CN106319074A - LNA probe for detecting KRAS gene mutation and detection method - Google Patents
LNA probe for detecting KRAS gene mutation and detection method Download PDFInfo
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Abstract
The invention relates to the technical field of gene detection, in particular to an LNA (Locked Nucleic Acid) probe, which is represented by sequences 1, 2, and 3 in a sequence table, for detecting KRAS gene mutation. A detection method for the KRAS gene mutation comprises the following steps: constructing a nanopore sensor by natural alpha-hemolysin, hybridizing the LNA probe with a gene to be detected to obtain a hybrid material, and generating a blocking signal through a nano channel of the nanopore sensor. By the hybridization of the LNA-modified probe and a KRAS mutant gene and detection of the alpha-hemolysin nanopore sensor, the specificity of target gene detection is greatly improved, and a concentration relation between the frequency of the signal and the KRAS mutant gene can be constructed to quantitatively analyze a target; the LNA probe has the advantages of simplicity, quickness and high sensitivity; DNA amplification is not needed; the operation is simple and specific; the LNA probe is high in applicability.
Description
Technical field
The present invention relates to technical field of gene detection, particularly to a kind of LNA probe detecting KRAS gene mutation, also relate to
And use this LNA probe detection method to KRAS gene mutation.
Background technology
Malignant tumor is the major disease of serious threat human health and life.The statistical data of World Health Organization (WHO) shows
Show in recent years, the most every 8 deaths in the whole world just have 1 people to die from cancer, causes than acquired immune deficiency syndrome (AIDS), tuberculosis and malaria
Death toll summation is taller.Report is had to point out, by current medical level, 5 years survival rates to cancer patient early treatment
More than 80%, and 5 years survival rates after patient with advanced cancer treatment can be less than 10%.Therefore, the early discovery and early of cancer
Phase diagnosis has particularly important meaning to prevention and the treatment of cancer.
At present, the methods for clinical diagnosis of cancer is still based primarily upon imaging examination tissue and cellular morphology, and its resolution is relatively
Low, it is difficult to realize the early diagnosis of cancer.Occurring of cancer is the most closely related with the unconventionality expression of gene, therefore, in recent years,
Gene test becomes the focus of clinical diagnosis and scientific research, and the crucial oncogene expression level by detection tumor patient can be real
The early diagnosis of existing cancer and treatment, and prognosis is played the most crucial effect.KRAS is the key gene of " startup " canceration
One of, when KRAS gene is undergone mutation, may result in malignant increment, break up and regulate and control disorder.Research shows, 20% is non-little
There is KRAS gene mutation in cell lung cancer, 30-35% PATIENTS WITH LARGE BOWEL.
The method of detection KRAS gene mutation is mainly DNA sequencing and polymerase chain reaction (PCR) at present.DNA sequencing
Method is currently the goldstandard of detection gene mutation, but order-checking often has higher background, and mutant allele ratio is less than
It is difficult to when 20% judge whether sudden change, and cost is high, the longest, it is unfavorable for timely treatment clinically.Round pcr holds
Easily occurring that fallibility expands, easily there is false positive or false negative result in the detection especially for point mutation.
Biological nano multichannel analysis is the technology directly studying molecule weak interaction at single molecules level.At extra electric field
Under the driving of power, single testing molecule is limited in the passage of nano-scale.Owing to single testing molecule is in nanochannel
Physics occupy-place effect etc., change the resistance of passage, thus cause the ion current flowing through nanochannel to change, formed
Block current signal.The ion flow resistance signal such as degree and Xining that breaks can reflect the information such as the sequence of molecule, architectural feature.
