CN1273609A - Method and compositions for detection or quantification of nucleic acid species - Google Patents

Abstract

The present invention provides a method for detecting a target nucleic acid species using an array of probes affixed to a substrate and a plurality of labeled probes. The invention also relates to oligonucleotide probes attached to discrete particles wherein the particles can be grouped into a plurality of sets based on a physical property. A different probe is attached to the discrete particles of each set, and the identity of the probe is determined by identifying the discrete particles from their physical property. The invention further relates to methods using agents which destabilize the binding of complementary polynucleotide strands (decrease the binding energy) or increase stability of binding between complementary polynucleotide strands (increase the binding energy). The figure is an illustration of an apparatus for mass producing probe arrays.

Description

Detect or quantize the method and composition of nucleic acid species

Related application

Patent application of the present invention is the Application No. No.08/912 that submitted on August 15th, 1997,885 the application that continues, be the Application No. No.08/947 that submitted on October 9th, 1997,779 the application that continues, be the Application No. No.08/892 that submitted on July 14th, 1997,503 the application that continues, be the Application No. No.08/812 that submitted on March 4th, 1997,951 the application that continues, be the Application No. No.08/784 that submitted on January 16th, 1997,747 the application that continues.

Invention field

The present invention relates to the method and apparatus of foranalysis of nucleic acids on the whole, in particular to the method and apparatus of foranalysis of nucleic acids.

Background technology

The speed of determining the sequence of 4 kinds of Nucleotide in the nucleic acid samples is the major technique obstacle that molecular biology, medical science and biotechnology further develop.Just used the method for nucleic acid sequencing that relates to isolated nucleic acid molecule in gel from 1978.Other certified method for nucleic acid sequencing are by hybridization check order (SBH).

The method of traditional definite nucleotide sequence (being the order of A in the sample, G, C and T) is that the nucleic acid fragment mixture of terminated, non-isolabeling carries out by preparing at random at the degraded of specific Nucleotide place or the dideoxy chain termination that duplicates chain.Then with gained 1 to 500bp between nucleic acid fragment on gel, separate, produce a gradient zone, wherein adjacent sample has the difference of a Nucleotide on length.

Based on the SBH method of array do not require separate, degraded, synthetic or when describing nucleic acid molecule to the resolution of single base.Use length to distinguish hybridization, can determine that the series of the K base sequence oligonucleotide of target nucleic acid is formed as the mispairing of the short chain oligonucleotide of K base.Oligonucleotide by unique overlapping scoring (uniquelyoverlapping scored) is assembled out target nucleic acid sequence.

There are several possibility approach can finish sequencing by hybridization.In the method that is called SBH Format 1, nucleic acid samples is arranged, and hybridized with label probe and sample.Have on the same group that the photomechanical printing film of nucleic acid samples can be used to several probes of parallel scoring and/or probe can be duplicated by multiple.Nucleic acid samples can be arranged on nylon membrane or other the suitable upholders and hybridize.Each membrane array can use repeatedly.1 pair of a large amount of sample of batch processing of Format are effective especially.

In SBH Format 2, probe is arranged in place, the corresponding matrix of sequence place separately, the nucleic acid sample fragment of a mark and the probe hybridization of arrangement.In this case, carry out simultaneously can determining fragments sequence information in the hybridization at probe with all arrangements.When other nucleic acid fragments of order-checking, can reuse same oligonucleotide arrays.Can prepare these Nucleotide arrays by drop (spotting) or original position synthesising probing needle.

In SBH Format 3, use two groups of probes.In one embodiment, can be arranged in probe array form with one group, the probe groups of another group echo is stored on the porous plate with known location.In this case, needn't tagged target nucleic acid.Target nucleic acid and one or more are labeled probe join the probe groups of having arranged.If an adhesion probe (attached probe) and a label probe all are close to hybridization on target nucleic acid, they produce checked order row that are equivalent to the linking probe length overall with covalently bound so.This method long nucleic acid fragment that can check order, as the intact bacterial genome, need not the nucleic acid subclone is small segment.

Among the present invention, SBH is used to discern effectively and check order one or more nucleic acid samples.This method is usually used in diagnostic nucleic acid, legal medical expert and the making gene mapping.It also can be used for discerning the sudden change that causes hereditary illness and other features, assesses species diversity and produce multiple other forms of data based on nucleotide sequence.

The invention summary

The invention provides a kind of method that is used to detect target nucleic acid thing class, may further comprise the steps: the probe array and a large amount of label probe that are fixed in a kind of matrix are provided, select each label probe to have complementary first nucleotide sequence with target nucleic acid first part, and be fixed in the second section complementation of the nucleotide sequence and the target nucleic acid sequence of at least one probe on the matrix, described second section and first part are adjacent; Under appropriate condition, the target nucleic acid application of sample is made probe sequence and complementary sequence hybridization on array; One label probe is introduced this array; Make the probe and the target nucleic acid hybridization of being fixed on the matrix; Make the hybridization of label probe and target nucleic acid; Label probe is fixed on the hybridization probe adjacent in the array; The label probe on the array middle probe is fixed in detection.According to the preferred method of the present invention, the probe array that is fixed on the matrix comprises one group of general probe.Other preferred versions according to the present invention are fixed in the overlap that at least two probes on the matrix limit target nucleic acid sequences, and more preferably at least two label probes limit the overlap of target nucleic acid sequences.Further, the present invention provides a kind of method that detects the known array target nucleic acid on the other hand, may further comprise the steps: nucleic acid samples is contacted under hybridization conditions with one group of immobilized oligonucleotide probe that is attached on the solid substrate, wherein the immobilization probe can with the different piece specific hybridization of described target nucleic acid sequence; Target nucleic acid is contacted under hybridization conditions in solution with the oligonucleotide probe of a group echo, wherein label probe can with the different piece specific hybridization adjacent to the immobilization probe of described target nucleic acid sequence; With immobilization probe and the label probe that just in time is adjacent to immobilization probe on the target sequence covalently bound (as using ligase enzyme); Remove the label probe on not having to connect; The existence that is connected in the described label probe on the immobilization probe by detection detects the existence of target nucleic acid.The present invention also provide a member of determining in one group of partial or complete row gene that checked order in cellular type, organize or organize the method for the expression in the mixture, it is right to comprise the following steps: to limit the fixing or label probe that is specific to sequenced genes; With unlabelled nucleic acid samples and corresponding label probe and an array or the hybridization of many arrays stationary probe; Between the hybridization label probe of adjacency and stationary probe, form covalent linkage; Remove not linking probe; Being bonded to the label probe on the predetermined position in the probe array by detection determines by the existence of order-checking gene.In the present invention's one preferred embodiment, the existence of target nucleic acid identification infectious.

In addition, the present invention also provides an oligonucleotide probe array, contains a nylon membrane; The inferior array of a large amount of oligonucleotide probes on this nylon membrane, these inferior arrays comprise many single spots (spot), every bit is made up of the oligonucleotide probe of many same sequences; At the many hydrophobic barriers between the inferior array on this nylon membrane, wherein these hydrophobic barriers prevent the crossed contamination between adjacent inferior array.

The present invention also provides a kind of sequence measurement that has the tumor-necrosis factor glycoproteins of first end and second end on target nucleic acid, comprises the following steps: that (a) provides the spacer oligonucleotide of many different lengthss, and wherein the spacer oligonucleotide comprises tumor-necrosis factor glycoproteins; (b) provide known and tumor-necrosis factor glycoproteins first terminal first an adjacent oligonucleotide; (c) provide many second oligonucleotide, one of them and tumor-necrosis factor glycoproteins second terminal adjacent, these second oligonucleotide are labeled; (d) with first oligonucleotide, a plurality of second oligonucleotide and spacer oligonucleotide and target nucleic acid hybridization; (e) connect the oligonucleotide of hybridizing; (f) isolate the oligonucleotide of connection in the oligonucleotide that never connects; (g) certification mark in the oligonucleotide that connects.

The present invention also provides the sequence measurement of the tapping point sequence that has first and second ends on a kind of target nucleic acid, comprise the following steps: that (a) provides complementary first oligonucleotide with tapping point sequence first part, wherein first oligonucleotide is from first terminal at least one Nucleotide that extends of tapping point sequence; (b) provide second oligonucleotide of many marks, the second section complementation of itself and tapping point sequence, wherein a plurality of second oligonucleotide are from second terminal at least one Nucleotide that extends of tapping point sequence, and partly comprise complementary sequence from many sequences of tapping point sequence from tapping point sequence second terminal second oligonucleotide that extends; (c) one of first oligonucleotide, second oligonucleotide and target DNA are hybridized; (d) connect the oligonucleotide of hybridizing; (e) oligonucleotide that never connects separates the oligonucleotide that connects; (f) mark in the oligonucleotide of detection connection.

The present invention also provides the sequence of method use prediction to determine to(for) the negative probe of target nucleic acid.Mate by target nucleic acid and " feminine gender " probe hybridization being determined these probes do not form fully with target nucleic acid, thereby determine target sequence.

The present invention also provides a kind of method of using the oligonucleotide probe analysis of nucleic acids, these oligonucleotide probes are compounded with different marks, there is not the loss (be that different probes has different marks, the hybridization of different probe and target can be differentiated like this) of sequence information like this in the repeated use of hybridization middle probe.In a preferred embodiment, be labeled as radio isotope or fluorescence molecule or enzyme and charge species mark (electrophore mass label).In a preferred embodiment, the oligonucleotide probe of isolabeling not is used for Format III SBH, and a plurality of probes (more than 2, a probe is the immobilization probe) are linked together.

When the amount of comparing target with homologous nucleic acid in the sample is very little, the method that the target nucleic acid that the present invention also provides a kind of detection to have known array exists.In a preferred embodiment, target nucleic acid is an allelotrope, exists with low-down frequency in the sample of the nucleic acid with different sources.In another preferred embodiment, target nucleic acid has a mutant nucleotide sequence, is present in the nucleic acid samples with low-down frequency.

The present invention also provides a kind of method of using the single gel to check order to determine target nucleic acid sequence.The sequence that the primer of single gel order-checking obtains from SBH, the mulberry lattice sequencing reaction that these primers are used for standard provides the gel sequence information for target nucleic acid.The sequence that the order-checking of single gel is obtained is recently determined sequence mutually with the SBH derived sequence then.

The present invention also provides a kind of method of using the order-checking of single gel to resolve tapping point.The primer of the end identification single gel sequencing reaction of the Sfs that obtains after SBH checks order the first round, these primers are used for standard mulberry lattice sequencing reaction so that the gel order-checking information by the Sfs tapping point to be provided.To relatively confirm adjacent Sfs by the mulberry lattice sequencing result and the Sfs of tapping point then, thereby Sfs will be lined up.

The present invention also provides a kind of and has prepared the method that contains the target nucleic acid sample by PCR, need not purified pcr product before the SBH reaction.In Format I SBH, the PCR crude product be need not in advance the purifying application of sample on matrix, but before introducing label probe rinsing matrix.

The present invention also provides a kind of apparatus and method that are used to analyze target nucleic acid.Described device comprises two array nucleic acid, and they were mixed together in the desirable time.In a preferred embodiment, wherein an array nucleic acid is labeled.In another embodiment preferred, a kind of material is positioned over prevents between two array probes that two array nucleic acid from mixing.When removing this material or making it permeable, two array nucleic acid are mixed together.In another preferred embodiment, an array nucleic acid is target nucleic acid, and another array nucleic acid is oligonucleotide probe.In another preferred embodiment, two array nucleic acid are oligonucleotide probe.In another preferred embodiment, an array nucleic acid is oligonucleotide probe and target nucleic acid, and another array nucleic acid is oligonucleotide probe.In another preferred embodiment, two array nucleic acid are oligonucleotide probe and target nucleic acid.

One of the present invention uses the method for said apparatus may further comprise the steps: the nucleic acid array that is fixed on the matrix is provided, second nucleic acid array is provided, the second array nucleic acid and the fixing contacted condition of array nucleic acid of making is provided, wherein a nucleic acid array is a target nucleic acid, another array is an oligonucleotide probe, analyzes results of hybridization.In a preferred embodiment, fixedly array is a target nucleic acid, and second array is the oligonucleotide probe of mark.In another embodiment preferred, between two arrays, placed a kind of material to prevent the mixing of nucleic acid, when removing material or making it permeable nucleic acid, two array nucleic acid could mix.

Second of the present invention uses the method for said apparatus may further comprise the steps: two array nucleic acid probes are provided, condition and target nucleic acid that two array probes are in contact with one another are provided, will link together by adjacent probe on target nucleic acid, analyze results of hybridization.In a preferred embodiment, an array probe is fixed, and another array probe is labeled.In embodiment preferred more, between two arrays, placed a kind of material to prevent the mixing of probe, when removing material or making it permeable probe, two array probes could mix.

In addition, the present invention also provides be fixed thereon matrix of oligonucleotide probe array, and wherein, the probe that each probe is adjacent is hindered the physical barriers that sample solution flows and separates by a kind of.In a preferred embodiment, physical barriers is made of hydrophobic material.

In addition, the present invention also provides the method for the oligonucleotide probe array that a kind of preparation separated by physical barriers.In a preferred embodiment, use a kind of ink gun that a kind of grid is placed on the matrix, a kind of material that reduces the array reaction volume is provided.

The present invention also provides oligonucleotide to be fixed and has formed the matrix of cubical array thereon.This cubical array will read probe result's very high resolution (the every cm of each aspect ²Have low-density relatively probe) combine with three-dimensional high information content (a plurality of aspects or probe).

The matrix that the present invention also provides a kind of oligonucleotide probe to be fixed thereon, wherein oligonucleotide probe has spacer, and spacer has increased the distance of matrix and oligonucleotide probe message part (as combine and provide the oligonucleotide probe part of sequence information with target).In a preferred version, spacer comprises ribose and phosphoric acid, thereby wherein phosphoric acid and ribose form ester and ribose covalent attachment by 5 ' and 3 ' hydroxyl, the formation polymkeric substance.

The present invention also provides a kind of method that the cDNA clone is divided into the similar sequences group or is equal to sequence set, can select a representational clone from each group like this and check order.In a preferred embodiment, when a large amount of clone of order-checking, use the method for this grouping, may further comprise the steps: survey each clone with a large amount of oligonucleotide probes; Determine the strength of signal of which probe and each clone's combination and each probe; To similar probe bonded clone these clones are divided into many groups with similar intensity by identification; The every group of clone that checks order at least.In embodiment preferred more, a plurality of probes comprise about 50 to about 500 different probes.In another embodiment preferred, a plurality of probes comprise about 300 different probes.In the most preferred embodiment, a large amount of clones are a large amount of cDNA clones.

The invention still further relates to the oligonucleotide probe with discrete particles compound (covalently or non-covalently), wherein particle is divided into many groups according to physical properties.In a preferred embodiment, different probes is attached on every group of dispersed particles, determines the identity of probe by the physical properties of identification discrete particles.In another embodiment, discern probe according to the physical properties of probe.Physical properties comprises arbitrary character that can be used to differentiate discrete particles, comprises for example size, fluorescence, radioactivity, Electromagnetic Charge or absorbancy, or attached to the mark on the particle such as dyestuff, radionuclide or EML.In a preferred embodiment, separate discrete particles by a flow-cytometer that detects granular size, electric charge, fluorescence or absorbancy.

The invention still further relates to the method for analyzing target nucleic acid with discrete particles compound probe of using.These probes can be used for above-mentioned arbitrary method, but will discern probe by the physical properties of discrete particles.These probes also can be used in the Format III step, and wherein free probe is by a mark identification, and the probe that is compound on the discrete particles is discerned by physical properties.In a preferred embodiment, probe is used for the target nucleic acid that checks order with SBH.

The invention still further relates to and use reagent that reduces complementary polynucleotide chain combination stability (reduction bound energy) and the compositions and methods that improves complementary polynucleotide chain combination stability (raising bound energy).In a preferred embodiment, this reagent is positively charged molecule, stain remover such as sodium laurylsulfonate, the sarcosyl of trialkyl ammonium salts, sodium-chlor, phosphoric acid salt, borate, organic solvent such as methane amide, ethylene glycol, dimethyl sulfoxide (DMSO) and dimethyl formamide, urea, Guanidinium salt, amino acid analogue such as trimethyl-glycine, polyamines such as spermidine and spermine or other neutralising phosphoric acid skeleton negative charges, little/the major groove wedding agent, positively charged polypeptide and intercalating agent such as acridine, ethidium bromide and anthracin.In a preferred embodiment, reduce or improve the right Tm of complementary polynucleotide with a kind of reagent.In a preferred embodiment, with the mixture reduction or the right Tm of raising complementary polynucleotide of reagent.In a most preferred embodiment, from the complementary polynucleotide of mispairing, debate the ability of the complementary polynucleotide of coupling not fully with the mixture raising of a kind of reagent or reagent.In a preferred embodiment, thus the bound energy that adds reagent or plurality of reagents AT base pair approximates the bound energy of GC base pair.Can improve the bound energy of these complementary polynucleotide by the negative charge that adds phosphate group in reagent neutralization or the shielding polynucleotide skeleton.

Brief description of the drawings

Fig. 1 is the vertical view that is used to produce in batches the device of probe array.

Fig. 2 is the side-view that is used to produce in batches the device of probe array.

Fig. 3 is the decomposition side view of allocation units that is used to produce in batches the device of probe array.

Detailed description of the preferred embodiments

Format I SBH is suitable for analyzing simultaneously many group samples. Independently make in the hybridization reaction at thousands of Can carry out at big array the parallel scoring of thousands of samples with many Small diaphragm-pieces. The identification of DNA Relate to 1-20 probe of each reaction, the identification that suddenlys change in some situation is related to the special selection of each sample Or more than 1000 probe of design. For identifying the character of the dna fragmentation that suddenlys change, can be in the first round hybridization Synthetic or the selection specific probe of detected every kind of sudden change.

The DNA sample preparation can be become little array, these little arrays can be separated by suitable sept, can Detect simultaneously with being selected from the one group of oligonucleotide probe that is arranged on the porous plate. Little array can be by one or more Sample composition. The DNA sample can comprise mutant or the individual sample of a sequence in each little array. Can With the synthetic bigger array of the little array group of adjacent continuous. The array that this class is bigger can comprise identical little array Repeat array maybe can comprise the array of different dna fragmentation samples. The common group probe comprises with accurately predetermined Enough probes of degree analyzing DNA fragment, as consider the redundancy that reads each base-pair (" bp ") The property. The probe that these groups comprise can be more than a necessary probe of specific fragment, but the probe that comprises can lack In thousands of necessary probes of different sequence DNA samples of test.

DNA or allelic identification and diagnosis sequence measurement can comprise the following steps:

1) screening probe subgroup from special use, representative or versatility group, with a plurality of little arrays Each hybridization array;

2) each the inferior array at each array of parallel analysis adds first probe;

3) hybridize and results of hybridization is marked;

4) peel off previous used probe;

5) the residue probe that will mark is repeated hybridization, scoring and strip step;

6) acquired results is processed obtained final analysis result or determine other probes that will hybridize;

7) some inferior array is hybridized again;

8) to a complete set of data analysis and obtain last analysis result.

This approach provides a small amount of of a kind of quick identification and check order a type (such as DAN, RNA) The method of nucleic acid samples also provides and has used pre-synthesis one group easily to control big or small probe parallel analysis and be inferior battle array The method of the several samples type of row form. With two kinds of approach in conjunction with produce a kind of be used for determining DNA with Effective and general method of one property, DNA diagnosis and identification sudden change.

For the identification known array, can use the shorter probe of a small group, replace long particular probe. At this In one approach, although will mark to more probe, can synthesize one group of general probe and cover arbitrary Type sequence. For example, a complete set of 6 base sequences only comprise 4096 probes, and 7 complete base sequences only Comprise 16384 probes.

Can use the hybridization of two kinds of levels to carry out the complete order-checking of dna fragmentation. A kind of level is to cover each One group of enough probe of base is hybridized once at least. For reaching this purpose, can be synthetic to a standard sample One group-specific probe. Use results of hybridization of this group probe whether to demonstrate in non-standard sample and at which In undergo mutation (difference). And this group probe can comprise " the moon of definite " positive " Probe Hybridization result The property " probe. Be the homogeneity of determining to change, can use the hybridization of additional specific probe and sample. This is attached Add the group probe have " positive " (mutant nucleotide sequence) and two kinds of probes of " feminine gender " probe, the variation of sequence by Positive probe identification is determined by negative probe.

In another embodiment, marked from all probes of common group. One group of common group probe allows With two-step method the probe of each sample relatively small amount is marked, avoided waste of time. Crossover process The one best subgroup probe that can relate to continuous detecting, at first hybridize with Computer Processing in the first step, right Rear second step is determined those the additional probes that will mark in the common group on the basis of acquired results. Two groups of probes The negative probe that all has positive probe in the affirmation group. And, can in an independent step, pass through subsequently Sample and one group of " feminine gender " Probe Hybridization identifying from SBH result are determined institute's calling sequence.

In the amalgamation of SBH sequence, because contingency or biological reasons repeat out when the analyzing DNA fragment Existing K-1 oligonucleotides can be considered especially. If there is not other information, relatively little dna fragmentation Can be by complete amalgamation, each base is read repeatedly.

