CN103993084A - Amplification and sequencing primers and kit for mutation detection of all coding regions of gene of androgen receptor - Google Patents
Amplification and sequencing primers and kit for mutation detection of all coding regions of gene of androgen receptor Download PDFInfo
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- CN103993084A CN103993084A CN201410223073.7A CN201410223073A CN103993084A CN 103993084 A CN103993084 A CN 103993084A CN 201410223073 A CN201410223073 A CN 201410223073A CN 103993084 A CN103993084 A CN 103993084A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
Abstract
The invention discloses amplification and sequencing primers and kit for the mutation detection of all coding regions of a gene of an androgen receptor The kit comprises a reaction solution A, reaction tubes and primers (E1-A, E1-B, E1-C, E1-D, E2, E3, E4, E5, E6, E7 and E8), wherein the reaction solution A is applied to PCR (Polymerase Chain Reaction) amplification, the reaction tubes are coated with the primers, and the primers are used for sequencing. According to the invention, all eight exons of a human androgen receptor can be amplified under the same reaction conditions once and then are subjected to sequencing by the sequencing primers; a PCR reaction system is provided with a fluorescent dye, so that amplification results can be learnt directly through the change of fluorescence values, and inconvenience and pollution resulting from electrophoresis are avoided; the primers are directly coated into the reaction tubes, so that the difficulty of primer allocation is avoided; the number of CAG replication in a first exon can be detected; the kit can be applied to the detection on clinical specimens, and more biological information can be provided.
Description
Technical field
The present invention relates to the molecular biology method based on polymerase chain reaction (PCR) and DNA sequencing technology, relate in particular to a kind of amplification and sequencing primer and test kit thereof detecting for the full coding region sudden change of androgen receptor (AR) gene.
Background technology
Androgen-insensitivity syndrome (AIS) is the stealthy inherited disease of X chromosome that a class causes sexual abnormality (DSD), and pathogenesis and AR transgenation are closely related.Patient's caryogram is 46, XY, the androgen receptor defect on male sex hormone target organ and cause all or part of forfeiture of androgenic normal effect.According to clinical manifestation, can be divided into two kinds of hypotypes: perfect form androgen-insensitivity syndrome (CAIS) and reifenstein syndrome (PAIS).
The androgen receptor gene of coding androgen receptor protein is positioned at Xq11-12, and androgen receptor gene overall length 75～90kb, comprises 7 introns and 8 exons.AR belongs to nuclear receptor superfamily member, by 910～919 amino acid, formed, molecular weight is 110～114kDa, transcriptional activation domain, the 2nd and the 3rd DNA land of exons coding and three of the ligand binding domains of the 4-8 exons coding functional domain of the 1st exons coding, consists of.At gene level, the Androgen receptor gene mutation of having found roughly has following several: 1. point mutation cause amino acid to replace or termination codon occurs in advance or normal shearing site suddenlys change 2. nucleotide deletion or insertion cause phase shift mutation 3. in exons 1 CAG tumor-necrosis factor glycoproteins length extend or shorten etc.CAIS, PAIS, MAIS and cancer (comprising prostate cancer, liver cancer, mammary cancer) that the AR transgenation that ends in April, 2013 and upgrade causes have kind more than 1000.Androgen receptor gene mutation order-checking detects, can be diagnosis androgen-insensitivity syndrome important diagnosis basis is provided, the research structure and function of androgen receptor,, the contact between the pathogenesis of androgen-insensitivity syndrome provides the basis of technical Analysis, is had to more important clinical meaning.
At present, also untappedly go out to detect comprehensively, result Androgen receptor gene mutation order-checking accurately detection kit, for clinical diagnosis.The present invention is based on polymerase chain reaction (PCR) and DNA sequencing technology, by design, screening, obtain a set of desirable primer being increased and checked order in the full coding region of androgen receptor gene, reach the object whether complete detection androgen receptor gene undergos mutation, carry out gene type accurately.
