CN109517819A - A kind of detection probe, method and kit modified for detecting multiple target point gene mutation, methylation modification and/or methylolation - Google Patents
A kind of detection probe, method and kit modified for detecting multiple target point gene mutation, methylation modification and/or methylolation Download PDFInfo
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Abstract
The present invention provides a kind of detection probes, the detection probe includes at least one capture probe, sequence of the capture probe from 5 ' ends to 3 ' ends successively includes preamble sequence, probe random tags sequence and target complementary series, the preamble sequence is as shown in SEQ ID NO:1, the probe random tags sequence is made of 0-12 randomized bases, and the target complementary series includes base of at least ten continuously with gene complementation to be detected.Detection of the detection probe for multiple target point gene mutation, methylation modification and/or methylolation modification, gene content to be detected is required low, effectively avoid the interference of mrna length to be detected and content to detection, be conducive to that degree of degradation is high, detection of the serious gene of fragmentation, and testing cost it is low, it is high-efficient, have a wide range of application.
Description
Technical field
It is the present invention relates to field of medical biotechnology, in particular to a kind of for detecting multiple target point gene mutation, methylation modification
And/or detection probe, method and the kit of methylolation modification.
Background technique
Multidigit point/region detection of hereditary variation, the methylation modification or methylolation modification of target gene, for disease
The molecular mechanisms of generation, the screening identification of molecular marked compound, auxiliary patient diagnosis and medication guide have important science
And clinical meaning.In addition, establishing hereditary information database for the hereditary information for analyzing and obtaining rare or endangered sample
It is same indispensable.But since the DNA amount to obtain of sample in most cases is lower, Fragment Differential is larger, is unfavorable for library
Foundation.For example, cfDNA (cell free DNA) is to be free on extracellular DNA, fragment length usually in 150bp or so,
From Apoptosis or the histopathology necrosis of internal Different Organs, therefore all heredity for carrying each organ or tissue become
Different information is science and medical field extensive concern hot spot.But every milliliter of blood of healthy individuals contains 0-100ng cfDNA, i.e.,
It is late tumor patient, apoptosis and necrosis due to massive tumor cell, cfDNA average content are also only 180ng/mL；
Secondly, inevitably pollution has other genomic DNAs in blood, traditional multiplexed PCR amplification skill to cfDNA during extraction
Art cannot exclude the pollution of genomic DNA, influence the interpretation of follow-up data.
In recent years, the target gene multidigit point/region detection technology being most widely used includes hybrid capture technology, PCR
Technology and molecular inversion probes technology.Hybrid capture technology includes solution hybridization technology (CN102796808B；CN103103624
B) and solid-phase hybridization technology, more demanding for the amount of DNA, it usually needs 1 μ g, capture probe synthesis cost is higher, entirely
Experimental period and process are longer.Multiple PCR technique (CN108103181A is mostly used in round pcr；CN108085395A), for
When the DNA fragmentation seriously degraded is expanded, it is difficult to ensure that upstream and downstream primer can be fallen in simultaneously on same DNA fragmentation；For
Contaminated segment can not judge practical source of its amplified production etc..Molecular inversion probes technology (WO2013173774A2)
It is a kind of extensive technological means of detection gene SNP mutation, can completes the amplification and detection of tens of thousands of DNA fragmentations, but its
Defective is essentially identical with PCR amplification, it is difficult to differentiate repetition that PCR introduces, double ended probes while fall in same DNA
Chain.
Therefore, it is necessary to it is a kind of can to avoid the multiple target point gene mutation that sample content and length and outside contamination interfere,
The detection method of methylation modification and/or methylolation modification.
Summary of the invention
In view of this, the present invention provides one kind for detecting multiple target point gene mutation, methylation modification and/or methylol
Change detection probe, connector, detection method and the kit of modification, it can efficient, accurate, specificity detection gene mutation, first
Base modification and/or methylolation modification interpret detection to medication guide, disease forecasting, disease prognosis assessment, a human genome
Etc. playing important booster action.
In a first aspect, the detection probe includes at least one capture probe, institute the present invention provides a kind of detection probe
Stating sequence of the capture probe from 5 ' ends to 3 ' ends successively includes preamble sequence, probe random tags sequence and target complementary series,
As shown in SEQ ID NO:1, the probe random tags sequence is made of the preamble sequence 0-12 randomized bases, described
Target complementary series includes base of at least ten continuously with gene complementation to be detected.
Optionally, the target complementary series includes the 18-60 bases with gene complementation to be detected.
In the present invention, when the detection probe captures the positive-sense strand of cls gene to be checked, the target complementary series packet
Include at least ten continuously base complementary with the positive-sense strand；When the detection probe captures the antisense strand of cls gene to be checked
When, the target complementary series includes at least ten continuously base complementary with the antisense strand；When the detection probe is same
When capture positive-sense strand and when antisense strand, the target complementary series includes at least ten continuously alkali complementary with the positive-sense strand
The continuous base complementary with the antisense strand of base and at least ten.That is, detection probe of the invention can only be caught
Obtain the positive-sense strand or the only antisense strand of Acquisition Detection gene or the simultaneously positive-sense strand of Acquisition Detection gene and negative justice of detection gene
Chain, to be detected to gene to be detected.
