CN107604067A - A kind of primer and kit for the mutation of testing goal gene low frequency - Google Patents
A kind of primer and kit for the mutation of testing goal gene low frequency Download PDFInfo
- Publication number
- CN107604067A CN107604067A CN201710976796.8A CN201710976796A CN107604067A CN 107604067 A CN107604067 A CN 107604067A CN 201710976796 A CN201710976796 A CN 201710976796A CN 107604067 A CN107604067 A CN 107604067A
- Authority
- CN
- China
- Prior art keywords
- sequence
- primer
- downstream
- specific primer
- dna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of primer and kit for the mutation of testing goal gene low frequency.The invention provides the catastrophe primer set for detecting sample to be tested target gene region to be checked, including Barcode primers Fs 1, sense primer F2, downstream outer primer R1, downstream inner primer R2.The primer set that the present invention designs is used to build storehouse, in addition to it can detect tissue samples, the different zones to dissociative DNA in blood, urine and cerebrospinal fluid equal samples that can also be quick, easy, sensitive, special carry out targeting amplification, and the mutation that efficient detection as little as 0.1% is horizontal, greatly simplify experimental implementation, effectively avoid library from losing and pollute, significantly reduce cost, improve efficiency.
Description
Technical field
The invention belongs to biological technical field, more particularly to a kind of primer and examination for the mutation of testing goal gene low frequency
Agent box.
Background technology
Tumour has height heterogeneity, and pathogenic mutation therein may exist with extremely low ratio, the blood of tumor patient,
The mutational site of the target gene in cfDNA in urine and cerebrospinal fluid or the frequency of mutation of sudden change region can influence tumour in future
Medication or the judgement in tumor development direction.Therefore, detect in the cfDNA in blood, urine and the cerebrospinal fluid of tumor patient
The mutational site of target gene or the frequency of mutation of sudden change region turn into research emphasis, and this is just needed to mutational site or mutation
Region is sequenced, and detects the frequency of mutation.
The most accurate Hiseq sequencings error rate of itself is sequenced just about 0.2% in current two generation, in addition, currently
The amplification error rate of archaeal dna polymerase is also 10-7-10-5Between, therefore, how in sequencing result exclude amplification mistake and
Mistake is sequenced, directly reflecting the low frequency variation situation of original template molecule in sample just becomes the key of problem.
The content of the blood of tumor patient, urine and the cfDNA in cerebrospinal fluid is seldom, for this detection to low frequency mutation
It is a problem.Mainly there is the detection mode of three kinds of low frequency mutation on Vehicles Collected from Market：Digital pcr, sequencing (next- of future generation
Generation sequencing, NGS) and mutation amplification system (amplification refractory mutation
System, ARMS) PCR.NGS has the advantage such as high flux, low cost, quick, easy to operate, is current domestic most popular low
Frequency mutation detection techniques.During NGS, structure gene library is first step in whole sequencing process, and is closed the most
Key step, the quality of gene library directly affect follow-up examining order.But the conventional banking process of in the market exist into
This height, detection cycle are long, flow is complicated, library is easily contaminated and requires the defects of high to testing staff, are not suitable for a large amount of
Storehouse is built in the sequencing of sample.
The content of the invention
It is an object of the present invention to provide the catastrophe for detecting sample to be tested target gene region to be checked into
Cover primer.
Primer set provided by the invention is including in Barcode primers Fs 1, sense primer F2, downstream outer primer R1, downstream
Primer R2；
The Barcode primers Fs 1 are successively by sequence measuring joints 1, barcode sequences for distinguishing different samples and general
Sequence 1 forms；
The sense primer F2 is successively by universal sequence 1, molecular label, specific base sequence and upstream specific primer sequence
Row composition；
The downstream outer primer R1 is made up of sequence measuring joints 2 and universal sequence 2 successively；
The downstream inner primer R2 is made up of universal sequence 2 and downstream specific primer sequence successively；
The sequence measuring joints 1 and the sequence measuring joints 2 are the sequence measuring joints according to corresponding to the selection of different microarray datasets；
The barcode sequences are that length is 8-12nt, without continuous base, and G/C content is 40-60% nucleotides；
The length of the universal sequence 1 and the universal sequence 2 is 16-25nt, and is without continuous base, G/C content
35-65%, without obvious secondary structure；
The specific base sequence is GAT；
The upstream specific primer sequence and the downstream specific primer sequence are that the amplification target gene is to be checked
The primer in region；
The molecular label is 10-12 positions randomized bases.
In above-mentioned primer set,
The microarray dataset is Illumina platforms, and the sequence measuring joints 1 are I5, and the sequence measuring joints 2 are I7；
Or the microarray dataset is Ion Torrent platforms, the sequence measuring joints 1 are A, and the sequence measuring joints 2 are P.
In above-mentioned primer set,
The Barcode primers Fs 1, the sense primer F2, the downstream outer primer R1 and the downstream inner primer R2
Mol ratio is 6:(10-6):(1-3):(1-3).
In above-mentioned primer set,
It is described to sport low frequency mutation, the specially frequency of mutation most as little as 0.1%.
In above-mentioned primer set,
The sample to be tested is cfDNA, the in vitro urine separation of tumor patient that the isolated blood of tumor patient separates
The genomic DNA that cfDNA, the cfDNA of in vitro cerebrospinal fluid separation of tumor patient or the tumor tissue in vitro of tumor patient are extracted.
