CN101251511B - Method for testing SNP using restriction enzymes double zyme cutting - Google Patents
Method for testing SNP using restriction enzymes double zyme cutting Download PDFInfo
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Abstract
The invention provides a novel method for checking the polymorphism of mononucleotide by a combination of a special restriction endonuclease and a mass spectrometric method. Key points of the invention are as follows: positive and negative primers taking part in the PCR reaction contain two special restriction endonuclease sites and recognition sites, and the recognition sites and the endonuclease sites are different in position. PCR products undergo the restriction endonuclease digestion twice, which generates a single chain nucleotide segment containing SNP sites; after processes of purification and point target, etc., a sample which can be directly used for the substrate auxiliary laser analytic ionization flight time mass spectrometric detection is prepared. Compared with the prior art, the novel method has the advantages of short time, low cost, high resolution and convenient and easy operation.
Description
Technical field
The present invention relates to the particularly mass spectrophotometry of nucleic acid molecules of organic-biological molecule in genomics and the molecular biology; Specifically provide a kind of Restriction Enzyme cutting method and mass spectroscopy of uniting to detect the method for SNP; More particularly, a kind of method that in ground substance assistant laser desorption ionization flight time mass spectrum (MALDI-TOF MS), the genome SNP is detected is provided.
Technical background
Human hereditary information is stored in the genome, and the final completion of the international cooperative project Human Genome Project in 2002 has been drawn out the meticulous figure of human genome structure, for correlative study provides reference sequences.Human genomic sequence is made up of 3,000,000,000 sites altogether, and research shows, any two person-to-person genome differences about 0.1%, the difference in promptly about 3,000,000 sites.These differences very likely are to cause the inherent cause of individual difference between two people.Find these sites, research fields such as research, the assessment individual inheritance that can be widely used in genetic marker concerns, assessment spore.
In human genome, about 90% difference is to come out with the embodied of single nucleotide, such difference site be known as SNP (single nucleotide polymorphism, SNP).SNP has two outstanding features: the one, and number is numerous; SNP number in the human genome is about about 1,100 ten thousand according to estimates; Include the SNP that derives from human genome among the international data center dbSNP at present and reached 1,083 ten thousand; Be 6,440,000 (by on Augusts 22nd, 2007) through what confirm wherein, so a large amount of SNP has made things convenient for and studies this and select suitable site to study, and also makes most genome areas in the coverage that SNP renders a service; The another one characteristics are that SNP site great majority are bifurcation, and two kinds of forms of expression (genotype) are promptly only arranged on a site, are easy to so just can design high throughput automated detection method.Just because of these two characteristics; Make the SNP site more and more receive researcher's favor; Being described as is RFLP (the Restriction Fragment Length Polymorphism that continues; RFLP) and STR (short tandem repeat, STR) afterwards third generation molecular marked compound.Before and after the Human Genome Project is accomplished; The emphasis of genomics research turns to the SNP field gradually; Therefore, SNP has important use in fields such as genetic marker, biotic population science of heredity, taxonomy, genetic thremmatology, species or anthroponomy, forensic science, pharmacogenomicses.
For example; Rich and the diallele characteristic of SNP makes crop or domestic animal genetic mapping meticulousr; Make the breeding work person can carry out marker assisted selection (MAS) and auxiliary import (MAI) of mark more accurately; Reduce or eliminated the harmful effect that the genetic background outside the genes of interest is brought these technology, evaluation brings brand-new situation with variety also to give variety source simultaneously.Jiang Yun very waits the people through 3 different loci of muscle growth suppressor of 3 kinds of " two flesh stern " pig varieties are analyzed, and obtains the new varieties that meat improves.People such as Monna utilize the SNP labelling technique successfully with paddy rice semi-short-stalked gene sd-1 map based cloning, and people such as Shen have made up the full genome snp database of paddy rice, and successfully screen a plurality of paddy rice functional genes.
For example, the SNP difference that exists in the Different Individual becomes the strong instrument that identity is differentiated in the forensic science.People such as Gabirel analyze the SNP in the Mitochondrial H VI/HVII of 105 samples zone, and purposes analyzed sample such as hair and bone in the real case, have proved that it is feasible that this method is used for legal medical expert's inspection case.The SNP of Human To Human such as Li Li, Li Chengtao HLA-DR gene tests, and in 25 case that are applied to the parent evaluation, success ratio reaches 96%.