When the relatively long fragment of amalgamation because the K-1 sequence in one group of positive score probe (namely than The sequence that probe length is short) repeat to cause mispronounced. If must determine sudden change or similar sequences this Can there be (being that the K-1 sequence is not to be repeated fully equally) in the problem of kind. Can utilize having of certain sequence Close knowledge as the sequence (such as the sequence that exists in the database) of " template " next correct amalgamation known similar, By lining up array to demonstrate the optimum Match on template for the positive probe of unknown nucleotide sequence.

Use a sample array to avoid on simple sample or a small group sample company to many oligonucleotides Continuous scoring. This approach allows by only a physical target operation being come the many probes of parallel scoring. Can Order-checking length is the inferior array of the DNA sample of 1000 bp in short time relatively. If with sample at one Drop is that 50 inferior arrays and array are repeated to detect 10 times in the array, 500 probes of can marking so. When the generation of examination sudden change, can use enough probes to cover each base three times. If there is sudden change, The probe of several coverings will be influenced. It is accurate to utilize negative probe homogeneity information to make to have two bases The mutation map of degree. For determine this mode map in the sudden change of single base, can add again 15 of uses Probe. These probes have covered for the combination of arbitrary base of two positions that have a question (supposes not disappearance And insert). These spies of scoring in a circulation on 50 inferior arrays of the sample of giving can contained Pin. In finishing multiple labelling cromogram (being multiple copying (multiplexing)), with 2 to 6 probes As a storehouse (pool), each probe has different marks such as different fluorescent dyes, reduces thus assorted Hand over period and shorten the order-checking process.

Under complicated situation more, two adjacent sudden changes or insertion may be arranged. Available more probe advances Row is processed. For example, available 64 probes are determined the insertion of 3 bases. Can be by hybridization, formerly assorted Hand over and to select the complicated situation of one group of several step process of new probe on result's the basis.

If the inferior array of analyzing comprises tens an or hundreds of sample of one type, can find them so In some contain one or more variations (sudden change, insert or disappearance). For each sheet of undergoing mutation Section, the one group of specific probe of can marking. The probe sum of one type of sample of scoring can be hundreds of. Counterweight The multiple parallel scoring of array is conducive to hundreds of probes of less circulation scoring. In addition, can collect compatible probe. Positive hybridization can belong to the probe for detection of specific DNA fragments, because these fragments are usually in its group Become on the base 75% difference is arranged.

Use bigger one group can analyze long target than long probe. These targets can represent a sheet phase library as The extron clone bank.

Can utilize a kind of specific hybridization methods of marking to determine the existence that suddenlys change from the genomic genomic fragment of waiting to check order of diploid.Two kinds of situations are arranged: i) represent a known allelotrope, represent a new sudden change from another chromosomal sequence from a chromosomal sequence; Or ii) two karyomit(e) all contains new but different sudden changes.In both cases, the designed scanning step that collection of illustrative plates is made in variation has provided the peak signal difference in sudden change position twice.And this method can be used for discerning that individuality carries is which allelotrope, for this gene individuality whether be isozygoty or heterozygosis.

By with corresponding signal with isozygoty and heterozygosis contrast is compared, can effectively obtain the scoring of twice signal difference required in first kind of situation.This approach can determine that each gives weakening relatively for each particular probe hybridization signal in the sample.This mainly be because for a particular probe of different IPs acid fragment hybridization with identical full coupling target, hybridization efficiency can have the many difference of twice.And according to the oligonucleotide probe number, different mutational sites can influence more than a probe.Weakening of two to four continuous probe signals demonstrates a mutational site comparatively significantly.Can use the selection probe of several groups to come detected result, one or several can provide full matched signal in these probes, and the signal of the recently self-contained mispairing duplex of signal averaging is strong 8 times.

The diaphragm of separating allows tissue test very neatly, with sample that holds the relatively large quantity of representing the sequence type of being given or the many dissimilar sample of representing with the relatively small amount sample.Control from view of profit 4-256 that can be a specific sample.Inferior array is designed to match with the shape size of the used standard porous plate of storage and labeled oligonucleotide in the scope of this can being counted.For the size that the sample of different quantities can be regulated inferior array, perhaps can use the inferior array of some normal sizes.If one type all samples is not suitable for an inferior array, can uses additional inferior array or film, and handle with same probe.In addition, can change the time of finishing identification or order-checking process by the repetition number of regulating each inferior array.

" intermediate segment " used herein refers to that length is the oligonucleotide of 5 to 1000 bases, 10 to 40 bases of preferred length.

In Format 3, first group of oligonucleotide probe of known array allow its with the condition with nucleic acid hybridization of complementary sequence separately under be fixed on a kind of solid support.Provide second group of oligonucleotide probe that is labeled in solution.In the probe groups and between probe groups can be equal length also can be different lengths.Nucleic acid to be checked order or its middle fragment can be offered first group of probe (when particularly existing recA albumen to hybridize under non-sex change condition with permission) with double chain form, or provide and allowing under the hybridization conditions of different complementary degree (for example, allowing to distinguish under the condition that coupling fully and base-pair mismatch hybridize) to carry out with single stranded form.After can be before using second group of probe or nucleic acid or its centre fragment that simultaneously will be to be checked order offer first group of probe.The probe that combines with adjacent site on the target be joined together (as by accumulative facies mutual effect or ligase enzyme or can between adjacent probe, form the additive method of chemical bond).Make adjacent probe in conjunction with after, flush away does not combine with a member in first group of probe by chemical bond and is fixed in the fragment and the probe on surface, for example makes and hybridizes high temperature (the reaching 100 ℃) rinsing solution that unwinds.Then, use the method (for example can be chemoluminescence, fluorescence, radioactivity, enzyme, optical density(OD) or charge species mark) that is suitable for used mark to detect bonded probe in second group.

Nucleotide base used herein " coupling " or " complementation " refer to that they form stable duplex by hydrogen bond under given conditions.For example under the condition that adopts usually in hybridization analysis, VITAMIN B4 (" A ") is mated with thymus pyrimidine (" T "), rather than guanine (" G ") or cytosine(Cyt) (" C ").Similarly, G mates C, rather than A or T.Other form the base of hydrogen bond such as xanthoglobulin or universal base (" M " base, Nichols etc. 1994) or other adorned bases such as methylated base and those in relatively poor special mode can form the base complementrity of stablizing duplex under given conditions.If each base all is to form duplex according to the basepairing rule of Watson and Crick and the nucleic acid base of waiting to check order by hydrogen bonding in the probe, think probe " fully complementary " or " coupling fully " (promptly without any the influence of sequence on every side, the duplex that forms for a particular probe has maximum bound energy) so." complementary fully " and " coupling fully " also refers to comprise the probe with analogue or modified nucleotide.According to " matching principle fully " selected for analogue or modified nucleotide judge analogue or modified nucleotide " coupling fully " (as the Nucleotide to a particular analog or modification have maximum combined can combination to).Do not form according to this principle that to be considered under the specific hybrid condition in conjunction with each base in the right probe be mispairing.

Can be when each probe mates fully with the nucleic acid of waiting to check order with a row probe amalgamation.Can analyze this row probe then, it is sorted with maximum reduplicative forms.The longest hold the identical base sequence of base sequence by other each probes in first probe and these row being come relatively to determine which probe has at 3 ' end, can finish this ordering with second probe 5 '.Afterwards, the one the second probes are overlapping, by with second probe 5 ' 3 ' end of all residue probes of end and other relatively and with 5 ' end of all residue probes of 3 ' end and other of first probe relatively comes this process of repetition.Can carry out this process does not continuously have probe not overlapping by other probes in these row.Perhaps, can from positive probe row, select more than a probe, and the parallel overlapping probe (" sequence nuclear (sequence nucleus) ") that produces more than a group.Probe in every kind of method of this sequence amalgamation row can be with all probes row of the complete complementary of nucleic acid of waiting to check order maybe can be its arbitrary subgroup.

Can be with 5 ' the overlapping sequence extensions that obtain than length of end and 3 ' end of probe.Carry out the process of this amalgamation probe continuously, until because tapping point (probe is repeated in fragment), be longer than the tumor-necrosis factor glycoproteins of probe or not cloned sequence produce and to mispronounce.The extension of sequence all is called subclone sequence fragment (Sfs) between any two dependencys.When overlapping owing to obtaining selectable suitable probe, in the sequence amalgamation, produce when mispronouncing, can use leap can select the long probe hybridization of overlapping bit point, competitive hybridization, right selected end of site probe mispronounced in leap be connected, maybe can use single gel analysis (non-ly mispronouncing ordering) with what Sfs was provided with end.

By adopting above-mentioned steps, from obtaining the sequence of arbitrary desired level until the crossing pattern of the full sequence of the dna molecular (as karyomit(e)) in the Sfs of amalgamation and intermediate segment or complete source (can relevant with as the feature of discerning nucleic acid samples) with nucleic acid samples identity with overlapping or non-overlapped probe.

Order-checking can may further comprise the steps usually:

(a) allowing a fragment and an immobilization probe to form under the condition for validity of elementary mixture, an array of immobilized oligonucleotide probe is being contacted with a nucleic acid fragment with complementary sequence;

(b) under the condition for validity of the oligonucleotide probe hybridization that allows elementary mixture and mark, the oligonucleotide probe of elementary mixture and this group echo is hybridized in solution, form the secondary mixture thus, wherein fragment is all hybridized with immobilization probe and label probe;

(c) from the secondary mixture, remove arbitrary not label probe adjacent of hybridization with the immobilization probe;

(d) detect the existence of adjacent label probe and unmarked probe by the existence of certification mark thing;

(e) be connected by known array and determine segmental nucleotide sequence immobilization probe and label probe.

Select hybridization and rinsing condition to detect basically the hybridization of coupling fully (wherein fragment and probe have 6 positions that the hybridization of hybridizing takes place as those) on 7 positions, can select to hybridize with rinsing condition and allow the variation and a pair of base-pair mismatch of mating fully, or select hybridization and rinsing condition to allow only to detect the hybridization of mating fully.

Can determine suitable hybridization conditions by optimization method or exploratory development according to a conventional method.This method and research are undertaken by those skilled in the art that those formulate experimental program usually.Referring to Current Protocols in Molecular Biology such as Ausubel, Vol.1-2, John Wiley ﹠amp; Sons (1989); Sambrook etc., Molecular Cloning A Laboratory Manual, second edition, Vols.1-3, ColdSprings Harbor Press (1989); With Maniatis etc., Molecular Cloning:A LaboratoryManual, Cold Spring Harbor Laboratory Cold Spring Harbor, New York (1982), all these draw at this and are reference.For example, temperature, concentration of component, hybridization and rinsing time, buffer reagent composition and its pH and these conditions of ionic strength all can change.

In the example that label probe and immobilization probe are not connected by physics or chemistry, can be only according to the rinse step detection of controlled stringency.In this case, because the accumulation between the adjacent probe, adjacent probe has the enhanced binding affinity.For the optimization said process can change experiment condition.

In immobilization and the connected example of label probe, can connect by a kind of chemical linking agent (as water-soluble carbodiimide or cyanogen bromide), maybe can adopt a kind of ligase enzyme such as commercially available T4DNA ligase enzyme.Utilize adjacent probe can select rinsing condition to distinguish adjacent and non-adjacent mark and immobilized probe with respect to the difference of non-adjacent probe stability.

Can use coordination that fluorescence dye, chemiluminescence system, radio-labeling (as 35S, 3H, 32P or 33P) or available mass spectroscopy detect labeled oligonucleotide probe usually.

When the nucleic acid molecule of unknown nucleotide sequence is longer than about 45 or 50 base pairs, this molecule fragmentization can be measured fragment sequence.By restriction enzyme digestion, shearing or NaOH processing carrying out fragmentation.Can obtain the preferred fragment length of about 10 to 40 base pairs according to molecular size (as by gel electrophoresis) isolated fragment.

Can be by several different methods immobilized oligonucleotide known in the art, as using the light deprotection absorption of nucleoside phosphoramidites (nucleoside phosphoramidite) or phosphonic acids hydrogenation nucleosides (nucleoside hydrogen phosphorate) reagent by the laser activation of a phosphate group.Can use glass, nylon, silica gel and fluorocarbon upholder.

Oligonucleotide can be lined up array, these arrays can comprise the subgroup of all probes all or given length or select the probe groups of length.

Can use hydrophobic separator to separate the inferior array of probe or probe.But array of designs is used for different purposes (as making target region order-checking, mRNA order-checking and the large scale sequencing that collection of illustrative plates, part checked order, were used for diagnostic purpose).Be exclusively used in a specific end use by selecting the combination and permutation of probe on matrix can design a kind of specific chip.

For example, can make up 1024 immobilization probe arrays that all oligonucleotide probes are 5 base length (each array contains 1024 different probes).Probe in this example says it is 5 base sequences (in fact they can be long probes) from the information meaning.Second group 1024 5 base sequence probes can be carried out mark, each label probe can be provided for the immobilization probe array with fragment to be checked order.In this example, 1024 arrays will be combined to form a big super array or " super chip ".In the example of and terminal hybridization terminal along immobilization probe of nucleic acid fragment and label probe at those, by for example connecting two probes are bonded together, and after removing unconjugated mark, relation between the material of being used on existence by a bit locating mark in the immobilization probe array with a known array and the known array label probe detects and sample fragment complementary 10 base sequences.The sample fragment sequence is exactly successive immobilization probe sequence in the label probe sequence in fact.By this way, can detect 1,000,000 kinds of all 10 possible base sequences by the combined method of only utilizing 5 base sequences, the synthetic required workload of oligonucleotide only is a thousandth.

In a preferred embodiment, will support the matrix of oligonucleotide probe array to be divided into mass part, each probe can be by for example being that the physical barriers and the adjacent probe of hydrophobic material separated in the array like this.In a preferred embodiment, the width of physical barriers is 100 μ m to 30 μ m.In a preferred embodiment, each probe core to the distance at any one adjacent probe center is 325 μ m.Can use non-moving fixedly matrix or be fixed in going barrel or the matrix of dish and suitable automatic handling system such as this probe array of a kind of anorad gantry mass production that has inkjet deposited device such as droplet amount head.

In another preferred embodiment, oligonucleotide probe is fixed in a cubical array.This cubical array is made up of multilayer, every layer can analyze separately and with other layers be what to separate.This cubical array can be a various ways, for example, array can be placed on the matrix with many grooves, and probe is arranged in the different depths (each aspect is made up of the probe at the similar degree of depth of groove place) of groove; Maybe array can be placed on the matrix with different depth recess, probe is positioned at bottom portion of groove or is positioned at the high spot that groove is separated, and perhaps can use projection and some of groove to make up (each aspect is made up of all probes in a certain degree of depth); Perhaps array can be positioned on the matrix of a plurality of lamellas compositions described lamella stratification cubical array.

Probe in these arrays can comprise the spacer that increases distance between stromal surface and the detecting probe information part.These spacers can be made up of the atom that can form two covalent linkage such as carbon, silicon, oxygen, sulphur, phosphorus etc. at least, or can be made up of the molecule that can form at least two covalent linkage such as sugared Monophosphate group, amino acid, peptide, nucleosides, Nucleotide, sugar, carbohydrate, aromatic nucleus, hydrocarbon ring, straight chain and branched paraffin etc.

Can hinder hybridization to form to avoid the sample secondary structure with waiting to check order nucleic acid sample fragmentization or carry out other processing (as using recA).For example can shear (as with ultrasonic) or handle and make sample fragmentization by restriction enzyme such as Cvi JI digestion, physics with NaOH.Can be by the separating obtained fragment of gel electrophoresis, and from gel, extract according to appointment the fragment of 10 bases to the appropriate length between about 40 bases.In a preferred embodiment, " fragment " of nucleic acid samples can not link to each other with other fragments in the storehouse.Can obtain this phase library by the nucleic acid of handling fragmentation with Phosphoric acid esterase (as calf small intestine Phosphoric acid esterase).In addition, in the mulberry lattice dideoxy sequencing reaction of nucleic acid samples, use random primer (as N ₅-N ₉, wherein N=A, G, T or C) can obtain nucleic acid samples can not junction fragment.This will produce has and the target nucleic acid complementary sequence and dna fragmentation that end at two deoxidation residues that can not be connected with other fragments.

By between fixing and label probe, introducing the cleavable key, then finish one take turns Format 3 analyze after this key of cracking can prepare recycling Format 3 SBH arrays.Label probe can be a ribonucleotide, or ribonucleotide can be used as the connection base in the label probe, handles by RNAse or uridylic-DNA glycosylation like this or NaOH handles and can subsequently this probe be removed.In addition, can select cracking to connect the key that produces by chemistry.

Other changes comprise uses the oligonucleotide of modifying to improve specificity or efficient, cyclical crosses is to strengthen hybridization signal, for example under the optimal condition (as temperature) that is the selection of the first group echo probe, hybridize circulation, under the optimal condition that is the selection of the second group echo probe, hybridize subsequently.By using end is respectively that the mixture mixture of equimolar amount (preferred) of the probe of one of four kinds of nucleotide base A, T, C and G determines to read moving in the frame.

For segmental ordering sequence, tapping point produces and mispronounces.Though sequence information determines by SBH, can use the single gel order-checking of the readable length that (i) grow with the part of the cost of complete gel order-checking; Or (ii) compare with correlated series, to the hybridization data ordering of this mispronouncing (" tapping point ") nidus.Discern the primer that is used for by the single gel order-checking of tapping point from the SBH sequence information or from the flanking sequence in known carrier information such as carrier insertion site, at the enterprising column criterion mulberry of nucleic acid samples lattice sequencing reaction.The sequence that obtains from the order-checking of this single gel with read in read tapping point Sfs relatively with the order of identification Sfs.Perhaps, by the contrast of Sfs sequence and correlated series and ordering Sfs are produced one with the immediate sequence of correlated series, Sfs can sort.

In addition, can determine the segmental number of series connection repetitive nucleic acid in the target fragment by the order-checking of single gel.Because series connection repeats rare encoding histone part at gene, so only just carry out the gel step that checks order when one of non-coding region is identified as when having special purpose (if be an important regulatory region as it).

The hybridization degree information that shows about the group of only about 200 oligonucleotide probes (being about 5% work of complete order-checking) defines unique feature of each gene, can be used to from the library sorting cDNA to determine whether the library contains a plurality of copies of same gene.By these features, can distinguish and investigate thoroughly identical, similar and different cDNA.

The method of nucleic acid and separation, clone and sequencing nucleic acid is well known to those skilled in the art.Referring to as Ausubel etc., Current Protocols in Molecular Biology, 1-2 volume, John Wiley ﹠amp; Sons (1989); Sambrook etc., Molecular Cloning A Laboratory Manual, the 2nd edition, 1-3 volume, Cold Spring Harbor Press (1989), these two parts of documents all draw at this and are reference.

SBH is a full-fledged technology, can be undertaken by several different methods well known to those skilled in the art.Especially, technology relevant with sequencing by hybridization in the following document is hereby incorporated by reference: Drmanac etc., U.S. patent 5,202,231 (drawing at this is reference), publication on April 13rd, 1993; Drmanac etc., Genomics, 4,114-128 (1989); Drmanac etc., Proceedings of the First Int ' l.Conf.ElectrophoresisSupercomputing Human Genome, volumes such as Cantor, World Scientific Pub.Co., Singapore, 47-59 (1991); Drmanac etc., Science 260,1649-1652 (1993); Lehrach etc., GenomeAnalysis:Genetic and Physical Mapping, 1,39-81 (1990), Cold Spring HarborLaboratory Press; Drmanac etc., Nucl.Acids Res.4691 (1986); Stevanovic etc., Gene, 79,139 (1989); Panusku etc., Mol.Biol.Evol., 1,607 (1990); Nizetic etc., Nucl.Acids Res., 19,182 (1991); Drmanac etc., J.Biomol.Struct.Dyn., 5,1085 (1991); Hoheisel etc., Mol.Gen., 4,125-132 (1991); Sterezoska etc., Proc.Nat ' l Acad.Sci. (USA), 88,10089 (1991); With Drmanac etc., Nucl.Acids Res., 19,5839 (1991); Drmanac etc., Int.J.Genome Res., 1,59-79 (1992).

Following embodiment describes the present invention in detail.According to the disclosure of invention, it will be appreciated by those skilled in the art that and to make many other embodiments and change within the scope of the present invention.Therefore, should think that relative broad range of the present invention is not limited in the open scope of following embodiment.

Embodiment 1

The preparation of probe groups

Can prepare two types common group probe.First group is one group of complete (or at least one incomplementarity subgroup) shorter probe, for example individual 6 base sequences of all 4096 (or about 2000 incomplementarities), or all 16,384 (or about 8000 incomplementarities), 7 base sequences.8 base sequences and whole incomplementarity subgroups of long probe not too be convenient to obtain because they comprise 32,000 or more probe.

Select one group second type probe, be a little probe subgroup, use at least one probe to be enough to read each bp in arbitrary sequence.For example, in 16 dimers 12 be enough.The little subgroup of 7 base sequences, 8 base sequences and 9 base sequences of double-stranded DNA of being used to check order 3000,10,000 and 30,000 probes of to have an appointment respectively.