In above-mentioned primer set,
The nucleotides sequence of the universal sequence 1 is classified as sequence 1；
The nucleotides sequence of the universal sequence 2 is classified as sequence 2；
The nucleotides sequence of the sequence measuring joints 1 is classified as sequence 3；
The nucleotides sequence of the sequence measuring joints 2 is classified as sequence 4；
The barcode sequences for being used to distinguish different samples are respectively sequence 5- sequences 14；
The testing gene is NRAS, and corresponding upstream specific primer sequence and downstream specific primer sequence are respectively
Sequence 15 and sequence 16 or sequence 17 or sequence 18；
The testing gene is ALK, and corresponding upstream specific primer sequence and downstream specific primer sequence are respectively
Sequence 19 and sequence 20 or sequence 21 and sequence 22 or sequence 23 and sequence 24 or sequence 25 and sequence 26 or sequence 27 and sequence
28 or sequence 29 and sequence 30 or sequence 31 and sequence 32；
The testing gene is PIK3CA, corresponding upstream specific primer sequence and downstream specific primer sequence difference
For sequence 33 and sequence 34 or sequence 35 or sequence 36；
The testing gene is ROS, and corresponding upstream specific primer sequence and downstream specific primer sequence are respectively
Sequence 37 and sequence 38；
The testing gene is EGFR, and corresponding upstream specific primer sequence and downstream specific primer sequence are respectively
Sequence 39 and sequence 40 or sequence 41 and sequence 42 or sequence 43 and sequence 44 or sequence 45 and sequence 46 or sequence 47 and sequence
48；
The testing gene is MET, and corresponding upstream specific primer sequence and downstream specific primer sequence are respectively
Sequence 49 and sequence 50 or sequence 51 and sequence 52 or sequence 53 and sequence 54 or sequence 55 and sequence 56；
The testing gene is BRAF, and corresponding upstream specific primer sequence and downstream specific primer sequence are respectively
Sequence 57 and sequence 58 or sequence 59 and sequence 60；
The testing gene is KRAS, and corresponding upstream specific primer sequence and downstream specific primer sequence are respectively
Sequence 61 and sequence 62 or sequence 63 and sequence 64；
The testing gene is TP53, and corresponding upstream specific primer sequence and downstream specific primer sequence are respectively
Sequence 65 and sequence 66 or sequence 67 and sequence 68 or sequence 69 and sequence 70 or sequence 71 and sequence 72 or sequence 73 and sequence
74 or sequence 75 and sequence 76；
The testing gene is ERBB2, corresponding upstream specific primer sequence and downstream specific primer sequence difference
For sequence 77 and sequence 78.
Another object of the present invention is to provide the examination of the catastrophe for detecting sample to be tested target gene region to be checked
Agent.
Reagent provided by the invention, including above-mentioned primer set and the body containing PCR amplification buffers and archaeal dna polymerase
System.
In mentioned reagent,
Concentration of the Barcode primers Fs 1 in the reagent in the primer set is 1.67 μM；
The concentration into sense primer F2 in the reagent is 0.28 μM -0.83 μM；
Concentration of the downstream outer primer R1 in the reagent is 1.67 μM -2.78 μM；
Concentration of the downstream inner primer R2 in the reagent is 0.28 μM -0.83 μM.
3rd purpose of the invention is to provide the reagent of the catastrophe for detecting sample to be tested target gene region to be checked
Box.
Kit provided by the invention, including above-mentioned primer set or above-mentioned reagent.
Mentioned reagent box also includes the extracts reagent and sequenator of sample cfDNA to be checked or genomic DNA.
The mutation of above-mentioned primer set or mentioned reagent or mentioned reagent box target gene in sample to be tested cfDNA is detected
Application in site or sudden change region catastrophe is also the scope of protection of the invention.
Or above-mentioned primer set or mentioned reagent or mentioned reagent box in sample to be tested cfDNA is detected target gene it is prominent
The application become in the frequency of mutation of site or sudden change region is also the scope of protection of the invention.
The catastrophe in above-mentioned detection sample to be tested target gene region to be checked is to be checked for detection sample to be tested target gene
The mutating alkali yl in the region either frequency of mutation in mutating acid or detection sample to be tested target gene region to be checked.
The computational methods of the frequency of mutation are as follows：
In sequencing result, the DNA molecular with same molecular label is a kind of amplified production of initial DNA profiling, is ordered
Entitled 1 family；
The mutation rate in the family is detected, if mutation rate >=80% of the family, the family is denoted as carrying molecular label
Mutation DNA families；
Mutation rate=(codon of coded amino acid residue has the quantity of the DNA molecular of mutation/same in same family
DNA molecular sum in race) * 100%；
Own in the frequency of mutation=sequencing result in quantity/sequencing result of the DNA families of the mutation with molecular label
Quantity * 100% with molecular label DNA families.