Optionally, the length of the sequence capture probe is 44nt-86nt.Further, the sequence capture probe
Length is 52nt-71nt.
Detection probe provided by the invention can be used for multiple target point gene mutation, methylation modification and/or methylolation and repair
The detection of decorations, it is low to gene content to be detected requirement, the interference of mrna length to be detected and content to detection is effectively avoided, favorably
High, the serious gene of fragmentation the detection in degree of degradation, and the synthesis cost of the probe is low, is conducive to the inspection of high-volume sample
It surveys.
Second aspect, the present invention provides a kind of connector, the connector includes the first connector, and first connector is held from 5 '
Sequence to 3 ' ends successively includes First ray, the first connector random tags sequence and T base, and 3 ' ends of the First ray are arrived
5 ' ends at least 10 continuous bases of the 3 ' ends to 5 ' ends, first connector in the sequence as shown in SEQ ID NO:2
Random tags sequence is made of 0-12 randomized bases, and 5 ' ends of first connector and cls gene to be checked connect.
Optionally, the length of the First ray is 12bp-46bp.Further, the length of the First ray is
20bp-42bp。
Specifically, the First ray can be, but not limited to as AATGATACGGCGACCACCGAGA TCTACACTCTTT
CCCTACACGACGCTCTTCCGATCTT。
Specifically, the sequence of first connector can be, but not limited to as the sequence as shown in SEQ ID NO:4.
Optionally, the connector further includes the second connector, and sequence of second connector from 5 ' ends to 3 ' ends successively includes
Second connector random tags sequence and the second sequence, the second connector random tags sequence are made of 0-12 randomized bases,
Second sequence continuous base of at least six in the sequence as shown in SEQ ID NO:3, second connector is for promoting
It is connected into 5 ' ends of first connector and the cls gene to be checked.
Further, 5 ' ends of second connector carry out amido modified.
Wherein, second sequence continuous base of at least six in the sequence as shown in SEQ ID NO:3, as
Selected from the sequence as shown in SEQ ID NO:3, from 5 ' ends, at least six into 3 ' ends is continuous from 5 ' ends to 3 ' ends for second sequence
Base.
Optionally, the sequence length of second connector is 6bp-40bp.Further, the sequence of second connector is long
Degree is 6bp-20bp.
Specifically, second sequence can be, but not limited to as AGATCGGAAGAGCGTCGTGTAG GGAAAGAGTGTAG
ATCTC。
Specifically, the sequence of second connector can be, but not limited to as the sequence as shown in SEQ ID NO:5.
Detection of the connector provided by the invention for multiple target point gene mutation, methylation modification and/or methylolation modification,
Wherein the first connector is used to connect with 5 ' ends of cls gene to be checked；According to the principle of DNA double chain link, the second connector is for promoting
The connection of first connector is not connect with cls gene to be checked, is obtained the cls gene to be checked of single-ended jointing, is conducive to subsequent sequencing
With the analysis of result.
The third aspect, the present invention provides a kind of multiple target point gene mutation, methylation modification and/or methylolation modifications
Detection method, including using detection probe described in first aspect, the detection probe and gene recombination to be detected.
In the present invention, the gene mutation can be, but not limited to become for single nucleotide polymorphism (SNP), gene copy number
Different (CNV), insertion and deletion mark (InDel).
Optionally, the detection method further includes using connector described in second aspect, and the connector includes the first connector,
5 ' ends of first connector and the cls gene to be checked connect.
Further, the connector further includes the second connector, and second connector is for promoting first connector and institute
State 5 ' end connections of cls gene to be checked.
Optionally, the detection method includes that genetic fragmentization to be detected is formed genetic fragment, by the genetic fragment
It carries out end reparation and in 3 ' ends plus A base, first connector is connected with 5 ' ends of the genetic fragment；The inspection is added
Probing needle, is extended linearly by PCR, and purifying obtains extension products；PCR amplification is carried out by template of the extension products,
Purifying obtains amplified production, and the amplified production is sequenced and is compared, and obtains sequencing result.
In the present invention, detection probe includes at least one capture probe, what the number of capture probe detected as needed
Target area is set.
In the prior art, the extraction process of the extraction of DNA, especially dissociative DNA is inevitably mixed into long segment base
Because of a group DNA, PCR amplification primer can be made in connection, so that amplified production is genomic DNA, subsequent dissociative DNA be interfered to survey
The analysis of sequence result, so that testing result is difficult have booster action to the judgement of the physiological conditions of individual.And the present invention mentions
The detection method of confession for the natural degradations such as dissociative DNA sample be not necessarily to artificial fragmentation, DNA 5 ' end connection the first connectors and
3 ' ends are hybridized by probe to be extended, it is possible to prevente effectively from the pollution of genome；Overcome short segment DNA double during PCR amplification
End primer falls in the defect on DNA fragmentation simultaneously, improves the sensitivity of detection accuracy and short segment DNA detection site.