Also can select the target nucleic acid of probe groups identification known array, and/or the allelotrope or the mutant of identification known array target nucleic acid.This class probe groups contains enough probes, and each position Nucleotide of target nucleic acid is read once at least thus.By lose with " positive " probe combine recognition allele or mutant.Then, by surveying the distinguished sequence that target nucleic acid is determined these allelotrope or mutant with containing the probe groups that changes combination on each possible Nucleotide variation and these probe locations.

Probe groups can also be made of (all probes with a certain length) 50 probes to common group probe, more preferably this common group is made up of 100-500 probe, and in a most preferred embodiment, probe groups contains 300 probes.In a preferred embodiment, probe groups is a 6-9 length of nucleotides, and is used to the cDNA clone is grouped into similar sequences or is equal to sequence, can treat to select the order-checking group single representative clone from each like this.

Utilize standard chemical process to prepare the probe that end has 1 to 3 unspecific (blended A, T, C and G) or general (as M base or inosine) base.If the use radio-labeling can have the 5 ' terminal hydroxyl that is used for kinasesization by the inferior phosphorus group of radio-labeling probe.Perhaps, can use probe with arbitrary compatible system such as fluorochrome label.Also can use other forms of probe, as contain the probe of the modified base of PNA (protein nucleic acid) or change duplex stability.

Probe can be left in the bar coding porous plate.The probe of small number can use 96 orifice plates; 10,000 or more probe preferably leave 384 or 864 orifice plates in.5 to 50 flaggies are enough to deposit all probes.About 5pg probe is enough to and a DNA sample hybridization.Therefore, the synthetic on a small quantity about 50mg of each probe can analyze 100,000,000 samples.If per three samples use a probe, and if each sample length be 1000bp, use one 5000 probe groups can check order so more than 30,000,000,000 bases (10 human genomes).

Embodiment 2

Probe with oligonucleotide of modification

Can be with the oligonucleotide probe introductive crossing probe and the use under suitable condition of modifying.For example, can use at C ⁵The position is that the pyrimidine of halogen improves duplex stability by influencing base stacking.Can use 2,6-diaminopurine in the base pair that forms with thymus pyrimidine, to provide the 3rd hydrogen bond, heat-stable DNA duplex thus.Use 2, the 5-diaminopurine can improve duplex stability, allowing more stringent condition annealing, thereby improves the specificity of duplex form, suppresses the background problem and allows the use of shorter oligomer.

Hoheisel ﹠amp; Lehrach (1990) discloses triphosphoric acid model synthetic of these modified nucleotides.

Also can use non-differentiation base analogue or universal base, designed as people such as Nichols (1994).Prepare this new analogue 1-(2-deoxidation-D-ribofuranoside)-3-nitro-pyrrole (representing) and be used in oligonucleotide probe and the primer with M, solving the design problem that causes by the genetic codon degeneracy, or in the time only can obtaining fragment peptide sequence data.This analogue makes minimized the making simultaneously of hydrogen bond action pile up maximization, and does not destroy DNA duplex from solid.

Design this M nucleoside analog, use the non-proton polar substituent be connected on the fragrant heterocycle so that the accumulation maximization, strengthen in the chain and the interchain accumulation to reduce the effect of hydrogen bond in the base pairing specificity.People such as Nichols (1994) agree with using 3-nitro-pyrrole 2-deoxyribosyl nucleosides, because itself and right-N-methyl-p-nitroaniline have structure and electric charge similarity, its derivative is the double-stranded DNA intercalating agent of known minimum.

The phosphoramidite that also can obtain the dimethoxy trityl protection of nucleosides M is inserted in the Nucleotide, as the primer of order-checking and polymerase chain reaction (PCR).People such as Nichols (1994) disclose that a considerable amount of Nucleotide can be replaced by M and the specificity of not losing primer.

The peculiar property of M is its long-chain that can replace continuous nucleosides, and still can produce effective sequencing primer.Reported that the sequence with 3,6 and 9 M replacements all provides the readable sequences ladder, carries out PCR with three different primers that contain M and all obtains correct amplification product (Nichols etc., 1994).

The oligonucleotide that contains the 3-nitro-pyrrole can show that effectively the duplex structure must be formed by complementary strand as primer.What the tool report obtained is used for oligonucleotide to d (5-C ₂-T ₅XT ₅G ₂-3) and d (5-C ₂A ₅YA ₅G2-3) the photo-thermal distribution plan of (wherein X and Y can be A, C, G, T or M) and dna double chain change observed normal S shape figure to strand and match.The Tm value tool report that contains the oligonucleotide of XM base pair (wherein X is A, C, G or T, and Y is M) all falls in 3 ℃ of scopes (Nichols etc., 1994).

Embodiment 3

Select and label probe

When the array of preparation an array Asia, be limited to the probe groups to be hybridized on each inferior array in each hybridization circulation.For example, can from common group, select one group of 384 probe, in each circulation of 4 round-robin, can carry out 96 probes and survey.Selected probe of hybridizing in a circulation preferably has similar G+C content.

Forward each round-robin probe that is used for of selecting to one 96 orifice plates, if they are not labeled then carry out mark by kinasesization or other mark programs (as using the stable fluorescence dyestuff) then before being stored.

On the basis of first round hybridization, can limit one group of new probe to each inferior array and be used for additional circulation.Some array may not be used in some circulation.For example, if in 64 patient's samples only 8 demonstrate sudden change, to each sudden change 8 probes of at first marking, can in a circulation, mark so to whole 64 probes, 32 inferior arrays are not used.Then, the available filter membrane exsiccant hybridization buffer that prevents is handled the inferior array that these are not used.

Can from deposit plate, search probe by any approach easily, resemble Beckman Biomek 1000 (Beckman Instruments as single track liquid-transfering device or a kind of automatic manoeuvring platform, Fullerton, California) or a kind of Mega Two (Megamation of robot, Lawrenceville, New Jersey).DAP and probe sequence of control can be combined on the automatic manoeuvring platform.

The inferior array that can search probe singly and the probe application of sample is covered to hybridization buffer.Preferably the probe that will find is added to the row labels or mix with hybridization buffer of going forward side by side on the new plate.Preferred lookup method is to deposit plate and each selects the particular bore of probe to an intermediate plate from each plate moves liquid (or shift by metallic plug (metalpins)) q.s by access singly.Can use an independent addressable transfer pipet or plug array to quicken search procedure.

Embodiment 4

The preparation label probe

Oligonucleotide probe be can prepare by automatic synthesis method, method well known to those skilled in the art and Applied Biosystems system for example used.Perhaps, use utilizes the GenosysBiotechnologies Inc. method of porous Teflon stack of wafers can prepare probe.

For example available radio-labeling with array that 100-200um or 100-400um order ( ³⁵S, ³²P, ³³P is also preferred ³³P), non radioactive isotope (Jacobsen etc., 1990) or fluorophore (Brumbaugh etc., 1988) labeled oligonucleotide probe.All this marking methods are ordinary methods in this area, for example in people's such as Sambrook (1989) relevant portion and the method described as (1991) such as (1990), Murakami etc. (1991) such as Schubert and Cate, these at this all by incorporated by reference.

About radio-labeling, common method is to use the T4 polynucleotide kinase to carry out end mark or uses Klenow or flush end T7 polysaccharase to carry out the high degree of specificity mark.Below this is described.

Synthetic oligonucleotide its 5 end when being synthesized does not have phosphate group, therefore by use phage T4 polynucleotide kinase from [ ³²P] ATP or [ ³³P] the ATP transfer- ³²P or- ³³P is easy to carry out mark.If the reaction effectively carry out, the activity specific of so this probe can with [ ³²P] ATP or [ ³³P] ATP itself is equally high.The reaction that describes below is that the oligonucleotide of mark 10pmol is to high specific acitivity.By improving or reduce to react size, keeping the constant mark that can obtain different amount oligonucleotide easily of all the components concentration.

With 1.0ul oligonucleotide (10pmol/ul); 2.0ul 10 x phage T4 polynucleotide kinase damping fluids; 5.0ul[- ³²P] ATP or [ ³³P] ATP (specific activity 5000Ci/mmol; Be 10mCi/ml in the solution) (10pmol) make reaction mixture with 11.4ul water.The phage T4 polynucleotide kinase that adds 8 units (about 1ul) in this reaction mixture, 37 ℃ are incubated 45 minutes.68 ℃ of reacting by heating 10 minutes so that phage T4 polynucleotide kinase inactivation.

Then, determine ³²P or ³³P is transferred to the efficient and the specific activity thereof of oligonucleotide.If the specific activity of probe is acceptable, be purified.If specific activity is too low, add the enzyme of 8 units again and be incubated 30 minutes again at 37 ℃, 68 ℃ of reacting by heating 10 minutes so that enzyme deactivation.

By as with ethanol sedimentation, with bromination cetyltrimethyl ammonium yl pyridines precipitation, by bio-gel P-60 chromatographically pure or at Sep-Pak C ₁₈But chromatogram or by the radiolabeled oligonucleotide of polyacrylamide gel electrophoresis purifying on the post.

Can use synthetic and DNA chain of synthetic oligonucleotide complementary, obtain the high specific acitivity probe from the Klenow fragment of E.coli dna polymerase i.The oligonucleotide templates hybridization that primer and of a weak point is required radiolabeled probe's complementary sequence.The Klenow fragment of using the E.coli dna polymerase i then by mode shown in the template insert [ ³²P] dNTP or [ ³³P] dNTP.After the reaction, by sex change, polyacrylamide gel electrophoresis separates template with product under the sex change condition subsequently.Make in this way and can produce the oligonucleotide probe that the per molecule oligonucleotide contains several radioactive atoms.

For making in this way, can in micro-centrifuge tube (microfuge), will obtain required than the essential and [a-that be enough to finish all template strand synthetic calculated amount that lives ³²P] dNTP or [a- ³³P] the dNTP mixing.The primer and the template DNA that in test tube, add appropriate amount then, the molar weight that primer is more excessive 3 to 10 times than template.

Add 10 x Klenow damping fluids of 0.1 volume then, and mix.Every 5ul reaction volume adds the E.Coli.DNA polysaccharase I Klenow fragment of 2-4 unit again, mixes to be incorporated in 4 ℃ of insulations 2-3 hour.If necessary, can become precipitable radioactive segment, monitoring reaction course by 10% trichoroacetic acid(TCA) (TCA) by shifting out a small amount of (0.1ul) sample aliquot and measuring.

React the gel loading buffer liquid dilution of an available equal-volume,, entire sample is loaded on the denaturing polyacrylamide gel in 80 ℃ of heating 3 minutes.After the electrophoresis, gel is by radioautograph, makes probe be positioned and shifts out from gel.Also can use the method for various fluorescent probe marks, describe the synthetic of fluorescent dye primer as (1988) such as Brumbaugh.The deoxyuridine analogue that has synthesized the primary amine " connecting arm " that on C-5, is connected with 12 atoms.The 2-deoxyuridine of deriving that synthesizes of analogue obtains 5 (methacryloyl)-2-deoxyuridines by the organo-metallic intermediate product.Produce two pairs of methoxy trityls of corresponding 5-adducts with the dimethoxy trityl chloride reaction.With methyl esters hydrolysis, activation and with suitable single acetyl alkyl diamine reaction.After the purifying, the connecting arm nucleosides of gained is converted into the nucleoside analog that is suitable for chemical synthetic oligonucleotide.

Then, use the phosphoridite chemical preparation of modifying to comprise the oligonucleotide of the base of one or two connecting arm.The dimethyl sulphoxide solution that adds 20ul 300mM FITC in the 50nmol connecting arm oligonucleotide solution in 25ul 500mM sodium bicarbonate (pH9.4).Stirred the mixture under the room temperature 6 hours.Go up separate oligonucleotides from free F ITC, the wash-out form combines with first ultraviolet absorption peak part for using 1 * 30cmSephadex G-25 post of 20mM amine acetate (pH6).

Usually, carry out initial fluorescent mark at oligonucleotide 5 '-end and comprised for two steps.At first, in automatic nucleic acid is synthetic, the aminoalkyl group phosphoramidite derivative of N-protected is added in oligonucleotide 5 '-end.After removing all blocking groups, suitable fluorescence dye NSH ester and 5 '-amino coupled are spent the night, then, use the oligonucleotide of anti-phase chromatogram HPLC or PAGE purifying mark from excess dye.

People such as Schubert (1990) have described the synthetic of phosphoramidite, and it can produce with fluorescein-labeled oligonucleotide in automated DNA is synthetic.

People such as Murakami have also described the preparation of fluorescein-labeled oligonucleotide.

People such as Cate (1991) have described the use of oligonucleotide probe, probe be directly connected in conjunction with on the alkaline phosphatase of a kind of direct chemical luminous substrate (AMPPD) to allow probe in detecting.

Can comprise that GENSET directly buys label probe, needn't synthesize from various commercial source.

Other marks comprise can be as the part, chemoluminescence agent, enzyme that are labeled the antibodies specific binding substance, can be used as and be labeled the ligand specificity in conjunction with right antibody or the like.Many marks have been used in the immunoassay that can use fast.But other marks also comprise antigen, have active group of specific reaction and electrochemistry detection moiety.

Usually, for example people such as Xu has described in J.Chromatography 764:95-102 (1997) with charged species mark (" EML ") labeling nucleic acid.Charged species (electrophore) is to use the compound of the highly sensitive detection of electron capture mass spectrum (EC-MS).Can use the chemical process (the Nucleotide synthetic chemistry has been instructed many methods that are used as blocking group on the Nucleotide that molecule is connected as the well-known) of reversibly modified Nucleotide well known in the art that EML is connected on the probe.Use the various electron capture mass spectrographs of knowing (as the instrument of Finnigan Corporation sale) to detect EML.The technology that can be used for detecting EML in addition comprises as fast atom bombardment MS (referring to as Koster etc., Biomedical Environ.Mass Spec.14:111-116 (1987)); The plasma desorption mass spectrum; Electrospray/ion sprays (referring to Fenn etc., J.Phys.Chem.88:4451-59 (1984), PCT apply for WO90/14148, Smith etc., Anal.Chem.62:882-89 (1990)); With the auxiliary laser desorption/ionization (Hillenkamp etc. of matrix, " Matrix AssistedUV-Laser Desorption/Ionization:A new Approach to Mass Spectrometry of LargeBiomolecules. " (auxiliary Ultra-Violet Laser parsing/ionization of matrix: biomacromolecule mass spectrum novel method) Biological Mass Spectrometry (biological mass spectrometry) (Burlingame and McCloskey compile), ElsevierScience Publishers, (Elsevier Science Press) Amsterdam, the 49-60 page or leaf, 1990); Huth-Fehre etc. " Matrix Assisted Laser Desorption Mass Spectrometry ofOligodeoxythymidylic Acids " (the matrix auxiliary laser of oligodeoxythymidylic acid is resolved mass spectrum), RapidCommunications in Mass Spectrometry, 6:209-13 (1992)).

In preferred embodiments, EML is connected on the probe by light activated covalent linkage.After the light source of launching required optical wavelength by laser or other and target nucleic acid hybridization, discharge EML from probe.Then EML is fed in GC-MS (gas chromatography-mass spectrum) or other instruments that is fit to and and is differentiated by its quality.

Embodiment 5

The preparation of sequence testing chip and array

A basic example is to use 6 base sequences that are attached to 50 microns surfaces to obtain the chip of size a 3 * 3mm, and it can combinedly obtain the array of a 20 * 20cm. and another example is to use and is attached to the 9 base sequence chips that 10 * 10 microns lip-deep 9 base sequence oligonucleotide produce size a 5 * 5mm.Can use 4000 this unit chips to produce the array of a 30 * 30cm.4,000 to 16,000 few chips are aligned to a quadrate array in each array.According to described also can be with the set of a plate or pipe with the part of this array assembling as sequencing kit.

Between the array with physical form or by hydrophobic surface separated.Use a kind of this technology of Iso-Grid Microbiology System that may mode be to use as Toronto QA Laboratory Production of hydrophobic separation.

Hydrophobic grid membrane filter used about ten years in analysis food microbiology field, and they present unique extension digital scope and the cluster that counts automatically.A kind of grid of buying is the ISO-GRID in the limited laboratory of QA (Toronto) ^TM, (60 * 60cm) polysulfone polymers (GelmanTuffryn HT-450, hole size 0.45u) constitute, and are printed on the hydrophobic black grid of a black that is made of 1600 (40 * 40) square pond above by a square for it.HGMF is originally by vacuum filtration and is inoculated by bacterial suspension, and is incubated on discriminating of selecting or selective medium.

Because microbial growth is limited in the grating tank of known location on the film and size, the effect of HGMF more resembles a kind of MPN device, and non-traditional plate or membrane filter.People such as Peterkin (1987) have reported that these HGMF can be used to duplicate and store gene library when using with a HGMF reproducer.A kind of this device duplicates growth from each pond, 1600 ponds of ISO-GRID, and can prepare many copies (Peterkin etc., 1987) of main HGMF.

Sharpe etc. (1989) have also used the breadboard ISO-GRID HGMF of QA, automatic HGMF counter (MI-100 interpreter) and RP-100 reproducer.They have reported a kind of method that keeps and screen many microorganisms cultures.

Peterkin and colleague after a kind of method (Peterkin etc., 1989) of using hydrophobic grid membrane filter screening DNA probe has been described.These authors have reported the directly method of the effective colony hybridization on HGMF.Because the epoxy sulfone polymer binding ability that DNA and HGMF print on it is poor, the result who obtains is bad during beginning.But, can improve combining of DNA and film surface with the HGMF that handles the insulation of duplicating with a kind of polycation of polymine before DNA contacts according to (1989) such as Peterkin.Although cell DNA absorption is used in this early stage work, different with the object of the invention, described method can be used among the Format3 SBH.

For the useful sequence of quick identification, Peterkin etc. (1989) use from various clones' radio-labeling plasmid DNA and test its specificity to the DNA on prepared HGMF.By this way, by xeroxing the DNA of the colony hybridization rapid screening of 100 microorganisms on the replica from recombinant plasmid with HGMF, wherein HGMF replica can be replicated easily.

Use little (2-3mm) chip to operate, parallelly carry out thousands of reactions.Solution of the present invention is used for preserving the probe of these chips and respective array.In one embodiment, synthesized the chip that contains 250,000 9 base sequences on a silicon chip, to be 8 * 8mM plate (15uM/ oligonucleotide, Pease etc., 1994) arrange with 8 * 12 forms (96 chip) form, between the 1mM groove is arranged.Add probe, a probe chip piece by multiple tracks transfer pipet or plug (pin) array.For to all 4000 6 base sequence scorings, must use 42 chip arrays, or use different chips or a core assembly sheet to reuse repeatedly.

In above-mentioned situation, use the early stage nomenclature of this application, F=9; P=6; F+P=15.Chip can have the probe that general formula is BxNn, and wherein x is the quantity of special base B, the quantity of the special base of n right and wrong, like this, x=4 to 10, n=1 to 4.For obtaining more effective hybridization and avoid the potential impact of arbitrary upholder oligonucleotide, special choline base is surrounded by non-special choline base, represents with general formula (N) nBx (N) m.

In another chip embodiment, will support the matrix of oligonucleotide probe array to be divided into several sections, like this in the array each probe by a kind of can be that the physical barriers and the adjacent probe of hydrophobic material separated.In a preferred embodiment, to 30 μ m, the distance at each physical barriers center to adjacent physical barriers center is at least 325 μ m to the width of physical barriers from 300 μ m.

In a preferred embodiment, the ink gun that uses connection to fit on a kind of suitable automatic handling system is deposited on hydrophobic material on the matrix, to form the barrier of required width.For example, a kind of droplet dosage head that is used to provide required hydrophobic material (as a kind of oil based material that after solvent evaporates, forms barrier) suspension or solution, can join and fit over a kind of nothing mouthful in stand (anorad gantry) system and be suitable for suitable holding and dispersion system, the grid of hydrophobic material can be placed on the required matrix like this and on matrix, form porous.After the hydrophobic material grid forms, use be similar to form grid used but the automatic handling system that is suitable for providing probe solution or suspension with different probe points (or be positioned in each hole probe mixture) in each hole.In one embodiment, use same automatic handling system that hydrophobic grid and probe are provided.In this scheme, hydrophobic grid is being provided and is being ready for supply probe backlash flush away dispersion system.

Embodiment 6

The preparation of the oligonucleotide that is connected with upholder

For example use automatic oligonucleotide synthesizer to operate direct synthetic oligonucleotide routinely by chemical process, can prepare oligonucleotide fast is little nucleic acid fragment.

Usually, oligonucleotide can be connected on the upholder by suitable reactive group.This group is well known in the art, for example comprises amino (NH ₂), hydroxyl (OH) or carboxyl (COOH).Use suitable upholder such as glass, polystyrene or teflon, can prepare the oligonucleotide that is connected with upholder by either party's method well known in the art.A kind of strategy is that accurate drop is by standard synthesizer synthetic oligonucleotide.Can reach immobilization by several different methods, comprise as using passive absorption (Inouye ﹠amp; Hondo, 1990), UV light (Nagata etc., 1985; Dahlen etc., 1987; Morriey ﹠amp; Collins, 1989) or covalent attachment (Keller etc., 1988 by the adorned DNA of base; 1989) or between probe and upholder, form amido linkage (Wall etc., 1995; Chebab etc., 1992; Zhang etc., 1991); All these all draw at this and are reference.