Remarks：Read (sequence the being sequenced out) numbers >=2 with same molecular label just have statistics in sequencing result
Meaning
The primer set that the present invention designs, it can be used for the amplification sublibrary of testing goal gene low frequency mutation, pertain only to
One_step PCR reacts and corresponding product purification steps, simplifies the operating process for building storehouse, saving is built the storehouse time (can be complete in two hours
Cheng Jianku, terminates from library construction to upper machine and the whole flow process of raw letter analysis completion can be completed in 24 hours).With the present invention
Primer carry out building storehouse, can detect as little as 0.1% mutation, sample to be checked can be blood, urine and cerebrospinal fluid etc. separation
The genomic DNA of the extractions such as the dissociative DNA or traditional frozen tissue, paraffin section and the fresh puncturing tissue that go out.Due to
The barcode sequences for distinguishing different samples are added in the primer of the present invention, are intersected between PCR startings just effectively prevent sample dirty
Dye.One-step method builds storehouse process and greatly reduces the usage amount of reagent consumptive material, and Laboratory Request locellus only needs 3 rooms, and (sample carries
Take, PCR amplification between, library purifying and sequencing), save space requirement, build Kucheng originally also it is more traditional capture banking process it is much lower.
In a word, the primer set that the present invention designs is used to build storehouse, in addition to it can detect tissue samples, additionally it is possible to quick, easy,
Sensitive, the special different zones to dissociative DNA in blood, urine and cerebrospinal fluid equal samples carry out targeting amplification, and efficiently
The horizontal mutation of detection as little as 0.1%, greatly simplifies experimental implementation, effectively avoids library from losing and pollute, significantly reduces into
This, improves efficiency.
Brief description of the drawings
Fig. 1 is the function of molecular label.A, B and C is respectively different mutational sites.
Fig. 2 is Agilent 2200TapeStation after the completion of the ctDNA library constructions that the blood sample of person under inspection 1 extracts
Systems detects obtained amplified production distribution.
Fig. 3 is the amplification sublibraries that are obtained by one-step method of ctDNA that the blood sample of person under inspection 1 extracts in Ion
Torrent platform sequencing results.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
Embodiment 1, build the amplification sublibrary being mutated for testing goal gene low frequency
The mutational site or sudden change region of the target gene in cfDNA in the blood of tumor patient, urine and cerebrospinal fluid
The frequency of mutation can influence tumour medication in future or the judgement in tumor development direction, the present embodiment is in order to detect tumor patient
The mutational site of the target gene in cfDNA or the frequency of mutation of sudden change region in blood, urine and cerebrospinal fluid, build and are used for
The amplification sublibrary of testing goal gene low frequency mutation, it is specific as follows：
First, the primer Combination Design for the amplification sublibrary of testing goal gene low frequency mutation synthesizes
One section of region synthesizes following primer as region to be checked design in target gene known to selection：
There is mutantional hotspot in the region to be checked, simply whether occur can be with, it is known that also not for gene mutation in sample to be checked
Know
Barcode primers Fs 1：Sequence measuring joints 1+barcode sequences+universal sequence 1；
Sense primer F2：Universal sequence 1+ molecular labels+specific base sequence+upstream specific primer sequence；
Downstream outer primer R1：Sequence measuring joints 2+ universal sequences 2；
Downstream inner primer R2：Universal sequence 2+ downstream specific primer sequences；
Wherein, barcode sequences are the sequences for distinguishing different samples, and a sample to be tested corresponds to a barcode
Sequence, this Barcode sequence length are 8-12nt, it is desirable to without continuous base, G/C content 40-60%, introduce Barcode sequences
Primer without obvious secondary structure etc..F1 be for distinguishing different samples, as long as same sample, F1 all sames, with detecting position
Point is unrelated.
The length of universal sequence 1 and 2 is 16-25nt, it is desirable to which, without continuous base, G/C content 35-65%, calling sequence draws
Thing can be varied as desired in without obvious secondary structure etc., the sequence, the present embodiment using
The GGCATACGTCCTCGTCTA of universal sequence 1 (sequence 1), size 18nt；
The CGACATCGCCTCTGCTGT of universal sequence 2 (sequence 2), size 18nt.
Sequence measuring joints 1 and sequence measuring joints 2 determine according to microarray dataset：
If microarray dataset is Illumina platforms, sequence measuring joints 1 and 2 are respectively I5 and I7, on joint sequence and chip
Primer sequence be complementary, adjunction head is in order to which nucleic acid fragment is connected on carrier.
If microarray dataset is Ion Torrent platforms, the respectively A and P (sequence 3 and 4) of sequence measuring joints 1 and 2, A joints
Complementary with specific primer for being sequenced, P joints are complementary with sequence on carrier, for template is connected with carrier.
Specific base sequence is GAT, is not the part of gene specific amplified fragments, and it acts on the life for being easy for sequencing result
Thing information analysis, by identifying that GAT sequences can improve the efficiency of data screening.
Upstream specific primer sequence and downstream specific primer sequence are to be designed for according to target gene region to be checked
Its primer is expanded, upstream specific primer sequence size is 15-30nt, and downstream specific primer sequence size is 15-30nt.
Molecular label is 10-12 positions randomized bases, for marking starting cfDNA templates.Every randomized bases of molecular label
There are tetra- kinds of base forms of ATCG, so 10 randomized bases have 1048576 kinds of different molecular labels altogether, with initial
Exemplified by 20ngDNA templates, its copy number is 6000, and cfDNA molecule fragment is shorter, so the effective template that can be expanded is copied
Shellfish number is less than 6000, and 1048576 kinds of molecular label form can be that each original template " is marked plus specific completely
Note ".The starting template of sequencing result is classified by molecular label, it is possible to exclude amplification mistake and sequencing mistake.