Lock nucleic acid (Locked Nucleic Acid, LNA) is a kind of novel nucleic acids analog, containing 2 '-O and 4 '-C's
Methylene connects, and limits the motility of ribofuranose ring, structure latches becomes the double-ring mode of rigidity.LNA can follow
Base pair complementarity principle is combined with nucleic acid, and, Heat stability is good, antienzyme strong with complementary natural acid affinity are cut and internal nothing
The advantages such as poison so that LNA has unique advantage highly sensitive, that specificity is good in terms of detection of nucleic acids, obtain extensively in recent years
Pay close attention to.LNA probe is combined with biological nano multichannel analysis technology, set up a kind of simplicity quickly, high specificity, highly sensitive
KRAS gene mutation new detecting method, the early diagnosis for cancer is significant.
Summary of the invention
In order to solve above prior art to utilize PCR false positive or false negative knot easily occur the detection of point mutation
Really, DNA sequencing method background is high, the problem of time-consuming length, this application discloses a kind of LNA probe detecting KRAS gene mutation.
Present invention also offers this LNA probe detection method to KRAS gene mutation of use.
The present invention is obtained through the following steps:
A kind of LNA probe detecting KRAS gene mutation, for one or more in following base sequence:
LNA-P135A: 5 '-ACGCCATCAGCTC-3 ',
LNA-P135C: 5 '-ACGCCAGCAGCTC-3 ',
LNA-P134T: 5 '-ACGCCACAAGCTC-3 '.
A kind of detection method of KRAS gene mutation, with natural alpha hemolysin build nanopore sensor, by LNA probe with
Gene recombination to be checked, obtains hybrid so that it is by the nanochannel of nanopore sensor, produces disabling signal；
Described LNA probe is one or more in the base sequence in claim 1.
Described detection method, Xining match value more than 100ms, in gene the most to be checked containing 135A, 135C and/or
134T suddenlys change.
Described detection method, LNA probe is as follows with gene recombination to be checked operation:
LNA probe and gene to be checked are made into stock solution with ultra-pure deionized water respectively, mixing, and 95 DEG C of degeneration 5 minutes are placed in
Room temperature 2 hours so that it is anneal.
Described detection method, it is as follows that natural alpha hemolysin builds nanopore sensor operation:
(1) phospholipid chloroformic solution addition n-decane is configured to 30mg mL-1Solution, with No. 000 marten hair pen at 1mL
Uniform application 30mg mL inside and outside the aperture of trans detection cell-1Phospholipid n-decane solution；
(2) it is separately added into 1mL 1M KCl electrolyte solution: pH=after cis detection cell and trans detection cell being assembled
7.8,10mM Tris-HCl, 1mM EDTA；
(3) a pair Ag/AgCl electrode is immersed in electrolyte solution, popped one's head in phospholipid bilayer by current amplifier
Film two ends apply+100mV voltage, use czochralski method to form phospholipid bilayer tunic at the aperture in trans pond；
(4) in cis detection cell, add 1 μ L 5 μ g mL-1Alpha hemolysin, when alpha hemolysin is at phospholipid bilayer tunic
On when being self-assembly of a stable nanochannel, ion stream is by quantization step, in+120mV voltage, the condition of 1M KCl
Under, the stable open pore current of single nanochannel is 130pA.
Described detection method, the hybrid nanochannel by nanopore sensor, the process producing disabling signal is:
Hybrid is injected in cis detection cell, by the driving displacement of applied voltage, sensed by alpha hemolysin one by one
The nanochannel of device, produces disabling signal, and the pA level electric current of generation amplifies collection by current amplifier, and passes through low-pass filtering
Device is filtered, and frequency filtering is 3kHz, and the analogue signal that amplifier collects is converted to digital quantity by digital to analog converter,
And be transferred on computer, by software real-time monitored and record nanochannel single molecule experiments data.
Described detection method, detectable concentration scope is 5nM-500nM.
Beneficial effects of the present invention:
1) probe that the present invention is modified by LNA and KRAS gene recombination, by alpha hemolysin nanopore sensor
Detection, is greatly improved the specificity of genes of interest detection, can set up the concentration relationship of signal frequency and KRAS gene,
Object is carried out quantitative analysis, there is advantage simple, quick, high-sensitive；
2) utilize LNA probe, in conjunction with nanopore sensor, the detection to KRAS difference mutant gene can be realized, have very
High single base discrimination rate, highly sensitive；Without nano-pore is modified, it is not necessary to DNA cloning, the opposite sex simple to operate, have
Good application.