In order to seize market, each mcroorganism is learned company and has all released the SNP detection method of oneself.Wherein mass spectrometer is considered to rising instrument, especially ground substance assistant laser desorption ionization flight time mass spectrum (MALDI-TOF MS).Based on MALDI-TOF MS, developed some SNP detection methods, like the hME and the iPLEX method of U.S. Sequenom company, the GOOD assay method of German Bruker company, the RFMP method of Korea S GeneMatrix company.The principle of these methods is one of design and the oligonucleotide probe of the genome sequence at the upper reaches, SNP to be detected site coupling, according to the genotypic difference in SNP site, probe will be in SNP site connection to be detected different deoxynucleotides.Wherein, the molecular weight of four kinds of deoxynucleotides of composition dna fragmentation is respectively: dAMP 313.21Da, dCMP 289.19Da, dGMP329.21Da, dTMP 304.20Da.There is molecular weight difference between the different deoxynucleotides; That wherein difference is minimum is the 9.01Da between dAMP and the dTMP; That difference is maximum is the 40.02Da between dCMP and the dGMP, can detect this species diversity through mass spectrometer, thereby the probe that molecular weight is different makes a distinction.Mass spectrometric detection effect is relevant with the molecular weight of probe, and probe fragment is short more, and molecular weight is more little, and it is obvious more to distinguish effect: for example; In Fig. 1, two peaks that A figure indicates, two corresponding oligonucleotide fragments are 5 '-ACGT-3 ' and 5 '-ACGA-3 ', molecular weight is respectively 1174.699 and 1183.770Da; Differ 9.071Da, two peaks that B figure indicates, two corresponding oligonucleotide fragments are 5 '-ACGTACGT-3 ' and 5 '-ACGTACGA-3 '; Molecular weight is respectively 2410.628 and 2419.821Da, differs 9.193Da, two peaks that C figure indicates; Two corresponding oligonucleotide fragments are 5 '-ACGTACGTACGT-3 ' and 5 '-ACGTACGTACGA-3 ', and molecular weight is respectively 3645.917 and 3654.736Da, differs 8.819Da; Differ about 9Da equally, but as shown in the figure, two peak-to-peak differences are the most obvious among the A figure; B figure takes second place, and two peak-to-peak differences are not obvious relatively among the C figure, that is to say; Molecular weight is more little, and mass spectrometric differentiation effect is obvious more, and promptly resolution is high more.In order to improve mass spectrometric resolution; The SNP site detected tend to the less oligonucleotide fragment of detection molecules amount; Like the RFMP method through the PCR product that contains the SNP site is carried out restriction enzyme digestion; The oligonucleotide fragment that produces about 2000～4000Da detects, and (Phosphodiesterase PDE) will contain the small fragment that the oligonucleotide fragment in SNP site cuts into about 1000～2000Da and detect and GOOD assay method is through phosphodiesterase.
Yet there are many defectives in the method that offshore company utilizes mass spectrometer that the SNP site is detected in practical application:
What (1) hME of Sequenom company, two kinds of methods of iPLEX adopted is two-step pcr; Promptly PCR amplifies the copy number in SNP to be detected site for the first time, and PCR detects SNP site to be detected for the second time, between twice PCR; Also to use shrimp alkaline phosphotase (Shrimp Alkaline Phosphatase; SAP) remove remaining dNTP in the PCR reaction system first time, so just cause the prolongation of total detection time, and the increase of single detection cost; In addition, these two kinds of methods are not cut the oligonucleotide fragment that contains the SNP site, and sensing range is between 4000～9000Da, thereby resolution is not high;
(2) GOOD assay method has also adopted two-step pcr, and, in order to improve resolution, adopt the phosphodiesterase cutting to contain the oligonucleotide fragment in SNP site, improved cost, prolonged the time, be unfavorable for fast detecting;
(3) the RFMP method has only a step PCR, but (1000～2000Da) compare, and producing fragment to be detected, long (2000～4000Da), corresponding, resolution is not as GOOD assay method with GOOD assay method.
Therefore, based on MALDI-TOF MS, need at present a kind of resolution of exploitation high, accurately, cost is low, method detects SNP site in the genome fast.
Summary of the invention
The present invention is to detected the corresponding solution that problems such as the slow or detection resolution of processing speed in early stage that the method in genome SNP site exists is high inadequately propose based on MALDI-TOF MS in the past.