Another kind of spendable method is to use the strong interaction of vitamin H-chain enzyme avidin as connecting arm.For example, Broude etc. (1994) have described the use of biotinylation probe, but these are the duplex probes that are immobilized onto on the magnetic bead of chain enzyme avidin coating.Can be from Dynal, Oslo buys chain enzyme avidin coating pearl.Certainly, same chemical connection method can be applicable to any surface of chain enzyme avidin coating.Can be from various sources such as Operon Technologies (Alameda CA) buys biotinylated probe.

(Naperville IL) also sells spendable suitable material to Nunc Laboratories.NuncLaboratories has developed a kind of method, and DNA can be covalently attached to the micropore surface that is called Covalink NH.Covalink NH is a kind of polystyrene surface, is grafted with as the secondary amine of further covalent coupling end of the bridge (NH-).Can buy Covalink Modules from Nunc Laboratories.Dna molecular can only be connected in Covalink at 5 '-end by the phosphoramidic acid ester bond, can make the dna molecular immobilization (Rasmussen etc., 1991) more than 1pmol.

Use Covalink NH bar in existing describe (Rasmussen etc., 1991) of 5 '-end covalent bonding dna molecular.In the method, utilized a phosphoramidic acid ester bond (Chu etc., 1983).When preferably only using single covalent linkage immobilization is favourable.The phosphoramidic acid ester bond is connected DNA with Covalink NH secondary amine, secondary amine is positioned at the spacerarm end, by the long spacerarm covalence graft of 2nm at polystyrene surface.For by a phosphoramidic acid ester bond oligonucleotide being connected with Covalink NH, the oligonucleotide end must have 5 '-end phosphate group.Even also vitamin H may be covalently bonded to Covalink, use chain enzyme avidin bonding probes then.

More particularly, method of attachment comprised with DNA (7.5ng/ul) soluble in water and in 95 ℃ of sex change 10 minutes, cooled on ice 10 minutes.Then with the 1-Methylimidazole (1-MeIm of ice-cold 0.1MpH7.0 ₇) join the 1-MeIm that a final concentration is 10mM ₇In.Again A ssDNA solution is dispersed in the Covalink NH bar (75ul/ hole) that is arranged on ice.

Prepare the fresh 10mM 1-MeIm that is dissolved in ₇0.2M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), every hole adds 25ul.Bar was in 50 ℃ of insulations 5 hours.The insulation back is used as Nunc-Immuno Wash rinsing bar; At first every hole is washed 3 times, then it is soaked 5 minutes with washing soln, and (rinsing solution is to be heated to 50 ℃ 0.4N NaOH, 0.25%SDS) to wash 3 times at last again.

Suitable method is described in PCT application WO 90/03382 (Southern ﹠amp to be used for another kind of the present invention; Maskos), be hereby incorporated by reference.This be bonded to the preparation method of oligonucleotide on the upholder comprise with the covalency phosphodiester bond with nucleosides 3 '-reagent by phosphate group and upholder with aliphatic hydroxide radical be connected.Then this with nucleosides that upholder is connected on synthetic oligonucleotide, under standard conditions, remove protecting group, and can be from oligonucleotide under the cracking on the upholder from the synthetic oligonucleotide chain.Suitable reagent comprises nucleoside phosphoramidites and phosphonic acids hydrogenation nucleosides.

Can adopt the chip top legal system of preparation dna probe to be equipped with the dna probe array.For example, the light deprotection of addressable laser activation can be used to directly at the glass surface chemical synthetic oligonucleotide, as (1991) as described in the people such as Fodor, is hereby incorporated by reference.As also can be on the nylon upholder as described in the people (1991) such as Van Ness the immobilization probe; Or use Duncan ﹠amp; The method of Cavalier (1988) is connected probe on the teflon; All these draw at this and are reference.

As described in people (1991) such as Van Ness,, require by alkylation with the nylon surface active, with 5 '-amine of cyanuryl chloride selection activation oligonucleotide for probe is connected with the nylon upholder.

The concrete grammar of the oligonucleotide that a kind of preparation is connected with upholder is to utilize described luminous synthesizing of people (1994, be hereby incorporated by reference) such as Pease.These authors make with the light lithographic techniques and prepare immobilized oligonucleotide probe array (DNA chip).These methods are utilized the N-ethanoyl-deoxynucleoside phosphoramidite of 5 ' of signal-protection, surperficial connecting arm chemistry and multiple combination synthesis method, and wherein, light is used to the directly synthetic oligonucleotide probe that is the microminiaturized array of highly dense.The matrix that can prepare 256 oligonucleotide probes of a space boundary by this way, this matrix as described herein are used in preferred Format 3 order-checkings.

Certainly, can buy a DNA chip from the market easily, as above-mentioned photoactivation chip.At this moment can with Santa Clara, the Affymetrix of CA 95051 and Beckman contact.

In a preferred embodiment, probe of the present invention comprises four bases promptly can be found in a message part (with target nucleic acid hybridization and provide the part of sequence information), the reaction active groups that links to each other with matrix (solid support) and randomization position on these positions any one.Preferred probe has sequence 5 '-(T) ₆-(N) ₃-(B) ₅, T=thymus pyrimidine (combining) wherein, N=A, C, G or T (randomization position), 5 information positions (message part) of B=probe with solid support.In a preferred embodiment, probe can be connected with upholder, and spacer is positioned at probe end or at probe and (N) ₃5 '.Spacer can be made up of the atom that can form two covalent linkage such as carbon, silicon, oxygen, sulphur, phosphorus etc. at least, or is made up of the molecule that can form at least two covalent linkage such as sugar-phosphate group, amino acid, peptide, nucleosides, Nucleotide, sugar, carbohydrate, aromatic nucleus, hydrocarbon ring, straight chain and branched paraffin etc.

Embodiment 7

The preparation of nucleic acid fragment

Can obtain nucleic acid to be checked order from arbitrary suitable source,, comprise and do not carry out any amplification step mRNA as chromosome band, clay or the YAC inset and the RNA of cDNA, genomic dna, chromosomal DNA, micro-dissection.For example, people (1989) such as Sambrook has described three schemes (p.9.14-9.23) of separation of high molecular weight DNA from mammalian cell.

Can prepare nucleic acid fragment as the clone in M13, plasmid or the λ carrier and/or use PCR or other amplification methods directly prepare from genomic dna or cDNA.Can in porous plate, prepare or dispersed sample.Preparation 100-1000ng DNA sample final volume is 2-500ml.Can be directly the target nucleic acid of PCR preparation be placed on the matrix that Format I SBH uses, need not purifying.In a single day target nucleic acid is fixed on the matrix, but rinsing matrix or direct and probe annealing.

Then, can use any method well known in the art with nucleic acid fragmentization, comprise the Restriction Enzyme described in (1989) 9.24-9.28 such as use Sambrook, ultrasonic shearing and NaOH handle.

It also is suitable that low pressure is sheared, as described in the Schriefer etc. (1990, this draw be with reference to).In this method, to middle pressure, the DNA sample is passed through a little French press in various low pressure.A kind of pull rod device may command is applied to low pressure on the cell to middle pressure.The result of these researchs shows that the low pressure shearing can be used for substituting the sound method and enzyme process obtains dna fragmentation.

A concrete method for preparing fragmentation DNA is to use two bases of (1992) descriptions such as Fitzgerald to discern endonuclease CviJI.These authors have described the method that a kind of quick fragmentation and fractional separation DNA are specific size, and these DNA are designed to be suitable shotgun cloning and order-checking.The inventor predict this method also can be used in particular for producing at random but less relatively dna fragmentation be used in the sequencing technologies of the present invention.

Restriction endonuclease CviJI usually between the G of recognition sequence PuGCPy and C cutting obtain flush end.Change this kind of enzyme (CviJI ^*) the specific atypia reaction conditions quasi-random that obtains a dna fragmentation form small molecules pUC19 (2688 base pair) distributes.People such as Fitzgerald (1992) use CMJI ^*Digestion pUC19 carries out size fractionation by the PhastGel filtration method and separates and do not carry out end reparation and be connected directly to lac Z and bear the M13 cloning vector, qualitative assessment the randomness of this fragmentation method.76 clones' sequential analysis shows CviJI ^*Restriction pyGCPy and PuGCPu, and PuGCPy site, the speed of gathering new sequence data is consistent with random fragmentation.

Report that as document compare with the sepharose fractional separation with ultrasonic method, the advantage of this method comprises: the more a spot of DNA of needs (0.2-0.5ug replaces 2-5ug); Comprise less step (need not pre-connection, terminal reparation, chemical extraction or agarose gel electrophoresis and wash-out).For Format 3 order-checking prepare dnas the time, point out that also these advantages are available.

In a preferred embodiment, prepare nucleic acid samples " fragment " so that they are not interconnected.Can obtain this phase library by the fragmentation nucleic acid of handling with Phosphoric acid esterase (as the calf intestinal Phosphoric acid esterase) through enzymic digestion or physics shearing acquisition.Perhaps, in reacting, the mulberry lattice dideoxy sequencing with sample nucleic acid use 5 '-end not have the random primer of phosphoric acid (as N ₅-N ₉, wherein N=A, G, T or C) but the non-junction fragment of sample nucleic acid can be obtained.This can produce and target nucleic acid sequence complementary dna fragmentation, is that end can not be connected with other fragments with two deoxidation residues.

As for the method that obtains or prepare nucleic acid fragment, the single-chain fragment that makes the DNA sex change obtain can be used for hybridizing is important.By dna solution was reached this purpose in 2-5 minute in 80-90 ℃ of insulation.Then solution is cooled to 2 ℃ rapidly so that in them and the renaturation that prevents dna fragmentation before chip contacts.

Embodiment 8

The preparation of DNA array

By the DNA sample spot is dropped in as preparing array on the upholder of nylon membrane.Using metallic plug array (array in the hole of its position corresponding on the droplet plate) can finish drop forwards on the nylon membrane by the dna solution with 20nl and duplicates.By offset printing, obtain the density height of the density of drop than the hole.Type according to used mark can be at 1mm ²1 to 25 point of interior adjusting.For avoiding a little dropping in the row and column of some preselected numbers, can form the inferior array (secondary arrangement) of separation.Sample in inferior array can be from the identical genomic fragment DNA (or homologous genes) of Different Individual or different eclipsed genomic clones.Each inferior array can be represented the spot that duplicates of same sample.In one embodiment, can be from the increase gene fragment of a selection of 64 patients.For each patient, the amplification gene fragment can be placed on one 96 orifice plate (96 all holes contain same sample).Each patient among 64 patients is prepared a sample.By using one 96 plug device all samples point can be dropped on the film of 8 * 12cm.Inferior array can comprise 64 samples, and each sample is from a patient.When 96 inferior arrays were identical situation, the some zone can be 1mm ², the space between the inferior array can be 1mm.

Another kind of approach be to use can by the physical separation thing as the plastic grill that on film, forms or hydrophobic separated film or plate (available from NUNC, Naperville, Illinois), grid is similar in kind with the film that is provided in the porous plate bottom.Preferred immobilization physics spacer not be used in exposure image on flat phosphorus storage screen or the x-ray film.

Embodiment 9

Hybridization and methods of marking

Label probe can mix with hybridization buffer, preferably moves liquid to inferior array with the multiple tracks transfer pipet.For the mixing (if not at hydrophobic of the marking on the film or physical barriers) that prevents probe between inferior array can closely be pressed in corresponding plastics, metal or ceramic grid on the film.And, the damping fluid volume can be reduced to every mm ²About 1ml or still less.Can use foregoing concentration and probe concentration and hybridization conditions, can be poured on the array of inferior array with the rapid dilution that allows probe except the rinsing damping fluid fast and prevent significant cross hybridization thus.Based on same reason, can use the probe of Cmin, hybridization time may extend to maximum real standard.When DNA detection and order-checking, known " normally " sequence allows to use successive accumulative facies mutual effect phenomenon with enhancing signal.Remove label probe, also can add other unmarked probe in hybridization, itself and label probe end are hybridized end.The hybridization amount can strengthen several times.By ligation probe is linked to each other.This approach is important for solving DNA district formation " compression ".

Under radiolabeled probe's situation, preferably use phosphorus storage (phosphorstorage)) technology can obtain the picture of filter membrane.By CCD camera, confocal microscopy or the additive method fluorescent mark of can marking.Calculate and the data of integration for correct, proofread and correct original signal according to the target amount on each point from different hybrid experiments.By each probe signals is proofreaied and correct the difference of every some target DNA amount divided by the average signal of all probes of scoring on each point.Can mark to correction signal, be generally 1-100 with data ratio from different experiments.And, in each inferior array, can use several contrast DNA, to determine not contain average background signal in the sample that mates target fully at these.For sample, can use the homozygote contrast, with the heterozygote in the identification sample available from diploid (polyploid) scoring.

Embodiment 10

With oligonucleotide hybridization

Oligonucleotide can be from Genosys Inc., and Houston, Texas buy or be synthetic on Applied Biosystems381A dna synthesizer.Employed most of probe is not through HPLC or gel electrophoresis purifying.For example, but designing probe has one to be in single complete complementary target in the Interferon, rabbit, to contain the M13 clone of 921bp EcoRI-Bgl II people B1-Interferon, rabbit fragment (Ohno and Tangiychi, Proc.Natl.Acad.Sci.74:4370-4374 (1981)) and at least at a target of a terminal bases mispairing of M13 carrier itself.

According to (Maniatis Molecular Cloning:A Laboratory Manual, Cold Spring HarborLaboratory Cold Spring Harbor, Mew York (1982)) described method 10ml contain the T4 polynucleotide kinase (5 units, Amersham), γ ³²(4pM carries out the end mark of oligonucleotide in 10ng) for p-ATP (3.3pM, 10mCi Amersham 3000Ci/mM) and oligonucleotide.The specific activity of probe is 2.5-5 * 109cpm/nM.

Drop single stranded DNA (at the 0.5NaOH of 2-4ml, among the 1.5M NaCl) on same solution wetted Gene Screen film, filter membrane is at 0.05M Na ₂HPO ₄Neutralize among the pH6.5, oven dry is 60 minutes in 80 ℃ baking box, uv irradiating 1 minute.Then, filter membrane is incubated at hybridization solution (0.5MNa ₂HPO ₄PH7.2,7% sodium lauroyl sareosine) room temperature is following 5 minutes in, is placed on the plastic culture dish surface.To contain 4nM concentration ³²A hybridization solution (10ml, the 0.5M Na of the end-labelled oligomer probe of P ₂HPO ₄PH7.2,7% sodium lauroyl sareosine) be added on every filter membrane 1-6 the point, (1 * 1cm) covers, and is incubated 3 hours under the indicated temperature in damp camera with a slice square polyethylene.Stop hybridization, filter membrane is placed in the 6X SSC rinsing solution 0 ℃ removed not hybridization probe down in 3 * 5 minutes.Dry or the further rinsing under instruction time and temperature with filter membrane, radioautograph.Be the measurement value of distinguishing, point (can use a kind of phosphorus imager (Molecular Dynamics.Sunnyvale, California)) to be placed in the liquid scintillation mixture and counting under the exsiccant filter membrane was sheared afterwards from radioautograph.The not correct ratio cpm that IF and M13 are ordered provides with the D value.

Conditions permit described here and very short oligonucleotide hybridization, but guarantee and target nucleic acid is complementary and bonded coupling and do not mate distinguishing between the oligonucleotide.The factor that influences the short sequence hybridization of effective detection specificity is based in complementary target fully with there is the other degree (D) of debating between the non-complete complementary target of mispairing to be determined in hybridization.In tentative test, finish the probe of 6 to 8 Nucleotide of 28 length and 2 M13 clone or with the spot marking hybridization that is combined in the model oligonucleotide on the filter membrane.Provided the principle of guiding experiment program below.

Oligonucleotide with combine the filter hybridization of target nucleic acid, only several Nucleotide longer than probe under the excessive condition of probe are pseudo first order reactions for target level.This reaction is represented as: S _t/ S _o=e ^{-kh[OP] t}

S wherein _TAnd S _OBe to be respectively t and t the time ₀The time target sequence concentration.(OP) be concentration and probe concentration, t is a temperature.The rate constants k of hybridization _hSmall increase (Porsclike and Eigen, J.Mol.Biol.62:361 (1971) are only arranged in 0 ℃ to 30 ℃ scope; Craig etc., J.Mol.Biol.62:383 (1971)).Hybridization is unwind for hybridization concentration (being replaced by quality owing to the filter membrane bonding state here) is first order reaction, is expressed from the next:

H _t/H _o＝e ^-kmt

In the formula, H _tAnd H _oBe to be respectively t and t the time ₀The time hybridization concentration; k _mBe to depend on rate constant (Ikuta etc., the Nucl.Acids Res.15:797 (1987) that the hybridization of temperature and salt concn is unwind; Porsclike and Eigen, J.Mol.Biol.62:361 (1971); Craig etc., J.Mol.Biol.62:303 (1971)).In hybridization, chain association process, reversed reaction, unwind or the chain dissociation reaction carries out equally.Therefore the hybridization amount that in time forms is the result of positive reaction and reversed reaction.By increasing concentration and probe concentration and/or reducing temperature and balance can be shifted to hybridization formation.But in the rinse cycle of a large amount of damping fluids, because there is not probe, it is main separating chain reaction, and reversed reaction hybridization is accessory.Short oligonucleotide hybridization (SOH) condition that this analysis revealed is suitable changes for concentration and probe concentration or temperature.

D or debate not to be worth and represent by following four equations:

D＝H _p(t _w)/H _i(t _w)

H _p(t _w) and H _i(t _w) be rinsing time t _wBe respectively the residue hybridization amount of the complete and non-complete complementary duplex of same amount afterwards.For a given temperature, debate the rinsing time change of other D with 10 double-length degree, work as H _iWhen being equation 5 ,=B reaches maximum value.

The minimum hybridization signal that on behalf of system, background B can survey.Because H _iAny further reducing be immesurable, D increases with continuous rinsing.Rinsing is through t _wOnly reduce H with respect to B _p, and be regarded as reducing of D.The optimization rinsing time t that obtains by equation 3 and equation 5 for imperfect hybridization _wFor:

t _w＝-ln(B/H _i(t _o))/K _m，i

Because H _pBy the same t of rinsing _w, in conjunction with these two equations, can obtain the optimization discriminant function:

D＝e ^{ln(B/Hi(t0))km，p/km，i}XH _p(t ₀)/B

As the function of T, because the change of the selection D of optimization rinsing temperature is important.By with Arhenius equation K-=Ae ^-Ea/RT

The equation of substitution front gets equation to the end:

D＝H _p((t ₀)/BX(B/H _i(t ₀)) ^{(Ap/Ai)e(Ea，i-Ea，p)/RT}；

Wherein B is less than H _i(t ₀).

Because Za Jiao activation energy E fully _{A, p}Activation energy E with imperfect hybridization _{A, i}Can equate, or E _{A, i}Less than E _{A, p}, D is respectively and is independent of temperature or reduces with temperature.This result means that it is irrational seeking strict temperature condition for the value of distinguishing good in SOH.By rinsing at a lower temperature, can obtain to be equal to or the better value of distinguishing, but the rinsing time reduce with temperature and be index and increase.If H _i(t _o) with respect to H _p(t ₀) proportional increase, the value of distinguishing significantly reduces with T.

D under the low temperature relies on H _p(t ₀The degree of)/B ratio is higher than H _p(t ₀)/H _i(t _o) ratio.This result shows the H that is preferably in acquisition q.s in the hybridization _p, and do not consider available value of distinguishing in this step.Can reach the better value of distinguishing by rinsing then, to be used for unwind time of demonstrating effect of differential just many more because hybridization amount fully is high more.Similarly, even use relatively large target nucleic acid can obtain the necessary value of distinguishing K _{M, p}And K _{M, i}Between very little difference is only arranged.

Extrapolate one than the more complicated situation that this naive model covered, the result is, has in the situation of probe hybridization of many terminal mispairing rinsing at a lower temperature even even more important with giving target nucleic acid inside.

Use described theoretic principle as experiment guide, obtained reliable hybridization with the probe of 6 to 8 Nucleotide of length.All experiments are all carried out with a plastic sheet that floats, and this plastic sheet provides a hybridization solution film that is placed on the strainer.This program allows the maximum of probe amount to reduce, and the mark of these minimizings is lost in the hybridization of the spot marking.In the phosphoric acid hybridization buffer, replace basic lauroyl sodium sulfate to allow temperature of reaction to reduce to 12 ℃ from room temperature with the high density sodium lauroyl sareosine.Similarly, the sodium lauroyl sareosine damping fluid of 4-6X SSC 10% allows to hybridize under 2 ℃ low temperature.Stain remover in these damping fluids is the label probe tolerable background that is used to obtain to have up to 40nM concentration.In 8 base sequence prototypes with 50%G+C content is that sequence is to determine the thermodynamics essential characteristic of short oligonucleotide hybridization on the TGCTCATG probe.Theoretical prediction is that this probe is in more unsettled 8 base sequences.It transforms the probe similar (Bresslauer etc., Proc.Natl.Acad.Sci.U.S.A.83:3746 (1986)) that enthalpy and more stable 7 base sequences even length are 6 oligonucleotide.At unwind 50% temperature parameter T of 1 minute unit time hybridization _dIt is 18 ℃.The result shows that hybridization is compared to a 11bp duplex T for 8bp _dLow 15 ℃ (Wallace etc., Nucleic Acids Res.6:3543 (1979)).

