CN108707658A - A kind of personalized medicine gene detecting kit and application - Google Patents
A kind of personalized medicine gene detecting kit and application Download PDFInfo
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- CN108707658A CN108707658A CN201810600578.9A CN201810600578A CN108707658A CN 108707658 A CN108707658 A CN 108707658A CN 201810600578 A CN201810600578 A CN 201810600578A CN 108707658 A CN108707658 A CN 108707658A
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- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses a kind of personalized medicine gene detecting kit and applications.The personalized medicine gene detecting kit of the present invention, suitable for a variety of sample types, including dried blood spot, whole blood, buccal swab, all drug gene polymorphic site information can disposably be obtained, 50% or more sequencing effective percentage, sequencing coverage 100%, it is more than 3000X that mean depth, which is sequenced, builds Kucheng's power >=98.5%, drug gene polymorphic site detection accuracy >=99.9%；Wherein, the primer sets high specificity of the present invention, non-specific amplification is few, interfering with each other between primer can be overcome well, in the case of up to 186 primer sequences, realize a pipe multi-PRC reaction, and amplicon homogeneity is high, still all can effectively be expanded in the case where amplification region G/C content difference is big；Kit, primer sets and the banking process of the present invention can be applied in personalized medicine genetic test, and guidance is provided for personalized medicine.
Description
Technical field
The invention belongs to gene sequencing fields, in particular to a kind of personalized medicine gene detecting kit and answer
With.
Background technology
According to the data of World Health Organization WHO, in global Died Patients, one third dies of Irrational Use of Drugs,
Rather than die of nature disease itself, this causes adverse reaction related to the organ irreversible damage of generation with drug improper use, sternly
It even causes death when weight, especially rate of adverse reactions is about 13% in children, newborn's incidence higher
Up to 24.4%.In addition, there is also deficiencies for the validity of clinical application, especially in hypertension, hyperlipidemia, diabetes chronic diseases
Treatment in, the effective percentage of drug is less than 50%, caused by this is drug individuation difference, i.e.,：Use the same medicine of equivalent
Object, can have the difference of invalid crowd, safe and effective crowd and toxic reaction crowd, fast metabolic pattern subject may because failing and
When the active ingredient that absorbs the drug, and cause therapeutic effect bad；Slow inactivation subject then may in vivo put aside because of drug
And there is more serious adverse drug reaction.Therefore, science, reasonable, accurate, safe and effective personalized medicine guidance, are to carry
One of the key factor of high whole people's survival rate and disease treatment efficiency.
Detection of the personalized medicine guidance dependent on related drugs gene, that is, pass through detection and drug metabolism, curative effect and poison
Property relevant gene polymorphism sites, understand the drug metabolic rate of individual, determine the suitable dose of drug, avoid practical diagnosis and treatment
In the problem of reagent adjusts, is delayed best occasion for the treatment repeatedly, while can also evade the drug that can cause toxic reaction, reach peace
Entirely, the purpose of effective medication.
Using multiplex PCR capture and two generation sequencing technologies carry out genetic test have it is easy to operate, at low cost, the short time is big
The significant advantages such as amount enrichment capture region, have been substantially shorter testing process and detection time；However, due to drug metabolism, treating
Effect and the relevant gene polymorphism sites of toxicity up to up to a hundred, it is uniform that amplification is often will appear using multiplex PCR capture technique
Property it is poor, amplification region G/C content difference is big, and influence each other big the problems such as being difficult to realize pipe operation between multiple PCR primer, from
And Detection accuracy is caused to be difficult to ensure, and need higher sequencing cost；Further, since extremely more there are quantity in genome
Mononucleotide polymorphism site (SNP), therefore within the scope of the limitation of two generation sequencing reading lengths, it is inevitable in a detection sites up to a hundred
Several site peripheries that have contain several non-purpose SNP, then require amplification efficiency and specificity for the primer in such site
It is high, it is otherwise difficult to obtain excellent amplification depth in multiplex PCR system.Based on above-mentioned technical problem, at present still
It there are no based on the personalized medicine gene detecting kit of multiplex PCR capture technique and its report of related application.
Invention content
The purpose of the present invention is to provide a kind of personalized medicine gene detecting kits；A kind of personalized medicine gene inspection
Survey primer sets；A kind of nucleic acid sequencing banking process of detection personalized medicine genetic test；The kit, primer sets, nucleic acid
Application in nondiagnostic and non-therapeutic the safe medication genetic test of sequencing library banking process.
The technical solution used in the present invention is：
A kind of personalized medicine gene detecting kit, the kit include for expanding drug gene polymorphic site
Primer sets；
Optionally, the primer sets include for expanding at least 10 in 110 drug gene polymorphic sites shown in table 1
The primer of a gene polymorphism sites；
Optionally, the primer sets include for expanding at least 30 in 110 drug gene polymorphic sites shown in table 1
The primer of a gene polymorphism sites；
Optionally, the primer sets include for expanding at least 50 in 110 drug gene polymorphic sites shown in table 1
The primer of a gene polymorphism sites；
Optionally, the primer sets include for expanding at least 70 in 110 drug gene polymorphic sites shown in table 1
The primer of a gene polymorphism sites；
Optionally, the primer sets include for expanding at least 90 in 110 drug gene polymorphic sites shown in table 1
The primer of a gene polymorphism sites；
Optionally, the primer sets include the primer for expanding 110 drug gene polymorphic sites shown in table 1.
It is preferred as mentioned reagent box, for expanding the primer of 110 drug gene polymorphic sites shown in table 1 such as
SEQ ID NO:1~SEQ ID NO:Shown in 186.
Further as mentioned reagent box, every primer 5 ' end of the primer sets is connected with nucleic acid sequencing library construction
Required universal sequence.
It is preferred as mentioned reagent box, the pooling systems of each primer in the primer mixing match such as table 1 of the primer sets
Shown in number.
Preferred as mentioned reagent box, the kit further includes connector, label connector, multiplex PCR buffer solution, DNA
Polymerase, DMSO.
A kind of personalized medicine genetic test primer sets, the primer sets for including for above-mentioned kit.
A kind of nucleic acid sequencing library constructing method of personalized medicine genetic test, includes the following steps：Utilize above-mentioned examination
Agent box or primer sets, according to nucleic acid sequencing platform used, amplification sublibrary is mixed after purification, is used by structure amplification sublibrary
In nucleic acid sequencing.
Further as the above method, the nucleic acid sequencing platform includes Ion torrent platforms, Illumina flat
Platform, Roche454 platforms, SoLID platforms, 4000 microarray datasets of BioelectronSeq.
Preferred as the above method, the method further includes steps：It is connected with drawing for universal sequence using 5 ' ends
Object group, 3 ' ends are connected with the joint sequence of universal sequence and 3 ' and hold the label connector for being connected with universal sequence, and it is multiple to carry out a step
PCR amplification obtains amplification sublibrary, amplification sublibrary is mixed after purification, for Ion torrent sequencings.