Optionally, when carrying out multiple target point detection in Gene Mutation, the detection method includes:
(1) genetic fragmentization to be detected is formed into genetic fragment, by the genetic fragment carry out end reparation and
In 3 ' ends plus A base；
(2) genetic fragment that step (1) obtains is mixed with first connector, so that first connector and the step
Suddenly the genetic fragment that (1) obtains is connected, and obtains the genetic fragment for being connected with first connector；
(3) using the genetic fragment for being connected with first connector as template, the detection probe is added, passes through PCR
It is extended linearly, purifying obtains extension products；
(4) using the extension products as template carry out PCR amplification, purifying obtain amplified production, by the amplified production into
Row sequencing and comparison, obtain sequencing result.
It further, further include that second connector is added in the step (2), to promote the company of first connector
It connects.
Optionally, when carrying out multiple target point methylation modification detection, the detection method includes:
(1) genetic fragmentization to be detected is formed into genetic fragment, by the genetic fragment carry out end reparation and
In 3 ' ends plus A base；
(2) genetic fragment that the C base in first connector obtain after methylation modification with step (1) is mixed
It closes, the genetic fragment for obtaining first connector with the step (1) is connected, then carries out sulphite processing；
(3) using sulphite treated genetic fragment as template, the detection probe is added, is carried out by PCR linear
Extend, purifying obtains extension products, wherein target complementary series in the detection probe is that treated is to be checked for sulphite
The complementary series of cls gene；
(4) using the extension products as template carry out PCR amplification, purifying obtain amplified production, by the amplified production into
Row sequencing and comparison obtain methylation decorating site.
It further, further include that second connector is added in the step (2), to promote the company of first connector
It connects.
Optionally, described when carrying out multiple target point methylation modification and methylolation modification or methylolation modification detection
Detection method includes:
(1) genetic fragmentization to be detected is formed into genetic fragment, by the genetic fragment carry out end reparation and
In 3 ' ends plus A base；
(2) experimental group is set, the C base in first connector is carried out to the base obtained after methyl modification with step (1)
Because segment mixes, the genetic fragment for obtaining first connector with the step (1) is connected, and successively uses perruthenate, sulfurous
Hydrochlorate processing；
Control group is set, the C base in first connector is carried out to the gene piece obtained after methyl modification with step (1)
Section mixing, the genetic fragment for obtaining first connector with the step (1) is connected, then is handled with sulphite；
(3) genetic fragment obtained respectively using the experimental group and the control group in step (2) is template, described in addition
Detection probe is extended linearly by PCR, and purifying obtains extension products, wherein the target complementation sequence in the detection probe
It is classified as sulphite treated the complementary series of cls gene to be checked；
(4) PCR amplification is carried out by template of the extension products, purifying obtains amplified production, by the experimental group and institute
The amplified production for stating control group is sequenced and is compared, and methylation and methylolation decorating site are obtained.
It further, further include that second connector is added in the step (2), to promote the company of first connector
It connects, the C base in second connector carries out methyl modification.
Optionally, when carrying out multiple target point gene mutation and methylation modification detection, the detection method includes:
(1) genetic fragmentization to be detected is formed into genetic fragment, by the genetic fragment carry out end reparation and
In 3 ' ends plus A base；
(2) experimental group a is set, the C base in first connector obtained after methylation modification with step (1)
Genetic fragment mixing, the genetic fragment for obtaining first connector with the step (1) is connected, then carries out at sulphite
Reason；
Control group a is set, first connector is mixed with the genetic fragment that step (1) obtains, makes first connector
The genetic fragment obtained with the step (1) is connected；
(3) institute is added as template in the genetic fragment obtained respectively using the experimental group a and control group a in step (2)
Detection probe is stated, is extended linearly by PCR, purifying obtains extension products, wherein detects and visits described in the experimental group a
Target complementary series in needle is the complementary series of sulphite treated cls gene to be checked；It is examined described in the control group a
Target complementary series in probing needle is the complementary series of cls gene to be checked.
(4) PCR amplification is carried out by template of the extension products, purifying obtains amplified production, by the experimental group a and institute
The amplified production for stating control group a is sequenced and is compared, and gene mutation and methylation decorating site are obtained.
It further, further include that second connector is added in the step (2), to promote the company of first connector
It connects, wherein when the experimental group a is added in second connector, the C base in second connector carries out methylation modification.