As shown in figure 1, Fig. 1 is in constructed library, 5 amplified productions with same molecular label, wherein A positions
The mutation of point all exists on 5 molecules, and the mutation in B and C sites only exists in wherein some amplified production, institute's accounting
Example is extremely low, it is possible to judge that sporting for A sites is mutated present in original template molecule, and the mutation in B and C sites is then to build
The mutation of the false positive occurred in the PCR amplifications in storehouse or in sequencing procedure.So the effect of molecular label is the original mould of mark
Plate molecule, identify and be mutated present in primary template, reject the false positive mutation occurred in PCR and sequencing procedure, improve detection
Sensitivity.
2nd, the foundation of detection method
1st, above-mentioned one primer (F2, R1 and R2) is mixed in specific proportions, thing mix is cited approvingly after fully mixing, it is stand-by.
2nd, the cfDNA of sample such as tumor patient blood, urine or cerebrospinal fluid to be checked is extracted.
3rd, barcode primers and primer mix corresponding to different samples enter performing PCR amplification to cfDNA, to the eight of 0.2ml
In platoon pipe or 96 orifice plates, reagent as shown in table 1 below is sequentially added, obtains PCR amplification system.
Table 1 is PCR amplification system
Wherein, in primer mix, it is 50 μM that R1, F2, R2 primer, which add initial concentration, and R1：F2：R2 (volume ratio)=
10:(1-5):(1-5)。
In PCR amplification system, Barcode primers Fs 1, sense primer F2, downstream outer primer R1 and downstream inner primer R2 rub
Your ratio is as follows：F1：R1：F2：R2 mol ratio=6:(10-6):(1-3):(1-3)
4th, in PCR instrument (PCR instrument uses Applied bio-system 2720Thermal Cycler), operation is as follows
Amplification program shown in table 2：
Table 2 is amplification program
The cycling condition for building the Gradient annealing temperature that the first two circulates during the PCR of storehouse is that primary template is tentatively expanded
Increase, it may also be said to it is to add specific molecular label for different primary templates, and the PCR conditions of subsequent 19 circulation are to original mould
Plate, which enters in molecular label, to be expanded, meanwhile, the primers F 2, R2 of the F1 of high concentration, R1 and low concentration, 19 circulations after also ensure that
Conducted in process in molecular label expand (other molecular labels will not be typically added in amplification procedure).
5th, the Agencourt AMPure XP Kit (BECKMAN of PCR 1.3 times of volumes of reaction solution are drawn with liquid-transfering gun
COULTER, A63882) purifying recovery PCR primer is carried out, obtain the DNA library for amplicon sequencing.Specific purification step is such as
Under：
1) shift to an earlier date 30 minutes and take out Agencourt AMPure XP Kit, after being fully vortexed, be stored at room temperature.
2) PCR reaction terminate after, magnetic bead is fully vortexed again, 24 μ l magnetic beads are added into system, repeatedly blow and beat 5 times with
Upper or abundant vortex, is stored at room temperature 5 minutes.
3) EP pipes are transferred to and be placed on magnetic frame, after standing is clarified for 5 minutes to solution, carefully removed with liquid-transfering gun
Clearly, it is careful not to touch magnetic bead.
4) often pipe adds 80% ethanol solution of the 100 fresh configurations of μ l, and EP pipes are placed on magnetic frame the slowly circle of rotation 2, quiet
Put 5m, supernatant discarding.
5) 4 steps are repeated once.
6) EP pipes are opened, be stored at room temperature, made to evaporate totally, be defined, be careful not to by magnetic bead surfaces tarnish
Divide and dry magnetic bead.
7) EP pipes are removed from magnetic frame, add 30 μ l PCR level purified waters, is vortexed after mixing, is stored at room temperature 10 minutes.
8) the EP pipes of upper step are placed on magnetic frame 2 minutes or until after solution clarification, with liquid-transfering gun away from magnetite
Simultaneously careful Aspirate supernatant, it is careful not to touch magnetic bead.
So far, amplicon library construction is completed, and quantitatively judges whether to successfully construct using QuBit.
6th, upper machine sequencing and interpretation of result
The library of above-mentioned different sample amplifications carries out equal proportion mixing according to the concentration of measure, is finally diluted to specific dense
Degree, with two generation sequencers, obtains sequencing result.
The result of sequencing obtains detecting the catastrophe of gene after data processing, bioinformatic analysis.Data
Processing procedure includes the changing of sequencing data, Quality Control, sequence alignment (reference gene group is into NCBI GRCh37/Hg19), is mutated position
The processes such as point analysis, by the catastrophe and the frequency of mutation that obtain detecting sample after Data Management Analysis.
Because amplified library process carries out molecular labeling to primary template, the computational methods of the frequency of mutation are as follows：
In sequencing result, the DNA molecular with same molecular label is a kind of amplified production of initial DNA profiling, is ordered
Entitled 1 family；
The mutation rate in the family is detected, if mutation rate >=80% of the family, the family is denoted as carrying molecular label
Mutation DNA families；
Mutation rate=(codon of coded amino acid residue has the quantity of the DNA molecular of mutation/same in same family
DNA molecular sum in race) * 100%；
Own in the frequency of mutation=sequencing result in quantity/sequencing result of the DNA families of the mutation with molecular label
Quantity * 100% with molecular label DNA families.