Preferred as the above method, 25 μ L systems of a step multiplexed PCR amplification are：80~200ng of sample DNA, primer
0.6 μ L of group, 0.25 μ L of connector, 0.25 μ L of label connector, 12.5 μ L of multiplex PCR buffer solution (2X), archaeal dna polymerase (0.8~1.2)
U/ μ L, DMSO 1.5%~8% (V/V), nuclease-free water are surplus.
In nondiagnostic and non-therapeutic individuation is used for mentioned reagent box, primer sets, nucleic acid sequencing library constructing method
Application in medicine genetic test.
The beneficial effects of the invention are as follows：
The present invention provides a kind of personalized medicine gene detecting kits, are suitable for a variety of sample types, including dry blood
Piece, whole blood, buccal swab, can disposably obtain the information of all drug gene polymorphic sites, 50% or more sequencing effective percentage,
Coverage 100% is sequenced, sequencing mean depth is more than 3000X, builds Kucheng's power >=98.5%, the inspection of drug gene polymorphic site
Survey accuracy >=99.9%, wherein personalized medicine genetic test primer sets high specificity, non-specific amplification is few, can be very well
Ground overcomes interfering with each other between primer, in the case of up to 186 primer sequences, realizes a pipe multi-PRC reaction, and
Amplicon homogeneity is high, still all can effectively be expanded in the case where amplification region G/C content difference is big, reagent of the invention
Box, primer sets and banking process can be applied in personalized medicine genetic test, and guidance is provided for personalized medicine.
Description of the drawings
Fig. 1 is upper machine sequencing peak figure；
Fig. 2 is amplicon homogeneity figure, and abscissa represents each amplicon, and ordinate indicates sequencing depth.
Specific implementation mode
Term is explained：" personalized medicine " of the invention includes at least safe medication and effective medication, can be according to drug gene
The testing result of polymorphic site instructs the suitable amounts of individual choice safety drug and drug；For example, carbamazepine is to control
One of the common medicine of epilepsy and arrhythmia cordis is treated, meanwhile, carbamazepine can result in Stevens-Johnson syndromes/poisoning
Property epidermal necrolysis (SJS/TEN), and carry human leukocyte antigen HLA-B genes individual occur SJS/TEN wind
Danger improves hundreds of or even thousands of times, therefore, when the detection individual HLA-B*1502 or HLA-B*3101 positives, the medication suggestion provided
It is：Forbid to use carbamazepine, guidance is provided for individual screening safety drug；For another example, warfarin is widely used anti-freezing
One of drug, can prevention of deep vein thrombosis formation, atrial fibrillation, recurrent apoplexy or valvular heart prosthesis thromboembolia type disease
Disease, while Fa Hualin is also a kind of VKORC1 inhibitor, and the vitamin K of the co-factor as blood coagulating protein can be caused to reduce, to the greatest extent
Pipe warfarin is very effective, but dosage appropriate is selected still to have challenge, the polymorphic site rs9923231 on VKORC1 genes
Related to warfarin dose, therefore, when detection individual carries CC genotype, then suggestion is used according to normal dose, when detection
Body carries CT or TT genotype, then suggests that reducing dosage uses, and the guidance of drug suitable amounts is realized with this.
A kind of personalized medicine gene detecting kit, the kit include for expanding drug gene polymorphic site
Primer sets；
Optionally, the primer sets include for expanding at least 10 in 110 drug gene polymorphic sites shown in table 1
The primer of a gene polymorphism sites；
Optionally, the primer sets include for expanding at least 30 in 110 drug gene polymorphic sites shown in table 1
The primer of a gene polymorphism sites；
Optionally, the primer sets include for expanding at least 50 in 110 drug gene polymorphic sites shown in table 1
The primer of a gene polymorphism sites；
Optionally, the primer sets include for expanding at least 70 in 110 drug gene polymorphic sites shown in table 1
The primer of a gene polymorphism sites；
Optionally, the primer sets include for expanding at least 90 in 110 drug gene polymorphic sites shown in table 1
The primer of a gene polymorphism sites；
Optionally, the primer sets include the primer for expanding 110 drug gene polymorphic sites shown in table 1.
It is preferred as mentioned reagent box, for expanding the primer of 110 drug gene polymorphic sites shown in table 1 such as
SEQ ID NO:1~SEQ ID NO:Shown in 186.
Further as mentioned reagent box, every primer 5 ' end of the primer sets is connected with nucleic acid sequencing library construction
Required universal sequence；It should be noted that the microarray dataset that is utilized is different, library constructing method can difference, only need
Demand is built according to sequencing library, adaptively hold connection universal sequence in primer 5 ', you can library purpose, the survey are built in realization
Sequence platform include but not limited to Ion torrent platforms, Illumina platforms, Roche454 platforms, SoLID platforms,
4000 microarray datasets of BioelectronSeq；Wherein, by taking Ion torrent platforms as an example, universal sequence used is 5 '-
AAATGGGCGGTAGGCTTG-3’。
It is preferred as mentioned reagent box, the pooling systems of each primer in the primer mixing match such as table 1 of the primer sets
Shown in number；
Preferred as mentioned reagent box, the kit further includes connector, label connector, multiplex PCR buffer solution, DNA
Polymerase, DMSO；Wherein, connector and label connector can be equipped with according to microarray dataset；By taking Ion torrent platforms as an example, connector：
5'-CCTCTCT ATGGGCAGTCGGTGATAAATGGGCGGTAGGCTTG-3'；Label connector：5'-
CCATCTCATCCCTGCGTGT CTCCGACTCAGNNNNNNNNGATAAATGGGCGGTAGGCTTG-3 ', N is indicated in sequence
The arbitrary bases of AGCT, the libraries that different samples are built for identification NNNNNNNN.
A kind of personalized medicine genetic test primer sets, the primer sets for including for above-mentioned kit.
A kind of nucleic acid sequencing library constructing method of personalized medicine genetic test, includes the following steps：
Using mentioned reagent box or primer sets, according to nucleic acid sequencing platform used, structure amplification sublibrary, by amplicon
Library mixes after purification, is used for nucleic acid sequencing.
Further as the above method, the nucleic acid sequencing platform includes Ion torrent platforms, Illumina flat
Platform, Roche454 platforms, SoLID platforms, 4000 microarray datasets of BioelectronSeq, but not limited to this.
Preferred as the above method, the method further includes steps：It is connected with drawing for universal sequence using 5 ' ends
Object group, 3 ' ends are connected with the joint sequence of universal sequence and 3 ' and hold the label connector for being connected with universal sequence, and it is multiple to carry out a step
PCR amplification obtains amplification sublibrary, amplification sublibrary is mixed after purification, for Ion torrent sequencings.
