CN104087657A - Kit for examining Liddle's related gene mutation - Google Patents
Kit for examining Liddle's related gene mutation Download PDFInfo
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- CN104087657A CN104087657A CN201410209085.4A CN201410209085A CN104087657A CN 104087657 A CN104087657 A CN 104087657A CN 201410209085 A CN201410209085 A CN 201410209085A CN 104087657 A CN104087657 A CN 104087657A
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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
Abstract
The invention relates to a kit made for detecting Liddle's syndrome related gene mutation samples, and concretely relates to a product for detecting Liddle's syndrome related gene by adopting a large scale parallel sequencing platform technology. A detection method comprises the following steps: 1, uniquely designing and making a capture probe against all exon fragments of gene SCNN1B and SCNN1G; 2, designing a unique joint with a labeling sequence; 3, carrying out PCR amplification on the sequence of the probe by using a general primer; and 4, designing a unique capture operation of mixed target fragments. The method and the kit have the advantages of large flux of prepared large scale parallel sequencing platform samples, high efficiency, simple operation, and great reduction of the sequencing examination cost.
Description
Technical field
The invention belongs to biology field, relate to medical diagnosis and biotechnology, the external diagnosis reagent case that relates to a kind of Liddle ' of detection s associated gene mutation is a kind of test kit of a kind of Liddle ' the s associated gene mutation that is applied to new-generation sequencing platform technology.
Technical background
Liddle ' s syndrome is a kind of single-gene essential hypertension of autosomal dominant inheritance, its Clinical symptoms is the hypertension of early sending out, hypokalemia, low renin, hypoaldosteronemia, to epithelium sodium channel (ENaC) inhibitor guanamprazine or triamterene sensitivity, and insensitive to aldosterone receptor antagonist spironolactone, conventional condition of medicine treatment for hypertension effect is not good enough.
Liddle ' s syndrome is a kind of orphan disease, but its sickness rate possibly than expection height, be monogenic inheritance essential hypertension most commonly encountered diseases because of one of.This disease all has a Case report not agnate, comprises Caucasian, Aisa people, African etc.Research of the U.S. is to 149 routine blood potassium lower than 4.0mmol/L and suffer from hypertensive veteran and investigate, and result shows that Liddle ' s syndrome sickness rate is up to 6%
[1].
The syndromic molecular basis of Liddle ' s is the acquired function mutation of gene (SCNN1B and SCNN1G) of coding ENaC, modal sudden change is the highly conserved sequence PY motif (P: proline(Pro) of β or the C of γ subunit C-terminal and proline rich, Y: tyrosine, L: leucine) fracture, missense, produce due to termination codon or phase shift mutation in advance.The transgenation of PY motif can cause ENaC continuous activation, and sodium heavily absorbs increase, elevation of blood pressure, and aldosterone and renin secretion are suppressed, thereby occur the syndromic a series of clinical symptom of Liddle ' s.Its critical pathophysiological change is: the ENaC of distal renal tubular increases the heavily absorption of sodium, ECFV expansion.
Liddle ' s syndrome patient clinical manifestation is hypertension, hypokalemia, metabolic alkalosis, and plasma renin and aldosterone level, in normal range or reduction, have clear and definite family history of hypertension mostly.Take guanamprazine and effectively can support the diagnosis to this disease to controlling hypertension.Also the report that has in recent years some light moderate hypertension companions or do not accompany hypokalemic Liddle ' s syndrome patient.
It is little that Liddle ' s syndrome patient has age of onset, easily there is target organ damage, therefore for the age < hyperpietic of 30 years old, once there be hypertension familial inheritance history, and while thering is the features such as low plasma renin activity, hypokalemia, should consider to carry out SCNN1B and SCNN1G gene screening, contribute to early stage etiological diagnosis, early stage guiding treatment, once make a definite diagnosis, limit salt and oral guanamprazine or triamterene can receive good therapeutic action, thereby avoid the generation of pernicious cardiovascular event.
Because Liddle ' s syndrome is regarded as a kind of rare disease always, patient, for a long time by mistaken diagnosis, causes controlling of blood pressure difficulty, uses in a large number depressor to cause drug side effect to increase, and in one's early years the not congruent target organ damage of cardiorenal function occurs, and cerebral apoplexy is even died suddenly.
[1]Tapolyai M，Uysal A，Dossabhoy NR，et al.High prevalence of Liddle’s syndrome phenotype among hypertensive US Veterans in Northwest Louisiana.J Clin Hypertens (Greenwich).2010；12(11)：856-860.
The method traditional for the syndromic differential diagnosis of Liddle ' s mainly relies on biochemical analysis and imaging examination.There is very large shortcoming in these means:
1. check that kind is many;
2. sensitivity and specific degree are low;
3. be subject to the impact of the factor such as medicine and mental status;
4. require detection time harsh;
The advantage of gene diagnosis is:
1. convenience, safety is quick: only to extract 2-4 milliliter blood:
2. can check any time;
3. dietary restriction not, takes medicine, and any physical appearance can detect;
4. compare repeatedly, multinomial tradition checks, more economical;
5. premorbid, the sole mode of disease utmost point early diagnosis;
6. make a definite diagnosis in source, and one-time detection is benefited all the life.
Everything has all disclosed gene diagnosis huge application prospect clinically.
In recent years, the extensive parallel order-checking platform of DNA (massively parallel DNA sequencing platform) has developed into the sequencing technologies of main flow, the appearance of this sequencing technologies not only makes DNA sequencing expense drop to one of percentage in the past, also allows this " privilege " that was specific in the past large-scale order-checking center of gene order-checking be shared by numerous researchists.
But the cost that directly uses the parallel order-checking detection of platform of extensive DNA still makes individual be difficult to bear at present.The present invention can catch multiple even tens patients' the accurate selectivity of Liddle ' s genes involved simultaneously, then for large scale sequencing platform, can greatly reduce the cost of gene diagnosis, makes it to be widely used in the clinical possibility that becomes.
Summary of the invention
The test kit that provides a kind of target acquistion of inspection Liddle ' s associated gene mutation to check order is again provided.This test kit has improved the flux of gene trap and the flux of the parallel order-checking of gene, easy and simple to handle, cost is low.
For achieving the above object, the present invention adopts the probe of the non-coding region of catching object SCNN1B, the whole exon fragments of SCNN1G gene and contiguous 50bp; For the universal primer of the fragment to be measured that increases; Sequence measuring joints sequence; Damping fluid; DNTP; Exo+ polymerase; Streptomycin sulphate magnetic bead.The technical solution used in the present invention is: a kind of new target acquistion sequence measurement again, comprises the following steps:
A. design and for the preparation of the probe of catching Liddle ' s associated gene mutation.And these probes are combined on magnetic bead, film or slide.Probe sequence is shown in sequence table.
B. by testing gene DNA sample, fragmentation is to 200-400bp respectively, and then by end-filling, the ATP adding makes fragment two ends introduce sticky end.
C. add respectively Illumina company
the one end designing in DNA Sample Prep Kit, with the joint sequence fragment of sequence label, adds ligase enzyme, make each fragment with pair of joint be connected, form new fragment.
D. regulate temperature of reaction to activate archaeal dna polymerase, with a pair of Illumina company
index Kit-PCR primers universal primer, the fragment sequence that step c gained different genes group is completed to connection mixes laggard performing PCR amplified reaction;
E. by the pcr amplification reaction product of the whole samples of steps d gained and the hybridization of the designed capture probe of step a, then, through wash-out purification reaction repeatedly, obtain fragment to be measured.
F. fragment to be measured is carried out to extensive parallel order-checking by the standard scheme of the Miseq of illumina company sequenator.
G. the sequencing result obtaining is compared with standard database, obtain diagnostic result.
For realizing technique scheme, the capture probe described in steps A is thymus nucleic acid (DNA), or Yeast Nucleic Acid (RNA) composition, but is not limited only to DNA and RNA.Probe is fragment composition 120bp and target complementation.Probe can be combined on magnetic bead, also can be combined on slide glass, but be not limited only to these media.Streptomycin sulphate magnetic bead material is Al-Ni-Co series permanent magnet alloy or barium ferrite, Nd-Fe-Bo permanent magnet material, Nd-Fe-Bo permanent magnet material, rubber magnet, but is not limited only to these materials.
Wherein, the capture probe described in steps A, comprises and covers the sequence that following gene SCNN1B and SCNN1G all encode within the scope of exon fragment and two ends 50bp.These probes design with stacked tile type, and fragment length is 50bp-180bp, and optimum is 75bp; Fragment length is 60bp-200bp, and optimum is 80bp; Fragment length is 65bp-220bp, and optimum is 85bp; Fragment length is 70bp-240bp, and optimum is 90bp; Between probe, lap is 5-20bp, and optimum is 8bp; The probe coverage of target area base is 1 × to 10 ×, optimum is 3 ×.
