CN101565746B - DNA connecting and sequencing method for signal combined codes with parity check - Google Patents
DNA connecting and sequencing method for signal combined codes with parity check Download PDFInfo
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- CN101565746B CN101565746B CN2009100268917A CN200910026891A CN101565746B CN 101565746 B CN101565746 B CN 101565746B CN 2009100268917 A CN2009100268917 A CN 2009100268917A CN 200910026891 A CN200910026891 A CN 200910026891A CN 101565746 B CN101565746 B CN 101565746B
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Abstract
The invention discloses a DNA connecting and sequencing method for signal combined codes with parity check, relates to a DNA sequencing method for the signal combined codes, and belongs to the field of biotechnology. The invention is characterized in that parity check is added into connecting and sequencing codes of the signal combined codes and effectively performed on the code result acquired bydetection, and two conditions of fully empty signals and no generation of connection reaction are successfully distinguished, so that the sequencing accuracy is improved. The method adds parity check markers on the outsides of signal combined code markers, utilizes two states of 'signaled' and 'no signal' of the parity check markers and the variety of acquired 'signaled' markers of the code marke rs for parity check, and performs DNA connecting and sequencing reaction of the signal combined codes through the 'signaled' and 'no signal' arrangement and combination of a plurality of markers when different code markers are detected after successful check, so that the accuracy of the sequencing reaction is improved on the basis of not reducing the sequencing flux.
Description
Technical field
The DNA connecting and sequencing method for signal combined codes of tape parity check of the present invention relates to a kind of employing signal combination coded DNA and connects sequence measurement, and combines parity scheme, is a kind of method that realizes dna sequence analysis, belongs to biological technical field.
Technical background
Along with the completion of going deep into of genome research, the particularly Human Genome Project and various model animals genome plans, biological study and medical research mode have produced dramatic change.From the difference of gene level understanding life, disease takes place, development law, the interaction of medicine and life entity, and the inner interindividual hereditary difference of hereditary difference between the different plant species and same species becomes possibility.Although the factor that causes disease to take place is numerous, gene order difference (comprising SNP, dna methylation etc.) is extensively thought an important internal factor.Most complex diseases take place and development, like cancer, mellitus, cardiovascular disorder, Psychiatric disorders etc., are the coefficient results of numerous genes and environment.Through to the extensive detection of transgenation in a certain specified disease number of genes group sample and with the comparison of non-ill crt gene group sample; Can obtain the genotype information relevant with this disease; To the examination in drug sensitive gene mutational site, can obtain that clinical treatment and medication are had the information of guiding significance through subsequently.No matter be hereditary difference between the nearer species of sibship, the hereditary difference between the inner Different Individual of still same species all mainly is to come out with the embodied of gene order difference.Therefore, how the gene order difference in the quick screening genome becomes one of major subjects of genome times afterwards comprehensively.
The detection method of existing gene order difference is mainly: traditional Sanger dna sequencing method, restriction enzyme digestion length polymorphism, single strand conformation polymorphism and based on oligonucleotide probe hybridization method of gene chip etc.In these methods, only there is traditional Sanger dna sequencing method can accomplish comprehensive sequencing to target fragment, all the other methods all can only be confirmed a part of sequence information seldom.But there are deficiencies such as flux is low, cost is high, length consuming time in traditional Sanger dna sequencing method.The expense that first human genomic sequence is measured is approximately 1,000,000,000 dollars.Although this expense has been reduced to 2,000 ten thousand dollars at present, still be the bottleneck of restriction functional genome research, the cost that reduces dna sequencing significantly will promote development of life science greatly.For this reason, U.S. Venter foundation proposed the goal in research of 1000 dollars of human genome sequencings in 2003.At the beginning of 2004, the U.S. drops into state-run commune hospital more than 4,000 ten thousand dollars and supports the plan of dna sequencing Research on New, and accumulative total is above 100,000,000 dollars.Its target is the human complete genome DNA sequencing technologies of 100,000 dollars of development, and final the attenuating is 1000 dollars.
At present, except that to the existing improvement based on electrophoretic sequencing technologies, the sequencing technologies of researching and developing can be divided into four types generally.The first kind is to extend PCR sequencing PCR, the base of signal mark is just joined in the DNA of polymerization reaction take place chain, detect, and second type is to connect PCR sequencing PCR, the oligonucleotide fragment of signal mark is joined in the DNA chain that ligation takes place detect; The 3rd type is Sequencing by hybridization, through preparing the hybridization signal of one group of high density oligonucleotide micro-array chip, carries out the Sequence Identification of target gene; The 4th type is a series of technology that can on monomolecular level, check order such as molecular image; Last type technology is that the inducing DNA molecule wriggles through very trickle aperture, in this process, by electronics or method of optics base is read, and also becomes the nano pore sequencing technologies.Have only at present and extend sequence measurement and be connected sequence measurement and be used for genome sequencing, and developed commercial instrument and reagent, improved the efficient of dna sequencing greatly, reduced the cost of dna sequencing significantly.Yet the dna sequencing of a new generation still can not satisfy the needs of life science at aspects such as order-checking cost, flux and speed at present.One of the main reasons be single in the signal marking method that detects nucleic acid, efficient is not high.For example; Connect in the sequence measurement at DNA, receive the restriction of affinity tag (like fluorophor) kind, each ligation generally only can be confirmed the information of a base; Confirm two and two above base information like need a ligation, then need carry out two dimension or multidimensional coding affinity tag.Yet existing coding method to multiple affinity tag all " no signal " state and successfully do not take place can't effectively differentiate the error rate that the existence of sequencing reaction is certain between " spacing wave " of ligation.
Summary of the invention
The object of the invention is exactly to the connection sequencing technologies that has DNA now is single in the signal marking method that detects nucleic acid, efficient is not high; Attempt to carry out the coding of two dimension through the combination of different affinity tag states; Composite signal coded DNA order-checking probe; And the introducing through the parity flag thing, differentiate " no signal " and " spacing wave ", improve the accuracy rate that connects the encoding scheme that checks order; The more base of markers tests of equal amts or the purpose of base combination are used in realization, set up quick, accurate, low-cost and high-throughout sequencing method.