Remarks：Read (sequence the being sequenced out) numbers >=2 with same molecular label just have statistics in sequencing result
Meaning
Embodiment 2, build the amplification sublibrary being mutated for testing goal gene low frequency
Target gene is as shown in table 5, and sample to be tested derives from 10 persons under inspection for being identified as patients with lung cancer, this implementation
The purpose of example is the gene mutation frequency with 10 patients shown in the method detection table 5 of the present invention.
First, the primer Combination Design for the amplification sublibrary of testing goal gene low frequency mutation synthesizes
Following primer is synthesized according to the mutational site of one target gene of embodiment 1 or sudden change region design, is specifically shown in
Table 3 and table 4：
Table 3 combines for primer
Specific primer design principle：55-65 DEG C of annealing temperature, as far as possible secondary structure less, G/C content 35%-65%,
Primer length 16-30nt, secondary structure should not be formed between primer, it is specific such as table 4.
Table 4 is that specific primer sequences corresponding to each gene are primer combination
2nd, detect
1st, it is identical with the 21 of embodiment 1, three kinds of primers (R1, F2 and R2) are mixed in specific proportions, claimed after fully mixing
Primer mix, F1:R1:F2:R2 mol ratio=6:10:1:1, it is stand-by.
The concentration of Barcode primers Fs 1 is 1.67 μM；
Downstream outer primer R1 concentration is 2.78 μM；
Sense primer F2 concentration is 0.28 μM；
Downstream inner primer R2 concentration is 0.28 μM.
2nd, (formalin is solid for FFPE samples corresponding to 10 persons under inspection (being the cancer patient made a definite diagnosis) actually collected
The tissue of FFPE after fixed) and blood sample, extract the genomic DNA of FFPE samples and the cfDNA of blood sample.
3rd, it is identical with the 23 of embodiment 1；
4th, it is identical with the 24 of embodiment 1；
5th, it is identical with the 25 of embodiment 1；
The testing result (containing 32 amplicons) in the library of person under inspection 1 is as shown in Fig. 2 Fig. 2 is specific sample library structure
Agilent 2200TapeStation Systems detect obtained amplified production distribution map after the completion of building, and abscissa is fragment
Length, ordinate are signal intensity (FU), and lower peaks are that 25bp positions marker, upper peak is 1500bp positions marker,
Gained PCR primer is concentrated in the range of 160-230bp after PCR is expanded as shown in Figure 2.
6th, upper machine sequencing and interpretation of result, it is identical with the 25 of embodiment 1, it is as a result as follows：
Fig. 3 is the ctDNA for the blood sample extraction for being diagnosed as patients with lung cancer (person under inspection 1) using this banking process
Ion Torrent platform sequencing results.
FFPE samples and blood sample testing result are as shown in table 5 below corresponding to 10 persons under inspection actually collected, wherein
The method one of FFPE samples and blood sample carries out capture using the SureSelect customization services of Agilent and builds storehouse, blood sample
This method two carries out building storehouse using this patent method, as a result shows, FFPE samples and related mutation detected by blood sample
As a result it is consistent.The practical application and good specificity of the present invention are absolutely proved.
Table 5 is the testing result of FFPE samples and blood sample corresponding to 10 persons under inspection
The implication that each mutational site represents in above-mentioned table 5 is exemplified below：
EGFR p.Glu746_Ala750del：The amino acids of EGFR gene the 746 to 750th lack, the 746th amino acids
Should be Glu, the 750th amino acids should be Ala.
EGFR p.Glu746_Thr751delinsAla：The amino acids of EGFR gene the 746 to 750th lack, and insert simultaneously
Ala.
TP53p.Arg282Trp：The amino acids of TP53 genes the 282nd sport Trp by Arg.
TP53p.Arg213X：Nonsense mutation occurs for the amino acids of TP53 genes the 213rd, and termination codon is sported by Arg
Son.