Major advantage of the present invention is:
1. the present invention can carry out the detection of the whole Liddle ' s of multiple different sources samples genes involved in once sequencing reaction simultaneously; Full sequence is directly measured, and accuracy rate can reach 99.99%.
2. test kit of the present invention can once complete the parallel of 1-48 sample and catches, and has improved capture rate, has greatly reduced the cost of preparation of samples.
3. test kit of the present invention can once complete whole Liddle ' s genes involveds of 1-1152 sample, and the Parallel testing of nearly 4000 sequences makes full use of the high-throughput characteristic of equipment, has greatly reduced the order-checking cost of each sample.
4. test kit of the present invention can primary first-order equation be differentiated missense, insertion and deletion mutantion simultaneously.Clinical recall rate and accuracy are greatly increased.
5. detection method step of the present invention is simple, reduces the link of repetitive operation, thereby has avoided the many uncertain primer existing in complex operations process, has improved Detection accuracy and stability.
6. detection method required time provided by the present invention is less than the sequencing technologies of sanger method greatly.
Example: complete same detection limit (detection limits of 100 person-portions), sanger method needs 2 and manually works one week simultaneously.Our method only needs a people in one day, to complete whole detection limits.
7. the accuracy detecting is better than biochip technology greatly, more meets clinical detection requirement.Gene chip can only detect base missense mutation, and insertion and deletion mutantion cannot detect simultaneously.Our method can detect the transgenation of missense, insertion, disappearance and variable shearing simultaneously.
Brief description of the drawings
Be illustrated as target area sequence and probe.
Embodiment
Be only the description for example to practical application of the present invention with embodiment below, practical application of the present invention is not limited only to following examples:
Embodiment mono-,
One, probe design:
By the probe of the synthetic Streptomycin sulphate mark of table.Probe solution adopts the SureSelect buffering system system of agilent company.
Two, genome extracts:
Adopt Qiagen FlexiGene DNA Kit (Code No:51204) to extract 96 increments to be measured genome originally, OD
260/280value reaches 1.8-2.0, respectively gets 1-3g as starting template.
Three, sample preparation before order-checking
1. goal gene fragmentation:
Get 270 parts of genomic dnas quantitatively crossing, be diluted to 20-35ng/L.Get 130L, carry out respectively fragmentation with Ultrasonic Cell Disruptor.
2.AMpure XP fragmentation is selected
1. get 96 orifice plates, add after 80-100L magnetic bead, then add sample DNA, pipettor is is repeatedly blown and beaten and is mixed;
2. 26 DEG C of placement 5min of mixed solution;
3. 96 orifice plates are rested on to 3min on magnetic sheet;
4. carefully pipette whole supernatants to new hole, carefully do not encounter magnet ring;
5. in the supernatant that moves to new hole, add 100-150L magnetic bead, pipettor is blown and beaten repeatedly, fully mixes again;
6. 26 DEG C of placement 5min of mixed solution;
7. 96 orifice plates are rested on to 3min on magnetic sheet, carefully remove supernatant;
8. add 70% ethanol 200L, leave standstill 30s, carefully remove supernatant;
9. repeating step 8 operates once;
10. 96 orifice plates are taken off from magnetic sheet, uncovered placement, fully volatilizees ethanol;
11. add 40L10mM pH=8.0Tris-HCl, and pipettor is is repeatedly blown and beaten and mixed;
96 orifice plates are rested on 1min on magnetic sheet by 12., becomes clear to solution;
13. carefully pipette supernatant to new PCR pipe, obtain the genomic dna after purifying.
3. the concordant and purifying of end
1. get 96 orifice plates, shown according to the form below, on ice chest, add all ingredients, keep ice bath state.
| Reagent | Each reaction consumption (L) |
| DNA sample | 19 |
| 10x Blunting Buffer | 2.5 |
| 1mM dNTP Mix | 2.5 |
| Blunt Enzyme Mix | 0.5 |
| Nuclease-Free Water | 0.5 |
| Total reaction system | 25 |
2. in PCR instrument (50 DEG C, heat lid), hatches 20min for 12 DEG C, then hatches 15min for 37 DEG C;
3. get 96 orifice plates, first add the magnetic bead 45L fully mixing, then add sample, pipettor piping and druming mixes;
4. 26 DEG C of mixed solutions, leave standstill 5min;
5. 96 orifice plates are placed on magnetic sheet, leave standstill 3min, abandon supernatant;
6. add 70% ethanol 200L, leave standstill 30s, abandon supernatant;
7. repeating step 6, then wash one time;
8. 96 orifice plates are taken off from magnetic sheet, uncovered placement, fully volatilizees ethanol;
9. add 40L10mM pH=8.0Tris-HCl, pipettor piping and druming mixes for 10 times;
10. 96 orifice plates are placed on magnetic sheet, leave standstill 1min; Carefully pipette supernatant interior for subsequent use to clean PCR pipe.
4. joint connects and purifying
1.Oligo uses respectively pH=7.5 60mM Tris-HCl (containing 150mM NaCl), is diluted to 400M; Shown according to the form below, preparation joint working fluid:
2. grade mole is mixed two oligo of each joint, after mixing, opens PCR instrument, and the program of setting is 95 DEG C and hatches 2min, and be cooled to 20 DEG C, cooling 0.1 per second is spent, after program completes, place on ice, and packing ,-20 DEG C are frozen;
3. get PCR pipe, shown according to the form below, on ice chest, add all ingredients, keep ice bath state;
| Reagent | Each reaction consumption (L) |
| DNA sample | 15 |
| 2x Quick ligation Reaction Buffer | 25 |
| Adapter oligo mix AP(200uM) | 2 |
| PE-AP(200uM) | 2 |
| Quick T4DNA ligase | 1.2 |
| Nuclease-free Water | 4.8 |
| Cumulative volume | 50 |
4. be put in interior 25 DEG C of Thermo comfort and hatch 15min;
5. get 96 orifice plates, first add the magnetic bead 90L fully mixing, then add sample, pipettor piping and druming mixes;
6. 26 DEG C of mixed solutions, leave standstill 5min;
7. 96 orifice plates are placed on magnetic sheet, leave standstill 3min, abandon supernatant;
8. add 70% ethanol 200L, leave standstill 30s, abandon supernatant;
9. repeating step 8, then wash one time;
10. 96 orifice plates are taken off from magnetic sheet, uncovered placement, fully volatilizees ethanol;
11. add 40L10mM pH=8.0Tris-HCl, and pipettor piping and druming mixes for 10 times;
12. are placed on 96 orifice plates on magnetic sheet, leave standstill 1min, carefully pipette supernatant interior for subsequent use to clean PCR pipe.
5. gap repair and purifying
1. get PCR pipe, shown according to the form below, on ice chest, add all ingredients, keep ice bath state.
| Component | Volume (L) |
| GDNA after jointing | 40 |
| 10x ThermoPol Reaction Buffer | 5 |
| 10mM dNTP mix | 1 |
| Bst DNA Polymerase，Large Fragment | 2.5 |
| Nuclease-free Water | 1.5 |
| Cumulative volume | 50 |
On 2.PCR instrument, (50 DEG C, heat lid) 37 DEG C hatched 15min;
3. get 96 orifice plates, first add the magnetic bead 90L fully mixing, then add sample, pipettor piping and druming mixes for 10 times;
4. 26 DEG C of mixed solutions, leave standstill 5min;
5. 96 orifice plates are placed on magnetic sheet, leave standstill 3min, abandon supernatant;
6. add 70% ethanol 200L, leave standstill 30s, abandon supernatant;
7. repeating step 6 is washed one time again;
8. 96 orifice plates are taken off from magnetic sheet, uncovered placement, fully volatilizees ethanol;
9. add 40L10mM pH=8.0Tris-HCl, pipettor piping and druming mixes for 10 times;
10. 96 orifice plates are placed on magnetic sheet, leave standstill 1min, carefully pipette supernatant interior for subsequent use to clean PCR pipe;
11. get 2L Qubit2.0 carries out quantitatively.