DNA connects order-checking and belongs to the synthetic sequence measurement of a kind of DNA.The basic step that DNA connects order-checking is: the sequencing primer of one of hybridization and specific region complementary pairing on dna profiling to be measured at first; Then in the reactor drum that contains template to be measured and sequencing primer, add a kind of or one group of oligonucleotide sequencing probe, each oligonucleotide probe is made up of three parts: degeneracy base or non-strict pairing base, affinity tag and order-checking base; Degeneracy base or non-strict pairing base are one group of nonspecific can combinations with the base of sequencing template hybridization, and affinity tag is used for labeled oligonucleotide probe, is convenient to after generation DNA ligation, detect; The order-checking base is one and a plurality of definite bases; Be used for making the order-checking probe selectively with part sequencing template-sequencing primer mixture generation ligation; The order-checking base complementrity pairing of the corresponding base of the sequencing template that reacts and order-checking probe; Simultaneously, there are certain corresponding relation in order-checking base and affinity tag; After having carried out DNA connection sequencing reaction; Utilize the relevant detection means that sequencing template-primer-probe complex is detected; Obtain corresponding signal information, utilize the base information of corresponding position in the corresponding relation interpretation sequencing template of predefined order-checking base and affinity tag again.
After completion once connects sequencing reaction, remove the affinity tag on sequencing template-primer-probe complex, carry out connection sequencing reaction next time with the order-checking probe as new connection source, carry out ligation continuously until accomplishing required order-checking length.Sequencing primer separates with template sex change to be measured, and the sequencing primer of of hybridization and the translation of article one primer sequence and a plurality of bases on template to be measured carries out repeatedly ligation of a new round subsequently; Repetition sex change, hybridization, connection procedure are all accomplished mensuration until the base information of all positions after accomplishing.
Technical scheme of the present invention: the signal combination coded DNA of tape parity check connects sequence measurement; It is characterized in that to a certain specific dna sequencing probe; Utilize the row labels that is combined into of a group coding affinity tag and a kind of signal encoding affinity tag state; For a collection of dna sequencing probe; Adopt the various combination scheme of a group coding affinity tag combinations of states signal encoding affinity tag to carry out mark, the signal combination coded DNA order-checking probe of preparation one cover tape parity check, thus be implemented in when detecting differentiation and discriminating to different dna sequencing probes:
The preparation of the signal combination coded DNA order-checking probe of A one cover tape parity check: at first prepare unlabelled dna sequencing probe, each unlabelled dna sequencing probe is by one or more order-checking bases, one or more degeneracy base N or non-strict pairing based composition.The order-checking base is used for measuring through base complementrity pairing rule the base information of DNA to be measured corresponding position, and degeneracy base N is any in A, T, C, four kinds of bases of G.After accomplishing the preparation of unlabelled dna sequencing probe; To each unlabelled dna sequencing probe; Adopt a kind of row labels that is combined into of multiple coded markings thing state, every kind of coded markings thing this dna sequencing probe marked whether by this coded markings thing combination at state in pairing state decision, combine the parity flag thing simultaneously; When the coded markings thing number of mark is odd number; Mark parity flag thing not then, when the coded markings thing of mark is even number, mark parity flag thing then.Adopt the different state assembled scheme and combine the parity flag thing that different unmarked dna sequencing probes is carried out mark, accomplish the preparation of the signal combination coded DNA order-checking probe of a cover tape parity check.
B utilizes the order-checking flow process of signal combination coded DNA order-checking probe of an above-mentioned cover tape parity check following:
A. choose a sequencing primer and dna profiling to be measured is hybridized according to the base complementrity pairing mechanism;
B. in reaction system, add signal combination coded DNA order-checking probe and the dna ligase and the reaction system thereof of an above-mentioned cover tape parity check, carry out the DNA ligation;
C. after accomplishing the DNA ligation; Detect the affinity tag of whole participant status combinations and the signal of parity flag thing, the residing state of interpretation coded markings thing signal, the quantity of the coded markings thing of calculating picked up signal; And utilize the signal of parity flag thing that this numerical value is carried out parity checking; Pass through like verification,, confirm the kind of the dna sequencing probe of generation ligation according to the combined situation of whole coded markings thing states; Thereby the type of confirming order-checking base on this order-checking probe with arrange and the base or the base sequence information of correspondence position on finally definite tested dna profiling;
D. after accomplishing detection, remove the affinity tag on the dna sequencing probe to the affinity tag signal;
E. repeating above-mentioned steps b-d reaches or to exceed dna profiling to be measured zone to be measured terminal until the dna sequencing probe;
F. this sequencing primer separates with dna profiling sex change to be measured, chooses the second sequencing primer;
G. repeat above-mentioned steps a-f, the full sequence information until the zone to be measured of whole unknown dna profilings is able to confirm.
Described multiple affinity tag carries out composite marking to unmarked dna sequencing probe; Its scheme is with one or more affinity tags of tense marker to same dna sequencing probe molecule; Perhaps use the differing mol of different markers substance markers respectively, subsequently above-mentioned different order-checking probe molecules are mixed with a kind of dna sequencing probe.
Described sequencing primer be meant one group can only with the oligonucleotide fragment of one section universal sequence hybridization of all dna profilings; One group of sequencing primer is hybridized the zone and is differed one or several bases each other on dna profiling, can in number wheel sequencing reaction, accomplish the examining order to the dna profiling total length; Universal sequence on the sequencing template is to prepare in the process in sequencing template to add through ligation, perhaps in amplification procedure, introduces through primer.
Described dna profiling is to increase the segmental amount of interested purpose in the genome through dna fragmentation to be measured being passed through the DNA cloning technology, and amplification is a substance, the purpose fragment that promptly once increases, or multiple, a plurality of purpose fragments promptly once increase; In the described dna profiling to be measured fixedly is to be fixed on the planar chip base through chemistry or physical method, or is fixed on the pearl carrier of " 96 orifice plate ", " 384 orifice plate " and various modifications.
Described ligation can be to begin 5 ' terminal ligation to 3 ' end from the primer that checks order, and also can be 3 ' terminal ligation to 5 ' end.
Described affinity tag is the fluorophor that adopts, or utilizes the change of order-checking probe character chemistry, physics, changes like resistance change, electric current.
Described detection is meant with described affinity tag character and adapts; When the fluorescent mark group of laser excitation; Laser excitation intensity can be a plurality of varying strengths such as 95%, 70%, 45%, and photomultiplier transit can be respectively between a plurality of detection zones such as 95%, 70%, 45%.