Sequence table
<110>The modern wound in Beijing pangen Laboratory of medical test Co., Ltd of Beijing pangen Gene Tech. Company Limited Chongqing
Fan Sheng Laboratory of medical test Co., Ltd
<120>A kind of primer and kit for the mutation of testing goal gene low frequency
<160> 78
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 1
ggcatacgtc ctcgtcta 18
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 2
cgacatcgcc tctgctgt 18
<210> 3
<211> 30
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 3
ccatctcatc cctgcgtgtc tccgactcag 30
<210> 4
<211> 23
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 4
cctctctatg ggcagtcggt gat 23
<210> 5
<211> 10
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 5
tcctcgaatc 10
<210> 6
<211> 10
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 6
taggtggttc 10
<210> 7
<211> 10
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 7
tctaacggac 10
<210> 8
<211> 10
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 8
ttggagtgtc 10
<210> 9
<211> 10
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 9
tctagaggtc 10
<210> 10
<211> 10
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 10
tctggatgac 10
<210> 11
<211> 10
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 11
tctattcgtc
<210> 12
<211> 10
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 12
aggcaattgc 10
<210> 13
<211> 10
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 13
ttagtcggac 10
<210> 14
<211> 10
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 14
cagatccatc 10
<210> 15
<211> 23
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 15
ggtgaaacct gtttgttgga cat 23
<210> 16
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 16
cttcgcctgt cctcatgtat tg 22
<210> 17
<211> 27
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 17
tggtgtgaaa tgactgagta caaactg 27
<210> 18
<211> 25
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 18
gttctggatt agctggattg tcagt 25
<210> 19
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 19
tccaggccct ggaagagt 18
<210> 20
<211> 25
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 20
tgaggcagtc tttactcacc tgtag 25
<210> 21
<211> 23
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 21
cctgtggctg tcagtatttg gag 23
<210> 22
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 22
acacagatca gcgacaggat g 21
<210> 23
<211> 16
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 23
aatccctgcc ccggtt 16
<210> 24
<211> 15
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 24
gggcgggtct ctcgg 15
<210> 25
<211> 27
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 25
gttaattttg gttacatccc tctctgc 27
<210> 26
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 26
gattgcaggc tcaccccaa 19
<210> 27
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 27
actggatttc ctcatggaag cc 22
<210> 28
<211> 28
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 28
agatatcgat ctgttagaaa cctctcca 28
<210> 29
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 29
cggactctgt aggctgcagt 20
<210> 30
<211> 23
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 30
ggaaatccag ttcgtcctgt tca 23
<210> 31
<211> 26
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 31
gtttgactct gtctcctctt gtcttc 26
<210> 32
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 32
cttgggtcgt tgggcattc 19
<210> 33
<211> 27
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 33
caaagaacag ctcaaagcaa tttctac 27
<210> 34
<211> 28
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 34
attttagcac ttacctgtga ctccatag 28
<210> 35
<211> 25
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 35
agcaagaggc tttggagtat ttcat 25
<210> 36
<211> 26
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 36
tgtgtggaag atccaatcca tttttg 26
<210> 37
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 37
cttccctcgg gaaaaactga c 21
<210> 38
<211> 23
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 38
gatgtccact gctgttcctt cat 23
<210> 39
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 39
ccaaccaagc tctcttgagg at 22
<210> 40
<211> 15
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 40
caccgtgccg aacgc 15
<210> 41
<211> 27
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 41
cccagaaggt gagaaagtta aaattcc 27
<210> 42
<211> 23
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 42
cacatcgagg atttccttgt tgg 23
<210> 43
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 43
ctctccctcc ctccagga 18
<210> 44
<211> 17
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 44
gaggcagatg cccagca 17
<210> 45
<211> 17
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 45
ctgcctcacc tccaccg 17
<210> 46
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 46
attgtctttg tgttcccgga ca 22
<210> 47
<211> 17
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 47
ggaggaccgt cgcttgg 17
<210> 48
<211> 26
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 48
cttctgcatg gtattctttc tcttcc 26
<210> 49
<211> 23
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 49
cttgtaagtg cccgaagtgt aag 23
<210> 50
<211> 24
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 50
gtcacaaccc actgaggtat atgt 24
<210> 51
<211> 26
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 51
ctaaccaagt tctttctttt gcacag 26
<210> 52
<211> 24
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 52
agcacagtga attttcttgc catc 24
<210> 53
<211> 23
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 53
cagtcaaggt tgctgatttt ggt 23
<210> 54
<211> 24
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 54
ctttgcacct gttttgttgt gtac 24
<210> 55
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 55
ggtgcaaagc tgccagtg 18
<210> 56
<211> 27
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 56
aaccaataca ttaccacatc tgacttg 27
<210> 57
<211> 29
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 57
cttcatgaag acctcacagt aaaaatagg 29
<210> 58
<211> 26
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 58
ctcaattctt accatccaca aaatgg 26
<210> 59
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 59
gggcagatta cagtgggaca 20
<210> 60
<211> 28
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 60
aatgtcacca cattacatac ttaccatg 28
<210> 61
<211> 27
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 61
ggagaaacct gtctcttgga tattctc 27
<210> 62
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 62
tcctcatgta ctggtccctc at 22
<210> 63
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 63
aggcctgctg aaaatgactg a 21
<210> 64
<211> 25
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 64
gaattagctg tatcgtcaag gcact 25
<210> 65
<211> 27
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 65
gtgtatatac ttacttctcc ccctcct 27
<210> 66
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 66
cctcattcag ctctcggaac at 22
<210> 67
<211> 28
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 67
cctatcctga gtagtggtaa tctactgg 28
<210> 68
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 68
ccctttcttg cggagattct c 21
<210> 69
<211> 24
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 69
tctcctaggt tggctctgac tgta 24
<210> 70
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 70
cctggagtct tccagtgtga t 21
<210> 71
<211> 25
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 71
gcatcttatc cgagtggaag gaaat 25
<210> 72
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 72
cctcccagag accccagt 18
<210> 73
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 73
ctgtgggttg attccacacc 20
<210> 74
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 74
ctcaccatcg ctatctgagc a 21
<210> 75
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 75
gcattctggg acagccaag 19
<210> 76
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 76
tacggccagg cattgaagt 19
<210> 77
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 77
tcccataccc tctcagcgt 19
<210> 78
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 78
ccagaaggcg ggagacatat g 21
Claims (10)
1. for detecting the catastrophe primer set in sample to be tested target gene region to be checked, including Barcode primers Fs 1, on
Swim primers F 2, downstream outer primer R1, downstream inner primer R2；
The Barcode primers Fs 1 are successively by sequence measuring joints 1, the barcode sequences for distinguishing different samples and universal sequence 1
Composition；
The sense primer F2 is successively by universal sequence 1, molecular label, specific base sequence and upstream specific primer sequence group
Into；
The downstream outer primer R1 is made up of sequence measuring joints 2 and universal sequence 2 successively；
The downstream inner primer R2 is made up of universal sequence 2 and downstream specific primer sequence successively；
The sequence measuring joints 1 and the sequence measuring joints 2 are the sequence measuring joints according to corresponding to the selection of different microarray datasets；
The barcode sequences are that length is 8-12nt, without continuous base, and G/C content is 40-60% nucleotides；
The length of the universal sequence 1 and the universal sequence 2 is 16-25nt, and without continuous base, G/C content 35-
65%, without obvious secondary structure；
The specific base sequence is GAT；
The upstream specific primer sequence and the downstream specific primer sequence are the amplification target gene regions to be checked
Primer；
The molecular label is 10-12 positions randomized bases.