6. amplified library (Herculase II Fusion DNA Polymerases)
1. in super clean bench, prepare PCR reaction system, get PCR pipe, shown according to the form below, on ice chest, add all ingredients, keep ice bath state, prepare after system, mix gently with pipettor;
2.PCR amplification condition
7. magnetic beads for purifying PCR product
1. get 96 orifice plates, first add the magnetic bead 90L fully mixing, then add sample, pipettor piping and druming mixes for 10 times;
2. 26 DEG C of mixed solutions, leave standstill 5min;
3. 96 orifice plates are placed on magnetic sheet, leave standstill 3min, abandon supernatant;
4. add 70% ethanol 200L, leave standstill 30s, abandon supernatant;
5. repeating step 4, then wash one time;
6. 96 orifice plates are taken off from magnetic sheet, uncovered placement, fully volatilizees ethanol;
7. add 40L10mM pH=8.0Tris-HCl, pipettor piping and druming mixes for 10 times;
8. 96 orifice plates are placed on magnetic sheet, leave standstill 1min; Carefully pipette supernatant interior for subsequent use to clean PCR pipe;
9. get 2L Qubit2.0 quantitative, get 1L Agilent2200 and detect.
Four, adopt designing probe to catch object fragment
(1) library hybridization
1, adopt the damping fluid of the Sureselect system of agilent company, preparing hybrid damping fluid:
| Reagent | Volume for1capture(L) |
| Sureselect hyb#1 | 25 |
| Sureselect hyb#2 | 1 |
| Sureselect hyb#3 | 10 |
| Sureselect hyb#4 | 13 |
| Total | 49 |
2, mixed solution is caught in preparation:
1. PCR plate (PCR plate 1) is placed on ice to precooling;
2. according to SureSelect RNase Block: Nuclease-free Water=1: the RNase Block diluent that 9 ratio preparation needs, place on ice, stand-by;
3. by reaction quantity, every sample well adds the SueSelect RNase Block (this part step 2 is prepared) of 5L dilution;
4. in each sample aperture of step 3, add the SureSelect Capture Library of 2L, and mix with liquid-transfering gun;
5. place on ice, for subsequent use.
3, prepare Sureselect Block Mix by the Sureselect system of agilent company:
| Reagent | Volume for1reaction(L) |
| Sureselect Indexing Block#1 | 2.5 |
| Sureselect Block#2 | 2.5 |
| Blocker for PE/SE | 0.6 |
| Total | 5.6 |
4, prepare the sample library of target enrichment:
1. the row of the B on PCR plate 2, adds the ready sample of 3.4L library (concentration is 147ng/L);
2. the B row of PCR plate 2, add the SureSelectBlockMix mixed solution of 5.6L, and mix up and down with liquid-transfering gun;
3. the B row of sealing PCR plate 2, and place it on PCR instrument;
4. according to the form below heats (105 DEG C of heat lids)
5. keep PCR plate 2 at 65 DEG C, add the Hybridization Buffer of 40L A row, sealing, hatches the experiment of carrying out again next step after 5min at least;
6. the SureSelect Capture Library Mix of PCR plate 1 is joined in PCR plate 2:
(1), the C of PCR plate 2 row, add the Capture Library Mix (PCR plate need keep 65 DEG C) of 7L;
(2) sealing lid;
Hatch 2min for (3) 65 DEG C;
7. get the Hybridization Buffer of 13L from A row, join in C row's SureSelect Capture Library Mix (PCR plate need keep 65 DEG C);
8. the sample library mixed solution of preparing in B row is all joined in C row's hybridization solution, mix gently (PCR plate need keep 65 DEG C) 8-10 time with liquid-transfering gun;
9. with new glued membrane, PCR plate is sealed;
10. on PCR instrument (105 DEG C, heat lid), hatches 24h by 65 DEG C of hybrid mixed liquid.
(2) prepare magnetic bead
1. in 1.5ml centrifuge tube, add the Streptavidin T1 magnetic bead of 50L;
2. magnetic bead wash-out:
(1) add the SureSelect Binding Buffer of 200L;
(2) on eddy mixer, mix 5s;
(3) centrifuge tube is placed on magnetic frame;
(4) remove supernatant;
(5) again repeating step (1) to (4) twice, co-elute 3 times;
3. add the SureSelect Binding Buffer of 200L again to hang magnetic bead.
(3) carrying out selective cross by SureSelecte system catches
1. after incubation 24h, determine and record and hybridize volume;
2. keep PCR plate on PCR instrument 65 DEG C, hybrid mixed liquid is directly joined in magnetic bead solution, put upside down and mix 3-5 time;
3. be placed on mixing tank room temperature incubation 30min by hybridizing-catch with the mixed solution of magnetic bead;
4. of short duration centrifugal;
5. be placed on magnetic frame, leave standstill 5min, remove supernatant;
6. add the SureSelect Wash Buffer#1 of 500L, whirlpool mixing 5s;
7. room temperature incubation, 15min altogether, but need mix 5s every 5min whirlpool;
8. of short duration centrifugal;
9. be placed on magnetic frame, leave standstill 5min, remove supernatant;
10. washing magnetic bead:
(1) add 500L at the SureSelect of 65 DEG C of preheatings Wash Buffer#2, whirlpool mixes 5s;
(2) at 65 DEG C of incubation 10min, mix every 3min;
(3) of short duration centrifugal;
(4) be placed on magnetic frame, leave standstill 5min, remove supernatant;
(5) again repeating step (1) to (4) twice, co-elute 3 times;
11. add the SureSelect Elution Buffer of 50L, and whirlpool mixes 5s;
12. room temperature incubation 10min, mix every 3min;
13. is of short duration centrifugal;
14. put on magnetic frame, leave standstill 5min;
Supernatant is transferred to new 1.5ml centrifuge tube (the DNA library that obtains catching) by 15.;
The 16. SureSelect Neutralization Buffer that add 50L are in the DNA library of catching, and of short duration whirlpool mixes.
(4) with AMPure magnetic beads for purifying sample
1. add 180L magnetic bead to 1.5ml centrifuge tube, then add the DNA library that 100L catches, whirlpool mixes;
2. 26 DEG C of mixed solutions, incubation 5min;
3. centrifuge tube is placed on magnetic frame, leaves standstill 10min, abandon supernatant;
4. add 70% ethanol 500L, leave standstill 1min, abandon supernatant;
5. repeating step 4, then wash one time;
6. centrifuge tube is placed on to 37 DEG C, incubation 5min, fully volatilizees ethanol;
7. the Nuclease-free Water that adds 30L, whirlpool mixes, incubated at room 2min;
8. centrifuge tube is placed on magnetic frame, leaves standstill 3min;
9. carefully pipette supernatant interior for subsequent use to new 1.5ml centrifuge tube.
(5) catch sample amplification
In Bechtop, get PCR pipe, shown according to the form below, on ice chest, add all ingredients, keep ice bath state;
| Reagent | Each reaction consumption (L) |
| Captured DNA | 14 |
| Nuclease-free water | 22.5 |
| 5X Herculase II Run Buffer(clear cap) | 10 |
| 100mM dNTP Mix(gteen cap) | 0.5 |
| Herculase II Fusion DNA Polymerase(red cap) | 1 |
| PosHyb-PEF | 1 |
| PosHyb-PER | 1 |
| Total reaction system | 50 |
Prepare after system, mix gently with liquid-transfering gun;
◆ pcr amplification condition
(6) AMPure magnetic beads for purifying PCR product
1. add 90L magnetic bead to 1.5ml centrifuge tube, then add the PCR product of 50L amplification, whirlpool mixes;
2. 26 DEG C of mixed solutions, incubation 5min;
3. centrifuge tube is placed on magnetic frame, leaves standstill 10min, abandon supernatant;
4. add 70% ethanol 500L, leave standstill 1min, abandon supernatant;
5. repeating step 4, then wash one time;
6. centrifuge tube is placed on to 37 DEG C, incubation 5min, fully volatilizees ethanol;
7. the Nuclease-free Water that adds 30L, whirlpool mixes, incubated at room 2min;
8. centrifuge tube is placed on magnetic frame, leaves standstill 3min;
9. carefully pipette supernatant interior for subsequent use to new 1.5ml centrifuge tube;
10. getting 1L Agilent2200 detects;
11. to get 2ul Qubit2.0 quantitative.
Five, upper machine order-checking:
96 sample machine order-checkings on the Miseq of illumina company carries out.
Six, data analysis:
Data results carries out bioinformatic analysis by CLC Genomics Workbench7.0.
Seven, result:
By 1 order-checking, within 2 days, complete the examining order of 170 sequences of 270 all 2 goal gene of sample, order-checking sum reaches 459006.Carry out the parallel control of 2 genes with ABI3730xl, sudden change recall rate is 65%.And the order-checking of this programme sudden change recall rate reaches 88%, inspection accuracy rate 100%.