Except that the oligonucleotide experiment that uses a model, select the actual verification system of M13 carrier as short oligonucleotide hybridization.Main purpose is to use and is similar to terminal mispairing value of distinguishing that target used in the inventive method various uses shows usefulness.Itself contain the oligonucleotide probe that this mode of terminal base mismatch selects to be used for the M13 model with the M13 carrier.A kind of M13 recombinant vectors IF of the 921bp of containing human interferon gene inset carries the single target that mates fully.Therefore, when itself comparing with the M13 carrier, TF has the mispairing target of equal number or comparatively high amts.

Use the lower temperature conditions and the spot marking, containing fully and the banded spot of mispairing target and only contain the difference that can obtain enough hybridization signals between the spot of mispairing target.This for large nucleic acids be correct to 6 base sequence oligonucleotide of IF-M13 hybridization, also be correct for 7 base sequences and 8 base sequence oligonucleotide.

Hybridization signal depend on the used filter membrane of probe reaction on obtainable target amount.Necessary contrast is used for showing that the difference of signal density is not the different reactions of two spot amplifying nucleic acid amounts.Probe hybridization with have the target of equal amts and kind in IF and M13 shows the DNA that has equivalent in spot.Because hybridization forms efficient and increases with hybridization length, use the oligonucleotide target optimum detection that is incorporated in a large number on the filter membrane to have the signal of the duplex of 6 oligonucleotide.Because its lower molecular weight, when comparing with the nucleic acid molecule that is used as target, a large amount of oligonucleotide target molecules can be incorporated into the surf zone of being given.

For measuring the susceptibility that purify DNA not detects, with the phage supernatant liquor point of difference amount on filter membrane and with ³²The 8 base sequences hybridization of P mark.The 5000 ten thousand a spot of so not purifying phages that contain no more than 0.5ng DNA have provided detectable signal, and the susceptibility that shows the short oligonucleotide hybridizing method is enough.Reaction times is short, has increased practicality.

Described as top theory part, the hybridization equilibrated produces and depends on concentration and probe concentration and/or temperature of reaction.For example, during for 40nM low 3 times for target 4nM 8 base sequences of the same amount signal level 13 ℃ the time, reduce by 4.5 times by improving hybridization temperature to 25 ℃ signal level than concentration and probe concentration.

Proved that the low temperature rinsing obtains the practicality of the maximization value of distinguishing.For making phenomenon visual, utilize the hybridization with the carrier specific probe, the DNA that uses in the M13 spot is than Duoing 50 times in the IF spot.By this way, with actual probes hybridization after signal strong than mispairing in match condition.H _p/ H _iRatio is 1: 4.Picked up signal density conversion after 7 ℃ of time-delay rinsings, not a large amount of losses of coupling fully, the gained ratio is 2: 1.On the contrary, can not obtain any distinguishing, because 2 minutes rinsing matched signal have just been reduced to background level at 25 ℃; Simultaneously, the mismatch hybridization signal is still detectable.The loss of the value of distinguishing is not so big during than 7 ℃ 13 ℃, but clear visual.If consider in the time of 7 ℃ 90 minutes and 13 ℃ the time 15 minutes when the mismatch hybridization signal near background level interval scale best rinsing time under the condition separately, Duo several times when the amount 7 ℃ the time is than 13 ℃ so.Be further proof this point, under two kinds of temperature, the time course that changes the value of distinguishing with the rinsing of identical initial hybridization amount shows that maximization D was higher when temperature was low.These results have proved conclusively with temperature and have reached the ratio that begins two types of hybridization amounts in rinse step, the change trend of D.

For showing the versatility of oligonucleotide hybridization condition, we have observed, and 47 base sequences, 10 8 base sequences and other length are the hybridization of 14 probes of 12 oligonucleotide in simple M13 system.These comprise represents two 9 extreme base sequence GTTTTTTAA of GC content and 8 base sequence GGCAGGCG.Though the stability (Bresslaue etc., Proc.Natl.Acad.Sci.U.S.A.83:3746 (1986)) of imagination GC content and the short hybridization of sequence influence, low temperature oligonucleotide condition is applicable to all probes to be measured in obtaining enough values of distinguishing.Because the best distinguish value that obtains with the probe of 13 oligonucleotide of length is 20, be to allow easily because sequence variation causes several times reduction.

The advantage of M13 system is the effect that can be presented at target DNA complicacy on the level of distinguishing.Wherein do not have the mispairing target or 5 mispairing targets are arranged and only a pair of GC difference for two 8 base sequences, the observed value of distinguishing is respectively 18.3 and 1.7.

Be the practicality of proof present method, on a collection of 51 plasmid DNA spots, tested 3 probes that length is 8 Nucleotide by the preparation of Bluescript vector library.To have a probe be special to Bluescript vector but it is not present among the M13, and other two probes to have be the target of known array inset.This system allows to use feminine gender or positive control dna and each probe hybridization.This probe sequence (CTCCCTTT) also has a complementary target in the Interferon, rabbit inset.Because the M13 spot is negative when the Interferon, rabbit inset is positive in M13 or Bluescript, so hybridization is sequence-specific.Similarly, if suitable target is present among the clone, only detect in 51 insets 1 or in inset to be measured, do not have the probe of target sequence to produce with the contrast of conclusive evidence hybridization so.

For the curve of the oligonucleotide hybridization of the thermostability curve ratio length 11-12 of the very short oligonucleotide of length 6-8 oligonucleotide hybridization low 15 ℃ (accompanying drawing 1 and Wallace etc., Nucleic AcidsRes.6:3543-3557 (1979)) at least.But the oligonucleotide probe with the 0.4-40nM actual concentrations carries out hybridization at low temperatures, allows to detect complementary sequence in a known or unknown nucleic acid target.For determining to use a whole set of 65,535 8 base sequence probes by a unknown nucleotide sequence fully.The capacity nucleic acid that is used for this purpose is present in suitable biological sample, as a few microlitre M13 cultures, from the plasmid of 10ml bacterial cultures or single bacterial colony or be less than the Standard PC R reaction of 1ml.The short oligonucleotide of 6-10 length of nucleotides provides the splendid value of distinguishing.Single terminal mispairing in the relative reduction of hybrid stability greater than long probe.The result of 8 base sequence TGCTCATG supports this conclusion.In experiment, have the terminal mispairing of G/T target, with the hybridization of this mispairing form target be that every other form oligonucleotide is the most stable.This value of distinguishing of gained is identical or big (Ikuta etc., Nucl.Acids res.15:797 (1987)) with being present in 19 inside G/T mispairing in the base pair duplex.Utilize these distinguishing characteristicss to use the hybridization conditions of short oligonucleotide hybridization, allow to determine very exactly the oligonucleotide target.Hybridize fully with easy detection and imperfect hybridization between difference opposite, the problem of using very short oligonucleotide to exist is the preparation of q.s hybridization.In fact, amount and/or the concentration and probe concentration by increasing DNA in the spot or reduce hybridization temperature and help distinguish H _pAnd H _iBut background improves in concentration and probe concentration high pass regular meeting.And the actual target nucleic acid amount of using is limited.Use the stain remover sarcosyl of higher concentration to solve this problem, can provide an effective background with the 4nM probe.Utilize the competitor of probe and filter membrane non-specific binding or change the hybridization support material and can obtain further improvement.Moreover, for E _aLess than the probe (as for many 7 base sequences and most of 6 base sequences) of 45 kilocalories/mol, the oligonucleotide of modification is than the hybridization of the opposite part of its unmodified more stable (Asseline etc., Proc.Natl.Acad.Sci.81:3297 (1984)).Hybridization conditions at short oligonucleotide hybridization of the present invention uses low temperature can distinguish all list entries and duplex hybridization preferably.To reach cost that the hybridization conditions consistence pays only for increasing to 24 hours from several minutes at different sequences according to the sequence rinsing time.In addition, can reduce the rinsing time again by reducing salt concn.

Though coupling hybridization and mismatch hybridization have the splendid value of distinguishing, in short oligonucleotide hybridization, the mismatch hybridization signal exists with the mismatch hybridization of great majority owing to terminal mispairing.This has limited the inset size that available a certain length probe effectively detects.

Sequence complexity can be left in the basket to the influence of the value of distinguishing.Yet when with short oligonucleotide hybridization the nonrandom sequence of specificity being limited sequence information, the complicacy influence is more significant, can use the probe of suitable target length ratio to address this problem.On statistical basis, select length ratio, make unlikely occur having can eliminate or the wrong specific sequence that transforms many terminal mispairing of the value of distinguishing.The result is presented at the target nucleic acid inset and is shorter than 0.6,2.5 and should use the oligonucleotide of 6,7 and 8 Nucleotide of length during 10kb respectively.

Embodiment 11

Dna sequencing

An array of a plurality of inferior arrays allows effectively, and order-checking is arranged in group's sample of xeroxing in the inferior array format that duplicates; For example, 64 samples can be arranged on the inferior array of one 8 * 8mm, 16 * 24 inferior arrays can be replicated on 15 * 23cm film that 1mm wide interval thing is arranged between the inferior array by photomechanical printing.Can prepare some and xerox film.For example, be on 32 96 orifice plates from a common group probe of 3072 7 base sequences, and use the kinases mark.But 4 films of parallel processing in a hybridization circulation.On every film, 384 probes of can marking.Hybridize all probes of to mark in the circulation at two.The hybridization density of can marking, the amalgamation sequence is as follows.

If an inferior array of simple sample or a plurality of inferior array contain several the unknowns particularly when using similar sample, the computer as a result of the probe of marking in advance selects if they are based on, a small amount of probe is enough.For example, if probe AAAAAAA is not a male, a little variation is arranged then, any one is male for 8 overlapping probes.If AAAAAAA is a male, two probes male normally so.Order-checking process in this case comprises that at first hybridizing a group minimizes overlapping probe to limit positive anchor, and the select progressively probe is proved conclusively one about anchor order and most probable hypothesis big or small and space type between them then.In the subordinate phase of this process, can use the storehouse of 2-10 probe, select each probe only in a DNA sample, to be positive, this DNA sample with think that other samples that other probes in the storehouse are positive are different.

This Asia array approach allows in the effective enforcement that solves branch problem process middle probe competition (overlapping probe) or probe collaborative (probe is piled up continuously).After one group of common group probe hybridization, the sequence hybrid process has been determined candidate sequence subfragment (SFs).Further during amalgamation SFs, must provide Additional Information (from dna fragmentation overlap, similar sequences, single gel sequence or from other hybridization or restriction map data).Discern the primer that passes through tapping point single gel sequence from the flanking sequence in SBH sequence information or known carrier information such as carrier insertion site, at the enterprising column criterion mulberry of sample DNA lattice sequencing reaction.Will be from the order-checking of single gel sequence that obtains and the SFs that reads in and read tapping point relatively to discern the order of SFs.And single gel order-checking can combine with SBH, de novo sequencing and sequencing nucleic acid again.

Also can use competitive hybridization and continuous accumulative facies mutual effect amalgamation SFs.These approach are limited for a large amount of nucleic acid samples commercial values that check order by SBH, are immobilized in a sample on the array if wherein use the array of Unified Form that label probe is applied to.Can use and xerox effective enforcement that inferior array analysis small amount of sample allows two kinds of approach fortunately.On the inferior array of each photomechanical printing, use probe library can test a tapping point of one or more DNA samples, be similar to the mutant nucleotide sequence (referring to above) that solves in the different sample that drop in identical inferior array.

In described 64 samples of present embodiment, 100 tapping points if each sample is had an appointment, and if in each inferior array 8 samples of parallel analysis, at least 800 inferior array detection could solve all tapping points so.This just means for 3072 basic detections will add 800 detections (25%).More preferably, detect twice for a tapping point.If inferior array is less, additional detected is less.For example, if inferior array is made of 200 additional detected (6%) of can marking 16 samples.Use 7 base sequence probe (N _1-2B ₇N _1-2) and competitiveness or synergetic property branch solution route or this two kinds of approach, the fragment of about 4000 about 1000bp of detection capable assembling.In addition, use 8 base sequence probe (NB ₈N) detect capable assembling 4kb or longer fragment 12,000 times.Breach probe such as NB ₄NB ₃N or NB ₄NB ₄N can be used to reduce branch and count.

Embodiment 12

Be attached to the inferior array of probe by moment and carry out being connected of DNA analysis and label probe

By the oligonucleotide probe of 4 to 40 bases of standard chemical process composite signal length, and be stored in test tube or the porous plate.Synthesize on the different piece of a upholder of separating or big upholder by deposition or original position, arrange out the specific probe group that contains 1 to 10,000 probe.In the kind situation of back, available physical or hydrophobic barrier are separated each several part or inferior array.Can be by the synthetic preparation of original position probe array.The DNA sample of suitable size and one or more specificity hybridization array.Many samples can be used as the storehouse and are detected on identical inferior array or survey separately with the different inferior array in the upholder.Simultaneously or subsequently sample is added single marking probe or label probe storehouse on each inferior array.If adhere to and the terminal and terminal hybridization of the complementary target of label probe in sample DNA, they then are connected so.By measure the connection of generation from the probe in detecting mark.

This approach is that wherein the DNA sample does not have the change method of the described DNA analysis process of permanent attachment under the upholder situation.Adhere to by probe stationary is obtained moment on upholder.In this case, need not to arrange the target DNA process.In addition, by short label probe is allowed to detect long oligonucleotide sequence with the immobilization probe of weak point in conjunction with being connected.

This method has some unique features.At first, instantaneous the adhering to of target allows it to re-use.After connect taking place, target can be discharged, stay mark by covalent attachment on upholder.These characteristics allow the target circulation and use a spot of target to produce detectable signal.Under optimal condition, target need not to be amplified, and can be directly used in diagnosis and order-checking purpose as the DNA sample of natural origin.Target can be discharged by circulating temperature between effectively hybridization and effective duplex unwind.Preferred not circulation.But limiting temperature and concentration of component are to have a balance, about 50: 50% level between the target of free target and participation hybridization.In this case, generation is connected product continuously.For the different balanced proportions of various objectives is optimized.

Electric field can be used to the use of intensifier target.During beginning, can be in each inferior array the usage level field pulse with sorting target faster.In this stage, balance forms to hybridization and moves, and can use unmarked probe.After the target sorting stage, can carry out suitable rinsing (can help rinsing) by the vertical electric field that a restriction sample moves.Can introduce and distinguish that hybridization is unwind, hybridizes and is connected and collect target and remove the several cycles of not using target, with the enhancing specificity.At next step, add label probe, and can use vertical electricimpulse.Carry by improving temperature, can obtain an optimized ratio of dissociating and hybridizing target.Vertical electric field prevents the diffusion of the target of sorting.

Can various mode arrange stationary probe and the inferior array of label probe group (referring in particular to or be selected from the general probe group).For example, if the short-movie section of a bacterial genomes (about 100-500bp) is partially or completely checked order, can use little probe array (length is 5-30 base) based on the known array design.Suppose that connected only 2 bases are marked, if survey with different sink of 10 label probes of each inferior array, each has 10 probes 10 inferior arrays, then allows to detect 200 bases.Under the condition that picks out whole hybridization mispairing, probe can be replaced more than a base, covers long target with the probe with equal amts.By using long probe, but direct detection target and need not amplification or from the sample surplus DNA, separate.Also can analyze the several targets in (screening) sample simultaneously.If the gained result has shown the generation (or a pathogenic agent) of sudden change, can re-use additional probe library and detect mutation type or pathogenic agent hypotype.This is the essential feature of present method, only has the small part patient to have to infect or during sudden change, this point is very effective in preventative diagnosis when thinking.

In the described method of embodiment, can use various detection method, as radio-labeling, fluorescent mark, enzyme or antibody (chemoluminescence), scattering of light or detectable macromole of interventional procedures or particle.

Embodiment 13

Use 8 base sequences and 9 base sequences order-checking target

Data results from 8 base sequences and 9 base sequence oligonucleotide hybridizations shows that sequencing by hybridization has pin-point accuracy.In this experiment, use known array to predict a series of continuous overlapping component 8 base sequences and 9 base sequence oligonucleotide.

Except mating oligonucleotide, mispairing oligonucleotide fully, also detected the mispairing oligonucleotide that occurs in the inner or terminal mispairing of duplex that oligonucleotide and target form.In these are analyzed, use the minimum operation temperature to form maximization hybridization.Carrying out rinsing under the low temperature equally or more, with by utilizing bigger mispairing dissociation rate to guarantee that with respect to oligonucleotide/target hybridization of coupling maximum distinguishes.Although absolute hybrid rate is a sequence independence, these conditions show and can be used for all sequences.

The instability mispairing of the minimum that can suppose is simple terminal mispairing, and like this, the sequencing by hybridization test can pick out the oligonucleotide/target duplex of coupling fully from the oligonucleotide/target duplex of terminal mispairing.

102 the value of distinguishing allows the generation of pin-point accuracy sequence greater than 2 in 105 hybridization oligonucleotides in the spot stamping form.This system also allows the effect of sequence on hybridization formation and hybridization unstable analyzed.

Produce 100 base pairs by people's one interferon gene known portions of PCR preparation, i.e. 100bp target sequence from the data results of 105 oligonucleotide probes of known array and target nucleic acid hybridization.Used oligonucleotide probe comprises 72 8 base sequences and 21 9 base sequence oligonucleotide, and its sequence and target are complementary fully.One group of 93 probe provides target sequence continuous overlapping frame, and e is by 1 or 2 bases replacements in the target sequence.

For assessment mispairing effect, detect the hybridization of 12 additional probes, when hybridizing, contain at least one terminal mispairing with 100bp target sequence to be measured.Also detected 12 probes of the terminal mispairing of target and the hybridization of 4 other selected contrast nucleotide sequences, like this, 12 oligonucleotide and 4 contrast DNA form and mate duplex hybridization fully.Thus, to each used oligonucleotide in the experiment, assess inside mismatch hybridization, the terminal mismatch hybridization of oligonucleotide and target and mate duplex fully hybridization.By limiting target DNA concentration by detecting different oligonucleotide probes and the single hybridization that appears at the non-target site in the common amplified plasmid dna, determine with 8 base sequences to be measured and 9 base sequence oligonucleotide hybridizations in the absolute effect of DNA target level.

This experimental result shows that all contain the oligonucleotide hybridization effect of mating complementary sequence fully with target or contrast DNA and are better than the oligonucleotide that those contain mispairing.For obtaining this conclusion, we have detected the H of each probe _pWith the D value.H _pDefine the amount of the heteroduplex body that forms between target to be measured and oligonucleotide probe.By the hybridization of 105 probes being distributed the numerical value between 0 and 10, demonstrate in 105 probes 68.5% and have H _pGreater than 2.

When D is defined as 1) and 2) between the signal density ratio time, acquisition value of distinguishing (D), wherein said 1) for containing the spot that mates duplex fully that forms between oligonucleotide to be measured and target or the contrast nucleic acid, 2) for containing the spot of the mispairing duplex that forms between the different loci in identical oligonucleotide and target or the contrast nucleic acid.The variation of D value is owing to 1) to allowing the interference of the hybridization efficiency of signal on the visual background, or 2) the mispairing type between oligonucleotide to be measured and target, found.In the oligonucleotide probe of 105 detections of D value that obtain in this experiment 102 between 2 and 40.These 102 oligonucleotide groups are as an overall calculation D value, and D mean value is 10.6.

Oligonucleotide/target duplex demonstrates the 20 kinds of situations that have of terminal mispairing.Wherein in 5 kinds of situations, D is greater than 10.Mostly big D value is the hybridization unstable that causes owing to non-the most stable (G/T and G/A) terminal matching in these situations.Other possibilities are that a mistake is arranged in oligonucleotide or target sequence.

Be present in low H _pMistake in the target of probe is excluded as a kind of possibility, because thisly wrong can influence each the hybridization of other 8 overlapping oligonucleotide.Do not have significantly because the unstable that other overlapping oligonucleotide sequence mispairing cause shows that target sequence is correct.After the hybridization that has detected 7 new synthetic oligonucleotide again, the mistake that is present in the oligonucleotide sequence is excluded as a kind of possibility.Have only 1 to obtain a D value preferably in 7 oligonucleotide.The hybridization unstable maybe can not form the heteroduplex body and can cause low hybridization formation value.Can not form the heteroduplex body and be because 1) selected probe from complementary or 2) target/target is from hybridization.If probe is from complementary, the formation of oligonucleotide/oligonucleotide duplex can be better than the formation of oligonucleotide/target heteroduplex body.Similarly, if target is maybe can form inner palindrome from complementary, target/target association is preponderated so.When these possibilities of assessment, probe analysis shows that suspicious probe discord himself forms hybridization.And, detect target/target hybridization do the time spent determined a suspicious oligonucleotide probe and two invalid hybridization of different DNA that contain identical target.Two different DNA cause such conclusion for the low possibility that identical target sequence has from complementary district, and promptly target/target hybridization forms low hybridization does not have help.Therefore these results show the hybridization unstable and can not form the low hybrid rate that hybridization causes specific oligonucleotides.The result also shows the distinguished sequence of low hybrid rate because of some oligonucleotide.And the result shows if use the oligonucleotide of 9 base sequences and 8 base sequences, can obtain more reliable sequence results.