The principle of the invention is: utilize restriction enzyme site to be positioned at the peculiar property of one type of particular restriction property restriction endonuclease in recognition site downstream; Through being introduced, recognition site is used to increase the forward in SNP site and/or 5 ' end of reverse primer; Amplification obtains comprising the double-stranded amplified production in the recognition site of two particular restriction property restriction endonucleases, two restriction enzyme sites, SNP site, and wherein the SNP site is between forward primer restriction enzyme digestion sites and reverse primer restriction enzyme digestion sites.Then product is carried out twice particular restriction property restriction endonuclease respectively and obtain two strand short segments that comprise the SNP site, thereby realize only promptly obtaining to be suitable for probe short segments with mass spectrometer (MALDI-TOF MS) detection SNP through a PCR.
Therefore, the present invention program comprises steps such as confirming SNP position, design forward primer, design reverse primer, pcr amplification, twice digestion with restriction enzyme, Mass Spectrometer Method.
In a specific embodiments, step of the present invention is following:
(1) confirm the SNP position: the SNP to nucleotide sequence to be checked possibly exist, confirm the position of SNP in sequence to be checked;
(2) design forward primer: according to the position of SNP, the design forward primer, wherein forward primer 3 ' end is positioned at 5 ' end 1-47bp place, the upper reaches, SNP site; Forward primer 5 ' end contains a kind of recognition site of particular restriction property restriction endonuclease; And in nucleotide sequence to be checked, the restriction enzyme site of described particular restriction property restriction endonuclease is positioned at recognition site 3 ' end 1-25bp place, downstream (preferred 1-10bp, more preferably 1-8bp; 1-4bp most preferably); And be positioned at the scope (preferably 1-10bp, more preferably 1-8bp, most preferably 1-4bp) of 5 ' the end upper reaches 1-25bp of SNP.Wherein, described position range depends on concrete restriction enzyme.For example, BsmFI is in forward primer, and restriction enzyme site is positioned at recognition site 3 ' end downstream 10bp; HgaI is in forward primer, and restriction enzyme site is positioned at recognition site 3 ' end downstream 5bp; MlyI is in forward primer, and restriction enzyme site is positioned at recognition site 3 ' end downstream 5bp;
(3) design reverse primer: according to the position of SNP, the design reverse primer, wherein reverse primer is positioned at 5 ' the end 1-47bp place, the upper reaches, SNP site on another nucleic acid chains; Reverse primer 5 ' end contains the recognition site of another kind of particular restriction property restriction endonuclease; And in nucleotide sequence to be checked, the restriction enzyme site of described particular restriction property restriction endonuclease is positioned at recognition site 3 ' end 1-25bp place, downstream (preferred 1-10bp, more preferably 1-8bp; 1-4bp most preferably); And be positioned at the scope (preferred 1-10bp, more preferably 1-8bp, most preferably 1-4bp) of 5 ' the upstream extremity 1-25bp of SNP.Wherein, described position range depends on concrete restriction enzyme.For example, BsmFI is in reverse primer, and restriction enzyme site is positioned at recognition site 3 ' end downstream 10bp; HgaI is in reverse primer, and restriction enzyme site is positioned at recognition site 3 ' end downstream 10bp; MlyI is in reverse primer, and restriction enzyme site is positioned at recognition site 3 ' end downstream 5bp;
(4) pcr amplification: through pcr amplification; Acquisition comprises the double-stranded amplified production in the recognition site of two particular restriction property restriction endonucleases, two restriction enzyme sites, SNP site, and wherein the SNP site is between forward primer restriction enzyme digestion sites and reverse primer restriction enzyme digestion sites; Wherein concrete PCR reaction system promptly contains the dna profiling in SNP site, special primer of the present invention, dNTPs, heat-resisting fidelity polymerase, damping fluid, Mg
2+, aseptic all ingredients such as deionized water volume, all consistent, and temperature of reaction condition and reaction time are according to primer Tm value with fragment reaction is big or small decides with conventional PCR reaction;
(5) restriction enzyme reaction: use described two kinds of particular restriction property restriction endonucleases respectively reaction product directly to be carried out twice restriction enzyme digestion reaction completely after the PCR reaction is accomplished, the endonuclease reaction condition is decided according to the concrete restriction enzyme that uses.Wherein use described particular restriction property restriction endonuclease to carry out the restriction enzyme reaction, obtain two strand short segments that comprise the SNP site.