Sequence table
SEQUENCE LISTING
<110> promise all generations (Beijing) medical science and technology company limited
Mono-kind of <120> checks the test kit of liddle's associated gene mutation
<130> 2014
<160> 170
<170> PatentIn version 3.3
<210> 1
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<213> artificial sequence
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<213> artificial sequence
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<213> artificial sequence
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<210> 8
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<212> DNA
<213> artificial sequence
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ccgtctgcgc cgcctcctct ggatcgggtt cacactgact gccgtggccc tcatcctctg 60
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<210> 9
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<213> artificial sequence
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caccgttcgc caccttctag ctgacttgga acaggagacc agagaggccc tgaagtccct 60
gtatggcttt ccagagtccc 80
<210> 14
<211> 80
<212> DNA
<213> artificial sequence
<400> 14
cttggaacag gagaccagag aggccctgaa gtccctgtat ggctttccag agtcccggaa 60
gcgccgagag gcggagtcct 80
<210> 15
<211> 80
<212> DNA
<213> artificial sequence
<400> 15
cctgaagtcc ctgtatggct ttccagagtc ccggaagcgc cgagaggcgg agtcctggaa 60
ctccgtctca gagggaaagc 80
<210> 16
<211> 80
<212> DNA
<213> artificial sequence
<400> 16
agagtcccgg aagcgccgag aggcggagtc ctggaactcc gtctcagagg gaaagcagcc 60
tagattctcc caccggattc 80
<210> 17
<211> 80
<212> DNA
<213> artificial sequence
<400> 17
ggagtcctgg aactccgtct cagagggaaa gcagcctaga ttctcccacc ggattccgct 60
gctgatcttt gatcaggatg 80
<210> 18
<211> 80
<212> DNA
<213> artificial sequence
<400> 18
gggaaagcag cctagattct cccaccggat tccgctgctg atctttgatc aggatgagaa 60
gggcaaggcc agggacttct 80
<210> 19
<211> 80
<212> DNA
<213> artificial sequence
<400> 19
ccggattccg ctgctgatct ttgatcagga tgagaagggc aaggccaggg acttcttcac 60
agggaggaag cggaaagtcg 80
<210> 20
<211> 80
<212> DNA
<213> artificial sequence
<400> 20
tcaggatgag aagggcaagg ccagggactt cttcacaggg aggaagcgga aagtcggcgg 60
tagcatcatt cacaaggctt 80
<210> 21
<211> 80
<212> DNA
<213> artificial sequence
<400> 21
ggacttcttc acagggagga agcggaaagt cggcggtagc atcattcaca aggcttcaaa 60
tgtcatgcac atcgagtcca 80
<210> 22
<211> 80
<212> DNA
<213> artificial sequence
<400> 22
tccttctgaa gagtagcgat aggaccgatg gcttcagcct cgcatctcct cttattcaca 60
gtgctcaaat gacacctccg 80
<210> 23
<211> 80
<212> DNA
<213> artificial sequence
<400> 23
ccgatggctt cagcctcgca tctcctctta ttcacagtgc tcaaatgaca cctccgactg 60
tgccacctac accttcagct 80
<210> 24
<211> 80
<212> DNA
<213> artificial sequence
<400> 24
ctcttattca cagtgctcaa atgacacctc cgactgtgcc acctacacct tcagctcggg 60
aatcaatgcc attcaggagt 80
<210> 25
<211> 80
<212> DNA
<213> artificial sequence
<400> 25
cacctccgac tgtgccacct acaccttcag ctcgggaatc aatgccattc aggagtggta 60
taagctacac tacatgaaca 80
<210> 26
<211> 80
<212> DNA
<213> artificial sequence
<400> 26
cttcagctcg ggaatcaatg ccattcagga gtggtataag ctacactaca tgaacatcat 60
ggcacaggtg cctctggaga 80
<210> 27
<211> 80
<212> DNA
<213> artificial sequence
<400> 27
tcaggagtgg tataagctac actacatgaa catcatggca caggtgcctc tggagaagaa 60
aatcaacatg agctattctg 80
<210> 28
<211> 80
<212> DNA
<213> artificial sequence
<400> 28
catgaacatc atggcacagg tgcctctgga gaagaaaatc aacatgagct attctgctga 60
ggagctgctg gtgacctgct 80
<210> 29
<211> 80
<212> DNA
<213> artificial sequence
<400> 29
tctggagaag aaaatcaaca tgagctattc tgctgaggag ctgctggtga cctgcttctt 60
tgatggagtg tcctgtgatg 80
<210> 30
<211> 80
<212> DNA
<213> artificial sequence
<400> 30
ctattctgct gaggagctgc tggtgacctg cttctttgat ggagtgtcct gtgatgccag 60
gtcaggagag aatgctgctc 80
<210> 31
<211> 80
<212> DNA
<213> artificial sequence
<400> 31
tgccctgccc aacttcagct aagatgcatg ggggagatcc ctttctgacc cattttcttc 60
ctccatagga atttcacgct 80
<210> 32
<211> 80
<212> DNA
<213> artificial sequence
<400> 32
tgcatggggg agatcccttt ctgacccatt ttcttcctcc ataggaattt cacgcttttc 60
caccacccga tgcatgggaa 80
<210> 33
<211> 80
<212> DNA
<213> artificial sequence
<400> 33
cccattttct tcctccatag gaatttcacg cttttccacc acccgatgca tgggaattgc 60
tatactttca acaacagaga 80
<210> 34
<211> 80
<212> DNA
<213> artificial sequence
<400> 34
ttcacgcttt tccaccaccc gatgcatggg aattgctata ctttcaacaa cagagaaaat 60
gagaccattc tcagcacctc 80
<210> 35
<211> 80
<212> DNA
<213> artificial sequence
<400> 35
catgggaatt gctatacttt caacaacaga gaaaatgaga ccattctcag cacctccatg 60
gggggcagcg aatatggtaa 80
<210> 36
<211> 80
<212> DNA
<213> artificial sequence
<400> 36
aacagagaaa atgagaccat tctcagcacc tccatggggg gcagcgaata tggtaaggaa 60
acctgtgcca aggagatctt 80
<210> 37
<211> 80
<212> DNA
<213> artificial sequence
<400> 37
ggtttacccc caggaaatgt tttcctagcc ttggatcaca gcaggttgtc ttatcctccc 60
agggctgcaa gtcattttgt 80
<210> 38
<211> 80
<212> DNA
<213> artificial sequence
<400> 38
ctagccttgg atcacagcag gttgtcttat cctcccaggg ctgcaagtca ttttgtacat 60
aaacgaagag gaatacaacc 80
<210> 39
<211> 80
<212> DNA
<213> artificial sequence
<400> 39
tcttatcctc ccagggctgc aagtcatttt gtacataaac gaagaggaat acaacccatt 60
cctcgtgtcc tccactggag 80
<210> 40
<211> 80
<212> DNA
<213> artificial sequence
<400> 40
cattttgtac ataaacgaag aggaatacaa cccattcctc gtgtcctcca ctggagctaa 60
ggtgatcatc catcggcagg 80
<210> 41
<211> 80
<212> DNA
<213> artificial sequence
<400> 41
atacaaccca ttcctcgtgt cctccactgg agctaaggtg atcatccatc ggcaggatga 60
gtatcccttc gtcgaagatg 80
<210> 42
<211> 80
<212> DNA
<213> artificial sequence
<400> 42
cactggagct aaggtgatca tccatcggca ggatgagtat cccttcgtcg aagatgtggg 60
aacagagatt gagacagcaa 80
<210> 43
<211> 80
<212> DNA
<213> artificial sequence
<400> 43
tcggcaggat gagtatccct tcgtcgaaga tgtgggaaca gagattgaga cagcaatggt 60
cacctctata ggaatgcacc 80
<210> 44
<211> 80
<212> DNA
<213> artificial sequence
<400> 44
cgaagatgtg ggaacagaga ttgagacagc aatggtcacc tctataggaa tgcacctggt 60
aagagaatat tctcatttcc 80
<210> 45
<211> 80
<212> DNA
<213> artificial sequence
<400> 45
aagcccccgc ctgggtccgg ggggaggaca gggctgagtg tgtgctgctg tgattgcaga 60
cagagtcctt caagctgagt 80
<210> 46
<211> 80
<212> DNA
<213> artificial sequence
<400> 46
aggacagggc tgagtgtgtg ctgctgtgat tgcagacaga gtccttcaag ctgagtgagc 60