These results show the described method of utilizing, and the maximization by forming oligonucleotide and unique overlapping can be measured the sequence of any special purpose longer nucleic acid.These sequence measurements depend on the composition of every kind of oligonucleotide, rather than their frequency and position.

The sequence of utilizing following algorithm to obtain has very high fidelity.In 105 hybridization values, there are 4 when unreliable, the sequence that obtains is entirely true, and this fact shows that this algorithm can eliminate the false positive signal that the hybridization point produces.Fidelity by sequencing by hybridization comes from the dynamics of " all or none " of short oligonucleotide hybridization, and the difference of the stability of the duplex of coupling and mispairing duplex fully.Coupling increases along with reducing of duplex length with the stable ratio of terminal mispairing duplex.And owing to duplex length reduces, thereby bound energy reduces, and causes hybridization efficiency to reduce.But the result who provides shows, when the oligonucleotide that uses 8 base sequences is hybridized, influences the factor of duplex stability and the factor of influence discriminating and reaches balance, and this moment, the method height of sequencing by hybridization was accurate.Result among other embodiment shows that the oligonucleotide of 6,7,8 Nucleotide can be used for the target sequence of 0.5kb (6 base sequence), 2kb (7 base sequence), 6kb (8 base sequence) is reliably checked order effectively.Can the sequence of long segment is overlapping to produce complete genome sequence.

Embodiment 14

The analysis of gained data

Utilize the pattern analysis program, as DOTS program (Drmanac etc., 1993) graphic file is analyzed, and utilized program, for example the statistical function in the SCORES program (Drmanac etc., 1994) carries out measurement for Evaluation.Can determine appropriate threshold from the distribution of signal, with conversion of signals be+/-output.Utilize the position of detected mark,, can determine segmental F+P nucleotide sequence in conjunction with the immobilization probe corresponding and the known array of label probe with mark position.Can amalgamation go out complete nucleotide sequence or initial molecule by the overlapping F+P sequence of determining of deriving of machine as calculated, as the sequence subfragment of human chromosome.

A kind of selection is, in sequence amalgamation process with hybridization signal (for example scoring), be converted to+/-output.Under this situation, from F+P sequence (for example F+P sequence A AAAAATTTTTT) the beginning amalgamation that very high scoring is arranged.Four possible overlapping probe AAAAATTTTTTA, AAAAATTTTTT, AAAAATTTTTTC and AAAAATTTTTTG, with the different probe (TAAAAATTTTTT of other three section starts, CAAAAATTTTTT, GAAAAATTTTTT) scoring is compared, and obtains 3 results: (I) compare with other 6 probes, one scoring in only set out probe and 4 the overlapping probes just is evident as.In this case, the AAAAAATTTTTT sequence will be extended a Nucleotide to the right.(II) except the probe that sets out, the scoring of none probe just is evident as, and amalgamation will stop, and for example, the AAAAAATTTTT sequence is at the end of dna molecular of waiting to check order.(III) in overlapping and/or other three probes, have more than one for stopping (Drmanac etc., 1989) because of mistake or branch on the occasion of, amalgamation.

In the computer derivation process, use to use computer program (Pevzner for example, 1989 of existing algorithm; Drmanac etc., 1991; Labat and Drmanac, 1993; Each literary composition is incorporated herein by reference).

Except that F+P, if detect F (1 interval) P, F (2 at interval) P, F (3 at interval) P or F (4 at interval) P should adopt the algorithm that is fit to all data sets so that correct latent fault or solution exist the ramose problem (see as Drmanac etc., 1989; Bains etc., 1988; Be incorporated herein by reference).

Embodiment 15

By two step sequencing by hybridization

Below be the embodiment that describes the contemplated sequence measurement of contriver.At first, with entire chip and the nearly DNA mixture hybridization of 100,000,000 bases (human chromosome).The implementation principle of hybridization can find in some papers, as (1990) such as Drmanac; Khrapko etc. (1991); With (1994) such as Broude.These articles have indicated hybridization temperature scope, damping fluid and the rinse step of the initial step that is applicable to Format 3 SBH.

Because available target DNA concentration is relatively low, the special imagination of the inventor is hybridized a few hours at low temperature (2 ℃ to 5 ℃), high salt concentration.In order to reach this purpose, using the SSC damping fluid to be substituted in 10 ℃ can sedimentary sodium phosphate buffer (Drmanac etc., 1990).Because second step was arranged, thus rinsing needn't be too thoroughly (several minutes), and when using cyclical crosses that the DNA sample of high complexity is checked order, can omit rinsing.Hybridization is used identical damping fluid with rinsing and is carried out the hybridization of second step so that can continue the applying marking probe.

After using simple mechanical means to the suitable rinsing of each array, add a label probe, for example, the array of 8 * 8mm adds the probe of 6 base sequences.Use the equipment of 96 or 96 pins to carry out 42 operations.Equally, described in former scientific literature, can adopt multiple different condition.

The inventor considers to use following condition especially.At first, adding label probe and only being incubated several minutes after (because add the concentration of oligonucleotide higher), according to the length of F+P, temperature is risen to 3-10 ℃, and add the rinsing damping fluid in low temperature (0-5 ℃).At this moment, used rinsing damping fluid should be applicable to any ligation (for example salt concn scope of 100mM).After adding ligase enzyme, temperature is risen to 15-37 ℃ so that connect (being less than 30 minutes) fast, further distinguish coupling and mismatched crossbreeds fully then.

In FORMAT 3 SBH, consider equally to use cationic detergent, as Pontius ﹠amp; Berg described (1991, be incorporated herein by reference).These authors have described and used two kinds of simple cationic detergents, Trimethyllaurylammonium bromide (DTAB) and cetyl trimethylammonium bromide (CTAB) in the DNA renaturation.

DTAB and CTAB are the variants of quaternary ammonium salt 4 bromide (TMAB), and promptly one of them methyl is replaced by the alkyl group of 12 carbon (DTAB) or 16 carbon (CTAB).TMAB is the bromine salt of tetramethyl-ammonium, and tetramethyl-ammonium is used in and eliminates the influence of G-C content to melting temperature(Tm) in the nucleic acid renaturation.DTAB and CTAB and sodium laurylsulfonate (SDS) structural similitude, but the electronegative sulfate radical of SDS is replaced by the tetramine of positively charged.Although SDS is usually used in hybridization buffer to reduce non-specific binding and to suppress nuclease, it can not obviously influence the efficient of renaturation.

When carrying out attended operation, can add enzyme or suitably add enzyme to reduce background interference after the rinsing with label probe.The ligase enzyme technology is very perfect in biology field, although it was not proposed to be used in the SBH method in the past.For example, Hood and colleague thereof have described a kind of technique of gene detection (Landergren etc., 1988) of ligase enzyme mediation, can be modified for use in FORMAT 3 SBH simply to this technology.Wu has also described with Wallance and has utilized bacteriophage T4 dna ligase to be connected two sections adjacent short synthetic oligonucleotides.Their ligation is at 50mM pH7.6 Tris hydrochloride buffer, 10mM MgCl ₂, 1mM ATP carries out among 1mM DTT and the 5%PEG.Add T4 dna ligase (1 unit; The Bethesda research laboratory) before, the ligation system is heated to 100 ℃, 5-10 minute postcooling to 0 ℃.Most of ligations are carried out in 30 ℃, and by being heated to 100 ℃ of 5 minutes termination reactions.

Carry out final rinsing subsequently, to be suitable for that the oligonucleotide of (F+P) length of hybridization product or connection is distinguished detection.This rinse step was carried out several minutes so that all label probes that do not connect of flush away and other compound in 40-60 ℃ in water, thereby removed background interference to greatest extent.Because covalently bound labeled oligonucleotide is arranged, detect and simplified (not free and low temperature limitation).

According to used marker, use different instruments to the chip developing.To radioactively labelled substance, use phosphorus storage shield technology (phosphor storage screen technology), and utilize phosphorus developing instrument as scanner (molecular dynamics, Sunnyvale, CA).Chip is put into box, cover a phosphorus screen.Expose after 1-4 hour, scan this screen, image file is stored in the hard disc of computer.When detecting fluorescent marker, adopt CCD camera and surface fluorescence microscopy or Laser Scanning Confocal Microscope art.Chip for directly generating on CCD camera pixel can detect (1994, be incorporated herein by reference) with the method for descriptions such as Eggers.

Be in the based analyzing method with the probe, utilize the charge-coupled device (CCD) detector as strong support so that detection by quantitative and developing are carried out in the distribution of mark molecules of interest.These equipment utilizations microelectronic characteristic, promptly be suitable for highly parallel detection, overdelicate detection, high throughput, data obtain and calculations incorporated.Eggers etc. (1994) are described in the detection method based on probe, use CCD among FORMAT 3 SBH as described herein and since highly sensitive with directly be connected, can in the several seconds, finish quantitative analysis.

Complete CCD detection method makes it possible to molecule on the detection chip in conjunction with situation.Detection instrument generates two-dimensional image rapidly, characteristic ground show sample.Use directly is fixed on different biology probes on the pixel of CCD, or is attached on the disposable cover slide that places the CCD surface during based on the molecular detection instrument of CCD.Can use radio isotope, chemoluminescence or fluorescence to come the mark sample molecule.

In the example of Format 3,, on sample and two complementary probe bonded pixel locations, will radiate photon or radio isotope descendant in case sample is exposed to probe array based on CCD.Then, during gate that charged particle that sends when the mark sample or radiation incide CCD, in silicon, generate electron-hole pair.Electronics is assembled under adjacent C CD gate subsequently, and shows on display element.The photoelectron number and the molecule that produce at each pixel are approximated to direct ratio in conjunction with the number of times that takes place.So, can measure molecule quantitatively in conjunction with (Eggers etc., 1994).

The developing array is placed near the sample, and collection rate improves 10 times at least than the technology (for example seeing the magazine technology of traditional C CD) of using lens.In other words, sample (radiation) closely contacts with detector (developing array), and this can eliminate traditional developing opticinstrument such as lens and mirror.

Radio isotope is attached on the molecules of interest as the indication group, just can detects energy particle.In little detector, successfully used several indication groups that can radiate the different-energy particle, comprised ³²P, ³³P, ³⁵S, ¹⁴C and ¹²⁵L.The higher particle of energy (as ³²P) Fang She ion, Molecular Detection sensitivity is the highest.And low-energy particle (as ³⁵S), resolving power is better.Therefore, can select different labelled with radioisotope for use by demand.In case selected radioisotopic tracer just can be predicted detection case by the calculating signal to noise ratio (snr) as (1994) as described in the Eggers etc.

Alternative luminous detection process comprises uses fluorescence or the chemoluminescence indication group that is connected on the molecules of interest.Fluorescent mark can covalency or is connected with molecule by interacting.Fluorescence dye, such as ethidium bromide, (300-350nm) has the intensive absorption band near ultraviolet region, (500-650nm) has main emission band in the visible region, this is best suited for used CCD instrument, because in excitation wavelength, its quantum yield is than low several orders of magnitude when fluorescent signals wavelengths.

From detecting luminous angle, polysilicon CCD gate has following intrinsiccharacteristic, and it can filter the incident light of UV scope, but extremely sensitive to the visible fluorescence of fluorescence indication group generation.This intrinsic makes CCD that high s/n ratio (greater than 100) be arranged to the very high resolution that UV excites, as described in the article of the Eggers that quotes etc. (1994).

For with probe stationary on detector, can be at the SiO of cheapness ₂Produce hybridization matrix on the thin slice, after hybridization and drying, place it in the CCD surface subsequently.This mode is comparatively economical, because DNA hybridization is at the disposable SiO of cheapness ₂Carry out on the thin slice, just make the higher CCD detection instrument of price to utilize again.In addition, probe directly can also be fixed on the CCD, as the probe matrix of special use.

For with probe stationary at SiO ₂Skin uses epoxy silane and standard SiO ₂Modify chemical method, at film surface bonding layer of even epoxy resin layer.By forming secondary amine, amine-modified oligonucleotide probe is connected to SiO then with oxirane ring ₂The surface.After the connection, at 3 bases and the SiO of oligonucleotide ₂The surface directly generates 17 isolating rotatable keys.For amine in the assurance coupling process takes off proton fully, and reduce the formation of secondary structure most possibly, be reflected among the 0.1M KOH and carry out, and in 37 ℃ of insulations 6 hours.

Usually in FORMAT 3 SBH, per 1,000,000,000 some recording signals.Needn't once hybridize (for example 4000 5 * 5mm), but can use the array of lesser amt continuously of all arrays.

A feasible method that strengthens hybridization signal is cyclical crosses.In a circulation, most of stationary probes and dna fragmentation hybridization, these segmental afterbody sequences and label probe are not complementary.By elevated temperature, the hybridization product can unwind.In next one circulation, wherein some (about 0.1%) meetings and suitable dna fragmentation hybridization, and can connect other label probe.In this case, the DNA of while and two groups of probe mispairing hybridization product can unwind.

In cyclical crosses, promptly add all the components before the circulation beginning, the starting temperature of T4 is 37 ℃, if then temperature is higher for thermally-stabilised ligase enzyme.Cool the temperature to 15-37 ℃ then, chip insulation 10 minutes, elevated temperature to 37 ℃ or higher maintenance number minute again, and then reduce temperature.But recirculation 10 times.In the method for a change, can use higher optimum temperuture (10-50 ℃), needn't circulate the ligation time longer (1-3 hour).

Utilize process described herein, can generate complicated chip with the synthetic method of standard, and can accurately locate oligonucleotide because required oligonucleotide number is less relatively.For example, if synthesized the oligonucleotide (16384 probes) of 7 all base sequences, just can determine the oligonucleotide of 256,000,000 14 base sequence.

Important change method of the present invention is that each base array uses more than one not probes of isolabeling.This can satisfy two purposes: variation is so that reduce the number of hybridizing array respectively; Perhaps measure a series of longer oligonucleotide sequences, oligonucleotide sequence such as 3 * 6 or 3 * 7.In the case,, almost can determine the distinguished sequence of 3 continuous oligonucleotide sequences, because must all there be enough signals in positive site to two kinds of marks if use two markers.

Also have one further to change and be to use the chip that contains the BxNy probe, wherein the scope of y is 1-4.These chips make the reading frame difference of calling sequence.Use each suitable group echo probe or the F and the P probe of non-special terminal position (being a certain terminal degeneracy composition) are arranged, also can reach effect same.Can also adopt the part of universal base, the probe of particular sequence is connected on the solid support as joint.Like this, probe is easier to hybridization, and structure is more stable.If a probe has 5 bases, can use, for example 3 universal base are as joint.

Embodiment 16

Determine sequence by hybridization data

When overlapping (N-1) base sequence of being given is replicated 2 times or repeatedly, will interrupts the sequence amalgamation.Can be with one of two different N base sequences of last Nucleotide sequence that extends, this tapping point has limited the single amalgamation of sequence.

In some cases, by will with the known oligonucleotide sequence amalgamation again of target nucleic acid hybridization, can not successfully obtain the complete sequence of target nucleic acid.This is because if the size of the clip size of target nucleic acid and the oligonucleotide that is used to hybridize is unsuitable mutually, can lose some information.The check order length of target nucleic acid of the amount of drop-out and waiting is directly proportional.If but used target nucleic acid is enough short, just can determine their sequence.

Tumor-necrosis factor glycoproteins is distributed in the amalgamation of the DNA meeting of the going up interference sequence of length-specific.Can calculate the possible frequency of these tumor-necrosis factor glycoproteinss.Need introduce definition during derivation, sequence subfragment (SF) to a parameter relevant with sequential structure.If any part of target nucleic acid sequence is with (N-1) base sequence starting and ending, this (N-1) base sequence has repeated in target sequence twice or repeatedly, will produce the sequence subfragment.Therefore, in the method for the invention, subfragment is the sequence between two tapping points in the sequence amalgamation process.Because exist eclipsed short terminal, the total length of all subfragments is longer than actual target sequence.Usually, if there is not other information, subfragment can not be pieced together linear order, because their initial sum end is common (N-1) base sequence.The subfragment number of different target nucleic acids depends on the repetition number of its (N-1) base sequence.This number depends on the value of N-1 and the length of target nucleic acid.

Calculability can be judged the mutual relationship of two factors.If pass through to use length to be N-1, or mean distance is A ₀Overlap successfully positive N base sequence has been sorted L _fThe segmental N-1 that individual base is long is drawn by formula 1:

N _sf＝1+A ₀XKXP(K，L _f)

Wherein K is more than or equal to 2, P (K, L _f) represent at L _fOn the fragment of individual base length, K time possibility appears in the N base sequence.Described a computer program in embodiment 18, it can form the subfragment of any given sequence by the content of N base sequence.

To the probe of a length-specific, the number of subfragment increases with the increase of fragment length.The gained subfragment may not be unique putting in order.Although not exclusively, this information to the functional performance of relatively sequential analysis and recognition sequence of great use.This category information can be called partial sequence.The another kind of method that obtains partial sequence is only to use the oligonucleotide probe of the given length of a subgroup.

Computer simulation to random dna sequence may coincide preferably with the sequence according to theoretical prediction.For example, (use one 8 base sequence or 16 5 ' (A, T, C, G) B for N-1=7 ₈(A, T, C, G) 10 base sequences of 3 ' type), the target nucleic acid of 200 bases on average has 3 subfragments.Yet because the disperse of average is arranged, should there be the insertion sequence of 500bp in the target nucleic acid library, thereby make less than 1/2000 target sequence subfragment more than 3 is arranged.So, when the longer nucleic acid to stochastic sequence checks order, ideally, should use those target nucleic acids that enough weak points are arranged to insert segmental typical library.By each insertion fragment of overlapping acquisition, can obtain the complete sequence of longer nucleic acid like this.

In order to reduce to too short segmental demand the fragment of 50 bases of 8 base sequence probes (for example at), can utilize the information that comprises in the overlapping fragments, these information are present in each DNA random fracture process (as clone or random PCR).Also can use short physics nucleic acid fragment storehouse.Use 8 base sequences or 5 ' (A, T, C, G) B ₈(A, T, C, G) 10 base sequences of 3 ' type when coming 1 megabasse checked order, do not need the fragment of 20,000 50bp, and as long as 2100 samples are just enough.This numeral comprises 700 7kb clones (basic library) at random, and there is the clone (subfragment ordering library) of 20 500bp in 1250 storehouses, each storehouse and from 150 clones in (or similar) library of jumping.Utilize the hybridization data of described sample, the algorithm of application enhancements (embodiment 18) checks order again.

Embodiment 17

Algorithm

Present embodiment has been described the algorithm that a long sequence is checked order, this sequence is written as the alphabet of one 4 letter, this alphabet is got by the character code of forming K tuple (K-tuple) in the separation of the nucleotide sequence minimal number that sets out, the random fragment, and wherein K is the length of oligonucleotide probe.This algorithm is mainly used in sequencing by hybridization (SBH) process.It is based on subfragment (SF), information segment (IF), and utilizes the physics nucleotide sequence to determine the possibility of information segment.

As previously mentioned, subfragment may be to be caused by the tapping point in the amalgamation process, and tapping point is because multiple K-1 oligonucleotide sequence is arranged in target nucleic acid.In a sequence, any two longly are subfragment for the sequence fragment between the repetition character code of K-1.In the order-checking process, the ordering that overlapping K character code repeatedly occurs having disturbed of K-1 character code causes sequence still to be in the form of subfragment.Therefore, the uncertain fragment of the order between tapping point is called the sequence subfragment.

Information segment is defined as the sequence fragment by the segmental most proximal end decision of overlapping physical sequence.

The ability that can compile the physical sequence fragment of some amount and not lose its decision information segment.The fragment total length that compiles at random depends on the length of the K tuple of using in the order-checking process.

This algorithm comprises two major portions.First part is used for forming subfragment from the K tuple set that sequence comprises.Subfragment may be located in a certain size the coding region of physics nucleotide sequence, or is positioned in the information segment that the longer nucleic acid sequence defines.Two types fragment all belongs to basic library.This algorithm is not described K tuple content in the information segment how to determine basic library, the i.e. preparation process of used information segment in the order-checking process.

The second section of algorithm is determined the linear precedence of gained subfragment, so that reappear the complete sequence of the nucleic acid fragment in basic library.For this purpose, used another library---the ordering library, it is made up of the fragment of the sequence of compiling at random of setting out.This algorithm does not comprise such step, and the fragment combination that is about to basic library is to reappear a complete big base sequence.Achieve this end, need to connect the fragment in basic library, this is a precondition of forming information segment.Another kind of selectable method is, exists on the basis of common end sequence, utilizes and searches the overlapping of them, determines basic library fragments sequence with this algorithm.

