CN108841924A - Peripheral white blood cells SNP site rapid detection method - Google Patents
Peripheral white blood cells SNP site rapid detection method Download PDFInfo
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- CN108841924A CN108841924A CN201810800571.1A CN201810800571A CN108841924A CN 108841924 A CN108841924 A CN 108841924A CN 201810800571 A CN201810800571 A CN 201810800571A CN 108841924 A CN108841924 A CN 108841924A
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Abstract
A kind of peripheral white blood cells SNP site rapid detection method, it is characterized in that the articles used using ammonium chloride solution, nano-pore ball, six oligonucleotides as detection, the detection of peripheral white blood cells SNP site is completed by the following steps, the first step takes patient's EDTA anticoagulation sample；Second step separates simultaneously enrichment of leukocytes；Third step, the leucocyte cutting section being enriched to；4th step, by leucocyte double-stranded DNA unwinding after segmentation, generate single stranded DNA makes single stranded DNA generate hybridized fragment in conjunction with six oligonucleotides of fluorescent marker with the base complementrity principle of DNA：5th step, the segment of hybridization are entered nano-pore ball, are detected using fluorescence detector, are established one group of complete gene by fluorescence label figure, are calculated using computer, final required SNP site genome sequence.The present invention does not need the laboratory of profession, does not need that PCR job qualification certificate, testing cost are low, process is simple, the time is short, result is accurate.
Description
Technical field
The present invention relates to technical field of biological, especially a kind of peripheral white blood cells SNP site rapid detection method.
Background technique
SNP, full name in English are single nucleotide polymorphism, and single nucleotide polymorphism is primarily referred to as
DNA sequence polymorphism caused by a single nucleotide variation at the genomic level, it is human heritable mutation base
The one of the most common type because in accounts for 90% or more of all known polymorphisms.SNP is widely present in human genome, average every
Just there is 1 in 500~1000 base-pairs, estimates that its sum is even more up to 3,000,000.Since human body is largely existing
SNP site, the Site discrepancy of everyone SNP form different genotype, determine people with disease different risks and
Differential responses to drug, and people is made to have an opportunity to find various diseases, wherein further including the relevant genome mutation of tumour；
From the point of view of experimental implementation, find that disease-correlative gene mutation is very useful by SNP site, therefore SNP under laboratory condition
Site primer has huge clinical demand.
In currently known technology, SNP site detection mainly includes following several method in the case of laboratory.The first：
One, the PCR amplification detection method under two generation gene sequencing, this method are needed to operate in the PCR Lab of profession, be tested
The shortcomings that room causes testing cost to increase there are high construction cost；Since required the scope of one's knowledge requires high, operator to have to pass through
Profession accompanies instruction that could operate（Operator needs to hold PCR job qualification certificate）；Program multioperation is cumbersome in detection, detection time compared with
It is long, all testing processes could be completed within most 72 hours；Also inevitably being contaminated in being detected leads to its detection
As a result it is affected.Second：Fluorescent PCR augmentation detection method：This method needs to grasp in the PCR Lab of profession
The shortcomings that work, laboratory causes testing cost to increase there are high construction cost；Since required the scope of one's knowledge requires high, operator must
It must accompany instruction that could operate by profession（Operator needs to hold PCR job qualification certificate）；Program multioperation is cumbersome in detection, when detection
Between it is longer, all testing processes could be completed by generally requiring 24 hours, be also inevitably contaminated and led in being detected
Its testing result is caused to be affected.The third：Hybridization color developing detection method under genetic chip：This method is needed in profession
The shortcomings that PCR Lab is interior to be operated, and laboratory causes testing cost to increase there are high construction cost；Due to required the scope of one's knowledge
Accompany instruction that could operate it is required that high, operator has to pass through profession（Operator needs to hold PCR job qualification certificate）；Program in detection
Multioperation is cumbersome, and detection time is longer, and all testing processes could be completed by generally requiring 8 to 24 hours；In being detected also
Inevitably being contaminated causes its testing result to be affected, and the result false positive of detection is high, the result inaccuracy of detection.
Above-mentioned several detection methods cause restriction to the detection of SNP site.
Summary of the invention
The drawbacks of in order to overcome existing a variety of detection methods to cause restriction to SNP site detection, the present invention provides one
The laboratory of profession kind is not needed, testing staff does not need that PCR job qualification certificate, testing cost are low, detection process is simple, detection time
A kind of accurate peripheral white blood cells SNP site rapid detection method of short, testing result.
The technical solution adopted by the present invention to solve the technical problems is：
A kind of peripheral white blood cells SNP site rapid detection method, it is characterised in that use ammonium chloride solution, nano-pore ball, six
The articles that oligonucleotide is used as detection complete the detection of peripheral white blood cells SNP site by the following steps, the
One step takes patient's EDTA anticoagulation sample；Second step separates simultaneously enrichment of leukocytes；Third step, the leucocyte being enriched to are cut
Cut segmentation；4th step, by leucocyte double-stranded DNA unwinding after segmentation, generating single stranded DNA with the base complementrity principle of DNA makes list
Chain DNA generates hybridized fragment in conjunction with six oligonucleotides of fluorescent marker：5th step, the segment of hybridization enter nano-pore ball,
It is detected using fluorescence detector, establishes one group of complete gene by fluorescence label figure, calculated using computer, final institute
The SNP site genome sequence needed.