Accompanying drawing explanation
Fig. 1 utilizes nanopore sensor to distinguish 135A, 135C and 134T mutant gene；
A:LNA-P135A/ 135A hybrid, b:LNA-P135A/ 135C-134T hybrid.C:LNA-P135C/ 135C hybridizes
Thing, d:LNA-P135C/ 135A-134T hybrid.E:LNA-P134T/ 134T hybrid, f:LNA-P134T/ 135A-135C hybridizes
Thing；
Fig. 2 carries out quantitative analysis to three kinds of KRAS genes；
A: to 135A quantitative analysis, b: to 135C quantitative analysis, c: to 134T quantitative analysis.The concentration of three kinds of mutant genes
All between 5nM and 500nM.Voltage :+120mV, electrolyte solution: 1M KCl (pH=7.8,10mM Tris-HCl, 1mM
EDTA)；
Fig. 3 detects the content of three kinds of KRAS genes in human serum；
A: utilize probe LNA-P135ADetection serum in 135A (on) and 135C-134T (under).B: have serum and depletion of blood
LNA-P time clear135AThe frequency of/135A hybrid.C: utilize probe LNA-P135C135C in detection serum (on) and 135A-
134T (under).D: LNA-P when having serum and serum-free135CThe frequency of/135C hybrid.E: utilize probe LNA-P134TDetection blood
134T in Qing (on) and 135A-135C (under).F: LNA-P when having serum and serum-free134TThe frequency of/134T hybrid.Blood
In clear, the concentration of determinand is 50nM, voltage :+120mV, electrolyte solution: 1M KCl (pH=7.8,10mM Tris-HCl,
1mM EDTA)。
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further described:
Embodiment 1: the foundation of detection method
1., for 3 kinds of mutant nucleotide sequences of KRAS gene 12,13 bit codon mutation, design corresponding LNA probe.
KRAS gene order is as follows:
| Title | Sequence (5 '-3 ') | Type |
| 135A | GTT GGA GCT GAT GGC GTA G | DNA |
| 135C | GTT GGA GCT GCT GGC GTA G | DNA |
| 134T | GTT GGA GCT TGT GGC GTA G | DNA |
Probe sequence is as follows:
| Title | Sequence (5 '-3 ') | Type | Position in sequence table |
| LNA-P135A | ACG CCA TCA GCT C | LNA | Sequence 1 |
| LNA-P135C | ACG CCA GCA GCT C | LNA | Sequence 2 |
| LNA-P134T | ACG CCA CAA GCT C | LNA | Sequence 3 |
2.LNA probe respectively with corresponding 3 kinds of KRAS gene recombinations, obtain 3 kinds of hybrids.
LNA probe and KRAS gene are made into 100 μMs of stock solutions with ultra-pure deionized water respectively.With LNA-P135AWith
As a example by 135A, taking 5 μ L mixing respectively, 95 ° of degeneration 5 minutes, as room temperature 2 hours so that it is slowly anneal.Remaining base that suddenlys change
Cause and corresponding LNA probe process equally.
3. build nanopore sensor with natural alpha hemolysin.
Phospholipid chloroformic solution is drained, adds n-decane and be configured to 30mg mL-1Solution.Exist with No. 000 marten hair pen
Uniform application 30mg mL inside and outside the aperture of 1mL trans detection cell-1Phospholipid n-decane solution.By cis detection cell and
Trans detection cell is separately added into 1mL 1M KCl electrolyte solution (pH=7.8,10mM Tris-HCl, 1mM after assembling
EDTA).A pair Ag/AgCl electrode is immersed in electrolyte solution, is popped one's head in phospholipid bilayer tunic two by current amplifier
End applies+100mV voltage, uses czochralski method to form phospholipid bilayer tunic at the aperture in trans pond.