Removing of described affinity tag is that labelling groups passes through method and dna sequencing probe separates chemistry, physics, and perhaps dna sequencing probe character chemistry, physics is recovered original state.Removing of affinity tag is only to remove affinity tag itself, perhaps removes simultaneously with affinity tag to link to each other or disjunct one or more base and relevant group.
Described second sequencing primer is any one that removes in one group of sequencing primer in the already used sequencing primer, might not be corresponding with the sequencing primer that has used on sequencing template the most approaching one in hybridization position.
Know-why: the purpose of this invention is to provide a kind of tape parity validation signal coded DNA and connect sequence measurement, in a ligation, detect a plurality of bases simultaneously, and pass through the introducing of parity flag thing, improve the accuracy rate of experiment.The principle of this method is whether to want to connect condition parity flag thing in the sequencing reaction according to the odevity decision that connects signal encoding affinity tag in the sequencing reaction; And the realization through the status check signal encoding affinity tag of parity flag thing when detecting improves the safety of the dna sequencing method of signal encoding circulation connection to no signal condition be the resolution that ligation successfully takes place.
The purpose of this invention is to provide a kind of band background verification signal combination coded DNA and connect sequence measurement, in a ligation, detect a plurality of bases simultaneously, and pass through the introducing of context marker thing, reduce the error rate of sequencing reaction.The principle of this method is through in connecting sequencing reaction, adding specific background verification affinity tag, effectively differentiate " no signal " and successfully do not connect two states, improving the accuracy rate of the connection sequencing reaction of signal combination coding.
The different affinity tags of the connection sequence measurement utilization of signal combination coding carry out binary coding or multilevel code at have or not (two dimension) and the strength difference (multidimensional) of signal to be detected.Here we when affinity tag is two kinds, " have signal " and " no signal " permutation and combination through two kinds of affinity tags with the example of inaction that has of signal, obtain 2 altogether
2=4 kinds of combined states, i.e. " affinity tag 1 has signal, affinity tag 2 that signal is arranged ", " affinity tag 1 has signal, affinity tag 2 no signals ", " affinity tag 1 no signal, affinity tag 2 have signal ", " affinity tag 1 no signal, affinity tag 2 no signals ".When affinity tag quantity is n (n is the integer more than or equal to 2), record mark thing combined state is whole n bits, the seized state of a kind of affinity tag of each record of bit, and " 1 " for detecting signal, " 0 " is not for detecting signal, whole 2
n2 of individual n bit and n signal tracer
nIt is corresponding one by one to plant combined state.Have 4 kinds owing to constitute the common base of DNA; Be respectively VITAMIN B4 (A), guanine (G), thymus pyrimidine (T), cytosine(Cyt) (C), the two kinds of affinity tags of 4 types of needs that therefore need accurately to detect a base " have signal " and 4 kinds of combined states of " no signal "; The combination of the base of 2 based compositions has 4 * 4=16 type, needs 4 kinds of affinity tags totally 2
4=16 kinds of combined states detect; The rest may be inferred, and the base combination of n (n for more than or equal to 2 integer) based composition needs 2n kind affinity tag totally 2 altogether
2nPlant the composite signal state and detect, every kind of combined state comes unique expression through a 2n bit, and corresponding one by one with the type of order-checking probe.
Yet; There is following problem in the connection sequence measurement of simple signal combination coding: when the complementation order-checking probe of base to be measured or base combination correspondence does not have corresponding with it modification affinity tag; Because the order-checking probe that adds in the reaction system does not all contain the modification affinity tag, therefore obtain one group and be the signal combination of " no signal " entirely through detecting behind whole affinity tags us; Simultaneously,, can obtain one group equally and be the signal combination of " no signal " entirely because ligation failure etc. or other reasons cause the sequencing reaction failure when the order-checking probe that adds reaction system through detecting us.One group of this moment be full the signal combination of " no signal " corresponding two kinds maybe, the sequence measurement of conventional combination coding can't effectively be differentiated these two kinds possibilities, thereby there is certain error rate in the order-checking process.Simultaneously, connect order-checking and belong to a kind of biochemical reaction, also there is certain error rate in itself.
The present invention has increased a kind of affinity tag as parity flag thing (seeing table 1) on the basis of existing signal encoding, participate in the modification of order-checking probe.The parity flag thing carries out according to following rule with the combination of order-checking probe type: when a kind of order-checking probe type during simultaneously by 0 kind or the combination of even number kind coded markings thing, then increase the modification of parity flag thing to this order-checking probe type; When a kind of order-checking probe type is made up by odd number kind coded markings thing simultaneously, then do not increase of the modification of parity flag thing to this order-checking probe type;
The main effect of parity flag thing is: 1, and whether the odevity quantity of the coded markings thing number that " signal arranged " is correct behind the checking sequencing reaction, to improve the accuracy of order-checking; 2, the state of the state of whole coded markings things " no signal " and ligation failure behind the resolution sequencing reaction.
The coding method 2n kind affinity tag 2 of table 1 tape parity check
2nPlant combined state and detect n base oligonucleotide prescription tabulation
(" 1 " expression probe mixture contains this oligonucleotide in the table, and " 0 " expression probe mixture does not contain this oligonucleotide probe)
| Base composite type to be measured | The parity flag | Affinity tag | 1 | |
|
…… | Affinity tag 2n-2 | Affinity tag 2n-1 | Affinity tag 2n |
| Order- |
0 | 1 | 1 | 1 | …… | 1 | 1 | 1 | |
| Order- |
1 | 1 | 1 | 1 | …… | 1 | 1 | 0 | |
| Order- |
1 | 1 | 1 | 1 | …… | 1 | 0 | 1 | |
| …… | …… | …… | …… | …… | …… | …… | …… | …… | |
| Order-checking probe type 2 2n-2 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | ||
| Order-checking probe type 2 2n-1 | 0 | 0 | 0 | 0 | …… | 0 | 0 | 1 | |
| Order- |
1 | 0 | 0 | 0 | …… | 0 | 0 | 0 |
Beneficial effect of the present invention:
1. biggest advantage of the present invention is through the introducing of parity flag thing; State to the signal encoding affinity tag carries out parity checking; Effectively differentiating the coded markings thing entirely for " no signal " with when two kinds of situation of ligation successfully do not take place; Improve the accuracy rate of sequencing reaction, make the circulation connection PCR sequencing PCR of signal encoding have practicality more.The composite signal coding of the coded markings thing that passes through is implemented in and detects more base or base combination in the ligation simultaneously.