(6) Mass Spectrometer Method: the strand short segments that will comprise SNP is carried out Mass Spectrometer Method, compares through the molecular weight with wild type strand short segments, confirms the genotype of SNP.
In another embodiment, described particular restriction property restriction endonuclease includes but not limited to BsmI, BsrI, EarI, SapI, BspQI, BsaI, BsmAI, BsmBI, BsrDI, BstF5I, BtsI, BtsCI, BbsI, AlwI, BccI, PleI, FauI, BfuAI, BspMI, AatII, BmrI, MlyI, SfaNI, HgaI, BciVI, HpyAV, MnlI, HphI, MboII, BbvI, BspCNI, FokI, BseRI, BsmFI, BtgZI, EciI, BceAI, BpmI, BpuEI, BsgI, AcuI, MmeI, NmeAIII, EcoP15I.The recognition site of these restriction enzymes and restriction enzyme site be not or not same place; With HgaI is example, 5 the nucleotide places of the restriction enzyme site of this restriction enzyme after recognition sequence 3 ' end; Restriction enzyme site can produce flat terminal, like MlyI, also can produce cohesive end and overlap, like HgaI.The particular location of recognition sequence in forward and reverse primer looked employed restriction enzyme in the practical operation and decided, and makes enzyme cut the downstream that SNP site, back is positioned at restriction enzyme site.For example, BsmFI is in forward and reverse primer, and recognition site all is positioned at restriction enzyme site 5 ' end upper reaches 10bp; HgaI is in forward primer, and recognition site all is positioned at restriction enzyme site 5 ' end upper reaches 5bp, but in reverse primer, then is 10bp; MlyI is in forward and reverse primer, and recognition site is positioned at restriction enzyme site 5 ' end upper reaches 5bp.
In another embodiment, the PCR reaction is accomplished the afterreaction product and is directly carried out restriction enzyme digestion reaction completely, and the endonuclease reaction condition is decided according to the concrete restriction enzyme that uses.If introduced the recognition sequence of different restriction enzymes in forward and the reverse primer respectively; Endonuclease reaction will carry out at twice; Use for the first time the wherein recognition sequence in primer of a kind of restriction enzyme identification; And the PCR product carried out endonuclease reaction, be for the second time to discern the recognition sequence in another primer with another kind of restriction enzyme, and to the first time endonuclease reaction product carry out the secondary enzyme and cut.If contain the recognition sequence of same restriction enzyme in forward and the reverse primer; Then only need carry out one time endonuclease reaction; Restriction enzyme is discerned the recognition sequence in forward and the reverse primer respectively, and the restriction enzyme site corresponding at two recognition sequences cuts the PCR product simultaneously.
In another embodiment, before carrying out Mass Spectrometer Method, comprise that also enzyme is cut product carries out purification process,, improve resolution to reduce interference to final detection result.Not purified product carries out Mass Spectrometer Method, with sodion (Na occurring containing
+), potassium ion (K
+) waiting various cationic mass spectra peaks, these kation mass spectra peaks differ dozens of Da (sodion: 23Da, potassium ion: 39Da), be easy to cause erroneous judgement with the main mass spectra peak of cation not.And the product behind the purifying, the ratio at kation peak will reduce greatly, in addition detect less than, thereby improve the accuracy that detects.With the purifying resin method is example, cuts at enzyme to add quantity of resin in the product, then with micropipettor piping and druming evenly, puts upside down the eppendorf pipe back and forth and makes abundant mixing, turns upside down 15 minutes.Centrifugal 3 minutes of 1600rpm gets supernatant then and carries out next step operation.