1 μ L 5 μ g mL is added in cis detection cell-1Alpha hemolysin.When alpha hemolysin on phospholipid bilayer tunic from
When assembling one stable nanochannel of formation, ion stream is by quantization step.Under conditions of+120mV voltage, 1M KCl,
The stable open pore current of single nanochannel is 130pA.
The most different hybrids is joined in electrolyzer, record and analyze the nano-pore electricity before and after addition hybrid
Flow resistance break signal changes.
10 μ L hybrids to be measured are injected separately in cis detection cell, now final concentration of in detection cell of determinand
500nM.Testing molecule is by the driving displacement of applied voltage, one by one by alpha hemolysin nanochannel, produces disabling signal.
The pA level electric current that experiment produces is amplified by ChemClamp current amplifier (Dagan Corporation company, the U.S.) to be adopted
Collection, and be filtered by low pass filter, frequency filtering is 3kHz.The analogue signal that amplifier collects is passed through
DigitalData1440A digital to analog converter (Axon Instruments company, the U.S.) is converted to digital quantity, and is transferred to electricity
On brain.By PClamp 10.6 software (Axon Instruments company, the U.S.) real-time monitored and record nanochannel list and divide
Sub-experimental data.
Embodiment 2: utilize LNA probe in detecting KRAS gene
Respectively by 3 kinds of probes and corresponding KRAS gene recombination, the 3 kinds of hybrid (135A/LNA-P obtained135A,
135C/LNA-P135CAnd 134T/LNA-P134T) detected by nanopore sensor respectively.As comparison, respectively by probe
Hybridize with the mixture of remaining 2 kinds of mutant gene, the same terms (voltage :+120mV, electrolyte solution: 1M KCl (pH=7.8,
10mM Tris-HCl, 1mM EDTA)) under with nanopore sensor detect, result is as shown in Figure 1.By probe LNA-P135AWith prominent
Become the hybrid (135A/LNA-P of gene 135A135A) inject after detection cell, create blocking-up event (Fig. 1 a) for a long time,
Xining match value is 452.17ms, and probe LNA-P135ADetect through nano-pore with the mixture of remaining 2 kinds of mutant gene
After, disabling signal is the shortest, and Xining is 23.55ms (Fig. 1 b).Under the same terms, hybrid 135C/LNA-P135CWith
134T/LNA-P134TXining after nanopore sensor is 420.35ms (Fig. 1 c) and 513.77ms (figure respectively
1e), the Xining of corresponding comparison is 26.31ms (Fig. 1 d) and 19.83ms (Fig. 1 f) respectively.The resistance that not only statistical analysis obtains
Break time obvious difference, the most also can make a distinction easily by observing, thus be detected by purpose mutant gene
Come.
Embodiment 3: KRAS gene is carried out quantitative analysis
As a example by KRAS gene 135A, take the 135A and isocyatic probe LNA-P of variable concentrations respectively135AMiscellaneous
Hand over, inject the final concentration after detection cell and be respectively 5nM, 25nM, 50nM, 100nM, 500nM, the frequency of statistics blocking-up event, build
Vertical frequency and the concentration relationship of mutant gene 135A, thus 135A is carried out quantitative analysis.Result is as shown in Figure 2 a.To 135C and
The quantitative analysis results of 134T is shown in Fig. 2 b and Fig. 2 c respectively.Three kinds of mutant genes between detectable concentration 5nM-500nM, in good
Good linear relationship.