Summary of the invention
The object of the invention is to overcome in existing molecular diagnostic techniques, detect androgen receptor mutation type many, detect incomplete defect, provide a set of optimization primer for pcr amplification and order-checking, can be under the same conditions disposablely carry out 11 PCR, increase smoothly and the full coding region of the androgen receptor gene that checks order.
Second object of the present invention is to provide a kind of supporting this primer and carries out the specific PCR response procedures that androgen receptor gene full coding region sudden change detects, and reaches the object of rapid amplifying.
The 3rd object of the present invention is to provide a kind ofly carries out to clinical samples the fluorescent PCR amplification kit that androgen receptor carries out sequential analysis.
Technical scheme of the present invention is summarized as follows:
The amplification and the sequencing primer that for androgen receptor gene full coding region sudden change, detect, the amplification and the sequencing primer that comprise First Exon are SEQ ID NO.1-SEQ ID NO.9, the amplification of Second Exon and sequencing primer are SEQ ID NO.10 and SEQ ID NO.11, the amplification of the 3rd exon and sequencing primer are SEQ ID NO.12 and SEQ ID NO.13, the amplification of the 4th exon and sequencing primer are SEQ ID NO.14-SEQ ID NO.16, the amplification of the 5th exon and sequencing primer are SEQ ID NO.17 and SEQ ID NO.18, the amplification of the 6th exon and sequencing primer are SEQ ID NO.19 and SEQ ID NO.20, the amplification of the 7th exon and sequencing primer are SEQ ID NO.21-SEQ ID NO.23, the amplification of the 8th exon and sequencing primer are SEQ ID NO.24 and SEQ ID NO.25.
The PCR reaction of supporting amplification androgen receptor exon, after being 95 ℃ of denaturation 3min, 94 ℃ of sex change 15s, 60 ℃ of annealing 15s, 72 ℃ are extended 35s, carry out 35 circulations, are then kept at 25 ℃; Take out and check order in time.
A kind of for amplification and sequencing kit to the sudden change detection of the full coding region of androgen receptor gene:
Reagent A: formed by 2mL damping fluid, comprise 2 * SYBR Green I dyestuff, 0.2mM dNTPs(comprises dATP, dCTP, dGTP, dTTP), 80mM Tris-HCl, 80mM KCl, 40mM (NH4) 2SO4,6mM MgSO4,100U Taq archaeal dna polymerase;
Reagent E 1-A: by the sequencing primer 30 μ L aqueous solution for AR First Exon E1-A, comprise 4 μ M SEQ ID NO.1;
Reagent E 1-B: by the sequencing primer 30 μ L aqueous solution for AR First Exon E1-B, comprise 4 μ M SEQ ID NO.3;
Reagent E 1-C: by the sequencing primer 30 μ L aqueous solution for AR First Exon E1-C, comprise 4 μ M SEQ ID NO.7;
Reagent E 1-D: by the sequencing primer 30 μ L aqueous solution for AR First Exon E1-D, comprise 4 μ M SEQ ID NO.8;
Reagent E 2: by the sequencing primer 30 μ L aqueous solution for AR Second Exon E2, comprise 4 μ M SEQ ID NO.11;
Reagent E 3: by the sequencing primer 30 μ L aqueous solution for AR the 3rd exon E3, comprise 4 μ M SEQ ID NO.12;
Reagent E 4: by the sequencing primer 30 μ L aqueous solution for AR the 4th exon E4, comprise 4 μ M SEQ ID NO.16;
Reagent E 5: by the sequencing primer 30 μ L aqueous solution for AR the 5th exon E5, comprise 4 μ M SEQ ID NO.18;
Reagent E 6: by the sequencing primer 30 μ L aqueous solution for AR the 5th exon E6, comprise 4 μ M SEQ ID NO.19;
Reagent E 7: by the sequencing primer 30 μ L aqueous solution for AR the 5th exon E7, comprise 4 μ M SEQ ID NO.23;
Reagent E 8: by the sequencing primer 30 μ L aqueous solution for AR the 5th exon E8, comprise 4 μ M SEQ ID NO.24;
The PCR reaction tubes of coated primer:
A reaction tubes: the amplimer containing for AR First Exon E1-A mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.1 and 1.5 * 10
-2nmol SEQ ID NO.2;