2. compare the decline that presents exponential type with traditional method owing in the single ligation, detect the required affinity tag kind of classification of a plurality of bases; Make the classification that in the single ligation, detects a plurality of bases simultaneously become possibility, shortened at double full sequence is measured the required time.
3. encode through signal combination, the minimizing of the ligation number of times that the sequence of detection equal length is required, the cost of order-checking also reduces at double.Simultaneously, through the signal combination coding, the required affinity tag kind of base type that detects equal amts significantly reduces,
Reduced demand, thereby reduced cost certification mark thing equipment.
Description of drawings
Below in conjunction with accompanying drawing the present invention is further specified.
Fig. 1 is when the affinity tag state is two dimension; When being " having " or " nothing " of signal; Two-dimensional state combine detection through two kinds of affinity tags is at the schematic diagram that once connects A in the sequencing reaction, T, C, four kinds of bases of G; Solid icon representation " has signal " among the figure, hollow icon representation " no signal ".1, when base to be measured is A, be that the assembled state of the pairing coded markings thing of order-checking probe of U is " affinity tag 1 has signal; affinity tag 2 has signal " by the order-checking base, i.e. " 1,1 "; Obtaining parity flag thing (square among the figure, solid in signal is arranged, hollow is no signal) signal subsequently is " signal is arranged "; I.e. " 1 ", with regard to the verification principle, coded markings thing state has two 1 according to preset; Be even number, corresponding parity flag thing should be 1, conforms to actual parity flag thing state; When base to be measured is C, be that the assembled state of the pairing coded markings thing of order-checking probe of G is " affinity tag 1 has signal, affinity tag 2 no signals " by the order-checking base, promptly " 1; 0 ", obtaining parity flag thing (square among the figure, solid in signal is arranged, hollow is no signal) signal subsequently is " no signal "; I.e. " 0 ", with regard to the verification principle, coded markings thing state has 11 according to preset; Be odd number, corresponding parity flag thing should be 0, conforms to actual parity flag thing state; When base to be measured is G, be that the assembled state of the pairing coded markings thing of order-checking probe of U is " affinity tag 1 no signal, affinity tag 2 has signal " by the order-checking base, promptly " 0; 1 ", obtaining parity flag thing (square among the figure, solid in signal is arranged, hollow is no signal) signal subsequently is " no signal "; I.e. " 0 ", with regard to the verification principle, coded markings thing state has 11 according to preset; Be odd number, corresponding parity flag thing should be 0, conforms to actual parity flag thing state; When base to be measured is T, be that the assembled state of the pairing coded markings thing of order-checking probe of A is " affinity tag 1 no signal, affinity tag 2 no signals " by the order-checking base, promptly " 0; 0 ", obtaining parity flag thing (square among the figure, solid in signal is arranged, hollow is no signal) signal subsequently is " signal is arranged "; I.e. " 1 ", with regard to the verification principle, coded markings thing state has 01 according to preset; Be 0, corresponding parity flag thing should be 1, conforms to actual parity flag thing state.
Fig. 2 is the workflow diagram of instance 2 and instance 3.1, the PCR product behind P1, the P2 primer amplification is fixed in surface of glass slide; 2, article one sequencing primer I1 and the PCR product hybridization of being fixed in surface of glass slide; 3, connected the oligonucleotide probe of order-checking base and PCR locations complementary to be measured on the sequencing primer I1; 4, completion removes the fluorescence modification group after detecting; 5, I1 connects the connection of the probe that carries out checking order for the second time; 6, completion removes the fluorescence modification group after detecting once more; 7, I1 accomplishes whole 4 ligations; 8, sequencing primer I1 separates with the sex change of PCR product; 9, second sequencing primer I2 and PCR product hybridization beginning new round sequencing reaction.
Fig. 3 is the detailed diagram that instance 2 sequencing primer I1 carry out preceding twice order-checking ligation.1, sequencing primer I1 and the PCR product hybridization of being fixed in surface of glass slide, whole 15 bases of I1 and 15 base complementrity pairings of PCR primer P2 complementary strand 5 ' end; 2; Carry out the ligation first time; The order-checking base is connected with the I1 of hybridizing on the PCR product for the oligonucleotide monomer (all the other positions of oligonucleotide probe are degeneracy base N) of " 5 '-AA-3 ' " in the mixture that 40 kinds of oligonucleotide probes (32 kinds of oligonucleotides coding probes, 8 kinds of parity checking oligonucleotide probes) are formed, through detecting the signal that whole affinity tags only obtain CY7; Coded markings thing state is whole " no signal "; The parity flag thing is " signal is arranged ", meets predefined parity checking rule, and parity checking is passed through.Look into 3 ' terminal the 3rd, No. 4 base that the oligonucleotide probe formula table learns when the coded markings thing is " no signal " DNA to be measured and be " 5 '-AA-3 ' "; 3, remove the terminal fluorescence modification group of oligonucleotide probe; 4, carry out connecting the second time, detect 3 ' terminal the 11st, No. 12 base of DNA to be measured.
Specific embodiments
Implementation method of the present invention is divided into two kinds:
First kind is to a certain order-checking probe, selects for use some kinds of coded markings things and a kind of parity flag thing simultaneously same order-checking probe molecule to be carried out mark.To a ligation; Each affinity tag presents the two states of " signal is arranged ", " no signal " respectively when to be detected; By the specific order-checking probe decision that in order-checking ligation system, adds, the order-checking probe that preparation contains different coding affinity tag and context marker thing is a key of the present invention.Add order-checking probe in the reaction system by a group echo oligonucleotide probe of different affinity tags form.Each oligonucleotide probe is by one or more order-checking bases (measuring the base information of corresponding position among the DNA to be measured through base complementrity pairing rule), one or more degeneracy base N (N is any in A, T, C, four kinds of bases of G) or non-strict pairing base (like xanthoglobulin etc.) and a kind of affinity tag formation with ability to be detected.Combination between order-checking base and the affinity tag is according to the listed state preparation of table 1; When the state of determined in the table 1 " order-checking probe type i " corresponding k kind affinity tag is " 1 "; Then when preparation order-checking base was the order-checking probe molecule of " base composite type i ", " order-checking probe type i " all states were the affinity tag of " 1 " in the flag table simultaneously; Synthetic line by line table 1 listed whole 2
2nPlant oligonucleotide probe, this probe mixture is the oligonucleotide probe mixture of required preparation.
