CN103993084B - For the amplification of androgen receptor gene full coding region abrupt climatic change and sequencing primer and test kit thereof - Google Patents
For the amplification of androgen receptor gene full coding region abrupt climatic change and sequencing primer and test kit thereof Download PDFInfo
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- CN103993084B CN103993084B CN201410223073.7A CN201410223073A CN103993084B CN 103993084 B CN103993084 B CN 103993084B CN 201410223073 A CN201410223073 A CN 201410223073A CN 103993084 B CN103993084 B CN 103993084B
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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 a kind of amplification for androgen receptor gene full coding region abrupt climatic change and sequencing primer and test kit thereof.Comprise pcr amplification reaction solution A in test kit, wrap by the reaction tubes of primer, and order-checking primer E1-A, E1-B, E1-C, E1-D, E2, E3, E4, E5, E6, E7, E8.The present invention can disposablely increase to all 8 exons of human androgenic receptor under the same reaction conditions, then is checked order by sequencing primer.Be with fluorescence dye in PCR reaction system, directly can understand amplification, the inconvenience avoiding electrophoresis to bring and pollution by fluorescent value change.Primer direct coated, in reaction tubes, avoids primer to configure difficulty.Can detect CAG multiplicity in First Exon, the present invention can be used for the detection to clinical samples, can provide more bioinformation.
Description
Technical field
The present invention relates to the molecular biology method based on polymerase chain reaction (PCR) and DNA sequencing technology, particularly relate to a kind of amplification for androgen receptor (AR) gene full coding region abrupt climatic change and sequencing primer and test kit thereof.
Background technology
Androgen-insensitivity syndrome (AIS) is that a class causes the stealthy inherited disease of the X chromosome of sexual abnormality (DSD), pathogenesis and AR transgenation 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, two kinds of hypotypes can be divided into: perfect form androgen-insensitivity syndrome (CAIS) and reifenstein syndrome (PAIS).
The androgen receptor gene of encoding androgen receptor albumen is positioned at Xq11-12, androgen receptor gene overall length 75 ~ 90kb, comprises 7 introns and 8 exons.AR belongs to nuclear receptor superfamily member, be made up of 910 ~ 919 amino acid, molecular weight is 110 ~ 114kDa, is made up of three, the ligand binding domain functional domain of the transcriptional activation domain of the 1st exons coding, the DNA land of the 2nd and the 3rd exons coding and the 4-8 exons coding.At gene level, the Androgen receptor gene mutation found roughly has following several: 1. point mutation causes amino acid to replace or termination codon occurs in advance or normal shearing site suddenlys change 2. nucleotide deletion or insertion causes phase shift mutation 3. CAG tumor-necrosis factor glycoproteins extension or shortening etc. in exons 1.CAIS, PAIS, MAIS that the AR transgenation ending in April, 2013 renewal causes and cancer (comprising prostate cancer, liver cancer, mammary cancer) have kind more than 1000.Androgen receptor gene mutation order-checking detects, can be diagnosis androgen-insensitivity syndrome and important diagnosis basis is provided, contact between the pathogenesis of the research structure and function of androgen receptor, androgen-insensitivity syndrome is provided to the basis of technical Analysis, there is 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, a set of desirable primer increased and checked order in the full coding region of androgen receptor gene is obtained by design, screening, 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, there is provided a set of optimization primer for pcr amplification and order-checking, disposablely under the same conditions can carry out 11 PCR, smoothly the full coding region of amplification and order-checking androgen receptor gene.
The present invention's second object is to provide the specific PCR response procedures that a kind of this primer supporting carries out androgen receptor gene full coding region abrupt climatic change, reaches the object of rapid amplifying.
The present invention's the 3rd object 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:
For amplification and the sequencing primer of androgen receptor gene full coding region abrupt climatic change, the amplification and the sequencing primer that comprise First Exon are SEQIDNO.1-SEQIDNO.9, the amplification of Second Exon and sequencing primer are SEQIDNO.10 and SEQIDNO.11, the amplification of the 3rd exon and sequencing primer are SEQIDNO.12 and SEQIDNO.13, the amplification of the 4th exon and sequencing primer are SEQIDNO.14-SEQIDNO.16, the amplification of the 5th exon and sequencing primer are SEQIDNO.17 and SEQIDNO.18, the amplification of the 6th exon and sequencing primer are SEQIDNO.19 and SEQIDNO.20, the amplification of the 7th exon and sequencing primer are SEQIDNO.21-SEQIDNO.23, the amplification of the 8th exon and sequencing primer are SEQIDNO.24 and SEQIDNO.25.