No. two reaction tubess: the amplimer containing for AR First Exon E1-B mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.3 and 1.5 * 10
-2nmol SEQ ID NO.4;
No. three reaction tubess: the amplimer containing for AR First Exon E1-C mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.5 and 1.5 * 10
-2nmol SEQ ID NO.6;
No. four reaction tubess: the amplimer containing for AR First Exon E1-D mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.8 and 1.5 * 10
-2nmol SEQ ID NO.9;
No. five reaction tubess: the amplimer containing for AR Second Exon E2 mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.10 and 1.5 * 10
-2nmol SEQ ID NO.11;
No. six reaction tubess: the amplimer containing for AR the 3rd exon E3 mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.12 and 1.5 * 10
-2nmol SEQ ID NO.13;
No. seven reaction tubess: the amplimer containing for AR the 3rd exon E4 mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.14 and 1.5 * 10
-2nmol SEQ ID NO.15;
No. eight reaction tubess: the amplimer containing for AR the 3rd exon E5 mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.17 and 1.5 * 10
-2nmol SEQ ID NO.18;
No. nine reaction tubess: the amplimer containing for AR the 3rd exon E6 mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.19 and 1.5 * 10
-2nmol SEQ ID NO.20;
No. ten reaction tubess: the amplimer containing for AR the 3rd exon E7 mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.21 and 1.5 * 10
-2nmol SEQ ID NO.22;
Ride on Bus No. 11 reaction tubes: the amplimer containing for AR the 3rd exon E8 mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.24 and 1.5 * 10
-2nmol SEQ ID NO.25.
Advantage of the present invention:
A kind of amplification and sequencing primer and test kit thereof detecting for androgen receptor gene full coding region sudden change is provided.Its advantage is: 1) a set of primer of fast PCR and sequencing primer of optimization of carrying out be provided, and the sequence of all 8 exons of all androgen receptor genes of disposable amplification is for follow-up order-checking; 2) in reaction system, be with fluorescence dye, can directly by quantitative fluorescent PCR, observe amplification, inconvenience and the pollution of avoiding electrophoresis to bring; 3) can find the CAG tumor-necrosis factor glycoproteins in androgen receptor First Exon, more bioinformation can be provided; 4) primer direct coated, in reaction tubes, is avoided primer configuration difficulty.By optimization experiment reaction system and reaction conditions, form simple, quick, sensitive, special detection kit, can be used for the detection to clinical samples, can reduce man power and material, more experiment information is provided.
Accompanying drawing explanation
Fig. 1 is the fluorescent value of human androgenic receptor fragment amplification.
Fig. 2 is human androgenic receptor sequencing fragment result.
Embodiment
In following embodiment, method therefor is ordinary method if no special instructions.
Amplification of the present invention and sequencing primer nucleotides sequence are classified as:
Embodiment 1
A set of Auele Specific Primer for androgen receptor amplification and order-checking use, the amplification and the sequencing primer that comprise First Exon are SEQ ID NO.1-SEQ ID NO.9, the amplification of Second Exon and sequencing primer are SEQ ID NO.10 and SEQ ID NO.11, the amplification of the 3rd exon and sequencing primer are SEQ ID NO.12 and SEQ ID NO.13, the amplification of the 4th exon and sequencing primer are SEQ ID NO.14-SEQ ID NO.16, the amplification of the 5th exon and sequencing primer are SEQ ID NO.17 and SEQ ID NO.18, the amplification of the 6th exon and sequencing primer are SEQ ID NO.19 and SEQ ID NO.20, the amplification of the 7th exon and sequencing primer are SEQ ID NO.21-SEQ ID NO.23, the amplification of the 8th exon and sequencing primer are SEQ ID NO.24 and SEQ ID NO.25.