Optionally, when progress multiple target point gene mutation and methylolation modification detection or multiple target point gene mutation, methylation
When modification and methylolation modification, the detection method includes:
(1) genetic fragmentization to be detected is formed into genetic fragment, by the genetic fragment carry out end reparation and
In 3 ' ends plus A base；
(2) experimental group b is set, by what is obtained after the C base progress methyl modification in first connector with step (1)
Genetic fragment mixing, the genetic fragment for obtaining first connector with the step (1) are connected, and successively use perruthenate, Asia
Sulfuric acid salt treatment；
Control group b is set, the C base in first connector is carried out to the gene obtained after methyl modification with step (1)
Segment mixing, the genetic fragment for obtaining first connector with the step (1) is connected, then is handled with sulphite；
Control group c is set, first connector is mixed with the genetic fragment that step (1) obtains, makes first connector
The genetic fragment obtained with the step (1) is connected；
(3) genetic fragment obtained respectively with the experimental group b, the control group b and the control group c in step (2)
For template, the detection probe is added, is extended linearly by PCR, purifying obtains extension products, wherein the experimental group b
It is the complementation of sulphite treated cls gene to be checked with the target complementary series in detection probe described in the control group b
Sequence；Target complementary series in detection probe described in the control group c is the complementary sequence of cls gene to be checked；
(4) using the extension products as template carry out PCR amplification, purifying obtain amplified production, by the amplified production into
Row sequencing and comparison obtain gene mutation, methylation modification and methylolation decorating site.
It further, further include that second connector is added in the step (2), to promote the company of first connector
It connects, wherein when the experimental group b and control group b is added in second connector, the C base in second connector carries out methyl
Change modification.
Optionally, the concentration of the sulphite is 1M-10M.
Optionally, the concentration of the perruthenate is 0.15mM-150mM.Further, the concentration of the perruthenate is
5mM-25mM.Specifically, the perruthenate can be, but not limited to as potassium perruthenate.
Optionally, when carrying out real-time quantitative PCR or digital pcr quantitative detection, the sequence of the capture probe includes mesh
Mark complementary series.Further, the sequence of the capture probe further includes probe random tags sequence, the probe random tags
Sequence is made of 6-50 randomized bases.Further, the length of the target complementary series is 12bp-60bp, 15bp-
35bp or 20bp-25bp, the length of the probe random tags sequence are 10bp-50bp, 15bp-35bp or 18bp-25bp.
Optionally, when carrying out real-time quantitative PCR or digital pcr quantitative detection, first joint sequence is by 6-50
Randomized bases composition, second joint sequence are made of 6-50 randomized bases.Further, first joint sequence by
12-40 randomized bases composition, second joint sequence are made of 12-40 randomized bases.Further, described first
Joint sequence is made of 15-35 or 20-25 randomized bases, and second joint sequence is random by 15-35 or 20-25
Base composition.
In the present invention, if what the joint sequence can be made according to the demand of Illumina, BGI, Life sequenator
Dry modification and improvement, belong to protection scope of the present invention.
In the present invention, the testing gene can be, but not limited to as dissociative DNA, Circulating tumor DNA, paraffin tissue sections
The genomic DNA of the genomic DNA fragment of sample extraction, animals and plants or microorganism, artificial synthesized oligonucleotide sequence, group
It knits, the genomic DNA of cell or body fluid separation, chloroplast DNA and mitochondrial DNA.Specifically, the dissociative DNA is present in blood
In the body fluid such as slurry, urine, cerebrospinal fluid, BAL fluid, hydrothorax hydrops, amniotic fluid and refining and Cell culture invitro liquid,
It can be extracted by kit.
Optionally, the content of the genetic fragment is not less than 1ng.
In the prior art, such as probe liquid phase hybridization technique is utilized to detect gene mutation, methylation modification and/or hydroxyl
When methylation modification, after jointing, needs DNA content to reach 1 μ g and be just able to satisfy subsequent experimental needs.And in the present invention
In, the gene content after fragmentation can carry out subsequent experimental not less than 1ng, avoid testing gene content to the shadow of detection
It rings.
Optionally, the length of genetic fragment described in the step (1) is 30bp-5000bp.Further, the gene
The length of segment is 100bp-1000bp.
Optionally, the method for fragmentation includes that ultrasonic wave interrupts or digestion interrupts in the step (1).
Optionally, in the step (1) end repair be included in PCR pipe be added T4DNA kinases, T4 archaeal dna polymerase,
Klenow polymerase, dNTP, high temperature resistant polymerase and the genetic fragment carry out end reparation to the genetic fragment.Into one
Step, the condition of the PCR reaction is 35 DEG C of -40 DEG C of reactions 20min-40min, 55 DEG C of -65 DEG C of reaction 20min-40min.
Optionally, it is included in addition Klenow (3 ' -5 ' exo-) in PCR pipe at 3 ' ends plus A base in the step (1)
Enzyme, dNTP and the genetic fragment repaired by end carry out PCR reaction to the genetic fragment repaired by end
3 ' ends plus A base.
Optionally, the step (2), which is included in PCR pipe, is added first connector and second connector, T4DNA
The genetic fragment that ligase and step (1) obtain carries out PCR reaction, the base for obtaining first connector and the step (1)
Because 5 ' ends of segment are connected.Further, the condition of the PCR reaction is 15 DEG C of -25 DEG C of reaction 30min-60min.