The PCR reaction of supporting amplification Androgen receptor Exon is after 95 DEG C of denaturation 3min, 94 DEG C of sex change 15s, 60 DEG C of annealing 15s, and 72 DEG C extend 35s, carry out 35 circulations, be then kept at 25 DEG C; Timely taking-up is checked order.
A kind of for the amplification of androgen receptor gene full coding region abrupt climatic change and sequencing kit:
Reagent A: be made up of 2mL damping fluid, comprise 2 × SYBR GreenI dyestuff, 0.2mMdNTPs(comprises dATP, dCTP, dGTP, dTTP), 80mMTris-HCl, 80mMKCl, 40mM (NH4) 2SO4,6mMMgSO4,100UTaqDNA polysaccharase;
Reagent E 1-A: by the sequencing primer 30 μ L aqueous solution for AR First Exon E1-A, comprise 4 μMs of SEQIDNO.1;
Reagent E 1-B: by the sequencing primer 30 μ L aqueous solution for AR First Exon E1-B, comprise 4 μMs of SEQIDNO.3;
Reagent E 1-C: by the sequencing primer 30 μ L aqueous solution for AR First Exon E1-C, comprise 4 μMs of SEQIDNO.7;
Reagent E 1-D: by the sequencing primer 30 μ L aqueous solution for AR First Exon E1-D, comprise 4 μMs of SEQIDNO.8;
Reagent E 2: by the sequencing primer 30 μ L aqueous solution for AR Second Exon E2, comprise 4 μMs of SEQIDNO.11;
Reagent E 3: by the sequencing primer 30 μ L aqueous solution for AR the 3rd exon E3, comprise 4 μMs of SEQIDNO.12;
Reagent E 4: by the sequencing primer 30 μ L aqueous solution for AR the 4th exon E4, comprise 4 μMs of SEQIDNO.16;
Reagent E 5: by the sequencing primer 30 μ L aqueous solution for AR the 5th exon E5, comprise 4 μMs of SEQIDNO.18;
Reagent E 6: by the sequencing primer 30 μ L aqueous solution for AR the 5th exon E6, comprise 4 μMs of SEQIDNO.19;
Reagent E 7: by the sequencing primer 30 μ L aqueous solution for AR the 5th exon E7, comprise 4 μMs of SEQIDNO.23;
Reagent E 8: by the sequencing primer 30 μ L aqueous solution for AR the 5th exon E8, comprise 4 μMs of SEQIDNO.24;
Bag is by the PCR reaction tubes of primer:
A reaction tubes: containing the amplimer mixing composition for AR First Exon E1-A, comprise 1.5 × 10
-2nmolSEQIDNO.1 and 1.5 × 10
-2nmolSEQIDNO.2;
No. two reaction tubess: containing the amplimer mixing composition for AR First Exon E1-B, comprise 1.5 × 10
-2nmolSEQIDNO.3 and 1.5 × 10
-2nmolSEQIDNO.4;
No. three reaction tubess: containing the amplimer mixing composition for AR First Exon E1-C, comprise 1.5 × 10
-2nmolSEQIDNO.5 and 1.5 × 10
-2nmolSEQIDNO.6;
No. four reaction tubess: containing the amplimer mixing composition for AR First Exon E1-D, comprise 1.5 × 10
-2nmolSEQIDNO.8 and 1.5 × 10
-2nmolSEQIDNO.9;
No. five reaction tubess: containing the amplimer mixing composition for AR Second Exon E2, comprise 1.5 × 10
-2nmolSEQIDNO.10 and 1.5 × 10
-2nmolSEQIDNO.11;
No. six reaction tubess: containing the amplimer mixing composition for AR the 3rd exon E3, comprise 1.5 × 10
-2nmolSEQIDNO.12 and 1.5 × 10
-2nmolSEQIDNO.13;
No. seven reaction tubess: containing the amplimer mixing composition for AR the 3rd exon E4, comprise 1.5 × 10
-2nmolSEQIDNO.14 and 1.5 × 10
-2nmolSEQIDNO.15;
No. eight reaction tubess: containing the amplimer mixing composition for AR the 3rd exon E5, comprise 1.5 × 10
-2nmolSEQIDNO.17 and 1.5 × 10
-2nmolSEQIDNO.18;
No. nine reaction tubess: containing the amplimer mixing composition for AR the 3rd exon E6, comprise 1.5 × 10
-2nmolSEQIDNO.19 and 1.5 × 10
-2nmolSEQIDNO.20;
No. ten reaction tubess: containing the amplimer mixing composition for AR the 3rd exon E7, comprise 1.5 × 10
-2nmolSEQIDNO.21 and 1.5 × 10
-2nmolSEQIDNO.22;
Ride on Bus No. 11 reaction tubes: containing the amplimer mixing composition for AR the 3rd exon E8, comprise 1.5 × 10
-2nmolSEQIDNO.24 and 1.5 × 10
-2nmolSEQIDNO.25.