Embodiment 2
For the test kit that androgen receptor is increased and checked order, by following reagent, formed:
Reagent A: formed by 2mL damping fluid, comprise 2 * SYBR Green I dyestuff, 0.2mM dNTPs(comprises dATP, dCTP, dGTP, dTTP), 80mM Tris-HCl, 80mM KCl, 40mM (NH4) 2SO4,6mM MgSO4,100U Taq archaeal dna polymerase;
Reagent E 1-A: by the sequencing primer 30 μ L aqueous solution for AR First Exon E1-A, comprise 4 μ M SEQ ID NO.1;
Reagent E 1-B: by the sequencing primer 30 μ L aqueous solution for AR First Exon E1-B, comprise 4 μ M SEQ ID NO.3;
Reagent E 1-C: by the sequencing primer 30 μ L aqueous solution for AR First Exon E1-C, comprise 4 μ M SEQ ID NO.7;
Reagent E 1-D: by the sequencing primer 30 μ L aqueous solution for AR First Exon E1-D, comprise 4 μ M SEQ ID NO.8;
Reagent E 2: by the sequencing primer 30 μ L aqueous solution for AR Second Exon E2, comprise 4 μ M SEQ ID NO.11;
Reagent E 3: by the sequencing primer 30 μ L aqueous solution for AR the 3rd exon E3, comprise 4 μ M SEQ ID NO.12;
Reagent E 4: by the sequencing primer 30 μ L aqueous solution for AR the 4th exon E4, comprise 4 μ M SEQ ID NO.16;
Reagent E 5: by the sequencing primer 30 μ L aqueous solution for AR the 5th exon E5, comprise 4 μ M SEQ ID NO.18;
Reagent E 6: by the sequencing primer 30 μ L aqueous solution for AR the 5th exon E6, comprise 4 μ M SEQ ID NO.19;
Reagent E 7: by the sequencing primer 30 μ L aqueous solution for AR the 5th exon E7, comprise 4 μ M SEQ ID NO.23;
Reagent E 8: by the sequencing primer 30 μ L aqueous solution for AR the 5th exon E8, comprise 4 μ M SEQ ID NO.24;
The PCR reaction tubes of coated primer:
A reaction tubes: the amplimer containing for AR First Exon E1-A mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.1 and 1.5 * 10
-2nmol SEQ ID NO.2;
No. two reaction tubess: the amplimer containing for AR First Exon E1-B mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.3 and 1.5 * 10
-2nmol SEQ ID NO.4;
No. three reaction tubess: the amplimer containing for AR First Exon E1-C mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.5 and 1.5 * 10
-2nmol SEQ ID NO.6;
No. four reaction tubess: the amplimer containing for AR First Exon E1-D mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.8 and 1.5 * 10
-2nmol SEQ ID NO.9;
No. five reaction tubess: the amplimer containing for AR Second Exon E2 mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.10 and 1.5 * 10
-2nmol SEQ ID NO.11;
No. six reaction tubess: the amplimer containing for AR the 3rd exon E3 mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.12 and 1.5 * 10
-2nmol SEQ ID NO.13;
No. seven reaction tubess: the amplimer containing for AR the 3rd exon E4 mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.14 and 1.5 * 10
-2nmol SEQ ID NO.15;
No. eight reaction tubess: the amplimer containing for AR the 3rd exon E5 mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.17 and 1.5 * 10
-2nmol SEQ ID NO.18;
No. nine reaction tubess: the amplimer containing for AR the 3rd exon E6 mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.19 and 1.5 * 10
-2nmol SEQ ID NO.20;
No. ten reaction tubess: the amplimer containing for AR the 3rd exon E7 mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.21 and 1.5 * 10
-2nmol SEQ ID NO.22;
Ride on Bus No. 11 reaction tubes: the amplimer containing for AR the 3rd exon E8 mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.24 and 1.5 * 10
-2nmol SEQ ID NO.25.