Preferred as the above method, 25 μ L systems of a step multiplexed PCR amplification are：80~200ng of sample DNA, primer
0.6 μ L of group, 0.25 μ L of connector, 0.25 μ L of label connector, 12.5 μ L of multiplex PCR buffer solution (2X), archaeal dna polymerase (0.8~1.2)
U/ μ L, DMSO 1.5%~8% (V/V), nuclease-free water are surplus.
In nondiagnostic and non-therapeutic individuation is used for mentioned reagent box, primer sets, nucleic acid sequencing library constructing method
Application in medicine genetic test.
Table 1,110 drug gene polymorphic site and amplimer and pooling coefficient ranges
The present invention is explained further by the following examples, but the scope of the present invention is not limited thereto.
On the basis of not departing from primer sequence of the present invention, kit and library constructing method, those skilled in the art are not
The technical solution that creativity labour obtains is carried out to all belong to the scope of protection of the present invention.In embodiment not specified reagent and
Instrument is commercially available, and not specified experimental implementation presses manufacturers instruction or this field routine techniques is implemented.
Embodiment 1, a kind of personalized medicine genetic test primer sets
A kind of personalized medicine genetic test primer sets, including it is used to expand 110 drug gene polymorphic sites in table 1
Primer sets, SEQ ID NO in primer sequence such as table 1:1~SEQ ID NO:Shown in 186.
The present invention with reference to defending planning commission's technique of gene detection guide, U.S. Food and Drug Administration's FDA drug labels with
And NIH pharmGKB pharmacogenomics databases, filter out 110 and drug metabolism, curative effect and the relevant drug base of toxicity
Because of polymorphic site (as shown in table 1), which is distributed on 54 genes as shown in Table 2；Invention
People is directed to 110 selected gene polymorphism sites, creatively designs, screens, and optimization finally obtains multiple shown in table 1
PCR primer, high specificity, non-specific amplification is few, interfering with each other between primer can be overcome well, at up to 186
In the case of primer sequence, a pipe multi-PRC reaction is realized, and amplicon homogeneity is high, in amplification region G/C content difference
It still all can effectively be expanded in the case of big.
Table 2, drug metabolism of the present invention, curative effect and toxicity related gene
| ABCB1 | APOE | CYP2D6 | HTR1A | NOS1AP | SLC47A1 |
| ACE | C11orf65 | CYP3A5 | IFNL3 | NUDT15 | SLCO1B1 |
| ADD1 | CHIA | CYP4F2 | IFNL4 | OPRM1 | STXBP1 |
| ADRB1 | COMT | DRD2 | ITPA | POLG | TPMT |
| ADRB2 | CRHR1 | EPHX1 | LDLR | PPARG | UGT1A |
| AGTR1 | CYP1A1 | G6PD | LTA4H | SCN1A | UGT1A1 |
| ALDH2 | CYP2B6 | GRIK4 | LTC4S | SCN2A | UGT1A4 |
| ALOX5 | CYP2C19 | HLA-A | MT-RNR1 | SLC22A1 | UGT2B15 |
| ANKK1 | CYP2C9 | HLA-B | NAT2 | SLC22A2 | VKORC1 |
In addition, it is worth noting that：With the presence of the several non-mesh in 3 sites periphery in 110 drug gene polymorphic sites
SNP, be respectively：The relevant detection site rs3134792 of Allopurinol in Treatment, organic anion transport polypeptide OATP1B1's
Polymorphic site SLCO1B1521T>C (rs4149056) and the relevant CHIA of aspirin adverse reaction:c.-21G>The sites A
(rs3818822), inventor attempts to avoid the primer (hereinafter referred to as " custom primer ") of non-purpose SNP designs, but amplification
Unsatisfactory, for this purpose, inventor is by creatively testing, improving, (" present invention draws the primer finally obtained for following detection
Object ") it is high to the amplification efficiency in these three sites, homogenization depth is ideal.
By taking rs3134792, rs4149056, rs3818822 as an example, custom primer is as shown in table 3 below, primer of the present invention
With reference to shown in table 1, test result is as shown in Table 4：In multiplex PCR system, a large amount of primer dimerization are will produce using custom primer
Body or small fragment, homogenization depth is low, cannot be satisfied sequencing analysis requirement；It is high using the amplification efficiency of primer of the present invention, and
One changes depth ideal.
Table 3, custom primer
Table 4, two class primer test results
Embodiment 2, a kind of personalized medicine gene detecting kit
A kind of personalized medicine gene detecting kit, including：
(1) it is used to expand the primer sets of 110 drug gene polymorphic sites in table 1, SEQ in primer sequence such as table 1
ID NO:1~SEQ ID NO:Shown in 186,5 ' ends of every primer sequence are also associated with universal sequence, and universal sequence is：5'-
AAATGGGCGGTAGGCTTG-3'(SEQ ID NO:193)；Kit is in use, each primer in each primer mixing match such as table 1
Shown in pooling coefficients；
(2) connector：5'-CCTCTCTATGGGCAGTCGGTGATAAATGGGCGGTAGGCTTG-3'(SEQ ID NO:
194)；
(3) label connector：5'-CCATCTCATCCCTGCGTGTCTCCGACTCAGNNNNNNNNGATAAATGGGCGGTAG
GCTTG-3'(SEQ ID NO:195), N indicates the arbitrary bases of AGCT in sequence, and different samples are built NNNNNNNN for identification
Library；
(4) multiplex PCR buffer solution can provide the multiplex PCR buffer solution of the high concentrations such as 2X, 5X, 10X as needed；
(5) archaeal dna polymerase, can be according to being diluted to suitable concentration needed for amplification system；
(6) DMSO, can be according to being diluted to suitable concentration needed for amplification system.
Above-mentioned personalized medicine gene detecting kit is suitable for a variety of sample types, including dried blood spot, whole blood, oral cavity are wiped
Son, can disposably obtain the information of all gene polymorphism sites, and 50% or more sequencing effective percentage, sequencing coverage 100% are surveyed
Sequence mean depth is more than 3000X, builds Kucheng's power >=98.5%, drug gene polymorphic site detection accuracy >=99.9%.