In another embodiment; Described Mass Spectrometer Method comprises: the enzyme behind matrix and the purifying is cut the product point to the used detection target of mass spectrometer; Supply next step Mass Spectrometer Method, the concrete prescription of matrix and look sample concentration, amount of samples, environmental baseline, machine state with consumption and decide.For example; Described Mass Spectrometer Method is to utilize high-resolution ground substance assistant laser desorption ionization flight time mass spectrum (MALDI-TOF MS) to detect the molecular weight of endonuclease bamhi; Because the molecular weight of single SNP changes 9 or 9 more than the Da; And high-resolution mass spectrometer can well be distinguished the difference of single base, therefore through with the comparison of wild type short segments molecular weight, can confirm the genotype of the SNP that detects in particular individual.Supernatant after centrifugal can be used for mass spectrometer and detects.On the sample target, put 0.5ul matrix earlier; Matrix group becomes ammonium citrate (Ammoniumcitrate) 1.6g/L, 3-pyridone carboxylic acid (3-hydroxypicolinic acid, 3-HPA) 6g/L; Acetonitrile-water mixed dissolution with 1: 1; Treat matrix under state of nature after the crystallization, some 1ul sample (being centrifuged supernatant) on the matrix after the crystallization, crystallization under the state of nature.Carry out MALDI-TOF MS then and analyze, obtain the mass spectrogram that enzyme is cut product, record enzyme through Mass Spectrometer Method and cut molecular weight of product that molecular weight of product infers according to experiment and compare and confirm corresponding SNP type, realize that SNP detects.
The present invention detects with conventional P CR or conventional mass spectrophotometry detection SNP compares, and has following beyond thought advantage:
(1) conventional PCR must enlarge the fragment that contains the SNPs site, promptly must carry out twice PCR, therefore needs very big template amount.And the present invention only carries out PCR one time, can detect to the SNP result of trace, has therefore simplified testing process;
(2) the primer of the present invention and condition are undertaken by conventional method, have than the highland versatility;
(3) PCR process of the present invention need not the high-fidelity enzyme, in digestion (enzyme is cut) process, also need not shrimp alkaline phosphotase, the enzyme cost of reduction;
(4) the present invention has deducted this step of single-basic extension, and the consumption of restriction enzyme also reduces, and detects cost about about 30～50% thereby reduce;
(5) enzyme is cut and can only be carried out an inscribe reaction in the process, can on the basis that keeps original accuracy in detection, effectively practice thrift about 1 hour detection time;
(6) dna fragmentation that is used for the mass spectrometer detection that the present invention produces can be 10 (molecular weight be lower than 4000Da) below the bp, and resolution is higher;
(7) for a SNP site, the present invention can produce two dna fragmentations, and their molecular weight is different, can confirm the accuracy of detection each other;
(8) for a plurality of positions relatively near the SNP site, traditional method detects the comparison difficulty, can carry a plurality of SNP site on the dna fragmentation that the present invention produces, and has therefore improved detection efficiency;
(9) the SNP site that disperses relatively for a plurality of positions; Traditional method will be carried out the secondary multiplex PCR; Disturb owing to exist between some primer, so some site can not participate in PCR simultaneously, and use the present invention only need carry out one time multiplex PCR; Interference between the primer is less relatively, can select the SNP site to detect simultaneously more easily;
Description of drawings
Fig. 1 is respectively the synoptic diagram of sequence molecular weight and mass spectrometer resolution relation with different length of identical SNP (T/A).Wherein, Legend A is the molecular weight Mass Spectrometer Method difference of 5bp length (5 '-ACGT-3 ' and 5 '-ACGA-3 '); Legend B is the molecular weight Mass Spectrometer Method difference of 8bp length (5 '-ACGTACGT-3 ' and 5 '-ACGTACGA-3 '), and legend C is the molecular weight Mass Spectrometer Method difference of 12bp length (5 '-ACGTACGTACGT-3 ' and 5 '-ACGTACGTACGA-3 ').
Fig. 2 is the full experiment process flow diagram of embodiment of the present invention.
Fig. 3 is that twice enzyme of restriction enzyme HgaI and restriction enzyme BsmFI cut schematic diagram.Wherein, Letter GACGC is the recognition sequence of restriction enzyme HgaI; Letter GGGAC is the recognition sequence of restriction enzyme BsmFI; Hollow triangle is the restriction enzyme site of restriction enzyme HgaI, and black triangle is the restriction enzyme site of restriction enzyme BsmFI, and lowercase is SNP site and genotype thereof in the square bracket.Through after twice enzyme inscribe, obtain two and comprise the SNP site but not complementary strand short segments.
Fig. 4 is according to the method for the invention, uses the Mass Spectrometer Method result of restriction enzyme HgaI and the resulting negative control A of BsmFI, testing sample B, testing sample C.