Embodiment 3
With test kit of the present invention, carry out all exons of real-time fluorescence quantitative PCR (real-time FQ PCR) amplification androgen receptor.First, calculate the requirement of each reagent by the sample number of experiment, experimental procedure is as follows:
1. amplification reaction solution configuration: configure in order reaction solution (1 is detected sample), add water 120 μ L, reagent A 150 μ L, DNA sample 30 μ L, totally 300 μ L; Mix.
2. each pipe is sequentially added into the amplification reaction solution of 25 μ L, builds lid.
3. by the PCR program of experimental design, increase: after being 95 ℃ of denaturation 3min, 94 ℃ of sex change 1min, 60 ℃ of annealing 1mins, 72 ℃ are extended 35s, and extended peroid obtains fluorescent value, carries out 35 circulations, is finally kept at 25 ℃; Take out and check order in time.
4. fluorescent value is 22-28 circulation take-off, and specific amplification is thought in each group take-off simultaneously, sees Fig. 1.
Embodiment 4
The primer supporting with test kit of the present invention checks order:
1. PCR product purification (ExoI/SAP method):
1.1 preparation purification system: exonuclease 1(ExoI) 6 μ L+shrimp solution enzyme (SAP) 12 μ L+water 6 μ L, totally 24 μ L, mix.
The 1.2 purification system 2 μ L that get preparation mix (totally 11) with each amplified production 8 μ L, add in another blank Sptting plate.
1.3 Sptting plate is placed on to PCR instrument more than, 37 ℃ of 30min, 80 ℃ of 20min can preserve about 1 week in 4 ℃ of refrigerators.
2. sequencing reaction:
In 2.1 10 μ L PCR purified products, every hole adds 20 μ L water, after fully mixing, centrifugal.
2.2 preparation sequencing reaction systems: get 4.8 μ L BDT Ready Reaction Premix (2.5 *) and add 21.6 μ L BDT damping fluids (5 *) and add 69.6 μ L Deionised water and mix.Get the above-mentioned mixed solution of 8 μ L at every turn and fully mix with E1-A, E1-B, E1-C, E1-D, E2, E3, E4, E5, E6, E7, each 1 μ L of E8 respectively, centrifugal fast.
2.3 every hole PCR product diluents are got 1 μ L (11 hole) to reacting hole, and every hole adds 9 μ L sequencing reaction mixed solutions, mixes, centrifugal fast.
2.4 are placed on Sptting plate on PCR instrument, and PCR sealing load pad is put at Sptting plate top, to prevent liquid evaporation, shut PCR instrument, operation sequencing reaction: after 96 ℃ of 10s, carrying out 25 circulations is 96 ℃ of 10s, 50 ℃ of 10s, 60 ℃ of 2min, finally preserve 15 ℃ and taking-up.
3. sequencing reaction product purification:
In 3.1 sequencing reaction hole, every holes, add 1 μ LEDTA, 1 μ L NaAc and 25 μ L dehydrated alcohols, fully concussion mixes 30s.Room temperature is placed 15min, the centrifugal 30min. of 3000rpm
3.2 are inverted Sptting plate, adjusting rotary speed, the centrifugal 1min of 500rpm
In 3.3 every hole sequencing reaction products, add 50 μ L 70% ethanol, the centrifugal 10min of 3000 rpm.
3.4 are inverted Sptting plate, the centrifugal 1min of 500rpm.
The standing 30min of 3.5 room temperature lucifuge, ethanol in the plate that fully volatilizees.
In 3.6 every holes, add methane amide 10 μ L, shrouding, is placed on the above 95 ℃ of 2min of PCR, puts into rapidly-20 ℃ of cooling 3-5min. of refrigerator
4. sequenator is read plate detection: use ABI3500 sequenator to detect sequencing result.The sequencing result software that makes to check order accordingly carries out data analysis, sees Fig. 2.