Embodiment 3, a kind of nucleic acid sequencing library constructing method of personalized medicine genetic test
A kind of nucleic acid sequencing library constructing method of personalized medicine genetic test, includes the following steps：
(1) one step multiplexed PCR amplification
Using the kit of embodiment 2,25 μ L systems of a step multiplexed PCR amplification are configured, including：Sample DNA 200ng,
0.6 μ L of primer sets, 0.25 μ L of connector, 0.25 μ L of label connector, 12.5 μ L of multiplex PCR buffer solution (2X), archaeal dna polymerase 1U/ μ L,
DMSO 5% (V/V), nuclease-free water are surplus, wherein each primer mixing match is by shown in table 5 in primer sets；System is filled
Divide mixing to be put into PCR instrument after of short duration centrifugation, runs PCR response procedures：95℃3min；35 cyclic amplifications (95 DEG C of 10s,
58 DEG C of 1.5min, 72 DEG C of 30s), 72 DEG C of 1min, 16 DEG C of Hold.
Table 5, primer pooling coefficients
(2) amplification sublibrary mixing purifying
It takes multiple amplification sublibraries to mix in equal volume, mixed amplification sublibrary is taken into 100 μ L purifying, according to
The standard purification procedures that Beckman purifies magnetic bead carry out segment screening purifying；Concrete operations are as follows
1) the mixed amplification sublibraries of 100 μ L are transferred in the centrifuge tube for purifying magnetic bead equipped with 70 μ L Beckman, are mixed
5min is stored at room temperature after even；2) after 3~5s of brief centrifugation, centrifuge tube is put into magnetic frame and is clarified up to liquid；3) supernatant is turned
It moves on in the centrifuge tube equipped with 20 μ L Beckman purifying magnetic beads, 5min is stored at room temperature after mixing；Centrifuge tube is put into magnetic frame
It is clarified up to liquid；4) supernatant is transferred to the centrifugation that magnetic bead and 10 μ L Low TE buffer are purified equipped with 20 μ L Beckman
Guan Zhong is stored at room temperature 5min after mixing；Supernatant is abandoned in suction, and the ethyl alcohol of 250 μ L 70%~80% is added, and stands 30s, then siphon away
Clearly；It is primary to repeat this step；5) centrifuge tube is uncapped on magnetic frame, is placed in drying at room temperature about 5min；6) 50 μ L are added
Low TE buffer, mixing are stored at room temperature 5min；Centrifuge tube is put into magnetic frame to clarify up to liquid；7) supernatant is transferred to
In the centrifuge tube for purifying magnetic bead equipped with 40 μ L Beckman, 5min is stored at room temperature after mixing；8) centrifuge tube is put on magnetic frame
It is clarified to liquid；Supernatant is abandoned in suction, and the ethyl alcohol of 250 μ L 70~80% is added, and stands 30s, then siphon away supernatant；Repeat this step 1
It is secondary；9) centrifuge tube is uncapped on magnetic frame, is placed in drying at room temperature about 5min；10) 20 μ L Low TE buffer are added, mix
It is even, it is stored at room temperature 5min；11) supernatant is sucked out, that is, obtains amplification sublibrary after purification.
(3) library concentration measures
Concentration mensuration is carried out to amplification sublibrary after purification, to be determined for compliance with the Quality Control requirement of the upper machine of semiconductor sequencing.
(4) semiconductor is sequenced
The amplification sublibrary of purifying is sequenced for Ion torrent, including：Sequencing template prepares and enrichment, refers to Ion
PITM Hi QTMOT2 200Kit (A26434) operate with specification, and the microballon with template molecule is loaded into Ion
ProtonTMIt is sequenced on the semiconductor chip of sequenator, detailed step is referring to Ion PITM Hi QTMSequence 200Kit
It operates with specification and obtains the lower machine data of sequencing.
Bioinformatic analysis, including step are carried out to the sequencing data of above-mentioned acquisition：From the initial data of lower machine just
After step filters out low quality data, compares, sorts and carry out secondary filter；Mutation parsing and mutation are carried out to filtered data
It searches, generates mutational site information, mutation is understood eventually by mutational site information and drug comment file, obtain sample
This personalized medicine tutorial message.
Embodiment 4, primer sets of the present invention and kit specific amplification height, homogeneity are good
The primer sets and kit of the present invention are tested using the banking process of embodiment 3, peak is sequenced in machine in acquisition
For figure as shown in Figure 1, the uniform implementations of each amplicon are as shown in Fig. 2, visible primer amplification specificity is good, non-specific amplification is few,
Illustrate to interfere with each other between primer small, can realize one step multi-PRC reaction of a pipe, greatly save reaction timeliness, and respectively expand
Sub- homogeneity is good, and Quality Control requirement (it is required that amplicon depth is more than 30X) can be met in the case of little data amount, is greatlyd save
Cost is sequenced.
Embodiment 5, primer sets of the present invention and kit can overcome the problems, such as that amplification region G/C content difference is big
Amplification region G/C content difference where 110 drug gene polymorphic sites of the present invention is big, G/C content
Statistical data is as shown in table 6；It is tested using the banking process of embodiment 3, successfully reaches Quality Control requirement (it is required that amplicon is deep
Degree be more than 30X) amplicon number be 91, gene polymorphism sites recall rate 100%.
Table 6, amplification region G/C content statistics
Embodiment 6, primer sets of the present invention and the applicable multisample type of kit
Using 345 samples of banking process pair (including whole blood 160, dried blood spot 105, the buccal swab 80 of embodiment 3
Example) it is tested, 340 samples can be detected, it is 98.55% to build Kucheng's power, refers to table 7.
Table 7, all types of samples
By taking 10 samples as an example (3 dried blood spots, 4 whole bloods, 3 buccal swabs), more specifically test result is provided：Respectively
DNA extraction kit MagPure Tissue& of the DNA extractions of class sample with reference to Magen;Blood DNA LQ Kit, using reality
The banking process for applying example 3 is tested, and it is quantitative to carry out Qubit to amplification sublibrary after purification, quantitative concentrations in 4.3ng/ μ L,
It is sequenced by machine in each sample 0.6M data, sample can reach quality control index shown in table 8：Effective percentage is up to 53.43%, coverage rate
It is 100%, amplicon mean depth is 4844, it is seen that Library Quality is good.