In the present invention, the capture probe is unique in the position of the positive minus strand fragment complementation of the testing gene.Optionally,
The Tm value of the capture probe is 55 DEG C -75 DEG C.Optionally, annealing temperature during capture probe extends linearly is 85
DEG C -37 DEG C, elongating temperature is 20 DEG C -75 DEG C.
In the prior art, the synthesis of probe is expensive, is unfavorable for the progress of many experiments.And it can root in the present invention
A variety of capture probes are designed according to single sample, matching is good, at low cost, is suitble to detect while high-volume, multidigit point.
Optionally, the step (3), which is included in PCR pipe, is added the detection probe, polymerase, dNTP and the step
Suddenly the genetic fragment that (2) obtain, carrying out PCR reaction extends linearly the detection probe.Specifically, the item of the PCR reaction
Part can be, but not limited to as 95 DEG C of 1min, 95 DEG C of 30s 10-20 circulations, 80 DEG C of -40 DEG C of gradient heating and cooling and digit rate heating and cooling
30s,72℃1min.Specifically, the polymerase can be, but not limited to for T4DNA polymerase, Klenow (3 ' -5 ' exo-) enzyme,
Bst archaeal dna polymerase, PCR polymerase or AmpliTaq DNA polymerase.
Optionally, the step (4), which is included in PCR pipe, is added amplification enzyme, the first primer, the second primer and the step
(3) genetic fragment obtained.PCR reaction condition be 94 DEG C 1min, 5-10 recycle 94 DEG C 30s/58 DEG C 30s/72 DEG C of 30s,
It is stored after 72 DEG C of 5min in 12 DEG C.
Optionally, the purifying includes column purification or magnetic beads for purifying.
Optionally, the length of the amplified production is 150bp-1000bp.
Optionally, the sequence of the first primer be the sequence as shown in SEQ ID NO:6, or with the detection probe
Sequence is all identical, and the sequence of second primer is the sequence as shown in SEQ ID NO:7, or with the connector
Sequence is all identical.Wherein, the Sequence with the detection probe is mutually all the sequence with the detection probe
Column are at least 30% identical, and described with the Sequence of the connector is mutually all identical as the sequence of the connector at least 30%.
In the present invention, A/G/C/T is routinely to understand in biological field, and A is the abbreviation of adenine, T is thymidine
Abbreviation, C are the abbreviation of cytimidine, the abbreviation that G is guanine；N is the abbreviation of randomized bases, is expressed as tetra- kinds of bases of A/G/C/T
Any one.
In the prior art, the first primer and the second primer can be designed according to the sequence of detection probe, for multiple
In the presence of capture probe, it is difficult to ensure that the first primer and the second primer with relative to capture probe fall into same molecular sequences
In, especially degradation or the serious sequence of fragmentation therefore lead to genetic fragment loss of learning to be detected.And the present invention provides
The detection method of a kind of multiple target point gene mutation, methylation modification and/or methylolation modification, by genetic fragment to be detected
5 ' terminate into connector, 3 ' ends are in conjunction with detection probe, so that detection probe is extended linearly, to obtain cls gene to be checked
All gene informations in segment, ensure that coverage rate.
Optionally, described amplified production to be subjected to sequencing and comparison specifically includes: sequencing data fractionation and pretreatment are carried out,
Sequence alignment, substrate statistics, Data correction, point mutation detection, the detection of small insertion/deletion, copy number change are carried out to sequencing data
Different detection, DNA modification detection.Specifically, the sequence alignment is to compare sequencing sequence and standard gene group canonical sequence
To and positioning；The basic statistics is calculating and technical parameter and quality control index in statistical experiment process and sequencing procedure；Institute
Stating Data correction is to be distributed spy by the sequencing fragment calculated between the data distribution characteristics and multiple samples in the different regions GC
Sign corrects the error introduced due to sequencing batch wise differences and regional sequence architectural difference；The point mutation detection be by with
Standard gene group canonical sequence and thousand human genome canonical sequences are compared, and obtain the point mutation of cls gene to be checked；It is described
Small insertion/deletion is detected as obtaining by being compared with standard gene group canonical sequence and thousand human genome canonical sequences
The small insertion/deletion of cls gene to be checked；The copy number variation is detected as by calculating cls gene to be checked and contrasting data collection
Degree of peeling off difference obtains the result of variations of different genes region copy number；The DNA modification is detected as converting by C-T, system
It is horizontal to count methylation or methylolation modification in cls gene to be checked.
Fourth aspect, the present invention provides one kind for detecting multiple target point gene mutation, methylation modification and/or methylol
Change the kit of modification, which is characterized in that including detection probe described in first aspect.
Optionally, the kit further includes the connector as described in second aspect.Further, the connector includes first
Connector and the second connector.
Optionally, the kit further includes the first primer and the second primer as described in second aspect.
Optionally, the kit further includes purified reagent.