Embodiment 4: the KRAS gene in detection serum
In order to verify whether the method can be used for the detection of KRAS gene in blood serum sample, three kinds of LNA probes are with prominent
The hybrid becoming gene is added separately in serum, and final concentration is 50nM, is then passed through nanopore sensor, and statistical nature is believed
Number frequency.As shown in Figure 3 a, when containing LNA-P135AThe serum of/135A hybrid, through nano-pore, creates the long period
Disabling signal, by contrast, containing LNA-P135AThe disabling signal that the serum of/135C-134T hybrid produces is the shortest.Serum
LNA-P in sample135AThe frequency when frequency of/135A hybrid and serum-free is substantially quite (Fig. 3 b).For other two kinds
LNA probe, equally can specific detection to the corresponding mutant gene (Fig. 3 c and 3e) in serum, and hybrid is through nanometer
Frequency during hole also is not apparent from being affected (Fig. 3 d and 3f) by serum.Therefore the method may be used for the life of specific detection complexity
KRAS gene in thing sample.
Above-described embodiment 1,2,3 describes 3 LNA probe LNA-P respectively135A、LNA-P135C、LNA-P134TThe most right
135A, 135C, 134T sudden change carries out the scheme detected, and in concrete detection, is and does not knows in gene to be checked which there is
Plant sudden change, so, detect after 3 probes mixing, situation about extending if there is Xining simultaneously, just illustrate to treat sample
There is 135A, 135C, 134T sudden change in product, which sudden change the most individually uses 3 probe in detecting to determine is；If 3
Detect after the mixing of individual probe simultaneously, the situation that Xining extends do not occur, then illustrate measuring samples does not contains 135A,
135C, 134T suddenly change, and this detects end-of-job, can shorten detection program and time.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not limited by embodiment
System, the change made, modifies, combines, substitutes, simplifies and all should be under other any spirit without departing from the present invention and principle
Equivalence substitute mode, within being included in protection scope of the present invention.
<110>Linyi University
<120>LNA probe and the detection method of KRAS gene mutation are detected
<160> 6
<210> 1
<211> 13
<212> DNA
<213>synthetic
<400> 1
ACGCCATCAG CTC 13
<210> 2
<211> 13
<212> DNA
<213>synthetic
<400> 2
ACGCCAGCAG CTC 13
<210> 3
<211> 13
<212> DNA
<213>synthetic
<400> 3
ACGCCACAAG CTC 13
<210> 4
<211>19
<212> DNA
<213>synthetic
<400> 4
GTTGGAGCTG ATGGCGTAG 19
<210> 5
<211> 19
<212> DNA
<213>synthetic
<400> 5
GTTGGAGCTG CTGGCGTAG 19
<210> 6
<211> 19
<212> DNA
<213>synthetic
<400> 6
GTTGGAGCTT GTGGCGTAG 19
Claims (7)
1. the LNA probe detecting KRAS gene mutation, it is characterised in that for one or more in following base sequence:
LNA-P135A: 5 '-ACGCCATCAGCTC-3 ',
LNA-P135C: 5 '-ACGCCAGCAGCTC-3 ',
LNA-P134T: 5 '-ACGCCACAAGCTC-3 '.
2. the detection method of a KRAS gene mutation, it is characterised in that build nanopore sensor with natural alpha hemolysin, will
LNA probe and gene recombination to be checked, obtain hybrid so that it is by the nanochannel of nanopore sensor, produces disabling signal；
Described LNA probe is one or more in the base sequence in claim 1.
Detection method the most according to claim 2, it is characterised in that Xining match value is more than 100 ms, base the most to be checked
Suddenly change containing 135A, 135C and/or 134T in Yin.
Detection method the most according to claim 2, it is characterised in that LNA probe is as follows with gene recombination to be checked operation:
LNA probe and gene to be checked are made into stock solution with ultra-pure deionized water respectively, mixing, and 95 DEG C of degeneration 5 minutes are placed in room temperature
2 hours so that it is anneal.