Table 8, quality control index
Table 9 provides the drug gene polymorphic position points that above 10 samples are measured and site primer result is tested with sanger
The coincidence rate situation of card, it is seen that the total 11000 site primer results of 10 samples are consistent with sanger, since this was sequenced in two generations
There are 99.9% base sniffing rates for body, therefore estimate that primer sets and kit detection drug gene polymorphic site of the present invention is accurate
True rate at least 99.9%.Table 9, sample detection number of sites and the coincidence rate with sanger verifications
SEQUENCE LISTING
<110>Dongguan Bo Aomuhua Gene Tech. Company Limited
<120>A kind of personalized medicine gene detecting kit and application
<130>
<160> 195
<170> PatentIn version 3.5
<210> 1
<211> 23
<212> DNA
<213>Primer
<400> 1
tctgaaagtg aactccctgc tac 23
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<211> 19
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<213>Primer
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ctgggactcc acagccatg 19
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<213>Primer
<400> 3
gagcatagta agcagtaggg agt 23
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<213>Primer
<400> 4
gcaatagcag gagttgttga aatg 24
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atcaacttct gtactgggct ctg 23
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<213>Primer
<400> 6
cccagcatcg acaatgtaat tcc 23
<210> 7
<211> 23
<212> DNA
<213>Primer
<400> 7
gtgggcttca tcctcaccta tag 23
<210> 8
<211> 23
<212> DNA
<213>Primer
<400> 8
tgggtgatac atacacaagg gtt 23
<210> 9
<211> 18
<212> DNA
<213>Primer
<400> 9
tgagcgcgtc acttcctg 18
<210> 10
<211> 21
<212> DNA
<213>Primer
<400> 10
caaccgagcc tttcctcttc c 21
<210> 11
<211> 23
<212> DNA
<213>Primer
<400> 11
ttcttttcta ggttgggagt ggg 23
<210> 12
<211> 23
<212> DNA
<213>Primer
<400> 12
ggccacctag agatgatttc ctt 23
<210> 13
<211> 23
<212> DNA
<213>Primer
<400> 13
tcttttctct tggaacaggt cgt 23
<210> 14
<211> 23
<212> DNA
<213>Primer
<400> 14
agggaaacag acacacagaa agt 23
<210> 15
<211> 20
<212> DNA
<213>Primer
<400> 15
attcctcctg ggacgctcaa 20
<210> 16
<211> 22
<212> DNA
<213>Primer
<400> 16
ctacgcttcc aaaaggcttt cc 22
<210> 17
<211> 21
<212> DNA
<213>Primer
<400> 17
ggctttgtcc aagagaccgt t 21
<210> 18
<211> 17
<212> DNA
<213>Primer
<400> 18
ttgtgccgcc ttcgccg 17
<210> 19
<211> 18
<212> DNA
<213>Primer
<400> 19
ccccaccctt tcctcacc 18
<210> 20
<211> 20
<212> DNA
<213>Primer
<400> 20
gcctcccaag ccatactatg 20
<210> 21
<211> 20
<212> DNA
<213>Primer
<400> 21
agtgcgtgag tgtctcagtg 20
<210> 22
<211> 18
<212> DNA
<213>Primer
<400> 22
ggtacctgga cgacccca 18
<210> 23
<211> 19
<212> DNA
<213>Primer
<400> 23
ccacctcagc accatgagg 19
<210> 24
<211> 22
<212> DNA
<213>Primer
<400> 24
catcatcgtg gagaagccct tc 22
<210> 25
<211> 23
<212> DNA
<213>Primer
<400> 25
ttgaagaagg gctcactctg ttt 23
<210> 26
<211> 20
<212> DNA
<213>Primer
<400> 26
ctggttacag ctgtgccctg 20
<210> 27
<211> 23
<212> DNA
<213>Primer
<400> 27
gtgcttagtt gaacagggcc ctg 23
<210> 28
<211> 23
<212> DNA
<213>Primer
<400> 28
ttaagctaca ctctggttcg tcc 23
<210> 29
<211> 23
<212> DNA
<213>Primer
<400> 29
gcatcactca ctttgtgacc att 23
<210> 30
<211> 23
<212> DNA
<213>Primer
<400> 30
ggagctgaag gactactacc tct 23
<210> 31
<211> 22
<212> DNA
<213>Primer
<400> 31
cttcacaaag cggaagaatg tg 22
<210> 32
<211> 23
<212> DNA
<213>Primer
<400> 32
ctcctgactg tcatccctat tgg 23
<210> 33
<211> 21
<212> DNA
<213>Primer
<400> 33
ttaaatggag ggagtcctga c 21
<210> 34
<211> 23
<212> DNA
<213>Primer
<400> 34
taaagggaca gtgatcacat ggg 23
<210> 35
<211> 21
<212> DNA
<213>Primer
<400> 35
ttaaatggag ggagtcatga c 21
<210> 36
<211> 23
<212> DNA
<213>Primer
<400> 36
taaagggaca gtgatcacgt ggg 23
<210> 37
<211> 23
<212> DNA
<213>Primer
<400> 37
ttggctgtgc tattttccat tcc 23
<210> 38
<211> 23
<212> DNA
<213>Primer
<400> 38
acatgttttg ttcaccggtt ctg 23
<210> 39
<211> 23
<212> DNA
<213>Primer
<400> 39
tcctactggc ggaatgaatt tga 23
<210> 40
<211> 23
<212> DNA
<213>Primer
<400> 40
ttgtggaaga aggctgttct cat 23
<210> 41
<211> 23
<212> DNA
<213>Primer
<400> 41
gacatccact tcatccacgt gaa 23
<210> 42
<211> 23
<212> DNA
<213>Primer
<400> 42
tcagtcagga gtgggatgat ctt 23
<210> 43
<211> 23
<212> DNA
<213>Primer
<400> 43
agcactttct tatgcaagga gct 23
<210> 44
<211> 23
<212> DNA
<213>Primer
<400> 44
tttctcttct gcaatgcgtt gtt 23
<210> 45
<211> 23
<212> DNA
<213>Primer
<400> 45
tttgtgagct ttgccaccta aac 23
<210> 46
<211> 22
<212> DNA
<213>Primer
<400> 46
atcacttacc ctgcatctga gc 22
<210> 47
<211> 21
<212> DNA
<213>Primer
<400> 47
aggtccacaa actctgtgac g 21
<210> 48
<211> 23
<212> DNA
<213>Primer
<400> 48
ttttcagctc ttcgcacttt cag 23
<210> 49
<211> 22
<212> DNA
<213>Primer
<400> 49
ctgagagtgg aaaggtgttg gt 22
<210> 50
<211> 23
<212> DNA
<213>Primer
<400> 50
ggctgtcagg gtgaaaaatt tct 23
<210> 51
<211> 23
<212> DNA
<213>Primer
<400> 51
cagcagaggg gacatgaaat agt 23
<210> 52
<211> 23
<212> DNA
<213>Primer
<400> 52
atgcccgaga ctaacaaaag act 23
<210> 53
<211> 21
<212> DNA
<213>Primer
<400> 53
tttgcataaa ttaatcagcc c 21
<210> 54
<211> 21
<212> DNA
<213>Primer