5th aspect, the present invention provides detection probes as described in relation to the first aspect, the connector as described in second aspect, such as
Detection method described in the third aspect or the kit as described in fourth aspect are in detection multiple target point gene mutation, methylation modification
And/or the application in methylolation modification.
Beneficial effects of the present invention:
The present invention provides a kind of for detecting multiple target point gene mutation, methylation modification and/or methylolation modification
Detection probe, connector, detection method and kit, it is low to gene content to be detected requirement, effectively avoid mrna length to be detected
Interference with content to detection is conducive to the detection of degree of degradation height, the serious gene of fragmentation, when detecting can be to be detected
Gene covers comprehensively, and testing cost it is low, it is high-efficient, have a wide range of application, to apparent/hereditary variation molecular marked compound screening, lose
It passes identification detection, the prediction of disease early screening, disease risks, medication and diagnosis and plays important booster action.
Detailed description of the invention
Fig. 1 is the sequencing depth map that detection probe provided in an embodiment of the present invention carries out detection in Gene Mutation.
Specific embodiment
The following is a preferred embodiment of the present invention, it is noted that for those skilled in the art
For, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also considered as
Protection scope of the present invention.
The modification detection of embodiment monomethylization
Step 1: it is extracted in vitro liver blood plasma using magnetic bead or DSP Blood Mini kit (Qiagen) kit
Dissociative DNA will extract product and analyze through the detection of Agilent 2100 library fragments distribution situation, and discovery is swum in 161bp
Then successively there is the multiple segment distribution of 161bp in main peak from DNA.
Step 2: it is poly- that 0.8 μ l T4DNA kinases, 0.8 μ l T4DNA polymerase, 0.2 μ l Klenow are added in PCR pipe
Synthase, 1 μ l dNTP (10mM), 70mM Tris-HCl, 10mM MgCl2, 5mM DTT, 0.2 μ l high temperature resistant polymerase and trip
From DNA and it is sterile exceed water so that PCR reaction total volume be 50 μ l.PCR reaction condition is 37 DEG C of reaction 30min, and 60 DEG C anti-
Answer 30min.1 μ l, first joint sequence as shown in SEQ ID NO:4,1 μ l are added after reaction as shown in SEQ ID NO:5
Second joint sequence, 8 μ l ATP (10mM), 3 μ l T4DNA ligases, 60mM Tris-HCl (pH 7.6), 10mM MgCl2、
1mM ATP, 1mM DTT and 7.5%PEG4000-8000, connector connection reaction total volume is 80 μ l.Reaction condition is 20 DEG C anti-
Answer 40min.Magnetic beads for purifying of the reaction product Jing Guo 0.9 times of volume is eluted in the ultrapure water of 20 μ l.To product after purification into
The processing of row sulphite, the product of processing are eluted in the ultrapure water of 20 μ l further across column purification or magnetic beads for purifying.
Step 3: the product of the step 2 sulphite of 3M is handled；
Step 4: designing detection probe to target area, include 10 capture probes, and wherein N is randomized bases, sequence
In C base carry out methylation modification, as shown in table 1 (5 ' → 3 '), 10 sequence capture probes are dissolved in for sequence capture probe
It in sterile ultrapure water and mixes, obtains mixed probe.
Table 1
Step 5: taking the product of 20 μ l step 3, is added into different PCR pipes, 1 μ l mixed probe (0.01 μ is added
M-10 μM), 5 μ l 10X PCR amplification buffers, 0.8 μ l PCR amplification enzyme, 1 μ l dNTP (10mM) mixing.PCR reaction condition
For 95 DEG C of 1min, 95 DEG C of 30s 20 circulations, 80 DEG C of -40 DEG C of gradient cooling 30s, 72 DEG C of 1min.PCR product is purified and eluted
Into the ultrapure water of 20 μ l.
Step 6: being added in the product of step 5 into different PCR pipes, and 1 μ l dNTP (10mM), 5 μ l is added
10X PCR amplification buffer, 0.8 μ l PCR amplification enzyme, 1 μ l the first primer sequence as shown in SEQ ID NO:6,1 μ l such as SEQ
Second primer sequence and sterile ultrapure water shown in ID NO:7, so that PCR reaction total volume is 50 μ l.PCR reaction condition is
94 DEG C of 1min, 10 94 DEG C 30s/58 DEG C 30s/72 DEG C of 30s, 72 DEG C of 5min recycled are stored in 12 DEG C.PCR product is through 2% fine jade
After lipolysaccharide electrophoresis, with the text of QIAquick gel extraction kit (Qiagen) recovery purifying 300-450bp segment
Library, or directly PCR product is recycled with Agencourt AMPure Beads (Beckman Coulter) magnetic beads for purifying.