Embodiment 5
Sequencing result and androgen receptor standard sequence are compared:
1. sequencing result splices;
2. the order-checking of androgen receptor standard and sequencing result Input Software are compared;
3. check emergent properties and analyze the repetition rate of First Exon CAG.
Sequence table
The attached First Hospital of <110> Wenzhou medical university
The test kit of <120> human androgenic receptor detection in Gene Mutation
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Claims (2)
1. a set of amplification and sequencing primer detecting for the sudden change of the full coding region of androgen receptor gene, it is characterized in that on the basis of optimize PCR design of primers, can be under the same conditions disposablely carry out 11 polymerase chain reactions, complete the amplification of AR gene complete penetrance, and can carry out all 8 exons in the full coding region of AR gene by sequencing primer and check order, the amplification and the sequencing primer that comprise First Exon are SEQ ID NO.1-SEQ ID NO.9, the amplification of Second Exon and sequencing primer are SEQ ID NO.10 and SEQ ID NO.11, the amplification of the 3rd exon and sequencing primer are SEQ ID NO.12 and SEQ ID NO.13, the amplification of the 4th exon and sequencing primer are SEQ ID NO.14-SEQ ID NO.16, the amplification of the 5th exon and sequencing primer are SEQ ID NO.17 and SEQ ID NO.18, the amplification of the 6th exon and sequencing primer are SEQ ID NO.19 and SEQ ID NO.20, the amplification of the 7th exon and sequencing primer are SEQ ID NO.21-SEQ ID NO.23, the amplification of the 8th exon and sequencing primer are SEQ ID NO.24 and SEQ ID NO.25.
2. for amplification and a sequencing kit that to androgen receptor gene, the sudden change of full coding region detects, by following reagent, formed:
Reagent A: formed by 2mL damping fluid, comprise 2 * SYBR Green I dyestuff, 0.2mM dNTPs, 80mM Tris-HCl, 80mM KCl, 40mM (NH
4)
2sO
4, 6mM MgSO4,100U Taq archaeal dna polymerase;
Reagent E 1-A: by the sequencing primer 30 μ L aqueous solution for AR First Exon E1-A, comprise 4 μ M SEQ ID NO.1;
Reagent E 1-B: by the sequencing primer 30 μ L aqueous solution for AR First Exon E1-B, comprise 4 μ M SEQ ID NO.3;
Reagent E 1-C: by the sequencing primer 30 μ L aqueous solution for AR First Exon E1-C, comprise 4 μ M SEQ ID NO.7;
Reagent E 1-D: by the sequencing primer 30 μ L aqueous solution for AR First Exon E1-D, comprise 4 μ M SEQ ID NO.8;
Reagent E 2: by the sequencing primer 30 μ L aqueous solution for AR Second Exon E2, comprise 4 μ M SEQ ID NO.11;
Reagent E 3: by the sequencing primer 30 μ L aqueous solution for AR the 3rd exon E3, comprise 4 μ M SEQ ID NO.12;
Reagent E 4: by the sequencing primer 30 μ L aqueous solution for AR the 4th exon E4, comprise 4 μ M SEQ ID NO.16;
Reagent E 5: by the sequencing primer 30 μ L aqueous solution for AR the 5th exon E5, comprise 4 μ M SEQ ID NO.18;
Reagent E 6: by the sequencing primer 30 μ L aqueous solution for AR the 5th exon E6, comprise 4 μ M SEQ ID NO.19;
Reagent E 7: by the sequencing primer 30 μ L aqueous solution for AR the 5th exon E7, comprise 4 μ M SEQ ID NO.23;
Reagent E 8: by the sequencing primer 30 μ L aqueous solution for AR the 5th exon E8, comprise 4 μ M SEQ ID NO.24;
The PCR reaction tubes of coated primer:
A reaction tubes: the amplimer containing for AR First Exon E1-A mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.1 and 1.5 * 10