This algorithm does not need to understand the occurrence number of specific K tuple in basis and ordering library amplifying nucleic acid sequence, does not need to understand segmental that terminal which K tuple character code what occur be yet.This algorithm can be applicable to the mixing of different lengths K tuple and forms.The notion of algorithm makes it may be applied to include the K tuple set of false positive and false negative K tuple.Only in special example, the content of false K tuple just can have a strong impact on integrity and the exactness of determining sequence.Algorithm can be used for optimizing the parameter in the simulation test, also can be used for the sequencing that carries out in the actual SBH test, for example order-checking of genomic dna.In the process of parameters optimization, select the practical and suitable oligonucleotide probe (K tuple) of fragment, and/or select to determine the fragment and the segments of the suitable length of probe, all be particularly important.

This part of algorithm is extremely important in be made of to determine the process of sequence the K tuple.It is based on by maximization eclipsed method the K tuple being carried out unique ordering.The major obstacle of order-checking is specific tumor-necrosis factor glycoproteins, and false positive and false negative K tuple.The purpose of this part of algorithm is the possible subfragment that obtains the correct sequence of tool of minimum quantity and maximum length.This part comprises a basic step and several controlled step.This must be the process in one two step, because some information only could be used behind all main subfragments of acquisition.

The subject matter of order-checking is to form from the character code of K tuple to obtain tumor-necrosis factor glycoproteins, and according to definition, these form the information of not load K tuple occurrence number.The notion of whole algorithm depends on the basis that addresses this problem.In brief, two kinds of opposite approach are arranged: 1) in the process of determining pSF, obtain tumor-necrosis factor glycoproteins during beginning, or 2) in the process of the last ordering of subfragment, the tumor-necrosis factor glycoproteins of reentrying.Under first kind of situation, pSF has unnecessary sequence, and under second kind of situation, they comprise sequence deletion.The former need remove unnecessary sequence, and under second kind of situation, in the process of amalgamation sequence in the end, needs to reuse some subfragment.

The difference of these two kinds of approach is, to the severity difference of the single eclipsed regulation of K tuple.Looser standard is: when the K-1 end of the low order end of and if only if K tuple X was present in the high order end of K tuple Y, K tuple X and Y were maximum clearly overlapping.This standard will cause the generation of tumor-necrosis factor glycoproteins and unnecessary sequence.

Another standard that is used for second kind of approach is strict, it has an additional explanation: the K-1 end of the low order end of and if only if K tuple X is present in the high order end of K tuple Y, and when the high order end K-1 end of Y did not appear at the low order end of any other K tuple, K tuple X and Y were maximum clearly overlapping.Algorithm based on strict standard is comparatively simple, is described at this.

When the K-1 end of last K tuple right-hand member does not appear at the left end of any one K tuple, or appear at the left end of a plurality of K tuples, the extension process of specific subfragment stops.If it appears at the left end of a unique K tuple, will check the second section of this standard.If also have a K tuple that is different from last tuple in addition, the amalgamation of specific subfragment ends at the position of first high order end; If there is no such K tuple just meets single eclipsed condition, and specific subfragment just can extend to the right.

Except basic standard, also has an additional standard so that can use the K tuple of different lengths.Maximum is overlapping to be that the short length of overlapping centering is the K tuple of K-1.The generation of pSF is from first K tuple of file, and the K tuple shows in this document at random, and with they sequence independences in nucleotide sequence.Therefore, the not necessarily beginning of sequence of first K tuple in the file, beginning that neither specific subfragment.Utilize the single overlapping of described standard definition, thereby produce subfragment the ordering of K tuple.Each used K tuple of deletion from file.When not having K tuple and last tuple clear and definite when overlapping again, the structure of subfragment stops, and begins to make up another pSF.Because be not to begin when determining most of subfragment, the pSF that forms be added K tuple file, as a longer K tuple from their actual zero position.Another kind may be to form subfragment from the K tuple that begins to both sides.Overlapping when forming, in the time of promptly can't carrying out the extension of any subfragment, process stops again.

PSF can be divided three classes: 1) when the K tuple set is correct, the subfragment of the tool maximum length of formation and correct sequence; 2) since with maximum and clear and definite overlapping standard application in incomplete K tuple set or the set of false positive K tuple is arranged, the short subfragment of formation; And 3) the incorrect pSF of sequence.2) the incomplete collection in is because the false negative result in the cross experiment causes, or owing to has used incorrect K tuple.Owing to use false positive or false negative K tuple, can form a) subfragment of incorrect link; B) have the subfragment of mistake end; C) show as the false positive K tuple of false minimum subfragment.

With regard to false positive K tuple, may there be such K tuple, they contain a plurality of wrong bases or contain a wrong base in middle somewhere, also a wrong base may be arranged endways.A kind of K tuple in back will cause forming the short wrong subfragment or the subfragment of incorrect link.The wrong pSF of preceding two kinds of K tuples formation and the same length of K tuple.

If a false negative K tuple is arranged, then overlapping because can not form maximum, can produce pSF.If the false positive K tuple of its high order end or the wrong base of low order end is arranged, then clear and definite overlapping because of forming, can produce pSF.Have the false positive and the false negative K tuple of the common K-1 sequence of tool simultaneously in file, will produce pSF, one of them pSF has the K tuple of mistake in associated end.

After forming subfragment, the subfragment of correct sequence mistake in the process of carrying out the subfragment ordering, and the pSF that will clearly connect couples together.The first step comprises the pSF that excises incorrect link, and obtains final subfragment by clearly connecting pSF, and this step is described below.

The subfragment that has two kinds of conditions meeting generation errors to connect.The first, when the K of mistake tuple appears at the amalgamation point of tumor-necrosis factor glycoproteins of long K-1, will make a mistake.The second, tumor-necrosis factor glycoproteins is shorter than K-1.These situations have two kinds of versions respectively again.First kind, one of tumor-necrosis factor glycoproteins is segmental end.Second kind of variation, tumor-necrosis factor glycoproteins occurs in segmental any position.In first kind of possibility, incorrect link occurring needs the disappearance of some K tuple in the file (false negative).In second kind of possibility situation, require to occur simultaneously hereof false positive and false negative K tuple.Consider the repeatability of K-1 sequence, when any end had inner the repetition, it was just enough only to lack a K tuple.And concerning repeat the inside of strictness, need two of disappearances, and this is because on information science, the end of a sequence can be considered to the unlimited linear array of false negative K tuple.In the situation of " being shorter than K-1 ", only consider that length is the tumor-necrosis factor glycoproteins of K-2, these sequences need 2 or 3 special wrong K tuples.Very possible these are detected unique situations of institute's energy in the actual tests, and it is much rare that other situation is wanted.

When tumor-necrosis factor glycoproteins is not segmental when terminal, stricter to the examination criteria of incorrect link subfragment.At this moment, can detect two subfragments in addition, one of them is at high order end, and another has the K-2 sequence respectively at low order end, and this K-2 sequence also is present in the subfragment of incorrect link.When tumor-necrosis factor glycoproteins is positioned at segmental end, have only a subfragment to contain the K-2 sequence, this K-2 sequence makes the mistake at high order end or right-hand member when subfragment forms.

Excise the subfragment of incorrect link according to general rule: if the high order end of the long K-2 of a subfragment or low order end sequence also are present in other any subfragment, this subfragment should be cut to two subfragments, each all contains the K-2 sequence.This rule does not comprise the sort of terminal rare cases that repeats, and promptly repeats end and is repeating on the K-1 sequence a plurality of false negative K tuples are arranged.But be used to the subfragment that information segment in the information of superimposed sheets section or basic library and ordering library is discerned this class incorrect link.In addition, when a plurality of false negative K tuple all occurring in two positions containing identical K-1 sequence, the subfragment of incorrect link will keep.This situation is rarely found, because it need have 4 special false negative K tuples at least.Combine to another subfragment head from a subfragment tail and can obtain specific sequence if will be shorter than the sequence of K-2, can introduce additional rule from long these subfragments of sequence excision for K.

Use described rule by strictness, for the accuracy that guarantees the result will be lost some integrities.Although some subfragment is not an incorrect link, and is still cut, because they meet the characteristic of misconnection subfragment.Several these class situations are arranged.For example, a fragment is except at least two identical K-1 sequences, it also contains the K-2 sequence from K-1, or a fragment, contains to repeat arbitrary K-2 sequence of twice at least, and have a false negative K tuple that contains specific K-2 sequence at least in the centre, or the like.

The purpose of this part algorithm is to reduce the number of pSF, obtains the long subfragment that correct sequence is arranged of minimal number.In both cases, might produce single long subfragment or complete sequence.First kind of situation relates to the special order of multiple K-1 character code.In some situation, some or all pSF (first kind pSF) of extending to greatest extent can be sorted uniquely.For example, in fragment S-R1-a-R2-b-R1-c-R2-E, S and E are segmental initial sum ends, a, b, c are to special different sequences of subfragment separately, R1, R2 are two series connection multiple K-1 sequences, have produced 5 subfragments (S-R1, R1-a-R2, R2-b-R1, R1-c-R2 and R2-E).Can in two ways they be sorted: above-mentioned original series or S-R1-c-R-b-R1-a-R2-E.On the contrary, identical at the number of a tumor-necrosis factor glycoproteins with type, but in the different fragment that puts in order, promptly S-R1-a-R1-b-R-c-R-E does not just exist other can comprise the sequence of all subfragments.Such example only after generating pSF, just can identify.They have shown the necessity of taking for two steps carried out in the pSF forming process.Second kind of situation is when file contains false negative and false positive K tuple, produces false short subfragment on non-repetitive K-1 sequence location, and this situation is more important.

The solution of two class pSF comprises two portions.At first, the also false positive K tuple deletion of non-existent minimum subfragment will be shown as.All are long to be the K tuple subfragment of K, if do not have such two overlapping, they at one end length greater than K-b, will delete in the other end length greater than K-a so that form the connection of maximum quantity.As if in our test, the numerical value of a and b is respectively 2 and 3, and this enough eliminates the false positive K tuple of quantity sufficient.

The subfragment that can connect uniquely in second step links together.The rule that connects is: the initial sum end of overlap what its subfragment not in office that and if only if is positioned at the corresponding initial or end of two subfragments occurs, and these two subfragments can connect clearly.

The situation of exception is that one of two subfragments have identical initial sum end.Even the same end that also have another subfragment to have in the file this moment still can connect.Here main problem is accurately defining of overlap.When overlap only has for a pair of subfragment, overlap is shorter than K-2, though or be not shorter than K-2, but also have the subfragment that has the overlap of being longer than K-4 in addition, then can not connect.Equally, the standard of pSF end terminal and (or minority) terminal bases of deletion all is counted as overlap.

After this step, may more residual false positive K tuples (as minimum subfragment) and some have the subfragment of mistake end.In addition, in rarely found situation, when the specific false K tuple of certain number exists simultaneously, connection can make a mistake.In subfragment sequencer procedure and other contrast step, can detect and address these problems, handle the subfragment of not cut misconnection simultaneously.

The short subfragment of gained is divided into two kinds.In the normal conditions,, these subfragments can be connected clearly because repeat the distribution of K-1 sequence.This can carry out after the process that generates pSF, and it also is a good example, illustrates that the pSF generative process must be in two steps.Contain in the situation of file of false positive and/or false negative K tuple in use, obtain short pSF in the position of non-repetition K-1 sequence.With regard to false positive K tuple, K tuple may contain a plurality of wrong bases (or have in middle somewhere a wrong base), also can be that the K tuple is arranged endways.The latter causes generating short mistake (or incorrect link) subfragment.The former can cause the wrong pSF identical with the K tuple length.

The part that connects pSF in the algorithm its objective is and reduces the pSF number, to generate the correct subfragment of sequence minimal number, longer.All K tuple subfragments, if do not have such two overlapping, they at one end length greater than K-b, will delete in the other end length greater than K-a so that form the connection of maximum quantity.Like this, most false positive K tuple is by reject.The rule that connects is: the initial and/or end of overlap what its subfragment not in office at the corresponding initial or end of and if only if two subfragments occurs, and two subfragments clearly can be connected.The situation of exception is that one of two subfragments have identical initial sum end.Even the same end that also have another subfragment to have in the file this moment still can connect.Here main problem is accurately defining of overlap.On K-1 or K-2 sequence multiple point, there are two specific false negative K tuples at least, and a combination of false negative and false positive K tuple, all can damage or " covering " some overlap, and can form clear and definite but the pSF of connection error.In order to prevent this situation, for accuracy must be sacrificed integrity: when end sequence is shorter than K-2, and when existing another one to be longer than the overlap of K-4, connection can not be carried out, overlap is limited by the end of pSF, or omits one or minority terminal bases.

Under very rare situation, because have some the specific false positive and the false negative K tuple of certain number, some subfragment and false positive K tuple (as minimum subfragment) that has the mistake end may remain, and connection perhaps can make a mistake.In subfragment sequencer procedure and other contrast step, can detect and address these problems, handle the subfragment of not cut misconnection simultaneously.

The process of subfragment ordering is similar with its generative process.If subfragment is regarded as long K tuple, thereby just can clearly connect ordering by overlapping end.The information that clearly connects is represented these segmental sections according to being that the subfragment that fragment produced with basic library is divided into each group.The biochemical method of this method and head it off is similar, and biochemical method is based on the hybridization that takes place with the long oligonucleotide that the relevant connection sequence is arranged.Utilize the K tuple of the segmental suitable section in basic library, make catenation sequence form subfragment.Relevant section by with basic library separately the fragment in fragment eclipsed ordering library represent.The shortest section is the information segment in ordering library.Long is several adjacent information segments, or whole laps of the respective segments in ordering library and the basic library.In order to reduce the number of independent sample, the fragment in ordering library is compiled at random, and determined the content of single K tuple.

Utilize the numerous fragments in the ordering library to generate very short section, therefore reduced the chance that the K-1 sequence repeatedly occurs, and the reason that subfragment repeatedly occurs producing just of K-1 sequence.In addition, those contain in the basic library the longer section of given segmental different zones and do not contain some and repeat K-1 fragment.In each section, come subfragment to be formed a catenation sequence (connection subfragment) to certain by given fragment.Sequencer procedure was divided into for three steps: the generation of the K tuple of (1) each section; (2) in each section, generate subfragment; And the connection of (3) section subfragment.Elementary section is defined as, in the basic library specific segmental K tuple form with the ordering library in K tuple main cross section and the difference part formed.Secondary (weak point) section is defined as, cross section and difference part that elementary section K tuple is formed.

Here have one in cross section and difference part, false positive and negative K tuple all have the accumulative problem.False negative K tuple from homing sequence is assembled at cross section, and occurs at random in two sequences but the false positive K tuple that is not present in relevant overlapping region all accumulates in cross section (lap).On the other hand, the most of false positive K tuples from arbitrary homing sequence all occupy at cross section.This is one and has utilized from reducing the example of the experimental mistake of individual chip with the segmental information of this fragment eclipsed.False K tuple is owing to Another reason accumulates in the difference part.To enlarge the false positive tuple that obtains from cross section with income from the false negative tuple set of homing sequence, the false positive tuple set is taken in those because of the wrong K tuple that is not included in cross section, i.e. the false negative tuple of cross section.If homing sequence comprises 10% false-negative message, the primary and secondary cross section will comprise 19% and 28% false negative K tuple respectively.On the other hand, if the length in basic fragment and storehouse is respectively 500bp and 10,000bp, then false-positive value of mathematical expectation is 77.Yet, might recover the K tuple that great majority lose and remove most of false positive K tuples.

At first, must determine the cross section that the essentially consist of K tuple is formed as a pair of K tuple to particular section.The K tuple that subsequently all is had the K tuple composition that sets out is included in the cross section, and this cross section contains the K-1 sequence at an end, and another end contains the K-+ sequence, and these sequences appear at the end of two K tuples of baseset.The generation of difference part is carried out after this, to prevent the assembling false positive tuple when generating the difference part.After this, in the same K tuple set that enlarges of difference part, the difference part is to carry out borrow from cross section.The K tuple of all incomes is deleted from the cross section file as the false positive tuple.

For each limits cross section (i.e. the set of a general K tuple) to (basic fragment) X (storehouse in ordering library).If in set, the number of K tuple is very big, just will gather by mentioned above principle income false negative tuple.Deducting gained cross part diversity from given basic fragment must be to elementary difference segment set.According to mentioned above principle, from the cross part diversity false negative K tuple is taken in the difference segment set, simultaneously, these K tuples are deleted from the cross part diversity as false positive.When basic fragment ratio compiled the sheet segment length, difference part can be represented two isolating sections, and this point can reduce its application in the step afterwards to a certain extent.Elementary section all is each cross section and the difference part that (basic fragment) X (a group ordering library) is generated that contains a large amount of K tuples.By the right K tuple set of more all possible elementary section, obtain the K tuple set of secondary section.By form cross section with a large amount of K tuples each to limiting two difference parts.The most of information that obtain from overlapping fragments all are resumed in this step, so gained is very little from the process of third round formation cross section and difference part.

(2) operation of the subfragment of generation section is segmental identical with above-mentioned basic library.

(3) method of connection subfragment comprises, from the given segmental subfragment in basic library that has some overlapping end, order determines that the correct subfragment that connects is right.In the situation that 4 relevant subfragments are arranged, two have identical initially, and two have identical end.Can connect into 4 pairs of different subfragments.Common 2 pairs is correct, and 2 pairs is wrong.Correct in order to find, in the subfragment that is generated by segmental all the primary and secondary sections in given basis, detect every pair and whether have catenation sequence.Select the length and the position of catenation sequence, to avoid randomness interference to sequence.The long K+2 of catenation sequence or longer has at least one element 2 on certain next door to overlap in the two of subfragment.Only found two catenation sequences, and remaining two do not have, just can connect.Two subfragments that link together replace original subfragment in the file, and this process moves in circles.

In this step, generate tumor-necrosis factor glycoproteins.This means that some subfragment repeatedly is contained in the subfragment that has linked together.By seeking the relevant connection sequence that a subfragment and two different subfragments are linked together, can identify these subfragments.

Discern the subfragment of the incorrect link that generates when those are linked as long subfragment at structure pSF with pSF, in the sequence of the subfragment that segmental section generated, whether exist from the segmental subfragment sequence in given basis based on detecting.This sequence can not found in the incorrect link position, this explanation subfragment connection error.

Except three steps of described subfragment ordering,, need some additional controlled step or be applicable to the step of distinguished sequence in order correctly to generate more complete sequence.

By the composition of the K tuple of section and subfragment relatively, determine which section certain subfragment belongs to.Because the mistake that the K tuple is formed (because the initial mistake in storehouse and mistake of statistics that the K tuple frequency of occurrences causes) can not accurately be divided subfragment.Therefore, do not adopt the division of " all or none ", but determine that certain subfragment belongs to the possibility (P (sf, s)) of certain given section.This possibility is the function of false K tuple per-cent in the size of K tuple length, subfragment length, ordering library fragment length, storehouse collection and the file:

(P(sf，s))＝(Ck-F)/Lsf

Wherein Lsf is the length of subfragment, and Ck is the right common K number of tuples of given subfragment/fragment, and F is a parameter, and this parameter comprises the relation between K tuple length, basic library fragment, storehouse collection size and the percentage of errors.

The subfragment that belongs to particular section is used as redundant short pSF, and clearly connects.The definition that clearly connects herein is slightly different because it is based on a kind of like this possibility, promptly have the subfragment of overlapping end belong to consider the possibility of section.In addition, according to the connection of these subfragments in other section, can control the accuracy that clearly connects.After in different sections, connecting, all subfragments that both got are linked together, delete the short subfragment that comprises in the long subfragment, the remaining connection procedure that enters routine.If sequence of complete is reappeared, use the standard of the affiliated section possibility of identical or looser judgement, repeat the division and the connection procedure of subfragment, clearly connect then.

Some information is not utilized when using strict standard to come definition clear-cut overlapping.What obtain like this is not complete sequence, but several subfragment, these subfragments define given segmental several possibilities.Use looser standard can obtain accurate and complete sequence.In some cases, for example make a mistake when connecting, may obtain complete but wrong sequence, or obtain " monster (monster) " subfragment, they do not connect each other.Therefore, each fragment in corresponding basic library can obtain: a) several possible results, one of them is correct, and b) the correct result of most probable.In addition, under few situation owing to generate mistake in the subfragment process, or since the ratio of affiliated possibility can not produce clear and definite result, or only produce a most probable result.These situations will remain imperfect sequence, perhaps contrast by other overlapping fragmentses with these data and basic library and obtain clear and definite result.

The above-mentioned algorithm of checking on a 50kb sequence that generates at random, this sequence contains 40%GC with the simulating human genome.At the middle part of sequence, different All and some other tumor-necrosis factor glycoproteins have been inserted, the about 4kb of total length.For the outer SBH test of analogue body, carry out following operation to obtain suitable data.