Second kind is that this kind order-checking probe is divided into several portions, and each part is a kind of affinity tag of mark (affinity tag comprises coded markings thing and parity flag thing) respectively.To a ligation, each affinity tag presents the two states of " signal is arranged ", " no signal " respectively when to be detected, by the specific order-checking probe decision that in order-checking ligation system, adds.Therefore preparing the order-checking probe that contains different coding affinity tag and context marker thing is key of the present invention.Add order-checking probe in the reaction system by a group echo oligonucleotide probe of different affinity tags form.Each oligonucleotide probe is by one or more order-checking bases (measuring the base information of corresponding position among the DNA to be measured through base complementrity pairing rule), one or more degeneracy base N (N is any in A, T, C, four kinds of bases of G) or non-strict pairing base (like xanthoglobulin etc.) and a kind of affinity tag formation with ability to be detected.Combination between order-checking base and the affinity tag is according to the listed state preparation of table 1; As determined in the table 1 " base composite type i " and " affinity tag j " when listed state be " 1 ", then preparation order-checking base is that " base composite type i ", affinity tag are the oligonucleotide probe of " affinity tag j "; Then not synthetic when being " 0 " like listed state, the oligonucleotide probe that the synthetic listed whole states of table 1 are " 1 ", this probe mixture is the oligonucleotide probe mixture of required preparation.
Embodiment 1: the signal combination coded DNA of tape parity check connects sequence measurement; It is characterized in that to a certain specific dna sequencing probe; Utilize the row labels that is combined into of a group coding affinity tag and a kind of signal encoding affinity tag state; For a collection of dna sequencing probe; Adopt the various combination scheme of a group coding affinity tag combinations of states signal encoding affinity tag to carry out mark, the signal combination coded DNA order-checking probe of preparation one cover tape parity check, thus be implemented in when detecting differentiation and discriminating to different dna sequencing probes:
The preparation of the signal combination coded DNA order-checking probe of A one cover tape parity check: at first prepare unlabelled dna sequencing probe, each unlabelled dna sequencing probe is by one or more order-checking bases, one or more degeneracy base N or non-strict pairing based composition.The order-checking base is used for measuring through base complementrity pairing rule the base information of DNA to be measured corresponding position, and degeneracy base N is any in A, T, C, four kinds of bases of G.After accomplishing the preparation of unlabelled dna sequencing probe; To each unlabelled dna sequencing probe; Adopt a kind of row labels that is combined into of multiple coded markings thing state, every kind of coded markings thing this dna sequencing probe marked whether by this coded markings thing combination at state in pairing state decision, combine the parity flag thing simultaneously; When the coded markings thing number of mark is odd number; Mark parity flag thing not then, when the coded markings thing of mark is even number, mark parity flag thing then.Adopt the different state assembled scheme and combine the parity flag thing that different unmarked dna sequencing probes is carried out mark, accomplish the preparation of the signal combination coded DNA order-checking probe of a cover tape parity check.
B utilizes the order-checking flow process of signal combination coded DNA order-checking probe of an above-mentioned cover tape parity check following:
A. choose a sequencing primer and dna profiling to be measured is hybridized according to the base complementrity pairing mechanism;
B. in reaction system, add signal combination coded DNA order-checking probe and the dna ligase and the reaction system thereof of an above-mentioned cover tape parity check, carry out the DNA ligation;
C. after accomplishing the DNA ligation; Detect the affinity tag of whole participant status combinations and the signal of parity flag thing, the residing state of interpretation coded markings thing signal, the quantity of the coded markings thing of calculating picked up signal; And utilize the signal of parity flag thing that this numerical value is carried out parity checking; Pass through like verification,, confirm the kind of the dna sequencing probe of generation ligation according to the combined situation of whole coded markings thing states; Thereby the type of confirming order-checking base on this order-checking probe with arrange and the base or the base sequence information of correspondence position on finally definite tested dna profiling;
D. after accomplishing detection, remove the affinity tag on the dna sequencing probe to the affinity tag signal;
E. repeating above-mentioned steps b-d reaches or to exceed dna profiling to be measured zone to be measured terminal until the dna sequencing probe;
F. this sequencing primer separates with dna profiling sex change to be measured, chooses the second sequencing primer;
G. repeat above-mentioned steps a-f, the full sequence information until the zone to be measured of whole unknown dna profilings is able to confirm.
Described multiple affinity tag carries out composite marking to unmarked dna sequencing probe; Its scheme is with one or more affinity tags of tense marker to same dna sequencing probe molecule; Perhaps use the differing mol of different markers substance markers respectively, subsequently above-mentioned different order-checking probe molecules are mixed with a kind of dna sequencing probe.
Described sequencing primer be meant one group can only with the oligonucleotide fragment of one section universal sequence hybridization of all dna profilings; One group of sequencing primer is hybridized the zone and is differed one or several bases each other on dna profiling, can in number wheel sequencing reaction, accomplish the examining order to the dna profiling total length; Universal sequence on the sequencing template is to prepare in the process in sequencing template to add through ligation, perhaps in amplification procedure, introduces through primer.
Described dna profiling is to increase the segmental amount of interested purpose in the genome through dna fragmentation to be measured being passed through the DNA cloning technology, and amplification is a substance, the purpose fragment that promptly once increases, or multiple, a plurality of purpose fragments promptly once increase; In the described dna profiling to be measured fixedly is to be fixed on the planar chip base through chemistry or physical method, or is fixed on the pearl carrier of " 96 orifice plate ", " 384 orifice plate " and various modifications.
Described ligation can be to begin 5 ' terminal ligation to 3 ' end from the primer that checks order, and also can be 3 ' terminal ligation to 5 ' end.
Described affinity tag is the fluorophor that adopts, or utilizes the change of order-checking probe character chemistry, physics, changes like resistance change, electric current.
Described detection is meant with described affinity tag character and adapts; When the fluorescent mark group of laser excitation; Laser excitation intensity can be a plurality of varying strengths such as 95%, 70%, 45%, and photomultiplier transit can be respectively between a plurality of detection zones such as 95%, 70%, 45%.
Removing of described affinity tag is that labelling groups passes through method and dna sequencing probe separates chemistry, physics, and perhaps dna sequencing probe character chemistry, physics is recovered original state.Removing of affinity tag is only to remove affinity tag itself, perhaps removes simultaneously with affinity tag to link to each other or disjunct one or more base and relevant group.