The ammonium chloride solution, nano-pore ball, six oligonucleotides acid constituents composition ratio be respectively：Ammonium chloride solution
0.74%, nano-pore ball 0.3%, six oligonucleotide 10UM.
The DTA anticoagulation sample for taking patient, sample size is 2ml.
Simultaneously enrichment of leukocytes process is as follows for the separation, and the DTA anticoagulation sample of ammonium chloride solution and 2 ml patients are put
Enter in test tube and mix well, is stored at room temperature after ten minutes, is centrifuged 10 minutes with revolving speed 13000 turns of centrifuge per minute, and go
Except supernatant, lower layer is required leucocyte.
It is described when carrying out the leucocyte cutting section process that will be enriched to, it is added in restriction nuclease using in leucocyte
Enzyme cutting, is catalyzed the chain fracture of polynucleotides in leucocyte genome, and leucocyte genome is cut into the double of 250bp or so at random
Chain DNA segment.
Described to carry out leucocyte double-stranded DNA unwinding after being segmented, generate single stranded DNA is made with the base complementrity principle of DNA
When single stranded DNA generates hybridized fragment process in conjunction with six oligonucleotides of fluorescent marker, pass through the temperature control system of fluorescence detector
System, double-stranded DNA unwinding and generates single stranded DNA.
Present invention has the advantages that：It is low that SNP site of the present invention detection process does not need special laboratory, testing cost；
Testing staff does not need to hold PCR job qualification certificate, only needs that there is certain relevant knowledge can be operated, more convenient to use；It is required
The articles that detection uses are few, and operating process is simple, overcomes the cumbersome disadvantage of existing detection method；It is not easy in the process dirty
The result contaminate, obtained is more accurate；Operating time is short, all process steps can be completed within general one hour and operates and finally detected
As a result；Overcome the disadvantage of existing detection method time length.The present invention can have found disease related gene to detect by SNP site
Mutation provides strong technical support, based on above-mentioned, so the application prospect that the present invention has had.
Detailed description of the invention
The present invention will be further described with reference to the accompanying drawings and examples.
Attached drawing 1 is testing process block diagram of the invention.
Specific embodiment
Shown in Fig. 1, a kind of peripheral white blood cells SNP site rapid detection method, using ammonium chloride solution, nano-pore
Ball, six oligonucleotides as the articles that use of detection, ammonium chloride solution, nano-pore ball, six oligonucleotides acid constituents composition
Ratio is respectively：Ammonium chloride solution 0.74%, nano-pore ball 0.3%, six oligonucleotide 10UM.
Shown in Fig. 1, a kind of peripheral white blood cells SNP site rapid detection method is completed outer by the following steps
The detection of all blood leukocytes SNP sites, the first step take patient's EDTA anticoagulation sample；The DTA anticoagulation sample size of patient is
2ml.Second step separates simultaneously enrichment of leukocytes；It separates and enrichment of leukocytes process is as follows, the patient of ammonium chloride solution and 2 ml
DTA anticoagulation sample be put into test tube and mix well, be stored at room temperature after ten minutes, with revolving speed 13000 turns of centrifugation per minute
Machine is centrifuged 10 minutes, and removes supernatant, and lower layer is required leucocyte.Third step, the leucocyte cutting section being enriched to；It adopts
Restriction endonuclease is added in leucocyte, the chain fracture of polynucleotides in leucocyte genome is catalyzed, by leucocyte
Genome is cut into the double chain DNA fragment of 250bp or so at random.4th step generates leucocyte double-stranded DNA unwinding after segmentation
Single stranded DNA makes single stranded DNA generate hybridization piece in conjunction with six oligonucleotides of fluorescent marker with the base complementrity principle of DNA
Section：By the temperature control system of fluorescence detector, double-stranded DNA unwinding and single stranded DNA is generated.The segment of 5th step, hybridization enters
Nano-pore ball（Using more in existing technique of gene detection）, detected using fluorescence detector, establish one group it is complete
Gene by fluorescence label figure, is calculated using computer, final required SNP site genome sequence.
It is low that SNP site of the present invention detection process does not need special laboratory, testing cost；Testing staff does not need PCR
Job qualification certificate only needs that there is certain relevant knowledge can be operated, more convenient to use；The required articles used that detect are few, operation
Process is simple, overcomes the cumbersome disadvantage of existing detection method；It is more accurate to be not easy contaminated obtained result in the process；Behaviour
It is short to make the time, all process steps can be completed within general one hour and operates and obtain final detection result；Overcome existing detection method
The disadvantage of time length.The present invention do not need profession laboratory, testing staff do not need PCR job qualification certificate, testing cost it is low, detection
Process is simple, detection time is short, testing result is accurate, can for by SNP find disease-correlative gene mutation powerful technique is provided
Support, based on above-mentioned, so the application prospect that the present invention has had.