cctacagtca gtgcacggag 80
<210> 47
<211> 80
<212> DNA
<213> artificial sequence
<400> 47
tgtgattgca gacagagtcc ttcaagctga gtgagcccta cagtcagtgc acggaggacg 60
ggagtgacgt gccaatcagg 80
<210> 48
<211> 80
<212> DNA
<213> artificial sequence
<400> 48
agctgagtga gccctacagt cagtgcacgg aggacgggag tgacgtgcca atcaggaaca 60
tctacaacgc tgcctactcg 80
<210> 49
<211> 80
<212> DNA
<213> artificial sequence
<400> 49
gcacggagga cgggagtgac gtgccaatca ggaacatcta caacgctgcc tactcgctcc 60
aggtaacaga ttggcagggg 80
<210> 50
<211> 80
<212> DNA
<213> artificial sequence
<400> 50
ggaaactccc tgacatccct gagcaaagac atgaatggca ttcctgggtc tcctctttca 60
gatctgcctt cattcatgct 80
<210> 51
<211> 80
<212> DNA
<213> artificial sequence
<400> 51
aaagacatga atggcattcc tgggtctcct ctttcagatc tgccttcatt catgcttcca 60
gacaaagatg gtggagaaat 80
<210> 52
<211> 80
<212> DNA
<213> artificial sequence
<400> 52
tctcctcttt cagatctgcc ttcattcatg cttccagaca aagatggtgg agaaatgtgg 60
gtgtgcccag tacagccagc 80
<210> 53
<211> 80
<212> DNA
<213> artificial sequence
<400> 53
ttcatgcttc cagacaaaga tggtggagaa atgtgggtgt gcccagtaca gccagcctct 60
acctcctgca gccaactact 80
<210> 54
<211> 80
<212> DNA
<213> artificial sequence
<400> 54
ggagaaatgt gggtgtgccc agtacagcca gcctctacct cctgcagcca actactgcaa 60
ctaccagcag caccccaact 80
<210> 55
<211> 80
<212> DNA
<213> artificial sequence
<400> 55
cagccagcct ctacctcctg cagccaacta ctgcaactac cagcagcacc ccaactggag 60
tgagtgagac ccagctccag 80
<210> 56
<211> 80
<212> DNA
<213> artificial sequence
<400> 56
tgacctgggg atggccagga agggtcctgt ggctccaaag ctcatgctgc cctctccctt 60
gtccctcagt gtattgttac 80
<210> 57
<211> 80
<212> DNA
<213> artificial sequence
<400> 57
tcctgtggct ccaaagctca tgctgccctc tcccttgtcc ctcagtgtat tgttactacc 60
aactgcatcg agcctttgtc 80
<210> 58
<211> 80
<212> DNA
<213> artificial sequence
<400> 58
gccctctccc ttgtccctca gtgtattgtt actaccaact gcatcgagcc tttgtccagg 60
aagagctggg ctgccagtct 80
<210> 59
<211> 80
<212> DNA
<213> artificial sequence
<400> 59
attgttacta ccaactgcat cgagcctttg tccaggaaga gctgggctgc cagtctgtgt 60
gcaaggaagc ctgcaggtat 80
<210> 60
<211> 80
<212> DNA
<213> artificial sequence
<400> 60
cctttgtcca ggaagagctg ggctgccagt ctgtgtgcaa ggaagcctgc aggtatgtgg 60
accccaaggg gtgagacggg 80
<210> 61
<211> 80
<212> DNA
<213> artificial sequence
<400> 61
aaggggtgag acgggtggct gggttgggtt gggctgccta cactcatgct gtcccctcct 60
cccttagctt taaagagtgg 80
<210> 62
<211> 80
<212> DNA
<213> artificial sequence
<400> 62
tgggttgggc tgcctacact catgctgtcc cctcctccct tagctttaaa gagtggacac 60
taaccacaag cctggcacaa 80
<210> 63
<211> 80
<212> DNA
<213> artificial sequence
<400> 63
ctgtcccctc ctcccttagc tttaaagagt ggacactaac cacaagcctg gcacaatggc 60
catctgtggt ttcggaggta 80
<210> 64
<211> 80
<212> DNA
<213> artificial sequence
<400> 64
aagagtggac actaaccaca agcctggcac aatggccatc tgtggtttcg gaggtaagtt 60
cttctgccca cccttcccca 80
<210> 65
<211> 80
<212> DNA
<213> artificial sequence
<400> 65
gaggcgggag gctggcccta gcctctcagg caccctcagg cccacgcttt ctctctccgt 60
tgtagaagtg gttgctgcct 80
<210> 66
<211> 80
<212> DNA
<213> artificial sequence
<400> 66
ctcaggcacc ctcaggccca cgctttctct ctccgttgta gaagtggttg ctgcctgttc 60
tcacttggga ccaaggccgg 80
<210> 67
<211> 80
<212> DNA
<213> artificial sequence
<400> 67
ttctctctcc gttgtagaag tggttgctgc ctgttctcac ttgggaccaa ggccggcaag 60
taaacaaaaa gctcaacaag 80
<210> 68
<211> 80
<212> DNA
<213> artificial sequence
<400> 68
tgctgcctgt tctcacttgg gaccaaggcc ggcaagtaaa caaaaagctc aacaagtaag 60
ttacctctac cctgttcctc 80
<210> 69
<211> 80
<212> DNA
<213> artificial sequence
<400> 69
aggactggta atctggtagg atgccaaggc tcttgattca cctgttggaa ttttgcagga 60
cagacttggc caaactcttg 80
<210> 70
<211> 80
<212> DNA
<213> artificial sequence
<400> 70
caaggctctt gattcacctg ttggaatttt gcaggacaga cttggccaaa ctcttgatat 60
tctacaaaga cctgaaccag 80
<210> 71
<211> 80
<212> DNA
<213> artificial sequence
<400> 71
aattttgcag gacagacttg gccaaactct tgatattcta caaagacctg aaccagagat 60
ccatcatgga gagcccagcc 80
<210> 72
<211> 80
<212> DNA
<213> artificial sequence
<400> 72
aactcttgat attctacaaa gacctgaacc agagatccat catggagagc ccagccaaca 60
gtgtgagtag agtggcttcc 80
<210> 73
<211> 80
<212> DNA
<213> artificial sequence
<400> 73
ggcctgtctt gcagattgag atgcttctgt ccaacttcgg tggccagctg ggcctgtgga 60
tgagctgctc tgttgtctgc 80
<210> 74
<211> 80
<212> DNA
<213> artificial sequence
<400> 74
ttctgtccaa cttcggtggc cagctgggcc tgtggatgag ctgctctgtt gtctgcgtca 60
tcgagatcat cgaggtcttc 80
<210> 75
<211> 80
<212> DNA
<213> artificial sequence
<400> 75
tgggcctgtg gatgagctgc tctgttgtct gcgtcatcga gatcatcgag gtcttcttca 60
ttgacttctt ctctatcatt 80
<210> 76
<211> 80
<212> DNA
<213> artificial sequence
<400> 76
ttgtctgcgt catcgagatc atcgaggtct tcttcattga cttcttctct atcattgccc 60
gccgccagtg gcagaaagcc 80
<210> 77
<211> 80
<212> DNA
<213> artificial sequence
<400> 77
aggtcttctt cattgacttc ttctctatca ttgcccgccg ccagtggcag aaagccaagg 60
agtggtgggc ctggaaacag 80
<210> 78
<211> 80
<212> DNA
<213> artificial sequence
<400> 78
ctatcattgc ccgccgccag tggcagaaag ccaaggagtg gtgggcctgg aaacaggctc 60
ccccatgtcc agaagctccc 80
<210> 79
<211> 80
<212> DNA
<213> artificial sequence
<400> 79
agaaagccaa ggagtggtgg gcctggaaac aggctccccc atgtccagaa gctccccgta 60
gcccacaggg ccaggacaat 80
<210> 80
<211> 80
<212> DNA
<213> artificial sequence
<400> 80
ggaaacaggc tcccccatgt ccagaagctc cccgtagccc acagggccag gacaatccag 60
ccctggatat agacgatgac 80
<210> 81
<211> 80
<212> DNA
<213> artificial sequence
<400> 81
aagctccccg tagcccacag ggccaggaca atccagccct ggatatagac gatgacctac 60
ccactttcaa ctctgctttg 80
<210> 82
<211> 80
<212> DNA
<213> artificial sequence
<400> 82
aggacaatcc agccctggat atagacgatg acctacccac tttcaactct gctttgcacc 60
tgcctccagc cctaggaacc 80
<210> 83
<211> 80
<212> DNA
<213> artificial sequence
<400> 83
acgatgacct acccactttc aactctgctt tgcacctgcc tccagcccta ggaacccaag 60
tgcccggcac accgcccccc 80
<210> 84
<211> 80
<212> DNA
<213> artificial sequence
<400> 84
ctgctttgca cctgcctcca gccctaggaa cccaagtgcc cggcacaccg ccccccaaat 60
acaatacctt gcgcttggag 80
<210> 85
<211> 80
<212> DNA
<213> artificial sequence
<400> 85
taggaaccca agtgcccggc acaccgcccc ccaaatacaa taccttgcgc ttggagaggg 60
ccttttccaa ccagctcaca 80
<210> 86
<211> 80
<212> DNA
<213> artificial sequence
<400> 86