Advantage of the present invention:
A kind of amplification for androgen receptor gene full coding region abrupt climatic change and sequencing primer and test kit thereof are provided.Its advantage is: 1) provide and a set ofly carry out the primer of fast PCR and the sequencing primer of optimization, and the sequence of all 8 exons of all androgen receptor genes of disposable amplification is used for follow-up order-checking; 2) be with fluorescence dye in reaction system, directly can observe amplification by quantitative fluorescent PCR, the inconvenience avoiding electrophoresis to bring and pollution; 3) the CAG tumor-necrosis factor glycoproteins in androgen receptor First Exon can be found, more bioinformation can be provided; 4) primer direct coated is in reaction tubes, avoids primer to configure 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 man power and material be reduced, 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 increasing for androgen receptor and check order, the amplification and the sequencing primer that comprise First Exon are SEQIDNO.1-SEQIDNO.9, the amplification of Second Exon and sequencing primer are SEQIDNO.10 and SEQIDNO.11, the amplification of the 3rd exon and sequencing primer are SEQIDNO.12 and SEQIDNO.13, the amplification of the 4th exon and sequencing primer are SEQIDNO.14-SEQIDNO.16, the amplification of the 5th exon and sequencing primer are SEQIDNO.17 and SEQIDNO.18, the amplification of the 6th exon and sequencing primer are SEQIDNO.19 and SEQIDNO.20, the amplification of the 7th exon and sequencing primer are SEQIDNO.21-SEQIDNO.23, the amplification of the 8th exon and sequencing primer are SEQIDNO.24 and SEQIDNO.25.
Embodiment 2
For the test kit increasing to androgen receptor and check order, be made up of following reagent:
Reagent A: be made up of 2mL damping fluid, comprise 2 × SYBR GreenI dyestuff, 0.2mMdNTPs(comprises dATP, dCTP, dGTP, dTTP), 80mMTris-HCl, 80mMKCl, 40mM (NH4) 2SO4,6mMMgSO4,100UTaqDNA polysaccharase;
Reagent E 1-A: by the sequencing primer 30 μ L aqueous solution for AR First Exon E1-A, comprise 4 μMs of SEQIDNO.1;
Reagent E 1-B: by the sequencing primer 30 μ L aqueous solution for AR First Exon E1-B, comprise 4 μMs of SEQIDNO.3;
Reagent E 1-C: by the sequencing primer 30 μ L aqueous solution for AR First Exon E1-C, comprise 4 μMs of SEQIDNO.7;
Reagent E 1-D: by the sequencing primer 30 μ L aqueous solution for AR First Exon E1-D, comprise 4 μMs of SEQIDNO.8;
Reagent E 2: by the sequencing primer 30 μ L aqueous solution for AR Second Exon E2, comprise 4 μMs of SEQIDNO.11;
Reagent E 3: by the sequencing primer 30 μ L aqueous solution for AR the 3rd exon E3, comprise 4 μMs of SEQIDNO.12;
Reagent E 4: by the sequencing primer 30 μ L aqueous solution for AR the 4th exon E4, comprise 4 μMs of SEQIDNO.16;
Reagent E 5: by the sequencing primer 30 μ L aqueous solution for AR the 5th exon E5, comprise 4 μMs of SEQIDNO.18;