Embodiment
Only further describe the present invention now with mode with reference to following non-restrictive example.But should be appreciated that following embodiment only as illustration, should be by any way when doing the restriction overall to the invention described above.Only if other explanation is arranged, embodiments of the invention use traditional molecular biology, cell biology, pcr amplification and mutating technology in this area or the like.These technology are that the technician knows, and in document, illustrated in detail are arranged.Referring to, for example,
Sambrook?and?Russell″Molecular?Cloning：A?Laboratory?Manual″(2001)；
Cloning：A?Practical?Approach，″Volumes?I?and?II(D.N.Glover，ed.，1985)″；
T.A?Brown“Genome”BIOS?Scientific?Publishers?Limited；
PY?Kwok?Annu.Rev.Genomics?Hum.Genet.(2001)，235-58.
Embodiment 1: the primer of 669 SNP of design amplification hepatitis B P gene
Hepatitis type B virus (hepatitis B virus, HBV) infecting is serious worldwide hygienic issues, annual have nearly million people to die from all kinds of diseases that hepatitis B virus infection causes approximately.But for the hepatitis B patient who has made a definite diagnosis, there is multiple SNP sudden change in the hepatitis B virus gene group in the Different Individual, causes the patient of same disease that same medicine is produced drug resistance.If the SNP mark in the hepatitis B patient body is studied, disclosed that Different Individual will help the researcher to screen more suitably medicine to the basic reason of the sensitivity differences of same medicine among the crowd.
Wherein, C → A sudden change takes place in last the 669th nucleotide of the P gene of hepatitis B virus gene group coding (NCBI Gene ID:944565); Make coded amino acid become methionine (L180M) by leucine, thus the virus drugs Lamivudine that creates antagonism (lamivudine, drug resistance LAM).
For the Mass Spectrometer Method in this SNP site, we design following experiment:
At first according to the sequence of hepatitis B virus gene group (NCBI accession number: NC_003977), design following PCR primer:
Forward primer is GGGCCTCAGTgacgcCCGTTTC; Reverse primer is CAAATGGCACTgggacAGTAAACTGAGC; Article two, lowercase gacgc in the primer and gggac are respectively the recognition sequence of restriction enzyme HgaI and BsmFI, and the restriction enzyme principle of the PCR product of this group primer is as shown in Figure 3.
Embodiment 2: amplification comprises 669 SNP fragments of hepatitis B P gene
PCR fragment reaction size is 55 nucleotide, contains the restriction endonuclease recognition sequence of 10 nucleotide.The PCR reaction system is: the 10ul system; Wherein hepatitis B virus gene group DNA 1.5ul (initial concentration 10ng/ul), forward primer and each 1ul of reverse primer (initial concentration 5pmol/ul), dNTPs 0.8ul (each 2.5mM of initial concentration), (initial concentration 1x contains Mg to damping fluid 1ul
2+), HS Taq enzyme 0.05ul (initial concentration 5U/ul), aseptic deionized water 4.65ul; The loop parameter of PCR reaction is: 95 ℃ of 5min, 95 ℃ of 20s, 56 ℃ of 20s, 72 ℃ of 20s circulate 30 times then, afterwards 72 ℃ of 7min.After the PCR reaction, the copy number that has that section nucleotide sequence in SNP site among the hepatitis B virus gene group DNA obtains amplifying.PCR product after reaction is accomplished can also can also can directly be used for follow-up endonuclease reaction-20 ℃ of freezing preservation several months immediately 4 ℃ of stored refrigerated a couple of days.
Embodiment 3: amplified production is carried out the digestion of particular restriction property restriction endonuclease
The PCR reaction is after corresponding restriction enzyme carries out restriction enzyme reaction completely, if wherein select other restriction enzymes for use, its reaction system is different slightly different because of the restriction enzyme of selecting with response parameter.
For the first time enzyme is cut to the 20ul system, wherein PCR product 10ul, enzyme buffer liquid 2ul (initial concentration 10x), HgaI enzyme 1ul (initial concentration 2U/ul), aseptic deionized water 7ul; The endonuclease reaction parameter is: 37 ℃ of 40min, 65 ℃ of 20min; For the second time enzyme is cut to the 30ul system, and wherein enzyme is cut product 20ul, enzyme buffer liquid 3ul (initial concentration 10x), BsmFI enzyme 1.5ul (initial concentration 2U/ul), aseptic deionized water 5.5ul for the first time; The endonuclease reaction parameter is: 65 ℃ of 40min, 95 ℃ of 20min, ice-water bath 20min.