<400> 54
acgtcctctg aaaaatggca c 21
<210> 55
<211> 20
<212> DNA
<213>Primer
<400> 55
aacatggcct gtttgggagt 20
<210> 56
<211> 22
<212> DNA
<213>Primer
<400> 56
gatccttaac caataatggt ca 22
<210> 57
<211> 23
<212> DNA
<213>Primer
<400> 57
cagtgtgaat tacagcaaac ccc 23
<210> 58
<211> 23
<212> DNA
<213>Primer
<400> 58
ccaatagccg tatctggaag gaa 23
<210> 59
<211> 23
<212> DNA
<213>Primer
<400> 59
tgaggttgag tgacatgttc gaa 23
<210> 60
<211> 23
<212> DNA
<213>Primer
<400> 60
agaaaagtcg gttcagtcca cat 23
<210> 61
<211> 23
<212> DNA
<213>Primer
<400> 61
ttactccttt gctagtgacg gtg 23
<210> 62
<211> 23
<212> DNA
<213>Primer
<400> 62
gatcgtgtgt aatgttcgtc cac 23
<210> 63
<211> 26
<212> DNA
<213>Primer
<400> 63
actgtagtca taatattccc aacaca 26
<210> 64
<211> 24
<212> DNA
<213>Primer
<400> 64
tgtcaatgcc agtaaatcat ctgc 24
<210> 65
<211> 23
<212> DNA
<213>Primer
<400> 65
cccccatttt tcagtttcac tgt 23
<210> 66
<211> 23
<212> DNA
<213>Primer
<400> 66
cggctctaac cttatcggat tca 23
<210> 67
<211> 23
<212> DNA
<213>Primer
<400> 67
tgagaaaagg aagcataggg agc 23
<210> 68
<211> 23
<212> DNA
<213>Primer
<400> 68
ttcgtttgtt tttggagacg gag 23
<210> 69
<211> 18
<212> DNA
<213>Primer
<400> 69
ctcacctgcc agactgcg 18
<210> 70
<211> 23
<212> DNA
<213>Primer
<400> 70
ggacgatgag agacatgacg atg 23
<210> 71
<211> 23
<212> DNA
<213>Primer
<400> 71
ggactccatt ctgaagccaa agg 23
<210> 72
<211> 20
<212> DNA
<213>Primer
<400> 72
cttgcagacc cccacatcat 20
<210> 73
<211> 23
<212> DNA
<213>Primer
<400> 73
tcctcaaaaa catgtcagtg tga 23
<210> 74
<211> 23
<212> DNA
<213>Primer
<400> 74
cgttgtcttg agaaggttga tgc 23
<210> 75
<211> 23
<212> DNA
<213>Primer
<400> 75
ggatgatgcc tcctgatcta tcg 23
<210> 76
<211> 23
<212> DNA
<213>Primer
<400> 76
gtctagggac aggaagatgg ttg 23
<210> 77
<211> 23
<212> DNA
<213>Primer
<400> 77
tatagtgtga gacagctgcc ttg 23
<210> 78
<211> 23
<212> DNA
<213>Primer
<400> 78
ttatgcctac acgaacacag aca 23
<210> 79
<211> 23
<212> DNA
<213>Primer
<400> 79
tggagtccag tgtttctctg atg 23
<210> 80
<211> 23
<212> DNA
<213>Primer
<400> 80
ctaaggttat cgcagcaaca gtg 23
<210> 81
<211> 21
<212> DNA
<213>Primer
<400> 81
ggatcatgga ggagaaggta g 21
<210> 82
<211> 23
<212> DNA
<213>Primer
<400> 82
tccctatttt tctgactgtc ccg 23
<210> 83
<211> 21
<212> DNA
<213>Primer
<400> 83
ctctggggag aatgtgagtc c 21
<210> 84
<211> 23
<212> DNA
<213>Primer
<400> 84
cgggtccttc ttcctgaata ctc 23
<210> 85
<211> 23
<212> DNA
<213>Primer
<400> 85
ctttcttttc cttccaacac ccc 23
<210> 86
<211> 23
<212> DNA
<213>Primer
<400> 86
gcccagagga gctcaaaaat ttt 23
<210> 87
<211> 19
<212> DNA
<213>Primer
<400> 87
cctgagcaac atttgaggc 19
<210> 88
<211> 20
<212> DNA
<213>Primer
<400> 88
atgtcttgat tccacctatc 20
<210> 89
<211> 19
<212> DNA
<213>Primer
<400> 89
cctcagcaac acttgaggc 19
<210> 90
<211> 23
<212> DNA
<213>Primer
<400> 90
tcagggtaac aagagcgaaa ctc 23
<210> 91
<211> 23
<212> DNA
<213>Primer
<400> 91
cagaatgtac tccagctgct cta 23
<210> 92
<211> 24
<212> DNA
<213>Primer
<400> 92
ggaaaataga ggaaaagatg ggca 24
<210> 93
<211> 23
<212> DNA
<213>Primer
<400> 93
gctctttcca tgaagttgtg tct 23
<210> 94
<211> 23
<212> DNA
<213>Primer
<400> 94
ggatctatta cctgtgcctg gag 23
<210> 95
<211> 23
<212> DNA
<213>Primer
<400> 95
agggtagaag gtcctggatt cta 23
<210> 96
<211> 23
<212> DNA
<213>Primer
<400> 96
tatgccacca cccatagcta atc 23
<210> 97
<211> 23
<212> DNA
<213>Primer
<400> 97
attctgaatt ggcctgagag agg 23
<210> 98
<211> 23
<212> DNA
<213>Primer
<400> 98
tcttgtgaac aggacatttg aca 23
<210> 99
<211> 24
<212> DNA
<213>Primer
<400> 99
ggacaagcta tcaaaatgac acca 24
<210> 100
<211> 21
<212> DNA
<213>Primer
<400> 100
cgtactcaag ttgctcccca g 21
<210> 101
<211> 22
<212> DNA
<213>Primer
<400> 101
gatggagtag agggccatga tc 22
<210> 102
<211> 23
<212> DNA
<213>Primer
<400> 102
ccttcatagc cctcatcacc att 23
<210> 103
<211> 23
<212> DNA
<213>Primer
<400> 103
caaagagtca caacactttc ccc 23
<210> 104
<211> 23
<212> DNA
<213>Primer
<400> 104
aggcagactt cttagcagaa taa 23
<210> 105
<211> 23
<212> DNA
<213>Primer
<400> 105
accaagttgc ctatacagtt ggg 23
<210> 106
<211> 23
<212> DNA
<213>Primer
<400> 106
gctcccaggc tgtttatttg aag 23
<210> 107
<211> 23
<212> DNA
<213>Primer
<400> 107
gcattgctga gaacattgcc tat 23
<210> 108
<211> 23
<212> DNA
<213>Primer
<400> 108
ccacccaagg cttcatatga tga 23
<210> 109
<211> 23
<212> DNA
<213>Primer
<400> 109
gagataccca cgtatgtacc acc 23
<210> 110
<211> 25
<212> DNA
<213>Primer
<400> 110
atgactccta tagcatccat agcaa 25
<210> 111
<211> 23
<212> DNA
<213>Primer
<400> 111
atttactggg catttgagca ctg 23
<210> 112
<211> 23
<212> DNA
<213>Primer
<400> 112
ttgcttttgc cacaggagat ttt 23
<210> 113
<211> 24
<212> DNA
<213>Primer
<400> 113
tgaaagctct aaactcaaag ggga 24
<210> 114
<211> 25
<212> DNA
<213>Primer
<400> 114
tgtttggaag ttgttttgtt ttgct 25
<210> 115
<211> 23
<212> DNA
<213>Primer
<400> 115
ggatttgagc tgaggtcttc tga 23
<210> 116
<211> 23
<212> DNA
<213>Primer
<400> 116
ctgcaatgtg atctgctcca tta 23
<210> 117
<211> 23
<212> DNA
<213>Primer
<400> 117
tgtaagtggt ttctcaggaa gca 23
<210> 118
<211> 23
<212> DNA
<213>Primer
<400> 118
ccagagcttg gcatattgta tct 23
<210> 119
<211> 23
<212> DNA
<213>Primer
<400> 119
tggtgttctt ttactttctc caa 23