Described second sequencing primer is any one that removes in one group of sequencing primer in the already used sequencing primer, might not be corresponding with the sequencing primer that has used on sequencing template the most approaching one in hybridization position.
Embodiment 2: four look fluorescence composite coding methods of tape parity check (respectively tagging scheme) detect on human No. 17 karyomit(e)s, 45332-45363 totally 32 base-pair sequences on the RP11-354P11 clone;
Extract human complete genome DNA, utilize PCR method amplification purpose fragment, primer is P1, P2 (sequence is seen table 2), 5 ' the terminal hydroxyl modified that adopts of primer P1.After PCR product after the amplification was fixed in aldehyde group modified surface of glass slide, heating slide to 95 ℃ made chain and fixedly chain disengaging of double-stranded PCR product, begins sequencing reaction subsequently.
Relevant oligonucleotide sequence information in table 2 instance 2
| The sequence name | Sequence information |
| Sequence to be measured | 5’-CACGGACCAGCTGCCCTGGACCAGCTGCAAGA-3’ |
| P1 | OH-5’-CGCTATACTACCTCATCTCCTCCTTCACG-3’ |
| P2 | 5’-GCAGTTGCCAGTGTTCCAGGAGT-3’ |
| I1 | 5’-CAGTGTTCCAGGAGT-3’ |
| I2 | 5’-GTGTTCCAGGAGTNN-3’ |
| I3 | 5’-GTTCCAGGAGTNNNN-3’ |
| I4 | 5’-GCCAGTGTTCCAGGA-3’ |
Add hybridization buffer and article one sequencing primer I1 (sequence is seen table 2) to surface of glass slide, hybridized renaturation 20 minutes for 37 ℃.In reaction tank, add the mixture that the listed whole 40 kinds of order-checking probes of table 3 are formed; Wherein CY7 is the parity flag thing; All the other four kinds of affinity tags are the coded markings thing; When the oligonucleotide probe of being made up of affinity tag CY7 only appears at corresponding order-checking base pair to answer oligonucleotides coding probe kind be 0 kind or even number kind, and the oligonucleotides coding probe that the order-checking base pair is answered is when being the odd number kind, the synthetic oligonucleotide probe that is made up of this order-checking base and CY7.Adopt the T4 nucleic acid ligase to carry out ligation, reaction conditions is 25 ℃ and connects 30 minutes.Utilize laser confocal microscope that slide is scanned after connecting completion, scanning adopts the wavelength corresponding to CY7, CY3, CY5, TXR and FTC to carry out respectively, the base information (accompanying drawing 2) corresponding according to the fluorescent signal interpretation that obtains.
Table 3 order-checking probe mixture Verbose Listing
In the ligation first time; On I1 primer and the P2 complementary strand " 5 '-AAGAACTCCTGGAACACTG-3 ' " sequence hybridization; After adding the oligonucleotide probe mixture; Hold the 3rd, No. 4 base in downstream (also being 3 ' terminal the 3rd, No. 4 base of DNA to be measured) complementary pairing if the base that checks order in the oligonucleotide probe mixture is positioned at I1 primer 3 ' with DNA to be measured, then these oligonucleotide probes and DNA to be measured are hybridized and with primer I 1 ligation are taken place.The the 3rd, 4 base that DNA to be measured is positioned at I1 primer 3 ' end downstream is " 5 '-AA-3 ' "; Its reverse complemental base is " 5 '-TT-3 ' "; Then all order-checking bases only have one for the oligonucleotide probe of " 5 '-TT-3 ' "; For " 5 '-NNTTNNNN-3 '-CY7 ", ligation (accompanying drawing 3-2) takes place with dna profiling to be measured and primer I 1.When carrying out signal detection, at first detected the combined state type of coded markings thing, be " CY3 no signal; CY5 no signal, TXR no signal, FTC no signal "; Having 0 kind of affinity tag has signal, detects parity flag thing state simultaneously for " signal is arranged ", when meeting the coded markings thing and be 0 kind or even number kind affinity tag and " signal being arranged "; The parity flag thing is the parity checking rule of " signal is arranged ", and parity checking is passed through.Learn through tabling look-up subsequently when coded markings thing state is " no signal "; The reverse complemental chain information of 3 ' terminal the 3rd, No. 4 base of DNA to be measured be " 5 '-TT-3 ' ", thereby 3 ' terminal the 3rd, No. 4 base that can obtain DNA to be measured is " 5 '-AA-3 ' ".
After the completion next round detects, utilize chemical process to excise the fluorophor of oligonucleotide probe 3 ' end on link and the primer I 1, carry out ligation once more (accompanying drawing 3-3) at free hydroxyl of 3 ' terminal acquisition.In reaction tank, add the listed whole thuja acid probe mixture of table 3 once more; Carry out second and take turns ligation; Ligation this time is to detect the 11st, No. 12 base (3 ' terminal the 11st, No. 12 base of DNA to be measured) that DNA to be measured is positioned at I1 primer 3 ' end downstream " 5 '-CC-3 ' "; Three kinds of order-checkings this moment bases be oligonucleotide probe " 5 '-NNGGNNNN-3 '-CY7 ", " 5 '-NNGGNNNN-3 '-CY5 ", " 5 '-NNGGNNNN-3 '-FTC " the generation ligation of " 5 '-GG-3 ' "; Carrying out fluoroscopic examination is to detect CY7, CY5, FTC simultaneously; The parity flag thing verifies successfully, the interpretation and carry out 3 ' terminal the 11st, No. 12 base that reverse complemental obtains DNA to be measured and be " 5 '-CC-3 ' " (accompanying drawing 3-4) of tabling look-up subsequently.Carry out the 3rd, 4 of I1 primer once more behind the terminal fluorophor of the oligonucleotide that the chemical process excision connects and take turns ligation, record 3 ' terminal the 19th, No. 20 base and the 27th, No. 28 base information of DNA to be measured respectively.
After accomplishing the four-wheel ligation, 95 ℃ make sequencing primer I1 separate with DNA to be measured, add hybridization buffer and second sequencing primer I2 (sequence is seen table 2) to surface of glass slide, hybridize renaturation 20 minutes for 37 ℃.Begin the four-wheel ligation subsequently and detect 3 ' terminal the 5th, No. 6 of DNA to be measured respectively, 13, No. 14,21, No. 22 and 29, No. 30 four groups of bases.
