triphosphate mixture is dATP, dTTP, dCTP, dGTP amount of substance is 1: 1: 1: 1 mixture.

5. method as claimed in claim 3 is characterized in that described PCR reaction solution final concentration is composed as follows:

PCR damping fluid final concentration is 1 *

Each 0.1～0.3 μ M of specificity amplification primer

dNTPs 0.1～0.3mM

Testing sample DNA 10～100ng/ μ L

Taq archaeal dna polymerase 0.05～0.5U/ μ L

SYBR Green I final concentration is 1 *

Solvent is ddH ₂O.

6. method as claimed in claim 3 is characterized in that described pcr amplification reaction condition is as follows: 95 ℃ of pre-sex change 5min at first, then 94 ℃ of sex change 1min, 59 ℃ of renaturation 1min, 72 ℃ extend 1min, totally 39 circulations, 72 ℃ are extended 10min again.