Basic principles and main features and advantages of the present invention of the invention have been shown and described above, for this field skill
For art personnel, it is clear that invention is not limited to the details of the above exemplary embodiments, and without departing substantially from spirit of the invention or
In the case where essential characteristic, the present invention can be realized in other specific forms.Therefore, in all respects, should all incite somebody to action
Embodiment regards exemplary as, and is non-limiting, the scope of the present invention by appended claims rather than on state
Bright restriction, it is intended that including all changes that fall within the meaning and scope of the equivalent elements of the claims in the present invention
It is interior.Any reference signs in the claims should not be construed as limiting the involved claims.
In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped
Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should
It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
The other embodiments being understood that.
Claims (6)
1. a kind of peripheral white blood cells SNP site rapid detection method, it is characterised in that using ammonium chloride solution, nano-pore ball,
The articles that six oligonucleotides are used as detection complete the detection of peripheral white blood cells SNP site by the following steps,
The first step takes patient's EDTA anticoagulation sample；Second step separates simultaneously enrichment of leukocytes；Third step, the leucocyte being enriched to
Cutting section；4th step, by leucocyte double-stranded DNA unwinding after segmentation, generate single stranded DNA is made with the base complementrity principle of DNA
Single stranded DNA generates hybridized fragment in conjunction with six oligonucleotides of fluorescent marker：The segment of 5th step, hybridization enters nano-pore
Ball is detected using fluorescence detector, is established one group of complete gene by fluorescence label figure, is calculated using computer, most
Required SNP site genome sequence eventually.
2. a kind of peripheral white blood cells SNP site rapid detection method according to claim 1, it is characterised in that the chlorine
Change ammonium salt solution, nano-pore ball, six oligonucleotides acid constituents composition ratio be respectively：Ammonium chloride solution 0.74%, nano-pore ball
0.3%, six oligonucleotide 10UM.
3. a kind of peripheral white blood cells SNP site rapid detection method according to claim 1, it is characterised in that take trouble
The DTA anticoagulation sample of person, sample size is 2ml.
4. a kind of peripheral white blood cells SNP site rapid detection method according to claim 1, it is characterised in that separation is simultaneously
Enrichment of leukocytes process is as follows, and the DTA anticoagulation sample of ammonium chloride solution and 2 ml patients is put into test tube and is mixed well, room
Temperature is stood after ten minutes, is centrifuged 10 minutes with revolving speed 13000 turns of centrifuge per minute, and remove supernatant, lower layer is institute
Need leucocyte.
5. a kind of peripheral white blood cells SNP site rapid detection method according to claim 1, it is characterised in that carry out by
When the leucocyte cutting section process being enriched to, using restriction endonuclease is added in leucocyte, it is catalyzed leucocyte base
Because of the chain fracture of polynucleotides in group, leucocyte genome is cut into the double chain DNA fragment of 250bp or so at random.
6. a kind of peripheral white blood cells SNP site rapid detection method according to claim 1, it is characterised in that carry out by
Leucocyte double-stranded DNA unwinding after segmentation generates single stranded DNA, with the base complementrity principle of DNA, makes single stranded DNA and fluorescent marker
When six oligonucleotides combine generation hybridized fragment process, by the temperature control system of fluorescence detector, double-stranded DNA unwinding and life
At single stranded DNA.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070190542A1 (en) * | 2005-10-03 | 2007-08-16 | Ling Xinsheng S | Hybridization assisted nanopore sequencing |
| CN104017800A (en) * | 2014-06-20 | 2014-09-03 | 益百尚(北京)生物技术有限责任公司 | Whole genome DNA (Deoxyribonucleic Acid) extraction kit for blood and method thereof |
| CN104894111A (en) * | 2015-05-18 | 2015-09-09 | 李卫东 | DNA targeted capture array for leukemia chromosome aberration high-throughput sequencing |
| US20170058335A1 (en) * | 2014-02-12 | 2017-03-02 | The Trustees Of Columbia University In The City Of New York | Single molecule electronic multiplex snp assay and pcr analysis |
-
2018
- 2018-07-20 CN CN201810800571.1A patent/CN108841924A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070190542A1 (en) * | 2005-10-03 | 2007-08-16 | Ling Xinsheng S | Hybridization assisted nanopore sequencing |
| US20170058335A1 (en) * | 2014-02-12 | 2017-03-02 | The Trustees Of Columbia University In The City Of New York | Single molecule electronic multiplex snp assay and pcr analysis |
| CN104017800A (en) * | 2014-06-20 | 2014-09-03 | 益百尚(北京)生物技术有限责任公司 | Whole genome DNA (Deoxyribonucleic Acid) extraction kit for blood and method thereof |
| CN104894111A (en) * | 2015-05-18 | 2015-09-09 | 李卫东 | DNA targeted capture array for leukemia chromosome aberration high-throughput sequencing |
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Application publication date: 20181120 |