ttatttaaac tgctgcatgg attcccgccg tggataatgc ctaccgatgg gaatttaggt 60
gacaaaaatt ttcaggttgg 80
<210> 87
<211> 80
<212> DNA
<213> artificial sequence
<400> 87
cgtccccatg cctctctgca ggtgccacta tgcacgtgaa gaagtacctg ctgaagggcc 60
tgcatcggct gcagaagggc 80
<210> 88
<211> 80
<212> DNA
<213> artificial sequence
<400> 88
ccactatgca cgtgaagaag tacctgctga agggcctgca tcggctgcag aagggccccg 60
gctacacgta caaggagctg 80
<210> 89
<211> 80
<212> DNA
<213> artificial sequence
<400> 89
tgctgaaggg cctgcatcgg ctgcagaagg gccccggcta cacgtacaag gagctgctgg 60
tgtggtactg cgacaacacc 80
<210> 90
<211> 80
<212> DNA
<213> artificial sequence
<400> 90
agaagggccc cggctacacg tacaaggagc tgctggtgtg gtactgcgac aacaccaaca 60
cccacggccc caagcgcatc 80
<210> 91
<211> 80
<212> DNA
<213> artificial sequence
<400> 91
aggagctgct ggtgtggtac tgcgacaaca ccaacaccca cggccccaag cgcatcatct 60
gtgaggggcc caagaagaaa 80
<210> 92
<211> 80
<212> DNA
<213> artificial sequence
<400> 92
acaacaccaa cacccacggc cccaagcgca tcatctgtga ggggcccaag aagaaagcca 60
tgtggttcct gctcaccctg 80
<210> 93
<211> 80
<212> DNA
<213> artificial sequence
<400> 93
agcgcatcat ctgtgagggg cccaagaaga aagccatgtg gttcctgctc accctgctct 60
tcgccgccct cgtctgctgg 80
<210> 94
<211> 80
<212> DNA
<213> artificial sequence
<400> 94
agaagaaagc catgtggttc ctgctcaccc tgctcttcgc cgccctcgtc tgctggcagt 60
ggggcatctt catcaggacc 80
<210> 95
<211> 80
<212> DNA
<213> artificial sequence
<400> 95
tcaccctgct cttcgccgcc ctcgtctgct ggcagtgggg catcttcatc aggacctact 60
tgagctggga ggtcagcgtc 80
<210> 96
<211> 80
<212> DNA
<213> artificial sequence
<400> 96
tctgctggca gtggggcatc ttcatcagga cctacttgag ctgggaggtc agcgtctccc 60
tctccgtagg cttcaagacc 80
<210> 97
<211> 80
<212> DNA
<213> artificial sequence
<400> 97
tcaggaccta cttgagctgg gaggtcagcg tctccctctc cgtaggcttc aagaccatgg 60
acttccctgc cgtcaccatc 80
<210> 98
<211> 80
<212> DNA
<213> artificial sequence
<400> 98
cagccctctc cccatccagg tattccaaaa tcaagcattt gctgaaggac ctggatgagc 60
tgatggaagc tgtcctggag 80
<210> 99
<211> 80
<212> DNA
<213> artificial sequence
<400> 99
ccaaaatcaa gcatttgctg aaggacctgg atgagctgat ggaagctgtc ctggagagaa 60
tcctggctcc tgagctaagc 80
<210> 100
<211> 80
<212> DNA
<213> artificial sequence
<400> 100
acctggatga gctgatggaa gctgtcctgg agagaatcct ggctcctgag ctaagccatg 60
ccaatgccac caggaacctg 80
<210> 101
<211> 80
<212> DNA
<213> artificial sequence
<400> 101
tcctggagag aatcctggct cctgagctaa gccatgccaa tgccaccagg aacctgaact 60
tctccatctg gaaccacaca 80
<210> 102
<211> 80
<212> DNA
<213> artificial sequence
<400> 102
agctaagcca tgccaatgcc accaggaacc tgaacttctc catctggaac cacacacccc 60
tggtccttat tgatgaacgg 80
<210> 103
<211> 80
<212> DNA
<213> artificial sequence
<400> 103
ggaacctgaa cttctccatc tggaaccaca cacccctggt ccttattgat gaacggaacc 60
cccaccaccc catggtcctt 80
<210> 104
<211> 80
<212> DNA
<213> artificial sequence
<400> 104
accacacacc cctggtcctt attgatgaac ggaaccccca ccaccccatg gtccttgatc 60
tctttggaga caaccacaat 80
<210> 105
<211> 80
<212> DNA
<213> artificial sequence
<400> 105
atgaacggaa cccccaccac cccatggtcc ttgatctctt tggagacaac cacaatggct 60
taacaagcag ctcagcatca 80
<210> 106
<211> 80
<212> DNA
<213> artificial sequence
<400> 106
tggtccttga tctctttgga gacaaccaca atggcttaac aagcagctca gcatcagaaa 60
agatctgtaa tgcccacggg 80
<210> 107
<211> 80
<212> DNA
<213> artificial sequence
<400> 107
tggctggggt cctgctagca gctcccacgc cacccacaaa aacccctctt ggcctccaca 60
gtgtagcctc aacaggaccc 80
<210> 108
<211> 80
<212> DNA
<213> artificial sequence
<400> 108
ccacgccacc cacaaaaacc cctcttggcc tccacagtgt agcctcaaca ggacccagtg 60
taccttccgg aacttcacca 80
<210> 109
<211> 80
<212> DNA
<213> artificial sequence
<400> 109
ttggcctcca cagtgtagcc tcaacaggac ccagtgtacc ttccggaact tcaccagtgc 60
tacccaggca ttgacagagt 80
<210> 110
<211> 80
<212> DNA
<213> artificial sequence
<400> 110
caggacccag tgtaccttcc ggaacttcac cagtgctacc caggcattga cagagtggta 60
catcctgcag gccaccaaca 80
<210> 111
<211> 80
<212> DNA
<213> artificial sequence
<400> 111
cttcaccagt gctacccagg cattgacaga gtggtacatc ctgcaggcca ccaacatctt 60
tgcacaggtg ccacagcagg 80
<210> 112
<211> 80
<212> DNA
<213> artificial sequence
<400> 112
gacagagtgg tacatcctgc aggccaccaa catctttgca caggtgccac agcaggagct 60
agtagagatg agctaccccg 80
<210> 113
<211> 80
<212> DNA
<213> artificial sequence
<400> 113
caccaacatc tttgcacagg tgccacagca ggagctagta gagatgagct accccggcga 60
gcagatgatc ctggcctgcc 80
<210> 114
<211> 80
<212> DNA
<213> artificial sequence
<400> 114
acagcaggag ctagtagaga tgagctaccc cggcgagcag atgatcctgg cctgcctatt 60
cggagctgag ccctgcaact 80
<210> 115
<211> 80
<212> DNA
<213> artificial sequence
<400> 115
ctaccccggc gagcagatga tcctggcctg cctattcgga gctgagccct gcaactaccg 60
gtgagagcca ccccaagccc 80
<210> 116
<211> 80
<212> DNA
<213> artificial sequence
<400> 116
cggcagacag tcgggggagg cattgcctgt ggtggaacct gccctgcagc tgatgctgtt 60
tcttttagga acttcacgtc 80
<210> 117
<211> 80
<212> DNA
<213> artificial sequence
<400> 117
gcctgtggtg gaacctgccc tgcagctgat gctgtttctt ttaggaactt cacgtccatc 60
ttctaccctc actatggcaa 80
<210> 118
<211> 80
<212> DNA
<213> artificial sequence
<400> 118
gctgatgctg tttcttttag gaacttcacg tccatcttct accctcacta tggcaactgt 60
tacatcttca actggggcat 80
<210> 119
<211> 80
<212> DNA
<213> artificial sequence
<400> 119
ttcacgtcca tcttctaccc tcactatggc aactgttaca tcttcaactg gggcatgaca 60
gagaaggcac ttccttcggc 80
<210> 120
<211> 80
<212> DNA
<213> artificial sequence
<400> 120
tatggcaact gttacatctt caactggggc atgacagaga aggcacttcc ttcggccaac 60
cctggaactg aattcggtga 80
<210> 121
<211> 80
<212> DNA
<213> artificial sequence
<400> 121
tggggcatga cagagaaggc acttccttcg gccaaccctg gaactgaatt cggtgagttt 60
tggtttatcg tggggccaga 80
<210> 122
<211> 80
<212> DNA
<213> artificial sequence
<400> 122
gtggggtctc ctttctgcct caggagaaag ttcaggcagc cctcacccca ccctccccac 60
aggcctgaag ttgatcctgg 80
<210> 123
<211> 80
<212> DNA
<213> artificial sequence
<400> 123
agaaagttca ggcagccctc accccaccct ccccacaggc ctgaagttga tcctggacat 60
aggccaggaa gactacgtcc 80
<210> 124
<211> 80
<212> DNA
<213> artificial sequence
<400> 124
caccctcccc acaggcctga agttgatcct ggacataggc caggaagact acgtcccctt 60
ccttgcgtcc acggccgggg 80
<210> 125
<211> 80
<212> DNA
<213> artificial sequence
<400> 125
gatcctggac ataggccagg aagactacgt ccccttcctt gcgtccacgg ccggggtcag 60