Same, utilize sequencing primer I3 to detect 3 ' terminal the 7th, No. 8 of DNA to be measured, 15, No. 16; 23, No. 24 and 31, No. 32 four groups of bases; Utilize sequencing primer I4 to detect 3 ' terminal the 1st, No. 2 of DNA to be measured, 9, No. 10,17, No. 18 and 25, No. 26 four groups of bases.
Splice the sequence information that can obtain whole 32 bases of DNA to be measured through the acquisition sequence information.
Embodiment 3: four look fluorescence composite coding methods of tape parity check (common tagging scheme) detect on human No. 17 karyomit(e)s, 45332-45363 totally 32 base-pair sequences on the RP11-354P11 clone;
Relevant oligonucleotide sequence information in table 4 instance 3
| The sequence name | Sequence information |
| Sequence to be measured | 5’-CACGGACCAGCTGCCCTGGACCAGCTGCAAGA-3’ |
| P1 | OH-5’-CGCTATACTACCTCATCTCCTCCTTCACG-3’ |
| P2 | 5’-GCAGTTGCCAGTGTTCCAGGAGT-3’ |
| I1 | 5’-CAGTGTTCCAGGAGT-3’ |
| I2 | 5’-GTGTTCCAGGAGTNN-3’ |
| I3 | 5’-GTTCCAGGAGTNNNN-3’ |
| I4 | 5’-GCCAGTGTTCCAGGA-3’ |
Extract human complete genome DNA, utilize PCR method amplification purpose fragment, primer is P1, P2 (sequence is seen table 4), 5 ' the terminal hydroxyl modified that adopts of primer P1.After PCR product after the amplification was fixed in aldehyde group modified surface of glass slide, heating slide to 95 ℃ made chain and fixedly chain disengaging of double-stranded PCR product, begins sequencing reaction subsequently.
Add hybridization buffer and article one sequencing primer I1 (sequence is seen table 4) to surface of glass slide, hybridized renaturation 20 minutes for 37 ℃.In reaction tank, add the mixture that the listed whole 16 kinds of oligonucleotide probes of table 5 are formed, a kind of oligonucleotide probe of every behavior in the form, wherein CY7 is the parity flag thing, all the other four kinds of affinity tags are the coded markings thing.On each oligonucleotide probe molecule with tense marker one or more signal tracer; Affinity tag CY7 only participate in mark those mark the order-checking probe of 0 kind or even number kind signal encoding affinity tag; And the order-checking probe has been when the signal tracer of mark has been the odd number kind, not mark CY7 group.Adopt the T4 nucleic acid ligase to carry out ligation, reaction conditions is 25 ℃ and connects 30 minutes.Utilize laser confocal microscope that slide is scanned after connecting completion, scanning adopts the wavelength corresponding to CY7, CY3, CY5, TXR and FTC to carry out respectively, the base information (accompanying drawing 2) corresponding according to the fluorescent signal interpretation that obtains.
In the ligation first time; On I1 primer and the P2 complementary strand " 5 '-AAGAACTCCTGGAACACTG-3 ' " sequence hybridization; After adding the oligonucleotide probe mixture; Hold the 3rd, No. 4 base in downstream (also being 3 ' terminal the 3rd, No. 4 base of DNA to be measured) complementary pairing if the base that checks order in the oligonucleotide probe mixture is positioned at I1 primer 3 ' with DNA to be measured, then these oligonucleotide probes and DNA to be measured are hybridized and with primer I 1 ligation are taken place.The the 3rd, 4 base that DNA to be measured is positioned at I1 primer 3 ' end downstream is " 5 '-AA-3 ' "; Its reverse complemental base is " 5 '-TT-3 ' "; Order-checking probe " 5 '-NNTTNNNN-3 ' " only mark CY7 fluorophor, ligation takes place with dna profiling to be measured and primer I 1.When carrying out signal detection, at first detected the combined state type of coded markings thing, be " CY3 no signal; CY5 no signal, TXR no signal, FTC no signal "; Having 0 kind of affinity tag has signal, detects parity flag thing state simultaneously for " signal is arranged ", when meeting the coded markings thing and be 0 kind or even number kind affinity tag and " signal being arranged "; The parity flag thing is the parity checking rule of " signal is arranged ", and parity checking is passed through.Learn through tabling look-up subsequently when coded markings thing state is " no signal "; The reverse complemental chain information of 3 ' terminal the 3rd, No. 4 base of DNA to be measured be " 5 '-TT-3 ' ", thereby 3 ' terminal the 3rd, No. 4 base that can obtain DNA to be measured is " 5 '-AA-3 ' ".
After the completion next round detects, utilize chemical process to excise the fluorophor of oligonucleotide probe 3 ' end on link and the primer I 1, carry out ligation once more at free hydroxyl of 3 ' terminal acquisition.In reaction tank, add the listed whole thuja acid probe mixture of table 5 once more; Carry out second and take turns ligation; Ligation this time is to detect the 11st, No. 12 base (3 ' terminal the 11st, No. 12 base of DNA to be measured) that DNA to be measured is positioned at I1 primer 3 ' end downstream " 5 '-CC-3 ' ", and the probe that checks order this moment " 5 '-NNGGNNNN-3 ' " has been gone up with tense marker CY7, CY5 and three kinds of groups of FTC.After ligation took place, carrying out fluoroscopic examination was to detect CY7, CY5, FTC simultaneously, and the parity flag thing verify successfully, the interpretation and to carry out the 11st, No. 12 base of 3 ' end that reverse complemental obtains DNA to be measured be " 5 '-CC-3 ' " of tabling look-up subsequently.Carry out the 3rd, 4 of I1 primer once more behind the terminal fluorophor of the oligonucleotide that the chemical process excision connects and take turns ligation, record 3 ' terminal the 19th, No. 20 base and the 27th, No. 28 base information of DNA to be measured respectively.
After accomplishing the four-wheel ligation, 95 ℃ make sequencing primer I1 separate with DNA to be measured, add hybridization buffer and second sequencing primer I2 (sequence is seen table 4) to surface of glass slide, hybridize renaturation 20 minutes for 37 ℃.Begin the four-wheel ligation subsequently and detect 3 ' terminal the 5th, No. 6 of DNA to be measured respectively, 13, No. 14,21, No. 22 and 29, No. 30 four groups of bases.