Reagent E 6: by the sequencing primer 30 μ L aqueous solution for AR the 5th exon E6, comprise 4 μMs of SEQIDNO.19;
Reagent E 7: by the sequencing primer 30 μ L aqueous solution for AR the 5th exon E7, comprise 4 μMs of SEQIDNO.23;
Reagent E 8: by the sequencing primer 30 μ L aqueous solution for AR the 5th exon E8, comprise 4 μMs of SEQIDNO.24;
Bag is by the PCR reaction tubes of primer:
A reaction tubes: containing the amplimer mixing composition for AR First Exon E1-A, comprise 1.5 × 10
-2nmolSEQIDNO.1 and 1.5 × 10
-2nmolSEQIDNO.2;
No. two reaction tubess: containing the amplimer mixing composition for AR First Exon E1-B, comprise 1.5 × 10
-2nmolSEQIDNO.3 and 1.5 × 10
-2nmolSEQIDNO.4;
No. three reaction tubess: containing the amplimer mixing composition for AR First Exon E1-C, comprise 1.5 × 10
-2nmolSEQIDNO.5 and 1.5 × 10
-2nmolSEQIDNO.6;
No. four reaction tubess: containing the amplimer mixing composition for AR First Exon E1-D, comprise 1.5 × 10
-2nmolSEQIDNO.8 and 1.5 × 10
-2nmolSEQIDNO.9;
No. five reaction tubess: containing the amplimer mixing composition for AR Second Exon E2, comprise 1.5 × 10
-2nmolSEQIDNO.10 and 1.5 × 10
-2nmolSEQIDNO.11;
No. six reaction tubess: containing the amplimer mixing composition for AR the 3rd exon E3, comprise 1.5 × 10
-2nmolSEQIDNO.12 and 1.5 × 10
-2nmolSEQIDNO.13;
No. seven reaction tubess: containing the amplimer mixing composition for AR the 3rd exon E4, comprise 1.5 × 10
-2nmolSEQIDNO.14 and 1.5 × 10
-2nmolSEQIDNO.15;
No. eight reaction tubess: containing the amplimer mixing composition for AR the 3rd exon E5, comprise 1.5 × 10
-2nmolSEQIDNO.17 and 1.5 × 10
-2nmolSEQIDNO.18;
No. nine reaction tubess: containing the amplimer mixing composition for AR the 3rd exon E6, comprise 1.5 × 10
-2nmolSEQIDNO.19 and 1.5 × 10
-2nmolSEQIDNO.20;
No. ten reaction tubess: containing the amplimer mixing composition for AR the 3rd exon E7, comprise 1.5 × 10
-2nmolSEQIDNO.21 and 1.5 × 10
-2nmolSEQIDNO.22;
Ride on Bus No. 11 reaction tubes: containing the amplimer mixing composition for AR the 3rd exon E8, comprise 1.5 × 10
-2nmolSEQIDNO.24 and 1.5 × 10
-2nmolSEQIDNO.25.
Embodiment 3
Carry out real-time fluorescence quantitative PCR (real-timeFQPCR) with test kit of the present invention to increase all exons of androgen receptor.First, sample number empirically calculates the requirement of each reagent, and experimental procedure is as follows:
1. amplification reaction solution configuration: configure reaction solution (1 is detected sample) in order, add water 120 μ L, reagent A 150 μ L, DNA sample 30 μ L, totally 300 μ L; Mixing.
2. each pipe is sequentially added into the amplification reaction solution of 25 μ L, builds lid.
3. the PCR program empirically designed increases: be after 95 DEG C of denaturation 3min, 94 DEG C of sex change 1min, 60 DEG C of annealing 1mins, and 72 DEG C extend 35s, and extended peroid obtains fluorescent value, carries out 35 circulations, is finally kept at 25 DEG C; Timely taking-up is checked order.