gctgatgctt cacgagcaga 80
<210> 126
<211> 80
<212> DNA
<213> artificial sequence
<400> 126
ctacgtcccc ttccttgcgt ccacggccgg ggtcaggctg atgcttcacg agcagaggtc 60
ataccccttc atcagagatg 80
<210> 127
<211> 80
<212> DNA
<213> artificial sequence
<400> 127
ggccggggtc aggctgatgc ttcacgagca gaggtcatac cccttcatca gagatgaggg 60
catctacgcc atgtcgggga 80
<210> 128
<211> 80
<212> DNA
<213> artificial sequence
<400> 128
cgagcagagg tcatacccct tcatcagaga tgagggcatc tacgccatgt cggggacaga 60
gacgtccatc ggggtactcg 80
<210> 129
<211> 80
<212> DNA
<213> artificial sequence
<400> 129
cagagatgag ggcatctacg ccatgtcggg gacagagacg tccatcgggg tactcgtggt 60
atggccggag cccaagggca 80
<210> 130
<211> 80
<212> DNA
<213> artificial sequence
<400> 130
agggaggtgc agaaagggct tcctggggtg accagtgtcc ccctcaagca acccctctaa 60
acacaggaca agcttcagcg 80
<210> 131
<211> 80
<212> DNA
<213> artificial sequence
<400> 131
ggggtgacca gtgtccccct caagcaaccc ctctaaacac aggacaagct tcagcgcatg 60
ggggagccct acagcccgtg 80
<210> 132
<211> 80
<212> DNA
<213> artificial sequence
<400> 132
caacccctct aaacacagga caagcttcag cgcatggggg agccctacag cccgtgcacc 60
gtgaatggtt ctgaggtccc 80
<210> 133
<211> 80
<212> DNA
<213> artificial sequence
<400> 133
cttcagcgca tgggggagcc ctacagcccg tgcaccgtga atggttctga ggtccccgtc 60
caaaacttct acagtgacta 80
<210> 134
<211> 80
<212> DNA
<213> artificial sequence
<400> 134
agcccgtgca ccgtgaatgg ttctgaggtc cccgtccaaa acttctacag tgactacaac 60
acgacctact ccatccaggt 80
<210> 135
<211> 80
<212> DNA
<213> artificial sequence
<400> 135
gaggtccccg tccaaaactt ctacagtgac tacaacacga cctactccat ccaggtggga 60
aggtggtgca cgcctcatgc 80
<210> 136
<211> 80
<212> DNA
<213> artificial sequence
<400> 136
gaatggggac atcactgacc atgcctgtgt tctctcctta tgaaccccct accctcccca 60
ggcctgtctt cgctcctgct 80
<210> 137
<211> 80
<212> DNA
<213> artificial sequence
<400> 137
ctgtgttctc tccttatgaa ccccctaccc tccccaggcc tgtcttcgct cctgcttcca 60
agaccacatg atccgtaact 80
<210> 138
<211> 80
<212> DNA
<213> artificial sequence
<400> 138
ctaccctccc caggcctgtc ttcgctcctg cttccaagac cacatgatcc gtaactgcaa 60
ctgtggccac tacctgtacc 80
<210> 139
<211> 80
<212> DNA
<213> artificial sequence
<400> 139
ctcctgcttc caagaccaca tgatccgtaa ctgcaactgt ggccactacc tgtacccact 60
gccccgtggg gagaaatact 80
<210> 140
<211> 80
<212> DNA
<213> artificial sequence
<400> 140
ccgtaactgc aactgtggcc actacctgta cccactgccc cgtggggaga aatactgcaa 60
caaccgggac ttcccagact 80
<210> 141
<211> 80
<212> DNA
<213> artificial sequence
<400> 141
cctgtaccca ctgccccgtg gggagaaata ctgcaacaac cgggacttcc cagactgggg 60
tgagcggggg cacgggggat 80
<210> 142
<211> 80
<212> DNA
<213> artificial sequence
<400> 142
caggggcacc taaaaagccc ccttaaacct cttggccgcc tttctgtctc ctgcgcagcc 60
cattgctact cagatctaca 80
<210> 143
<211> 80
<212> DNA
<213> artificial sequence
<400> 143
aaacctcttg gccgcctttc tgtctcctgc gcagcccatt gctactcaga tctacagatg 60
agcgtggcgc agagagagac 80
<210> 144
<211> 80
<212> DNA
<213> artificial sequence
<400> 144
tcctgcgcag cccattgcta ctcagatcta cagatgagcg tggcgcagag agagacctgc 60
attggcatgt gcaaggagtc 80
<210> 145
<211> 80
<212> DNA
<213> artificial sequence
<400> 145
gatctacaga tgagcgtggc gcagagagag acctgcattg gcatgtgcaa ggagtcctgc 60
aagtgagtgc gggtgggcgg 80
<210> 146
<211> 80
<212> DNA
<213> artificial sequence
<400> 146
cagcagcggg caggcatgga ggggcatcac tggcagggac cacaacaggc ctggccttct 60
ctttcagtga cacccagtac 80
<210> 147
<211> 80
<212> DNA
<213> artificial sequence
<400> 147
catcactggc agggaccaca acaggcctgg ccttctcttt cagtgacacc cagtacaaga 60
tgaccatctc catggctgac 80
<210> 148
<211> 80
<212> DNA
<213> artificial sequence
<400> 148
gcctggcctt ctctttcagt gacacccagt acaagatgac catctccatg gctgactggc 60
cttctgaggc ctccgaggtg 80
<210> 149
<211> 80
<212> DNA
<213> artificial sequence
<400> 149
cccagtacaa gatgaccatc tccatggctg actggccttc tgaggcctcc gaggtgagac 60
agttgggggc caagctcctg 80
<210> 150
<211> 80
<212> DNA
<213> artificial sequence
<400> 150
acagagccat gactgggagg gatgctgcag atggcaactt ttgcaaccac cttcttgggt 60
tccaggactg gattttccac 80
<210> 151
<211> 80
<212> DNA
<213> artificial sequence
<400> 151
ctgcagatgg caacttttgc aaccaccttc ttgggttcca ggactggatt ttccacgtct 60
tgtctcagga gcgggaccaa 80
<210> 152
<211> 80
<212> DNA
<213> artificial sequence
<400> 152
accttcttgg gttccaggac tggattttcc acgtcttgtc tcaggagcgg gaccaaagca 60
ccaatatcac cctgagcagg 80
<210> 153
<211> 80
<212> DNA
<213> artificial sequence
<400> 153
ttttccacgt cttgtctcag gagcgggacc aaagcaccaa tatcaccctg agcaggtgag 60
cctgagcctg ggcggggctg 80
<210> 154
<211> 80
<212> DNA
<213> artificial sequence
<400> 154
gtctgtctgt ttggaagggg gatacattag tcccggccct tctcgctgcc tcctgcagga 60
agggaattgt caagctcaac 80
<210> 155
<211> 80
<212> DNA
<213> artificial sequence
<400> 155
cattagtccc ggcccttctc gctgcctcct gcaggaaggg aattgtcaag ctcaacatct 60
acttccaaga atttaactat 80
<210> 156
<211> 80
<212> DNA
<213> artificial sequence
<400> 156
cctcctgcag gaagggaatt gtcaagctca acatctactt ccaagaattt aactatcgca 60
ccattgaaga atcagcagcc 80
<210> 157
<211> 80
<212> DNA
<213> artificial sequence
<400> 157
agctcaacat ctacttccaa gaatttaact atcgcaccat tgaagaatca gcagccaata 60
acgtgagttt aggagtctcc 80
<210> 158
<211> 80
<212> DNA
<213> artificial sequence
<400> 158
cctgttcccc acagatcgtc tggctgctct cgaatctggg tggccagttt ggcttctgga 60
tggggggctc tgtgctgtgc 80
<210> 159
<211> 80
<212> DNA
<213> artificial sequence
<400> 159
tgctctcgaa tctgggtggc cagtttggct tctggatggg gggctctgtg ctgtgcctca 60
tcgagtttgg ggagatcatc 80
<210> 160
<211> 80
<212> DNA
<213> artificial sequence
<400> 160
ttggcttctg gatggggggc tctgtgctgt gcctcatcga gtttggggag atcatcatcg 60
actttgtgtg gatcaccatc 80
<210> 161
<211> 80
<212> DNA
<213> artificial sequence
<400> 161
tgctgtgcct catcgagttt ggggagatca tcatcgactt tgtgtggatc accatcatca 60
agctggtggc cttggccaag 80
<210> 162
<211> 80
<212> DNA
<213> artificial sequence
<400> 162
agatcatcat cgactttgtg tggatcacca tcatcaagct ggtggccttg gccaagagcc 60
tacggcagcg gcgagcccaa 80
<210> 163
<211> 80
<212> DNA
<213> artificial sequence
<400> 163
tcaccatcat caagctggtg gccttggcca agagcctacg gcagcggcga gcccaagcca 60
gctacgctgg cccaccgccc 80
<210> 164
<211> 80
<212> DNA
<213> artificial sequence
<400> 164
tggccaagag cctacggcag cggcgagccc aagccagcta cgctggccca ccgcccaccg 60