Through the digestion of corresponding restriction enzyme, the PCR product will be cut into a plurality of dna fragmentations, and the single stranded DNA fragment below two 10bp is wherein arranged, all or part of SNP site (shown in the lowercase in the square bracket) of carrying of these two single stranded DNA fragments.
Article two, the single stranded DNA fragment is respectively 5 '-TCTC [c/a]-3 ' and 5 '-GCCA-3 '; Their length is inconsistent, is respectively 5bp and 4bp, also complementary pairing not, and only have one to carry the SNP site;
Enzyme after reaction is accomplished is cut product can also can also can directly be used for follow-up purification reaction-20 ℃ of freezing preservation several months immediately 4 ℃ of stored refrigerated a couple of days.
Embodiment 4: purifying enzyme is cut product
Add the 3mg resin in the product after two steps, enzyme was cut, with micropipettor piping and druming evenly, put upside down the eppendorf pipe back and forth and make abundant mixing, turned upside down 15 minutes.Through this step reaction, resin will be abundant with reaction system in kation combine, and make the reaction system desalination.Purified product after reaction is accomplished can be 4 ℃ of stored refrigerated a couple of days, also can be-20 ℃ of freezing preservation several months, but also 1600rpm takes out supernatant and directly is used for follow-up Mass Spectrometer Method after centrifugal 3 minutes.
Embodiment 5: ground substance assistant laser desorption ionization flight time mass spectrum (MALDI-TOF MS) detects SNP
First some 1ul matrix on the sample target is treated matrix under state of nature after the crystallization, some 1ul centrifuged supernatant on the matrix after the crystallization, crystallization under the state of nature.Carry out MALDI-TOF MS then and analyze, obtain the mass spectrogram that enzyme is cut product.Matrix group becomes: ammonium citrate (Ammonium citrate) 1.6g/L, 3-pyridone carboxylic acid (3-hydroxypicolinic acid, 3-HPA) 6g/L is with 1: 1 acetonitrile-water mixed dissolution, lucifuge room temperature preservation; Perhaps directly carry out MALDI-TOF MS and analyze, obtain the mass spectrogram 4 that enzyme is cut product.
Criterion is following as a result:
As medicament-resistant mutation do not take place; Enzyme is cut product and should be 5 '-TCTCC-3 ' and 5 '-GCCA-3 ', and molecular weight is respectively 1413.92 and 1158.75, if medicament-resistant mutation takes place; Enzyme is cut product and should be 5 '-TCTCA-3 ' and 5 '-GCCA-3 ', and molecular weight is respectively 1437.94 and 1158.75; The contrast mass spectrogram just can learn whether medicament-resistant mutation takes place; If the molecular weight that promptly occurs in the mass spectrogram is 1413.92 and 1158.75; Expression is not undergone mutation, if the molecular weight that occurs in the mass spectrogram is 1437.94 and 1158.75, then expression is undergone mutation; And all in mass spectrogram, represent as if 3 molecular weight, show that then wild strain that medicament-resistant mutation does not take place and the hepatitis B strain that medicament-resistant mutation takes place exist jointly in patient's body.
Testing result is following:
Wherein, the negative check sample of sample A, sample B and sample C are sample to be tested.
(1) for negative control sample A; Because the Strain of medicament-resistant mutation for not taking place in it; Therefore enzyme is cut product and should be 5 '-TCTCC-3 ' and 5 '-GCCA-3 '; And the molecular weight of actual Mass Spectrometer Method is 1413.815 and 1158.559, with the expection molecular weight (1413.92 and 1158.75) only deviation 0.2Da less than, this belongs to the acceptable error range.Therefore, confirm the negative check sample of sample A (referring to Fig. 4 A).
(2) for sample B; Actual Mass Spectrometer Method molecular weight is 1437.825 and 1158.727; This with the expection positive findings molecular weight (1437.94 and 1158.75) only deviation 0.2Da less than (belonging to the acceptable error range); Therefore confirm that sample B has the SNP sudden change, its enzyme is cut product and should be 5 '-TCTCA-3 ' and 5 '-GCCA-3 ' (referring to Fig. 4 B);
(3) for sample C; Actual Mass Spectrometer Method molecular weight is 1413.525,1437.641 and 1158.536Da; This negative molecular weight (1413.92 and 1158.75) and positive molecular weight (1437.94) with expection differs 0.2Da respectively less than (belonging to the acceptable error range), confirms that therefore sample C is Strain that medicament-resistant mutation takes place and the Strain (referring to Fig. 4 C) that medicament-resistant mutation does not take place.