Step 7: above-mentioned product is sequenced, and to lower machine data filtering after, count each capture probe capture target
The depth of gene, the methylation level of analysis statistics target gene related locus, the results are shown in Table 2, wherein positive minus strand represents
Positive-sense strand (+) or antisense strand (-) when detection, C type indicates type of the C base in genetic fragment, using CG as detection site,
CG is important methylation decorating site, and C number illustrates that the number that C base methylates, C&T number illustrate C base
Occur methylation and the number that does not methylate and.It is good to the responsiveness of 10 target areas detection as can be seen from Table 2,
The methylation modification for reflecting 10 target areas is horizontal.
Table 2
Embodiment two gene abrupt climatic change
Step 1: extracting in vitro Whole Blood Genomic DNA using magnetic bead or DSP Blood Mini kit (Qiagen),
DNA is eluted to 30 μ l's after purified by genomic DNA fragment to 100bp-1000bp by the method interrupted by ultrasonic wave
In ultrapure water.
Step 2: it is poly- that 0.8 μ l T4DNA kinases, 0.8 μ l T4DNA polymerase, 0.2 μ l Klenow are added in PCR pipe
Synthase, 1 μ l dNTP (10mM), 70mM Tris-HCl, 10mM MgCl2, 5mM DTT, 0.2 μ l high temperature resistant polymerase and trip
From DNA and it is sterile exceed water so that reaction total volume be 50 μ l.End repairs and A reaction condition is added to be 37 DEG C of reaction 30min,
50 DEG C of reaction 30min.1 μ l, first joint sequence as shown in SEQ ID NO:4,1 μ l such as SEQ ID will be added in product again
Second joint sequence shown in NO:5,8 μ l ATP (10mM), 3 μ l T4DNA ligases, 60mM Tris-HCl (pH 7.6),
10mM MgCl2, 1 mM ATP, 1mM DTT and 7.5%PEG4000-8000, reaction total volume is 80 μ l.Connector connection reaction
Condition is 20 DEG C of reaction 50min.Reaction product is purified by QIAquick PCR Purification Kit (Qiagen), is washed
It takes off into the ultrapure water of 20 μ l.Sulphite processing is carried out to product after purification, the product of processing is further across column purification
Or magnetic beads for purifying, it is eluted in the ultrapure water of 20 μ l.
Step 3: 21 capture probes, sequence such as 3 institute of table are devised for thalassemia gene HBA1, HBA2 and HBB
Show (5 ' → 3 '), 21 sequence capture probes are dissolved in sterile ultrapure water and are mixed, mixed probe is obtained.
Table 3
Step 4: product, the 1 μ l mixed probe (0.01 μM -10 μM), 5 μ l of 20 μ l step 2 are added in PCR pipe
10X PCR amplification buffer, 0.8 μ l PCR amplification enzyme, 1 μ l dNTP (10mM) mixing.PCR reaction condition is 95 DEG C of 1min, 95
DEG C of 30s 15 circulation, 80 DEG C of -40 DEG C of gradient cooling 30s, 72 DEG C of 1min.PCR product is purified and is eluted to the ultrapure of 20 μ l
In water.
Step 5: product, 1 μ l dNTP (10mM), 5 μ l 10X the PCR amplification of 20 μ l step 4 are added in PCR pipe
Buffer, 0.8 μ l PCR amplification enzyme, 1 μ l the first primer sequence as shown in SEQ ID NO:6,1 μ l such as SEQ ID NO:7 institute
The second primer sequence and sterile ultrapure water shown, so that PCR reaction total volume is 50 μ l.PCR reaction condition is 94 DEG C
1min, 8 94 DEG C 30s/58 DEG C 30s/72 DEG C of 30s, 72 DEG C of 5min recycled are stored in 12 DEG C.PCR product is through 2% agarose
After electrophoresis, with QIAquick gel extraction kit (Qiagen) recovery purifying 150bp-1000bp segment.
Step 6: above-mentioned product is sequenced, and to lower machine data filtering after, count the sequencing depth point of target area
Cloth, as a result as shown in Figure 1.It can be seen that target area (between 220000bp-230000bp, 5247000bp attachment and
Near 5248000bp) sequencing depth it is high, show probe and target area efficiently, specific binding, be conducive to target gene
It is analyzed.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Sequence table
<110>Shenzhen's mutable gene Science and Technology Ltd.
<120>a kind of for detecting multiple target point gene mutation, the detection probe of methylation modification and/or methylolation modification, side
Method and kit
<160> 7
<170> SIPOSequenceListing 1.0
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<213> Artificial Sequence
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gtgactggag ttcagacgtg tgctcttccg atct 34
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<211> 58
<212> DNA
<213> Artificial Sequence
<400> 2
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatct 58
<210> 3
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<213> Artificial Sequence
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<213> Artificial Sequence
<400> 5
tgacatagat cggaagagc 19
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<213> Artificial Sequence
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<213> Artificial Sequence
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Claims (10)
1. a kind of detection probe, which is characterized in that the detection probe includes at least one capture probe, the capture probe from
The sequence at 5 ' ends to 3 ' ends successively includes preamble sequence, probe random tags sequence and target complementary series, the preamble sequence
As shown in SEQ ID NO:1, the probe random tags sequence is made of 0-12 randomized bases, the target complementary series packet
Include base of at least ten continuously with gene complementation to be detected.