tggccgagct ggtggaggcc 80
<210> 165
<211> 80
<212> DNA
<213> artificial sequence
<400> 165
gagcccaagc cagctacgct ggcccaccgc ccaccgtggc cgagctggtg gaggcccaca 60
ccaactttgg cttccagcct 80
<210> 166
<211> 80
<212> DNA
<213> artificial sequence
<400> 166
caccgcccac cgtggccgag ctggtggagg cccacaccaa ctttggcttc cagcctgaca 60
cggccccccg cagccccaac 80
<210> 167
<211> 80
<212> DNA
<213> artificial sequence
<400> 167
tggaggccca caccaacttt ggcttccagc ctgacacggc cccccgcagc cccaacactg 60
ggccctaccc cagtgagcag 80
<210> 168
<211> 80
<212> DNA
<213> artificial sequence
<400> 168
tccagcctga cacggccccc cgcagcccca acactgggcc ctaccccagt gagcaggccc 60
tgcccatccc aggcaccccg 80
<210> 169
<211> 80
<212> DNA
<213> artificial sequence
<400> 169
gccccaacac tgggccctac cccagtgagc aggccctgcc catcccaggc accccgcccc 60
ccaactatga ctccctgcgt 80
<210> 170
<211> 80
<212> DNA
<213> artificial sequence
<400> 170
gtgagcaggc cctgcccatc ccaggcaccc cgccccccaa ctatgactcc ctgcgtctgc 60
agccgctgga cgtcatcgag 80
Claims (11)
1. a test kit that detects Liddle ' s syndrome associated gene mutation, it comprises: the probe of catching the non-coding region of object SCNN1B, the whole exon fragments of SCNN1G gene and contiguous 50bp; For the universal primer of the fragment to be measured that increases; Sequence measuring joints sequence; Damping fluid; DNTP; Exo+ polymerase; Streptomycin sulphate magnetic bead.
2. " a kind of test kit that detects Liddle ' s syndrome associated gene mutation " according to claim 1, all probe adopts biotin modification 3 '.
3. according to claim 1 " a kind of test kit that detects Liddle ' s syndrome associated gene mutation " this probe is oligomerization Yeast Nucleic Acid (RNA), or DNA oligo (DNA), but be not limited only to RNA and DNA, can comprise DNA or RNA after vitamin H, fluorescence or isotropic substance etc. are modified.
4. according to claim 1 the probe of " a kind of test kit that detects Liddle ' s syndrome associated gene mutation " is combined on magnetic bead, or is combined on slide glass, but is not limited only to these media.
5. according to claim 1 the probe of " a kind of test kit that detects Liddle ' s syndrome associated gene mutation " is for target area with the design of high-density stacked tile type, and probe sequence and SCNN1B, SCNN1G are complementary sequence.
6. according to claim 1 " a kind of test kit that detects Liddle ' s syndrome associated gene mutation " probe length is at 50-180bp, optimum is 75bp.
7. according to claim 1 " a kind of test kit that detects Liddle ' s syndrome associated gene mutation " probe length is at 60-200bp, optimum is 80bp.
8. according to claim 1 " a kind of test kit that detects Liddle ' s syndrome associated gene mutation " probe length is at 65-2200bp, optimum is 85bp.
9. according to claim 1 " a kind of test kit that detects Liddle ' s syndrome associated gene mutation " probe length is at 70-240bp, optimum is 90bp.
10. according to claim 1 " a kind of test kit that detects Liddle ' s syndrome associated gene mutation " probe lap optimum range is 5bp-20bp, and optimum is 8bp.
11. according to claim 1 " a kind of test kit that detects Liddle ' s syndrome associated gene mutation " Streptomycin sulphate magnetic bead material is Al-Ni-Co series permanent magnet alloy or barium ferrite, Nd-Fe-Bo permanent magnet material, Nd-Fe-Bo permanent magnet material, rubber magnet, but is not limited only to these materials.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410209085.4A CN104087657A (en) | 2014-05-19 | 2014-05-19 | Kit for examining Liddle's related gene mutation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410209085.4A CN104087657A (en) | 2014-05-19 | 2014-05-19 | Kit for examining Liddle's related gene mutation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104087657A true CN104087657A (en) | 2014-10-08 |
Family
ID=51635416
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410209085.4A Pending CN104087657A (en) | 2014-05-19 | 2014-05-19 | Kit for examining Liddle's related gene mutation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104087657A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110656112A (en) * | 2019-11-04 | 2020-01-07 | 百世诺（北京）医疗科技有限公司 | Liddle syndrome gene detection kit |
| CN113957141A (en) * | 2021-12-01 | 2022-01-21 | 长沙艾迪康医学检验实验室有限公司 | Oligonucleotide for detecting hypertension-related gene SCNN1B mutation and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102344961A (en) * | 2011-09-30 | 2012-02-08 | 康旭基因技术（北京）有限公司 | Economical multi-target multi-gene detection method applying large-scale parallel sequencing technology |
| WO2014052855A1 (en) * | 2012-09-27 | 2014-04-03 | Population Diagnostics, Inc. | Methods and compositions for screening and treating developmental disorders |
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2014
- 2014-05-19 CN CN201410209085.4A patent/CN104087657A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102344961A (en) * | 2011-09-30 | 2012-02-08 | 康旭基因技术（北京）有限公司 | Economical multi-target multi-gene detection method applying large-scale parallel sequencing technology |
| WO2014052855A1 (en) * | 2012-09-27 | 2014-04-03 | Population Diagnostics, Inc. | Methods and compositions for screening and treating developmental disorders |
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| Title |
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| ILLUMINA: "TruSeq® DNA Sample Prep Kits", 《DATA SHEET: SEQUENCING》 * |
| ILLUMINA: "TruSeq® DNA Sample Prep Kits", 《DATA SHEET: SEQUENCING》, 6 July 2012 (2012-07-06) * |
| ILLUMINA: "TruSeqTM Exome Enrichment Kit", 《DATA SHEET: SEQUENCING》 * |
| WANG LIN-PING: "Genetic diagnosis of Liddle’s syndrome by mutation analysis of SCNN1B and SCNN1G in a Chinese family", 《CHINESE MEDICAL JOURNAL》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110656112A (en) * | 2019-11-04 | 2020-01-07 | 百世诺（北京）医疗科技有限公司 | Liddle syndrome gene detection kit |
| CN113957141A (en) * | 2021-12-01 | 2022-01-21 | 长沙艾迪康医学检验实验室有限公司 | Oligonucleotide for detecting hypertension-related gene SCNN1B mutation and application thereof |
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Application publication date: 20141008 |