4. fluorescent value is 22-28 circulation take-off, and specific amplification is thought in the take-off simultaneously of each group, 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, mixing.
The 1.2 purification system 2 μ L getting preparation mix (11) with each amplified production 8 μ L totally, add in another blank Sptting plate.
Sptting plate is placed on more than PCR instrument by 1.3,37 DEG C of 30min, 80 DEG C of 20min, can preserve about 1 week in 4 DEG C of refrigerators.
2. sequencing reaction:
In 2.110 μ LPCR purified products, every hole adds 20 μ L water, fully after mixing, centrifugal.
2.2 preparation sequencing reaction systems: get 4.8 μ LBDTReadyReactionPremix (2.5 ×) and add 21.6 μ LBDT damping fluids (5 ×) and add 69.6 μ LDeionisedwater and mix.Get the above-mentioned mixed solution of 8 μ L each 1 μ L fully mixes with E1-A, E1-B, E1-C, E1-D, E2, E3, E4, E5, E6, E7, E8 respectively at every turn, centrifugal fast.
2.3 every hole PCR primer diluents get 1 μ L to (11 hole) in reacting hole, and every hole adds 9 μ L sequencing reaction mixed solutions, mixing, centrifugal fast.
Sptting plate is placed in PCR instrument by 2.4, and PCR sealing load pad is put at Sptting plate top, to prevent liquid evaporation, shuts PCR instrument, runs sequencing reaction: after 96 DEG C of 10s, carrying out 25 circulations is 96 DEG C of 10s, 50 DEG C of 10s, 60 DEG C of 2min, finally preserves 15 DEG C and takes out.
3. sequencing reaction product purification:
1 μ LEDTA, 1 μ LNaAc and 25 μ L dehydrated alcohols are added, fully concussion mixing 30s in 3.1 sequencing reaction hole, every holes.Room temperature places the centrifugal 30min. of 15min, 3000rpm
3.2 are inverted Sptting plate, adjusting rotary speed, the centrifugal 1min of 500rpm
50 μ L70% ethanol are added, the centrifugal 10min of 3000rpm in 3.3 every hole sequencing reaction products.
3.4 are inverted Sptting plate, the centrifugal 1min of 500rpm.
3.5 room temperature lucifuges leave standstill 30min, fully ethanol in volatilization plate.
Add methane amide 10 μ L, shrouding in 3.6 every holes, be placed on more than PCR 95 DEG C of 2min, put into rapidly-20 DEG C of refrigerator cooling 3-5min.
4. sequenator reads 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
<110> The First Affiliated Hospital of 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 for androgen receptor gene full coding region abrupt climatic change and sequencing primer, it is characterized in that on the basis of optimize PCR design of primers, disposablely under the same conditions can carry out 11 polymerase chain reactions, complete the amplification of AR gene complete penetrance, and carry out all 8 exons order-checkings in the full coding region of AR gene by sequencing primer, the amplification and the sequencing primer that comprise First Exon are SEQIDNO.1-SEQIDNO.9, the amplification of Second Exon and sequencing primer are SEQIDNO.10 and SEQIDNO.11, the amplification of the 3rd exon and sequencing primer are SEQIDNO.12 and SEQIDNO.13, the amplification of the 4th exon and sequencing primer are SEQIDNO.14-SEQIDNO.16, the amplification of the 5th exon and sequencing primer are SEQIDNO.17 and SEQIDNO.18, the amplification of the 6th exon and sequencing primer are SEQIDNO.19 and SEQIDNO.20, the amplification of the 7th exon and sequencing primer are SEQIDNO.21-SEQIDNO.23, the amplification of the 8th exon and sequencing primer are SEQIDNO.24 and SEQIDNO.25.