2. primer set according to claim 1, it is characterised in that：
The microarray dataset is Illumina platforms, and the sequence measuring joints 1 are I5, and the sequence measuring joints 2 are I7；
Or the microarray dataset is Ion Torrent platforms, the sequence measuring joints 1 are A, and the sequence measuring joints 2 are P.
3. primer set according to claim 1 or 2, it is characterised in that：
The Barcode primers Fs 1, the sense primer F2, the downstream outer primer R1 and the downstream inner primer R2 mole
Than for 6:(10-6):(1-3):(1-3).
4. according to any described primer set in claim 1-3, it is characterised in that：It is described to sport low frequency mutation.
5. according to any described primer set in claim 1-4, it is characterised in that：The sample to be tested is tumor patient
The cfDNA of isolated blood separation, the cfDNA of the in vitro urine separation of tumor patient, the in vitro cerebrospinal fluid of tumor patient separate
The genomic DNA of the tumor tissue in vitro of cfDNA or tumor patient extraction.
6. according to any described primer set in claim 1-5, it is characterised in that：
The nucleotides sequence of the universal sequence 1 is classified as sequence 1；
The nucleotides sequence of the universal sequence 2 is classified as sequence 2；
The nucleotides sequence of the sequence measuring joints 1 is classified as sequence 3；
The nucleotides sequence of the sequence measuring joints 2 is classified as sequence 4；
The barcode sequences for being used to distinguish different samples are respectively sequence 5- sequences 14；
The testing gene is NRAS, and corresponding upstream specific primer sequence and downstream specific primer sequence are respectively sequence
15 and sequence 15 or sequence 17 or sequence 18；
The testing gene is ALK, and corresponding upstream specific primer sequence and downstream specific primer sequence are respectively sequence
19 and sequence 20 or sequence 21 and sequence 22 or sequence 23 and sequence 24 or sequence 25 and sequence 26 or sequence 27 and sequence 28 or
Sequence 29 and sequence 30 or sequence 31 and sequence 32；
The testing gene is PIK3CA, and corresponding upstream specific primer sequence and downstream specific primer sequence are respectively sequence
Row 33 and sequence 34 or sequence 35 or sequence 36；
The testing gene is ROS, and corresponding upstream specific primer sequence and downstream specific primer sequence are respectively sequence
37 and sequence 38；
The testing gene is EGFR, and corresponding upstream specific primer sequence and downstream specific primer sequence are respectively sequence
39 and sequence 40 or sequence 41 and sequence 42 or sequence 43 and sequence 44 or sequence 45 and sequence 46 or sequence 47 and sequence 48；
The testing gene is MET, and corresponding upstream specific primer sequence and downstream specific primer sequence are respectively sequence
49 and sequence 50 or sequence 51 and sequence 52 or sequence 53 and sequence 54 or sequence 55 and sequence 56；
The testing gene is BRAF, and corresponding upstream specific primer sequence and downstream specific primer sequence are respectively sequence
57 and sequence 58 or sequence 59 and sequence 60；
The testing gene is KRAS, and corresponding upstream specific primer sequence and downstream specific primer sequence are respectively sequence
61 and sequence 62 or sequence 63 and sequence 64；
The testing gene is TP53, and corresponding upstream specific primer sequence and downstream specific primer sequence are respectively sequence
65 and sequence 66 or sequence 67 and sequence 68 or sequence 69 and sequence 70 or sequence 71 and sequence 72 or sequence 73 and sequence 74 or
Sequence 75 and sequence 76；
The testing gene is ERBB2, and corresponding upstream specific primer sequence and downstream specific primer sequence are respectively sequence
Row 77 and sequence 78.
7. for detect sample to be tested target gene region to be checked catastrophe reagent, including in claim 1-6 it is any
Described primer set and the system containing PCR amplification buffers and archaeal dna polymerase.
8. reagent according to claim 7, it is characterised in that：
Concentration of the Barcode primers Fs 1 in the reagent in the primer set is 1.67 μM；
The concentration into sense primer F2 in the reagent is 0.28 μM -0.83 μM；
Concentration of the downstream outer primer R1 in the reagent is 1.67 μM -2.78 μM；
Concentration of the downstream inner primer R2 in the reagent is 0.28 μM -0.83 μM.
9. the kit of the catastrophe for detecting sample to be tested target gene region to be checked, including appoint in claim 1-6
The reagent described in primer set or claim 7 or 8 described in one.