Detection method the most according to claim 2, it is characterised in that natural alpha hemolysin builds nanopore sensor operation
As follows:
(1) phospholipid chloroformic solution addition n-decane is configured to 30 mg mL-1Solution, with No. 000 marten hair pen at 1 mLtrans
Uniform application 30 mg mL inside and outside the aperture of detection cell-1Phospholipid n-decane solution；
(2) willcisDetection cell andtransDetection cell is separately added into 1 mL 1 M KCl electrolyte solution: pH=7.8 after assembling, and 10
MM Tris-HCl, 1 mM EDTA；
(3) a pair Ag/AgCl electrode is immersed in electrolyte solution, popped one's head in phospholipid bilayer tunic two by current amplifier
End applies+100 mV voltages, uses czochralski method to existtransPhospholipid bilayer tunic is formed at the aperture in pond；
(4) existcisDetection cell adds 1 μ L 5 μ g mL-1Alpha hemolysin, when alpha hemolysin is on phospholipid bilayer tunic
When being self-assembly of a stable nanochannel, ion stream is by quantization step, in+120 mV voltages, the condition of 1 M KCl
Under, the stable open pore current of single nanochannel is 130 pA.
Detection method the most according to claim 5, it is characterised in that the hybrid nanochannel by nanopore sensor,
The process producing disabling signal is:
Hybrid is injectedcisIn detection cell, by the driving displacement of applied voltage, one by one by alpha hemolysin sensor
Nanochannel, produces disabling signal, and the pA level electric current of generation amplifies collection by current amplifier, and is entered by low pass filter
Row filtering, frequency filtering is 3 kHz, and the analogue signal that amplifier collects is converted to digital quantity by digital to analog converter, and
It is transferred on computer, by software real-time monitored and record nanochannel single molecule experiments data.
Detection method the most according to claim 3, it is characterised in that detectable concentration scope is 5 nM-500 nM.
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Cited By (6)
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| CN107436317A (en) * | 2017-07-21 | 2017-12-05 | 河南金泰生物技术股份有限公司 | A kind of method using nanochannel technology for detection cancer |
| CN109295187A (en) * | 2018-10-31 | 2019-02-01 | 南京大学 | It is a kind of based on nano-pore to the dislocation sequencing approach of unnatural nucleic acids direct Sequencing |
| CN110408677A (en) * | 2019-07-27 | 2019-11-05 | 苏州丽纳芯生物科技有限公司 | One kind being based on solid nano hole colon cancer kras detection method of gene mutation |
| CN110408701A (en) * | 2019-07-27 | 2019-11-05 | 苏州丽纳芯生物科技有限公司 | A kind of colon cancer K-ras oncogene mutation detection system |
| CN110988345A (en) * | 2019-12-20 | 2020-04-10 | 临沂大学 | Method for detecting thrombin by using nanopore |
| CN112280875A (en) * | 2019-07-22 | 2021-01-29 | 四川大学华西医院 | Method, device and system for rapidly detecting bacterial drug resistance by using nanopore |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107436317A (en) * | 2017-07-21 | 2017-12-05 | 河南金泰生物技术股份有限公司 | A kind of method using nanochannel technology for detection cancer |
| CN109295187A (en) * | 2018-10-31 | 2019-02-01 | 南京大学 | It is a kind of based on nano-pore to the dislocation sequencing approach of unnatural nucleic acids direct Sequencing |
| CN109295187B (en) * | 2018-10-31 | 2021-02-09 | 南京大学 | Dislocation sequencing method for directly sequencing non-natural nucleic acid based on nanopore |
| CN112280875A (en) * | 2019-07-22 | 2021-01-29 | 四川大学华西医院 | Method, device and system for rapidly detecting bacterial drug resistance by using nanopore |
| CN110408677A (en) * | 2019-07-27 | 2019-11-05 | 苏州丽纳芯生物科技有限公司 | One kind being based on solid nano hole colon cancer kras detection method of gene mutation |
| CN110408701A (en) * | 2019-07-27 | 2019-11-05 | 苏州丽纳芯生物科技有限公司 | A kind of colon cancer K-ras oncogene mutation detection system |
| CN110988345A (en) * | 2019-12-20 | 2020-04-10 | 临沂大学 | Method for detecting thrombin by using nanopore |
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