2., for the amplification of androgen receptor gene full coding region abrupt climatic change and a sequencing kit, be made up of following reagent:
Reagent A: be made up of 2mL damping fluid, comprises 2 × SYBR GreenI dyestuff, 0.2mMdNTPs, 80mMTris-HCl, 80mMKCl, 40mM (NH
4)
2sO
4, 6mMMgSO4,100UTaqDNA polysaccharase;
Reagent E 1-A: by the sequencing primer 30 μ L aqueous solution for AR First Exon E1-A, comprise 4 μMs of SEQIDNO.1;
Reagent E 1-B: by the sequencing primer 30 μ L aqueous solution for AR First Exon E1-B, comprise 4 μMs of SEQIDNO.3;
Reagent E 1-C: by the sequencing primer 30 μ L aqueous solution for AR First Exon E1-C, comprise 4 μMs of SEQIDNO.7;
Reagent E 1-D: by the sequencing primer 30 μ L aqueous solution for AR First Exon E1-D, comprise 4 μMs of SEQIDNO.8;
Reagent E 2: by the sequencing primer 30 μ L aqueous solution for AR Second Exon E2, comprise 4 μMs of SEQIDNO.11;
Reagent E 3: by the sequencing primer 30 μ L aqueous solution for AR the 3rd exon E3, comprise 4 μMs of SEQIDNO.12;
Reagent E 4: by the sequencing primer 30 μ L aqueous solution for AR the 4th exon E4, comprise 4 μMs of SEQIDNO.16;
Reagent E 5: by the sequencing primer 30 μ L aqueous solution for AR the 5th exon E5, comprise 4 μMs of SEQIDNO.18;
Reagent E 6: by the sequencing primer 30 μ L aqueous solution for AR the 5th exon E6, comprise 4 μMs of SEQIDNO.19;
Reagent E 7: by the sequencing primer 30 μ L aqueous solution for AR the 5th exon E7, comprise 4 μMs of SEQIDNO.23;
Reagent E 8: by the sequencing primer 30 μ L aqueous solution for AR the 5th exon E8, comprise 4 μMs of SEQIDNO.24;
Bag is by the PCR reaction tubes of primer:
A reaction tubes: containing the amplimer mixing composition for AR First Exon E1-A, comprise 1.5 × 10
-2nmolSEQIDNO.1 and 1.5 × 10
-2nmolSEQIDNO.2;
No. two reaction tubess: containing the amplimer mixing composition for AR First Exon E1-B, comprise 1.5 × 10
-2nmolSEQIDNO.3 and 1.5 × 10
-2nmolSEQIDNO.4;
No. three reaction tubess: containing the amplimer mixing composition for AR First Exon E1-C, comprise 1.5 × 10
-2nmolSEQIDNO.5 and 1.5 × 10
-2nmolSEQIDNO.6;
No. four reaction tubess: containing the amplimer mixing composition for AR First Exon E1-D, comprise 1.5 × 10
-2nmolSEQIDNO.8 and 1.5 × 10
-2nmolSEQIDNO.9;
No. five reaction tubess: containing the amplimer mixing composition for AR Second Exon E2, comprise 1.5 × 10
-2nmolSEQIDNO.10 and 1.5 × 10
-2nmolSEQIDNO.11;
No. six reaction tubess: containing the amplimer mixing composition for AR the 3rd exon E3, comprise 1.5 × 10
-2nmolSEQIDNO.12 and 1.5 × 10
-2nmolSEQIDNO.13;
No. seven reaction tubess: containing the amplimer mixing composition for AR the 3rd exon E4, comprise 1.5 × 10
-2nmolSEQIDNO.14 and 1.5 × 10
-2nmolSEQIDNO.15;
No. eight reaction tubess: containing the amplimer mixing composition for AR the 3rd exon E5, comprise 1.5 × 10
-2nmolSEQIDNO.17 and 1.5 × 10
-2nmolSEQIDNO.18;
No. nine reaction tubess: containing the amplimer mixing composition for AR the 3rd exon E6, comprise 1.5 × 10
-2nmolSEQIDNO.19 and 1.5 × 10
-2nmolSEQIDNO.20;
No. ten reaction tubess: containing the amplimer mixing composition for AR the 3rd exon E7, comprise 1.5 × 10
-2nmolSEQIDNO.21 and 1.5 × 10
-2nmolSEQIDNO.22;
Ride on Bus No. 11 reaction tubes: containing the amplimer mixing composition for AR the 3rd exon E8, comprise 1.5 × 10
-2nmolSEQIDNO.24 and 1.5 × 10
-2nmolSEQIDNO.25.
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