10. in claim 1-6 described in any described primer set or reagent described in claim 7 or 8 or claim 9
Kit in the mutational site of target gene in detecting sample to be tested cfDNA or the application in the catastrophe of sudden change region；
Or, in claim 1-6 described in any described primer set or reagent described in claim 7 or 8 or claim 9
Kit in the mutational site of target gene in detecting sample to be tested cfDNA or the application in the frequency of mutation of sudden change region.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710976796.8A CN107604067A (en) | 2017-10-19 | 2017-10-19 | A kind of primer and kit for the mutation of testing goal gene low frequency |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710976796.8A CN107604067A (en) | 2017-10-19 | 2017-10-19 | A kind of primer and kit for the mutation of testing goal gene low frequency |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107604067A true CN107604067A (en) | 2018-01-19 |
Family
ID=61077449
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710976796.8A Pending CN107604067A (en) | 2017-10-19 | 2017-10-19 | A kind of primer and kit for the mutation of testing goal gene low frequency |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107604067A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110359096A (en) * | 2018-04-09 | 2019-10-22 | 深圳华大智造科技有限公司 | A method of library is targeted using biological sample direct construction |
| CN111073961A (en) * | 2019-12-20 | 2020-04-28 | 苏州赛美科基因科技有限公司 | High-throughput detection method for gene rare mutation |
| CN112301430A (en) * | 2019-07-30 | 2021-02-02 | 北京泛生子基因科技有限公司 | Library building method and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105441580A (en) * | 2016-01-26 | 2016-03-30 | 绍兴华因生物科技有限公司 | Method and primers for detecting heterozygosity DMD gene deletion |
| CN106906210A (en) * | 2017-04-05 | 2017-06-30 | 北京泛生子医学检验实验室有限公司 | A kind of fusion primer combination of rapid build amplification sublibrary |
-
2017
- 2017-10-19 CN CN201710976796.8A patent/CN107604067A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105441580A (en) * | 2016-01-26 | 2016-03-30 | 绍兴华因生物科技有限公司 | Method and primers for detecting heterozygosity DMD gene deletion |
| CN106906210A (en) * | 2017-04-05 | 2017-06-30 | 北京泛生子医学检验实验室有限公司 | A kind of fusion primer combination of rapid build amplification sublibrary |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110359096A (en) * | 2018-04-09 | 2019-10-22 | 深圳华大智造科技有限公司 | A method of library is targeted using biological sample direct construction |
| CN112301430A (en) * | 2019-07-30 | 2021-02-02 | 北京泛生子基因科技有限公司 | Library building method and application |
| CN111073961A (en) * | 2019-12-20 | 2020-04-28 | 苏州赛美科基因科技有限公司 | High-throughput detection method for gene rare mutation |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107604045A (en) | A kind of construction method of amplification sublibrary for the mutation of testing goal gene low frequency | |
| CN108004301B (en) | Gene target region enrichment method and library construction kit | |
| CN108893466B (en) | Sequencing joint, sequencing joint group and detection method of ultralow frequency mutation | |
| CN107406885A (en) | Use the size and number Distortion Detect cancer of plasma dna | |
| CN107475375A (en) | A kind of DNA probe storehouse, detection method and kit hybridized for microsatellite locus related to microsatellite instability | |
| CN106834275A (en) | The analysis method of the construction method, kit and library detection data in ctDNA ultralow frequency abrupt climatic changes library | |
| CN106906211A (en) | A kind of molecular adaptor and its application | |
| CN105861710A (en) | Sequencing joint and preparation method and application thereof in ultra-low frequency mutation detection | |
| CN108753954B (en) | Capture probe set of dementia-related gene, kit, library construction method and application | |
| CN111073961A (en) | High-throughput detection method for gene rare mutation | |
| CN107475403A (en) | The analysis method of the method for detection Circulating tumor DNA, kit and its sequencing result from peripheral blood dissociative DNA | |
| CN112176057B (en) | Marker for detecting pancreatic duct adenocarcinoma by using CpG site methylation level and application thereof | |
| CN102952877A (en) | Method and system for detecting alpha-globin gene copy number | |
| CN109266744A (en) | Multiple PCR primer, kit and the method for targeting sequencing detection lung cancer gene based on UMI unimolecule label noise reduction technology | |
| CN108085395A (en) | Primer sets, kit and the method for cervical carcinoma polygenes DNA methylation assay based on high-flux sequence | |
| CN109952381A (en) | Method for Multiple detection methylate DNA | |
| CN108998508A (en) | The construction method and primer sets and kit of amplicon sequencing library | |
| CN107604067A (en) | A kind of primer and kit for the mutation of testing goal gene low frequency | |
| CN106520917A (en) | Gene large fragment deletion/duplication detection method | |
| CN110468211A (en) | Bladder cancer tumor mutant gene specific primer, kit and library constructing method | |
| CN106755486A (en) | The poor detection in Gene Mutation library constructing method in noninvasive prenatal foetal β types ground, detection method and kit | |
| CN108728515A (en) | A kind of analysis method of library construction and sequencing data using the detection ctDNA low frequencies mutation of duplex methods | |
| WO2023226939A1 (en) | Methylation biomarker for detecting colorectal cancer lymph node metastasis and use thereof | |
| CN108315425A (en) | PCR specific primers, kit and its application method of metastasis of thyroid carcinoma related gene detection | |
| CN108359723A (en) | A method of reducing deep sequencing mistake |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180119 |