<210> 120
<211> 23
<212> DNA
<213>Primer
<400> 120
gaaagaaatg gaaggagatc cgg 23
<210> 121
<211> 23
<212> DNA
<213>Primer
<400> 121
acccttggtt tttctcaact cct 23
<210> 122
<211> 23
<212> DNA
<213>Primer
<400> 122
gattgaacgt gtgattggca gaa 23
<210> 123
<211> 23
<212> DNA
<213>Primer
<400> 123
acaaacttac cttgggaatg aga 23
<210> 124
<211> 20
<212> DNA
<213>Primer
<400> 124
agccctgcgc gcgcagcaga 20
<210> 125
<211> 20
<212> DNA
<213>Primer
<400> 125
ccttcaaccc catcatctac 20
<210> 126
<211> 24
<212> DNA
<213>Primer
<400> 126
ggcaacaaac aggaaacaat taca 24
<210> 127
<211> 23
<212> DNA
<213>Primer
<400> 127
aaagtgggtt gcttgtggat aac 23
<210> 128
<211> 23
<212> DNA
<213>Primer
<400> 128
acctcctaga acatcacgca aat 23
<210> 129
<211> 20
<212> DNA
<213>Primer
<400> 129
cagctcactc catcctggac 20
<210> 130
<211> 22
<212> DNA
<213>Primer
<400> 130
gattccgcca tcccttgttt tg 22
<210> 131
<211> 23
<212> DNA
<213>Primer
<400> 131
cttggcttct gagtcctcaa agg 23
<210> 132
<211> 23
<212> DNA
<213>Primer
<400> 132
actggaaaga agtggactgg ttt 23
<210> 133
<211> 23
<212> DNA
<213>Primer
<400> 133
aaagtaaacc cacctcttcc ctc 23
<210> 134
<211> 20
<212> DNA
<213>Primer
<400> 134
ttaaaatgaa acactctctt 20
<210> 135
<211> 23
<212> DNA
<213>Primer
<400> 135
agaaagcccc aatggtacta tgg 23
<210> 136
<211> 23
<212> DNA
<213>Primer
<400> 136
agaaagcccc agtggtacta tgg 23
<210> 137
<211> 23
<212> DNA
<213>Primer
<400> 137
caaaccacaa acaaaaacag ccc 23
<210> 138
<211> 22
<212> DNA
<213>Primer
<400> 138
cttgaacacc ttggttcagc at 22
<210> 139
<211> 23
<212> DNA
<213>Primer
<400> 139
agggtcaact gctatgatgt gtt 23
<210> 140
<211> 19
<212> DNA
<213>Primer
<400> 140
ctccgagcca ccagcagac 19
<210> 141
<211> 23
<212> DNA
<213>Primer
<400> 141
ctggtgagtt tttagggact ggt 23
<210> 142
<211> 23
<212> DNA
<213>Primer
<400> 142
tcctagacag ctgggacaat aga 23
<210> 143
<211> 20
<212> DNA
<213>Primer
<400> 143
agtcccctgc ctagatcctg 20
<210> 144
<211> 22
<212> DNA
<213>Primer
<400> 144
ctacctgcct gtcaaccaga ac 22
<210> 145
<211> 23
<212> DNA
<213>Primer
<400> 145
aatggaaaga gctttggaga cca 23
<210> 146
<211> 23
<212> DNA
<213>Primer
<400> 146
ccctagatgt ggggcttcta gat 23
<210> 147
<211> 23
<212> DNA
<213>Primer
<400> 147
aaaggtgatt tccaagaagc cac 23
<210> 148
<211> 23
<212> DNA
<213>Primer
<400> 148
atagggtcag tgacatggaa tcc 23
<210> 149
<211> 23
<212> DNA
<213>Primer
<400> 149
aaactcctga cctcaagtga tcc 23
<210> 150
<211> 23
<212> DNA
<213>Primer
<400> 150
agaagggtag gtgcaacagt aag 23
<210> 151
<211> 23
<212> DNA
<213>Primer
<400> 151
cccagtttgt gctaagcatc gta 23
<210> 152
<211> 23
<212> DNA
<213>Primer
<400> 152
tggtgggaaa acttggtcct aaa 23
<210> 153
<211> 23
<212> DNA
<213>Primer
<400> 153
cgattgtcta gagccttctc ctc 23
<210> 154
<211> 20
<212> DNA
<213>Primer
<400> 154
gcctctgaga gcaaaggagg 20
<210> 155
<211> 23
<212> DNA
<213>Primer
<400> 155
gaaatgatct ggctgaatcc gtg 23
<210> 156
<211> 20
<212> DNA
<213>Primer
<400> 156
gtcctcacgg ccaaggtaac 20
<210> 157
<211> 23
<212> DNA
<213>Primer
<400> 157
gagggaggtg atgttggata ctc 23
<210> 158
<211> 23
<212> DNA
<213>Primer
<400> 158
ttctctccca caggcattat ctg 23
<210> 159
<211> 22
<212> DNA
<213>Primer
<400> 159
gattcctgga cgtggatggg ta 22
<210> 160
<211> 22
<212> DNA
<213>Primer
<400> 160
cagggtcaat cacagaaggg ag 22
<210> 161
<211> 23
<212> DNA
<213>Primer
<400> 161
agtcttgtat ttcacctcct gga 23
<210> 162
<211> 23
<212> DNA
<213>Primer
<400> 162
tgtgggagaa tgcaaatgag aga 23
<210> 163
<211> 17
<212> DNA
<213>Primer
<400> 163
ctgatgttcc ccaggca 17
<210> 164
<211> 23
<212> DNA
<213>Primer
<400> 164
tcagcatctt caggaactct tga 23
<210> 165
<211> 21
<212> DNA
<213>Primer
<400> 165
ctggaggaac aactgacccc g 21
<210> 166
<211> 19
<212> DNA
<213>Primer
<400> 166
gtgctctggc cgagcatgg 19
<210> 167
<211> 20
<212> DNA
<213>Primer
<400> 167
gctgcgtaag cggctcctcc 20
<210> 168
<211> 20
<212> DNA
<213>Primer
<400> 168
cacgcggccc tgttccacca 20
<210> 169
<211> 21
<212> DNA
<213>Primer
<400> 169
atcaccatcg agatcaaccc c 21
<210> 170
<211> 23
<212> DNA
<213>Primer
<400> 170
ttttcagtga acgtggtgtg aac 23
<210> 171
<211> 23
<212> DNA
<213>Primer
<400> 171
gggactaggt accccattct agc 23
<210> 172
<211> 23
<212> DNA
<213>Primer
<400> 172
gagctcttcc tcttcttcac ctc 23
<210> 173
<211> 23
<212> DNA
<213>Primer
<400> 173
tatgttggag gaggtcaggc tta 23
<210> 174
<211> 23
<212> DNA
<213>Primer
<400> 174
ctcctgctca tgatcctaca tcc 23
<210> 175
<211> 18
<212> DNA
<213>Primer
<400> 175
cgtggtcgag gtggtcac 18
<210> 176
<211> 22
<212> DNA
<213>Primer
<400> 176
gttctgtccc gagtatgctc tc 22
<210> 177
<211> 23
<212> DNA
<213>Primer
<400> 177
tatggtgtgt tctggaagtc cac 23
<210> 178
<211> 22
<212> DNA
<213>Primer
<400> 178
tttggtagtg aggcaggtat gg 22
<210> 179
<211> 23
<212> DNA
<213>Primer
<400> 179
ctgaggggac atagtatggc ttg 23
<210> 180
<211> 21
<212> DNA
<213>Primer
<400> 180
cagaacgtga agctccctga c 21
<210> 181
<211> 20
<212> DNA
<213>Primer
<400> 181
aggaccacat tgttggcctg 20
<210> 182
<211> 19
<212> DNA
<213>Primer
<400> 182
agtcatccct gcaccccaa 19
<210> 183
<211> 23
<212> DNA
<213>Primer
<400> 183
ttagttggaa aagctgaggc atg 23
<210> 184
<211> 22
<212> DNA
<213>Primer
<400> 184
gatcctgcgg gaagagcttt tc 22