Table 5 oligonucleotide probe mixture Verbose Listing
Same, utilize sequencing primer I3 to detect 3 ' terminal the 7th, No. 8 of DNA to be measured, 15, No. 16; 23, No. 24 and 31, No. 32 four groups of bases; Utilize sequencing primer I4 to detect 3 ' terminal the 1st, No. 2 of DNA to be measured, 9, No. 10,17, No. 18 and 25, No. 26 four groups of bases.
Splice the sequence information that can obtain whole 32 bases of DNA to be measured through the acquisition sequence information.
Claims (8)
1. the signal combination coded DNA of a tape parity check connects sequence measurement; It is characterized in that to a certain specific dna sequencing probe; Utilize the row labels that is combined into of a group coding affinity tag and a kind of signal encoding affinity tag state; For a collection of dna sequencing probe; Adopt the various combination scheme of a group coding affinity tag combinations of states signal encoding affinity tag to carry out mark, the signal combination coded DNA order-checking probe of preparation one cover tape parity check, thus be implemented in when detecting differentiation and discriminating to different dna sequencing probes:
The preparation of the signal combination coded DNA order-checking probe of A one cover tape parity check: at first prepare unlabelled dna sequencing probe, each unlabelled dna sequencing probe is by one or more order-checking bases, one or more degeneracy base N or non-strict pairing based composition; The order-checking base is used for measuring through base complementrity pairing rule the base information of DNA to be measured corresponding position, and degeneracy base N is any in A, T, C, four kinds of bases of G; After accomplishing the preparation of unlabelled dna sequencing probe; Use a group coding affinity tag and a kind of parity flag thing that the dna sequencing probe is carried out mark; By the combination of various coded markings things " mark " or " not mark " two states in the unlabelled dna sequencing probe each is carried out mark; Every kind of corresponding a kind of dna sequencing probe of combination; When a certain dna sequencing probe is carried out mark, be " mark " like the state of a certain coded markings thing in the employed combination, then use this dna sequencing probe of this coded markings substance markers; If the state of this coded markings thing is in the employed combination " not mark ", then do not use this dna sequencing probe of this coded markings substance markers; Combine the parity flag thing simultaneously, when the coded markings thing number of mark is odd number, mark parity flag thing not then, when the coded markings thing of mark is even number, mark parity flag thing then;
B utilizes the order-checking flow process of signal combination coded DNA order-checking probe of an above-mentioned cover tape parity check following:
A. choose a sequencing primer and dna profiling to be measured is hybridized according to the base complementrity pairing mechanism;
B. in reaction system, add signal combination coded DNA order-checking probe and the dna ligase and the reaction system thereof of an above-mentioned cover tape parity check, carry out the DNA ligation;
C. after accomplishing the DNA ligation; Detect the affinity tag of whole participant status combinations and the signal of parity flag thing, the residing state of interpretation coded markings thing signal, the quantity of the coded markings thing of calculating picked up signal; And utilize the signal of parity flag thing that this numerical value is carried out parity checking; Pass through like verification,, confirm the kind of the dna sequencing probe of generation ligation according to the combined situation of whole coded markings thing states; Thereby the type of confirming order-checking base on this order-checking probe with arrange and the base or the base sequence information of correspondence position on finally definite tested dna profiling;
D. after accomplishing detection, remove the affinity tag on the dna sequencing probe to the affinity tag signal;
E. repeating above-mentioned steps b-d reaches or to exceed dna profiling to be measured zone to be measured terminal until the dna sequencing probe;
F. this sequencing primer separates with dna profiling sex change to be measured, chooses the second sequencing primer;
G. repeat above-mentioned steps a-f, the full sequence information until the zone to be measured of whole unknown dna profilings is able to confirm.
2. the signal combination coded DNA of tape parity check according to claim 1 connects sequence measurement; It is characterized in that described multiple affinity tag carries out composite marking to unmarked dna sequencing probe; Its scheme is with one or more affinity tags of tense marker to same dna sequencing probe molecule; Perhaps use the differing mol of different markers substance markers respectively, subsequently above-mentioned different order-checking probe molecules are mixed with a kind of dna sequencing probe.
3. the signal combination coded DNA of tape parity check according to claim 1 connects sequence measurement; It is characterized in that described sequencing primer be meant one group can only with the oligonucleotide fragment of one section universal sequence hybridization of all dna profilings; One group of sequencing primer is hybridized the zone and is differed one or several bases each other on dna profiling, can in number wheel sequencing reaction, accomplish the examining order to the dna profiling total length; Universal sequence on the sequencing template is to prepare in the process in sequencing template to add through ligation, perhaps in amplification procedure, introduces through primer.
4. the signal combination coded DNA of tape parity check according to claim 1 connects sequence measurement; It is characterized in that described dna profiling is through dna fragmentation to be measured is increased the segmental amount of interested purpose in the genome through the DNA cloning technology; Amplification is a substance; The purpose fragment that promptly once increases, or multiple, a plurality of purpose fragments promptly once increase; In the described dna profiling to be measured fixedly is to be fixed on the planar chip base through chemistry or physical method, or is fixed on the pearl carrier of " 96 orifice plate ", " 384 orifice plate " and various modifications.
5. the signal combination coded DNA of tape parity check according to claim 1 connects sequence measurement; It is characterized in that described ligation; Can be to begin 5 ' terminal ligation to 3 ' end from the primer that checks order, also can be 3 ' terminal ligation to 5 ' end.
6. the signal combination coded DNA of tape parity check according to claim 1 connects sequence measurement; It is characterized in that described affinity tag; Be the fluorophor that adopts, or utilize the change of order-checking probe character chemistry, physics, change like resistance change, electric current.
7. the signal combination coded DNA of tape parity check according to claim 1 connects sequence measurement; It is characterized in that removing of described affinity tag; Be that labelling groups passes through method and dna sequencing probe separates chemistry, physics, perhaps dna sequencing probe character chemistry, physics is recovered original state; Removing of affinity tag is only to remove affinity tag itself, perhaps removes simultaneously with affinity tag to link to each other or disjunct one or more base and relevant group.
8. the signal combination coded DNA of tape parity check according to claim 1 connects sequence measurement; It is characterized in that described second sequencing primer is any one that removes in one group of sequencing primer in the already used sequencing primer, might not be corresponding with the sequencing primer that has used on sequencing template the most approaching one in hybridization position.
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