-2nmol SEQ ID NO.2;
No. two reaction tubess: the amplimer containing for AR First Exon E1-B mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.3 and 1.5 * 10
-2nmol SEQ ID NO.4;
No. three reaction tubess: the amplimer containing for AR First Exon E1-C mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.5 and 1.5 * 10
-2nmol SEQ ID NO.6;
No. four reaction tubess: the amplimer containing for AR First Exon E1-D mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.8 and 1.5 * 10
-2nmol SEQ ID NO.9;
No. five reaction tubess: the amplimer containing for AR Second Exon E2 mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.10 and 1.5 * 10
-2nmol SEQ ID NO.11;
No. six reaction tubess: the amplimer containing for AR the 3rd exon E3 mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.12 and 1.5 * 10
-2nmol SEQ ID NO.13;
No. seven reaction tubess: the amplimer containing for AR the 3rd exon E4 mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.14 and 1.5 * 10
-2nmol SEQ ID NO.15;
No. eight reaction tubess: the amplimer containing for AR the 3rd exon E5 mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.17 and 1.5 * 10
-2nmol SEQ ID NO.18;
No. nine reaction tubess: the amplimer containing for AR the 3rd exon E6 mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.19 and 1.5 * 10
-2nmol SEQ ID NO.20;
No. ten reaction tubess: the amplimer containing for AR the 3rd exon E7 mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.21 and 1.5 * 10
-2nmol SEQ ID NO.22;
Ride on Bus No. 11 reaction tubes: the amplimer containing for AR the 3rd exon E8 mixes composition, comprises 1.5 * 10
-2nmol SEQ ID NO.24 and 1.5 * 10
-2nmol SEQ ID NO.25.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108018342A (en) * | 2017-12-04 | 2018-05-11 | 合肥艾迪康临床检验所有限公司 | Detect the primer and method of AR gene mutations |
| CN108060232A (en) * | 2017-12-13 | 2018-05-22 | 安徽师范大学 | For expanding tortoise androgen receptor（AR）The RT-qPCR primers and method of gene |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101905788A (en) * | 2010-07-14 | 2010-12-08 | 中冶赛迪工程技术股份有限公司 | Self-locking bottom opening trough |
| CN101990578A (en) * | 2008-04-04 | 2011-03-23 | 固德基因公司 | New skin sampling kit which stores nucleic acids in stable status, genetic test methods by using the kit and their practical application |
| CN102439454A (en) * | 2009-02-11 | 2012-05-02 | 卡里斯Mpi公司 | Molecular profiling of tumors |
-
2014
- 2014-05-26 CN CN201410223073.7A patent/CN103993084B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101990578A (en) * | 2008-04-04 | 2011-03-23 | 固德基因公司 | New skin sampling kit which stores nucleic acids in stable status, genetic test methods by using the kit and their practical application |
| CN102439454A (en) * | 2009-02-11 | 2012-05-02 | 卡里斯Mpi公司 | Molecular profiling of tumors |
| CN101905788A (en) * | 2010-07-14 | 2010-12-08 | 中冶赛迪工程技术股份有限公司 | Self-locking bottom opening trough |
Non-Patent Citations (1)
| Title |
|---|
| 李亚丽等: "男性不育的遗传学研究及在ICSI中的意义", 《中国优生与遗传杂志》, vol. 14, no. 5, 31 December 2006 (2006-12-31), pages 19 - 21 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108018342A (en) * | 2017-12-04 | 2018-05-11 | 合肥艾迪康临床检验所有限公司 | Detect the primer and method of AR gene mutations |
| CN108060232A (en) * | 2017-12-13 | 2018-05-22 | 安徽师范大学 | For expanding tortoise androgen receptor（AR）The RT-qPCR primers and method of gene |
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