<210> 185
<211> 21
<212> DNA
<213>Primer
<400> 185
gagccactcc catcctttct c 21
<210> 186
<211> 23
<212> DNA
<213>Primer
<400> 186
ttagctcacc tctgcttgta agg 23
<210> 187
<211> 23
<212> DNA
<213>Primer
<400> 187
ggagttagag tttagcctga gca 23
<210> 188
<211> 23
<212> DNA
<213>Primer
<400> 188
tccacctatc tctgcattgc tac 23
<210> 189
<211> 20
<212> DNA
<213>Primer
<400> 189
aaattaatgt ttaaaatgaa 20
<210> 190
<211> 20
<212> DNA
<213>Primer
<400> 190
gtactatggg agtctcccct 20
<210> 191
<211> 22
<212> DNA
<213>Primer
<400> 191
gcaatgtatt ttaaatggag gg 22
<210> 192
<211> 19
<212> DNA
<213>Primer
<400> 192
ggggctaact tgttttcca 19
<210> 193
<211> 18
<212> DNA
<213>Universal sequence
<400> 193
aaatgggcgg taggcttg 18
<210> 194
<211> 41
<212> DNA
<213>Connector
<400> 194
cctctctatg ggcagtcggt gataaatggg cggtaggctt g 41
<210> 195
<211> 59
<212> DNA
<213>Label connector
<220>
<221> misc_feature
<222> (31)..(38)
<223> n is a,c,g,or t
<400> 195
ccatctcatc cctgcgtgtc tccgactcag nnnnnnnnga taaatgggcg gtaggcttg 59
Claims (11)
1. a kind of personalized medicine gene detecting kit, the kit includes for expanding drug gene polymorphic site
Primer sets；
Optionally, the primer sets include for expanding at least ten base in 110 drug gene polymorphic sites shown in table 1
Because of the primer of polymorphic site；
Optionally, the primer sets include for expanding at least 30 bases in 110 drug gene polymorphic sites shown in table 1
Because of the primer of polymorphic site；
Optionally, the primer sets include for expanding at least 50 bases in 110 drug gene polymorphic sites shown in table 1
Because of the primer of polymorphic site；
Optionally, the primer sets include for expanding at least 70 bases in 110 drug gene polymorphic sites shown in table 1
Because of the primer of polymorphic site；
Optionally, the primer sets include for expanding at least 90 bases in 110 drug gene polymorphic sites shown in table 1
Because of the primer of polymorphic site；
Optionally, the primer sets include the primer for expanding 110 drug gene polymorphic sites shown in table 1.
2. personalized medicine gene detecting kit according to claim 1, it is characterised in that：For expanding shown in table 1
The primer of 110 drug gene polymorphic sites such as SEQ ID NO:1~SEQ ID NO:Shown in 186.
3. personalized medicine gene detecting kit according to claim 1, it is characterised in that：Every of the primer sets
Primer 5 ' holds the universal sequence being connected with needed for nucleic acid sequencing library construction.
4. according to claims 1 to 3 any one of them personalized medicine gene detecting kit, it is characterised in that：It is described to draw
In the primer mixing match such as table 1 of object group shown in the pooling coefficients of each primer.
5. according to claims 1 to 3 any one of them personalized medicine gene detecting kit, it is characterised in that：The examination
Agent box further includes connector, label connector, multiplex PCR buffer solution, archaeal dna polymerase, DMSO.
6. a kind of personalized medicine genetic test primer sets are drawn for what Claims 1 to 5 any one of them kit included
Object group.
7. a kind of nucleic acid sequencing library constructing method of personalized medicine genetic test, includes the following steps：Utilize claim 1
Primer sets described in~5 any one of them kits or claim 6, according to nucleic acid sequencing platform used, structure amplification
Amplification sublibrary is mixed after purification, is used for nucleic acid sequencing by sublibrary.
8. nucleic acid sequencing library constructing method according to claim 7, it is characterised in that：The nucleic acid sequencing platform includes
Ion torrent platforms, Illumina platforms, Roche454 platforms, SoLID platforms, the sequencings of BioelectronSeq 4000 are flat
Platform.
9. nucleic acid sequencing czzz library constructing methods according to claim 7, it is characterised in that：The method is further wrapped
Include step：Be connected with the primer sets of universal sequence using 5 ' ends, 3 ' ends are connected with the connector of universal sequence and 3 ' ends be connected with it is logical
With the label connector of sequence, a step multiplexed PCR amplification is carried out, obtains amplification sublibrary, amplification sublibrary is mixed after purification, is used
It is sequenced in Ion torrent.
10. nucleic acid sequencing library constructing method according to claim 9, it is characterised in that：The 25 of one step multiplexed PCR amplification
μ L systems are：80~200ng of sample DNA, 0.6 μ L of primer sets, 0.25 μ L of connector, 0.25 μ L of label connector, multiplex PCR buffering
12.5 μ L of liquid (2X), archaeal dna polymerase (0.8~1.2) U/ μ L, DMSO 1.5%~8% (V/V), nuclease-free water are surplus.
11. the primer sets, claim 7~10 described in Claims 1 to 5 any one of them kit, claim 6 are any
The application in nondiagnostic and non-therapeutic the personalized medicine genetic test of nucleic acid sequencing library constructing method described in.
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