Therefore, according to testing result, disclosed sample A, B and the sample C individuality basic reason to the sensitivity differences of same medicine, this helps the researcher to screen more suitably medicine.
Claims (8)
1. unite Restriction Enzyme cutting method and mass spectroscopy to detect the method for SNP for one kind, may further comprise the steps:
(1) confirm the SNP position: the SNP to nucleotide sequence to be checked possibly exist, confirm the position of SNP in sequence to be checked;
(2) design forward primer: according to the position of SNP, the design forward primer, wherein forward primer 3 ' end is positioned at 5 ' end 1-47bp place, the upper reaches, SNP site; Forward primer 5 ' end contains a kind of recognition site of particular restriction property restriction endonuclease; And in nucleotide sequence to be checked, the restriction enzyme site of described particular restriction property restriction endonuclease is positioned at recognition site 3 ' end 1-25bp place, downstream, and is positioned at the scope of 5 ' the end upper reaches 1-25bp of SNP; Wherein, described position range depends on concrete restriction enzyme;
(3) design reverse primer: according to the position of SNP; The design reverse primer; Wherein reverse primer is positioned at 5 ' the end 1-47bp place, the upper reaches, SNP site on another nucleic acid complementary strand, and reverse primer 5 ' end contains the recognition site of another kind of particular restriction property restriction endonuclease, and in nucleotide sequence to be checked; The restriction enzyme site of described particular restriction property restriction endonuclease is positioned at recognition site 3 ' end 1-25bp place, downstream; And be positioned at the scope of 5 ' the upstream extremity 1-4bp of SNP, wherein, described position range depends on concrete restriction enzyme;
(4) pcr amplification: through pcr amplification; Acquisition comprises the double-stranded amplified production in the recognition site of two particular restriction property restriction endonucleases, two restriction enzyme sites, SNP site, and wherein the SNP site is between forward primer restriction enzyme digestion sites and reverse primer restriction enzyme digestion sites;
(5) restriction enzyme reaction: use described two kinds of particular restriction property restriction endonucleases to carry out twice restriction enzyme reaction respectively, obtain two strand short segments that comprise the SNP site;
(6) Mass Spectrometer Method: the strand short segments that will comprise SNP is carried out Mass Spectrometer Method, compares through the molecular weight with wild type strand short segments, confirms the genotype of SNP.
2. the described method of claim 1 wherein in step (5), can be carried out twice restriction enzyme reaction simultaneously.
3. the described method of claim 1 if wherein described two kinds of particular restriction property restriction endonucleases are with a kind of particular restriction property restriction endonuclease, then only need be carried out a restriction enzyme reaction.
4. claim 2 or 3 described methods, wherein said particular restriction property restriction endonuclease is selected from Bsm I, Bsr I, Ear I, Sap I, BspQ I, Bsa I, BsmA I, BsmB I, BsrD I, BstF5 I, Bts I, BtsC I, Bbs I, Alw I, Bcc I, Ple I, Fau I, BfuA I, BspM I, Aat II, Bmr I, Mly I, SfaNI, Hga I, BciV I, HpyA V, Mnl I, Hph I, Mbo II, Bbv I, BspCN I, Fok I, BseRI, BsmF I, BtgZ I, Eci I, BceA I, Bpm I, BpuE I, Bsg I, Acu I, Mme I, NmeAIII or EcoP15 I.
5. the described method of claim 4 wherein before carrying out Mass Spectrometer Method, comprises that also enzyme is cut product carries out purification process, to reduce the interference to final detection result, improves resolution.
6. the described method of claim 5, wherein purification process comprises purifying resin.
7. the described method of claim 5; Wherein said Mass Spectrometer Method comprises: the enzyme behind matrix and the purifying is cut the product point to the used detection target of mass spectrometer; Supply next step Mass Spectrometer Method, the concrete prescription of matrix and look sample concentration, amount of samples, environmental baseline, machine state with consumption and decide.
8. method according to claim 7, wherein said Mass Spectrometer Method are to utilize high-resolution ground substance assistant laser desorption ionization flight time mass spectrum to detect the molecular weight of endonuclease bamhi.
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| CN104268441B (en) * | 2014-09-23 | 2017-04-12 | 中国科学院海洋研究所 | Method for obtaining double DNA restriction endonuclease combinations based on bioinformatics |
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