2. the detection method of a kind of multiple target point gene mutation, methylation modification and/or methylolation modification, which is characterized in that packet
It includes using detection probe as described in claim 1, the detection probe and gene recombination to be detected.
3. detection method as claimed in claim 2, which is characterized in that the detection method further includes using the first connector, institute
Stating sequence of first connector from 5 ' ends to 3 ' ends successively includes First ray, the first connector random tags sequence and T base, described
3 ' ends of the First ray continuous alkali of at least ten of 3 ' ends to 5 ' ends in the sequence as shown in SEQ ID NO:2 to 5 ' ends
Base, the first connector random tags sequence are made of 0-12 randomized bases, first connector and the cls gene to be checked
5 ' end connection.
4. detection method as claimed in claim 3, which is characterized in that the detection method further includes using the second connector, institute
Stating sequence of second connector from 5 ' ends to 3 ' ends successively includes the second connector random tags sequence and the second sequence, and described second connects
Head random tags sequence is made of 0-12 randomized bases, and second sequence is in the sequence as shown in SEQ ID NO:3
The continuous base of at least six, second connector are used to that 5 ' ends of first connector and the cls gene to be checked to be promoted to connect.
5. detection method as claimed in claim 3, which is characterized in that when carrying out multiple target point detection in Gene Mutation, the inspection
Survey method includes:
(1) genetic fragmentization to be detected is formed into genetic fragment, the genetic fragment is subjected to end reparation and 3 '
End plus A base；
(2) genetic fragment that step (1) obtains is mixed with first connector, so that first connector and the step
(1) genetic fragment obtained is connected, and obtains the genetic fragment for being connected with first connector；
(3) using the genetic fragment for being connected with first connector as template, the detection probe is added, is carried out by PCR
It extends linearly, purifying obtains extension products；
(4) PCR amplification is carried out by template of the extension products, purifying obtains amplified production, the amplified production is surveyed
Sequence and comparison obtain sequencing result.
6. detection method as claimed in claim 3, which is characterized in that described when carrying out multiple target point methylation modification detection
Detection method includes:
(1) genetic fragmentization to be detected is formed into genetic fragment, the genetic fragment is subjected to end reparation and 3 '
End plus A base；
(2) genetic fragment that the C base in first connector obtain after methylation modification with step (1) is mixed, is made
First connector is connected with the genetic fragment that the step (1) obtains, then carries out sulphite processing；
(3) using sulphite treated genetic fragment as template, the detection probe is added, is extended linearly by PCR,
Purifying obtains extension products, wherein target complementary series in the detection probe is sulphite treated base to be detected
The complementary series of cause；
(4) PCR amplification is carried out by template of the extension products, purifying obtains amplified production, the amplified production is surveyed
Sequence and comparison obtain methylation decorating site.
7. detection method as claimed in claim 3, which is characterized in that repaired when carrying out multiple target point methylation modification with methylolation
When decorations or methylolation modification detection, the detection method includes:
(1) genetic fragmentization to be detected is formed into genetic fragment, the genetic fragment is subjected to end reparation and 3 '
End plus A base；
(2) experimental group is set, the C base in first connector is carried out to the gene piece obtained after methyl modification with step (1)
Section mixing, the genetic fragment for obtaining first connector with the step (1) are connected, and successively use perruthenate, sulphite
Processing；
Control group is set, the genetic fragment that the C base in first connector obtain after methyl modification with step (1) is mixed
It closes, the genetic fragment for obtaining first connector with the step (1) is connected, then is handled with sulphite；
(3) detection is added as template in the genetic fragment obtained respectively using the experimental group and the control group in step (2)
Probe is extended linearly by PCR, and purifying obtains extension products, wherein the target complementary series in the detection probe is
The complementary series of sulphite treated cls gene to be checked；
(4) PCR amplification is carried out by template of the extension products, purifying obtains amplified production, by the experimental group and described right
It is sequenced and is compared according to the amplified production of group, obtain methylation and methylolation decorating site.
8. a kind of for detecting the kit of multiple target point gene mutation, methylation modification and/or methylolation modification, feature exists
In, including detection probe as described in claim 1.
9. kit as claimed in claim 8, which is characterized in that further include the first connector, first connector from 5 ' end to
The sequence at 3 ' ends successively includes First ray, the first connector random tags sequence and T base, and the 3 ' of the First ray are held to 5 '
Hold in the sequence such as SEQ ID NO:2 shown in 3 ' end to 5 ' hold the continuous base of at least ten, first connector with
Machine sequence label is made of 0-12 randomized bases.
10. detection probe as described in claim 1, such as described in any item detection methods of claim 2-7 or as right is wanted
Ask the described in any item kits of 8-9 answering in detection multiple target point gene mutation, methylation modification and/or methylolation modification
With.
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