The basic library of preparation is simulated in-the position that limits 60 5kb overlapping " clone " at random:

-determine that at random the position of 1,000 500bp " clone " is to simulate the preparation in ordering library.These fragments extract from sequence.Set up 20 segmental with the hangar collection, determine the K tuple set of storehouse collection and be stored on the hard disk.These data will be used for the subfragment phase sorting: for whole human genome, and the clone of same density, basic library needs 400 ten thousand clones, and there are 300 ten thousand clones in the ordering library.Be used for nearly all genomic dna clone at random and the clone's of several kb when passing through number based on the order-checking of the method for gel than 700 ten thousand big several times of clone.

By the information at the segmental initial sum of 5kb end, can determine in sequence, to have 117 " information segments ".Determine the overlapping K tuple set that single " information segment " contains subsequently.Only the identical K tuple Asia of use and predetermined tabulation collects.This tabulation comprises 65% 8 base sequences, 30% 9 base sequences and 5% 10-12 base sequence.Carry out the generation and the ordering of subfragment according to these data.

In two tests, algorithm is verified with the mimic data.The sequence of 50 information segments is reappeared, data set (surpass 20,000bp) 100% is correct, and 26 information segments (about 10,000bp) 10% false K tuple (5% false positive and 5% false negative) is arranged.

In first test, all subfragments all are correct, only have the sequence of 1/50 information segment to reappear fully, but keep the form of 5 subfragments.The position of overlapping fragments to the ordering library is analyzed, and shows that they lack 5 subfragments are carried out the required information of single ordering.Based on overlapping end, can connect subfragment: 1-2-3-4-5 and 1-4-3-2-5 in two ways.Unique difference is subfragment 2 and 4 switches.Because subfragment 2,3,4 is relatively lacked (about altogether 100bp), bigger chance is just arranged here, and occurred this phenomenon really, do not have fragment to originate in the library or end at subfragment 3 districts in ordering exactly.

In order to simulate real order-checking, in many tests, some vacations (" hybridization ") data are included in the input data.In the oligonucleotide hybridization test, under the suggestion condition, unique situation that can produce suspicious data is with respect to the terminal mispairing of coupling hybridization fully.Therefore, in simulated experiment, only the K tuple that differs an element in arbitrary end and actual K tuple is considered to false positive.These false tuple set prepare as follows.In the initial K tuple set of information segment, add the Asia collection that 5% false positive K tuple is arranged.From set, choose a K tuple at random, copy and at it initial or end changes a Nucleotide, obtain false positive K tuple.Deduct an Asia collection that the K tuple of 5% random choose is arranged subsequently.In this way, obtained the statistics expection number of complexcase, in this case, correct K tuple is had the K tuple of a wrong base to replace by end.

Prepare the K tuple set as stated above, cause 10% false data.Because the K tuple that the selection of randomness ground will copy, changes and remove, this value becomes with situation.But this per-cent surpasses 3-4 times of the quantity of suspicious data in the actual cross experiment.10% mistake of introducing causes basic library fragment (basic library information segment) and section Central Asia number of fragments to increase by 2 times.As to containing false-positive K tuple set desired (seeing the generation of elementary subfragment), about 10% final subfragment has a wrong base endways.Do not observe the incorrect link of subfragment, find no the subfragment of wrong sequence yet.In sequencer procedure, there are 4 to fail to reappear complete sequence in 26 detected information segments.In these 4 examples, the form of the sequence of acquisition is to be contained in several long subfragment and several short subfragment of same section.This result shows that this algorithm principle can admit of the misdata of big per-cent.

Be made up of its K tuple and successfully generated sequence, this can describe with integrity and accuracy.In the process of formation sequence, can define two particular cases: 1) lost a part of information in the sequence of Sheng Chenging, but known its position, and known the type that they are affiliated, 2) the reproduction sequence of gained does not match with the sequence that obtains K tuple composition, does not make mistake but detect.Suppose algorithm development to its theoretical limit,, then have only first kind of situation to take place such as the definite K tuple set of use.The imperfect subfragment that can not clearly sort that causes certain number, and cause the definite length that is difficult to determine unique sequence, the multiple quantity of promptly connecting fully.

False K tuple can cause the generation error sequence.The reason of mistake is not the defective owing to algorithm, but because the given composition of K tuple is clearly being represented the sequence different with initiation sequence.According to the kind of the K tuple that exists in the file, can define three class mistakes.False negative K tuple (not following false positive) causes " disappearance ".False positive causes " extending (not waiting exchange) ".With false-negative false positive is to cause independent " insertion " or " disappearance " and " insertion " bonded reason.All K tuples (or most of K tuple) between two possibilities of subfragment are initial are false negatives, will cause disappearance.Because each position in the sequence is all limited by the K tuple, generally, disappearance takes place needs K successive false negative.(false negative as 10%, during K=8, per 108 elements of this situation can occur once).Even use the random library that contains 10 genome Equivalents that the mammalian genes group is checked order, this situation also seldom takes place.

The terminal extension of the sequence that false positive K tuple causes is a special case of " insertion ", and this is because the end of sequence can be counted as the unlimited linear array of false negative K tuple.Can consider to generate the single K group leader's of unit of ratio that a group false positive K tuple produces subfragment.If produce subfragment in overlapping fragments, the physical segments at random as in the ordering library just can detect this situation.False positive and false negative K tuple specific combination can cause insertion, or replace disappearance by inserting.In first kind of situation, continuous false-negative number is less than K.Either way need several eclipsed false positives K tuple.Insert and deletion mainly is theoretic possibility, do not have a lot of actual reflections because to the quantity of false K tuple and specific require so high.

In other situation, if false positive and false-negative kind and minimum quantity backlog demand, the mistake on the K tuple is formed only can cause generating incomplete sequence.

By sample nucleic acid is contacted with the immobilization probe of known array and the label probe in the solution, with SBH, the nucleic acid samples order-checking.In case the probe ligase enzyme is added the mixture of probe and sample, that is to say that in case with upholder a stationary probe and label probe and sample are hybridized in succession, two probes can chemically be coupled together by the effect of ligase enzyme.After the rinsing, under the situation that label probe exists, the stationary probe and the label probe that have only chemistry to link together can be detected.Be tested and appraised the immobilization probe and the identifying mark probe of array specific position, under a situation that probe is a bit arranged that is positioned at the array on the Format 3,, can determine a part of sequence of sample with the sample of one 3 substrate.What play a decisive role is the right maximum overlaps of all probes that link together, the sequence that can rebuild sample.Sample to be checked order can not be the nucleic acid fragment or the oligonucleotide of 10 base pairs (bp).Long 4 to 1,000 bases of preferred sample.

Probe is the fragment of length less than 10 bases, and 4-9 base preferably.Like this, the stationary probe array can comprise the oligonucleotide of all given lengths, perhaps only comprises the oligonucleotide that is used for particular detection.When using the oligonucleotide of all given lengths, the number of center oligonucleotide is 4 ^N, N is the length of probe.

Embodiment 18

Again utilize sequence testing chip

When adopting attended operation in the order-checking process, common oligonucleotide chip can not utilize immediately again.The inventor thinks can overcome this shortcoming in many ways.

For second probe (probe P), can use ribonucleotide, this probe can be handled with the RNA enzyme subsequently and remove like this.The RNA enzyme can use RNA enzyme A when handling, and this enzyme is an endonuclease, but specific action in single stranded RNA 3 ' pyrimidine, and excision is connected with the phosphoric acid that is close to Nucleotide.End product is pyrimidine 3 phosphoric acid and the oligonucleotide that has terminal pyrimidine 3 phosphoric acid.The effect of RNA enzyme does not need cofactor and divalent positively charged ion.

In order to utilize the RNA enzyme, as (1989, be hereby incorporated by) as described in the Sambrook etc. chip is incubated in the suitable damping fluid that contains the RNA enzyme usually.Suitable condition is that the array of each 8 * 8mm or 9 * 9mm uses 30-50 μ l to contain the damping fluid of RNA enzyme, keeps 10-60 minute in 37 ℃.Use the hybridization buffer rinsing then.

Although its application is not extensive, can also use uridylic (, being hereby incorporated by) in specific embodiments as (1989) as described in Craig etc.Remove the probe of connection, so that the recycling chip can be degraded with intestinal bacteria repair enzyme (uridylic-DNA glycosylase), this enzyme can be removed uridylic from DNA.

Can also between probe, form a kind of key that can special excision, after detection, excise it.For example, the chemistry of describing by (1988) such as Shabarova etc. (1991) and Dolinnaya is connected to form.Two pieces of documents all are incorporated herein by reference in detail at this.

Shabarova etc. (1991) describe, and concentrate oligodeoxynucleotide with cyanogen bromide as enriching agent.In their a chemical ligation of step, oligonucleotide is heated to 97 ℃, slowly be cooled to 0 ℃, add the BrCN acetonitrile solution of 1 μ l10mM then.

Dolinnaya etc. (1988) have shown how to introduce the internucleotide linkage between phosphoramidite and the tetra-sodium in DNA duplex.They have also used chemical connection process to come the sugared phosphate backbone of modifying DNA, wherein use water miscible carbodiimide (CDI) as coupling agent.The selection of phosphamide key excision comprised with 15% acetate contact 5 minutes at 95 ℃.The selection excision of focusing phosphate bond comprises and pyridine: water mixture (9: 1) and fresh distillatory (CF ₃CO ₂) the O contact.

Embodiment 19

Known sudden change of diagnosis-scoring or full-length gene check order again

In a simple case, target may be to seek whether specific known mutations has taken place in the DNA section.Probe below 12 has enough reached this purpose, allelic 5 positive probes for example, another allelic 5 positive probes, each allelic 2 negative probe.Because the number of probes that every duplicate samples need be marked is few, can analyze a large amount of samples abreast.For example, in 3 hybridization circulations, use 12 probes, can analyze 64 patients' 96 different genes group sites or constant gene segment C, analyze containing on 6 * 9in film of 12 * 24 inferior arrays and carry out, each inferior array has 64 points, and each represents 64 patients' same DNA section.In the present embodiment, can in 64 96 orifice plates, prepare sample.Each plate is represented a patient, each hole representative dna fragmentation to be detected.The sample of 64 plates is repeated a little 4 times, and point is in 4 directions of same film.

Utilize single track to move liquid or single needle transfer device (perhaps row the transfer pipet or the pin of control) respectively, can select in 96 sections 12 probes of each.The probe of selecting can be arranged in 12 96 orifice plates.If probe does not have prior mark, then can label probe, will mix from the probe and the hybridization buffer of 4 plates then, and preferably join inferior array with 96 road liquid-transfering devices.After the hybridization circulation, preferably with film in undiluted hybridization buffer or rinsing damping fluid in 37-55 ℃ of insulation, the probe that adds before can peeling off.

May an allelic positive probe be positive probe, and another allelic positive probe is negative probe, in 2 allelotrope of definite existence which this can be used to.In this redundant computation system, allow the hybridization of each probe to have the to a certain degree mistake of (about 10%).

Especially when less redundancy is just enough, can calculate most allelotrope, for example, can prove 1 or two probes whether there being one of two allelotrope in the sample with one group of incomplete probe.For example, use one group of 4000 8 base sequence, give on one of two allelotrope to select the site to find the possibility of at least 1 positive probe at random be 91%.One group of imperfect probe be can optimize and the G+C content of sample product and other influence are subjected to reflection.

When full-length gene is checked order, can be in the section of suitable number amplification gene.To each section, can select one group of probe (approximately each probe 2-4 base) to hybridize.Whether these probes can identify certain position of analyzing in the section sudden change.If detect section (the inferior array that promptly contains these sections) one or more mutational sites are arranged, section and other probe hybridization can be sought the definite sequence in mutational site.If use 6 base sequences to detect the DNA sample every two Nucleotide, and determining the probe TGCAAA and the TATTCC of just being hybridized the sudden change position surrounds, also covered by 3 negative probes: CAAAAC, AAACTA and ACTATT, then Tu Bian Nucleotide this position in normal sequence must be A and/or C.They may be by single base mutation, or 1 or 2 nucleotide deletion between AA, AC or CT and/or insert change.

An approach is to select such probe, and the probe TGCAAA that it will just hybridized extends 1 Nucleotide to the right, and probe TATTCC is extended a Nucleotide left.Utilize this 8 probes (GCAAAA, GCAAAT, GCAAAC, GCAAAG and ATATTC, TTATTC, CTATTC, GTATTC), determined two suspicious Nucleotide.

Can determine about the most probable hypothesis of suddenling change.For example, find that A sports G.Such result can cause two kinds of possibilities.A kind of is the displacement that A → G only takes place, and also having a kind of is except that displacement, has also inserted some bases between G that has just determined and C.If with the result of bridge joint probe be negative, can detect these possible reasons, at first use at least one bridge joint probe (AAGCTA) that contains the position that suddenlys change and other 8 probes (CAAAGA, CAAAGT, CAAAGC, CAAAGG and ACTATT, TCTATT, CCTATT, GCTATT).There are many other methods to select to solve the probe of sudden change.

In diplontic situation, the scoring of test sample and homozygote contrast is compared especially, to determine heterozygote (seeing above).If the section that the continuous probes probes of minority is covered at one of two karyomit(e)s sudden change has taken place, the signal estimation of these probes can weaken about twice.

Embodiment 20

Evaluation causes the gene (sudden change) of genetic diseases and other proterties

On immobilized sample array, use general long probe (8 base sequences or 9 base sequences) group, can not carry out the dna fragmentation order-checking that subclone will reach 5-20kb.In addition, the speed of order-checking is about 1,000 ten thousand bp/ days/hybridization instrument fast.This just can repeat order-checking to people's gene and the big fragment of genome that science and medical significance are arranged.50% people's gene to be checked order again, need to check 100,000,000 bp.This can finish with rational cost in the short time.

The sudden change and/or the gene that can in several ways this huge ability of order-checking again be used for identification code disease and other proterties.Basically, can will derive from specified disease patient's the genomic dna or the mRNA of particular organization (can be converted into cDNA) as the material that sets out.Prepare the isolated genes or the genomic fragment of suitable length through clone's process or amplification in vitro process (as PCR) by the DNA in these two kinds of sources.If the use clone technology before order-checking, should filter out one group of minimum clone to be measured from the library.By the hybridization of a small amount of probe, can screen effectively, in the time of especially will selecting the clone who is longer than 5kb on a small quantity.The clone can make the hybridization data amount increase twice, does not but need PCR primer up to ten thousand.

This process has an improved method, can prepare gene or genomic fragment with the restricted shearing of enzyme DNA, for example shears DNA:FACFC (N5 ')/CTGCG (N10 ') as follows with Hga I.5 base protruding terminus differences of different fragments.An enzyme can generate suitable fragment with the gene of some amount.By using several enzymes in dividing other reaction, to shear cDNA or genomic dna, each goal gene can be done suitably and shear.In one approach, the DNA that sheared sieves with size.The dna fragmentation of (also optional with exonuclease III processing, this enzyme can excise Nucleotide one by one from 3 ' end, and terminal length and the specificity of increase) preparation like this can be suspended in test tube or the porous plate.From the DNA joint of the less one group variable protruding terminus with common ground and suitable length, be that each gene fragment that need increase is selected pair of joint.These joints are connected, utilize universal primer to be PCR then.Can generate 100 ten thousand butt junctions by 1000 joints, thus under identical condition, a pair of universal primer of common terminal complementary of utilization and joint, 100 ten thousand different fragments specifically can increase.

If repeat to find a DNA difference in several patients, and this sequence variation is nonsense, perhaps can change the function of corresponding protein, and the gene of sudden change may be exactly the reason of disease.By analyzing the individuality that special proterties is arranged in a large number, the variation of function allelotrope and the specific trait of specific gene can be linked together.

This method makes needn't carry out gene mapping fully to a large amount of pedigrees, and when not having this class genetic data or information, this method more has special value.

Embodiment 21

Single nucleotide polymorphism in the marker gene collection of illustrative plates

Disclosed technology is applicable to and identifies the genomic fragment with single nucleotide polymorphism (SNUPs) effectively among the application.In 10 individualities, described order-checking process is applied to the known genomic fragment of a large amount of sequences (can through clone or amplification in vitro technology these fragments that increase), can identify the dna fragmentation with SNUPs of q.s.Further with these polymorphic bandses as the SNUP mark.These markers or former just by mapping (for example their represent the STSs that has been mapped) perhaps can be mapped by the screening process of the following stated.

Be arranged in the array of forming by inferior array by the amplification label thing and with them, in the future each individual SNUPs scoring of autocorrelation family or colony.Inferior array comprises the identical marker that obtains from tested individual amplification.For each marker, known mutations is the same with analyzing, and gives two allelotrope each one group positive probe of selecting and mark respectively, and every group is 6 or still less.Utilize the obvious relation between persistence of 1 or 1 group echo and disease, can determine the position of genes involved on karyomit(e).Because it is efficient and at a low price, can obtain thousands of mark of thousands of individuality.This data volume makes resolving power to an assignment of genes gene mapping less than 100 ten thousand bp, and can locate the gene that participates in multigenic disease.By being checked order in the specific region of coming autocorrelation normal individual and diseased individuals, can identify that the gene that is positioned is so that the scoring sudden change.

Preferably use the marker of pcr amplification from genomic dna.Each marker all needs a pair of special primer.Existing mark can be changed, and perhaps can shear genomic dna by using HgaI type restriction enzyme, and the connection pair of joint prepares new mark.

The SNUP mark can be increased, or point sample becomes the storehouse collection so that reduce the number of times of independent amplified reaction.In this situation, each sample all has more probe to be marked.When having compiled 4 markers and o'clock on 12 parts of duplicating films, can having obtained 48 probes (12 of each marks) after 4 circulations.

Embodiment 22

The detection of dna fragmentation identity and affirmation

Through the dna fragmentation that restricted shearing, clone or amplification in vitro (as PCR) obtain, can in single test, be identified usually.Can identify fragment by the DNA band of confirming specific size in the gel electrophoresis.Selectively, can prepare special oligonucleotide, be confirmed examining the DNA sample by hybridization.The step of Jian Liing can more effectively be identified a large amount of samples herein, and need not to be the special oligonucleotide of each produced in fragments.On the basis of known array, from each segmental general probe, filter out one group of positive and negative probe.The positive probe that filters out can form 1 or several eclipsed group usually, and negative probe intersperses among in the whole insertion sequence.

In the process to the STS mapping on yac clone, this technology can be used for identifying STS.On several storehouses of about 100 yac clones or yac clone, detect each STS.Can be in an inferior array with DNA o'clock of this 100 reactions.Different STS may represent the inferior array of successive.In several hybridization circulations, each DNA sample all can produce a sign, and this sign enough proves or negates to have specific STS in specifying yac clone.

In order to reduce the number of times or the sample number of putting of independent PCR reaction, several STS that can in a reaction, increase simultaneously respectively, or with the PCR sample mix.In this case, each point must have more multiprobe accept scoring.The set of STS does not rely on and compiles YAC, can be used for single YAC or YAC storehouse collection.When mark the probe of different colours when hybridizing together, this system is especially attractive.

Except having certain dna fragmentation in the proof sample, can also utilize the intensity for hybridization of several independent probes or probe sets to estimate the amount of DNA.The intensity of gained intensity and DNA being measured known control sample compares, and determines the DNA amount in all point sample samples simultaneously.Because the identification of dna fragment only needs a small amount of probe, and N the long DNA of base can have N possible probe, so this application does not need enough to identify any dna fragmentation once big group probe.For the fragment of a 1000bp, on average can select 30 probes of coupling fully from 1000 8 base sequences.

Embodiment 23

Identify communicable disease biology and their mutation

Detect the intravital virus of patient, bacterium, fungi and other Parasites based on DNA, more more reliable and cheap than other method usually.The major advantage of DNA detection is to identify special strain and mutation, and finally can carries out more effective treatment.Two examples are described below to be used.

By 12 the known antibiotic resistance geneses that increase, detect in infectation of bacteria, whether there are these genes.Can with from 128 patients' amplified production o'clock in 2 inferior arrays, on 8 * 12cm film, 24 of 12 genes can be repeated 4 times by inferior array then.To each gene, select 12 probes to make positive and negative mark.Carry out 3 round-robin hybridization.One group of most likely much smaller probe of general probe in these tests.For example, for the fragment of a 1000bp, in 1000 8 base sequences of a cover, average 30 probes are male, and common 10 probes are just enough identified highly reliably.As described in embodiment 9, can increase several genes also/or point sample simultaneously, and can determine the amount of specific DNA.The amount of amplification gene can indicate gradient of infection.

Another example comprises and may the gene or the whole genome of HIV virus be checked order.Because virus changes rapidly, give and select suitable methods of treatment to cause a lot of difficulties.Can remove amplification of DNA fragments by isolated viral, and utilize described process to check order again from 64 patients.On the basis of the sequence that obtains, can select the optimal treatment method.If the virus of two types is arranged mixes mutually, one of them contains basic sequence (situation of similar heterozygote), by quantitative comparison is made in the hybridization scoring of mutant and the hybridization scoring of other sample (control sample that especially and only contains basic virus type), can confirm mutant.If one of two Virus Types morph in certain site in the sample, the scoring that covers 3 to 4 probes in this site has only 1/2 of other sample.

Embodiment 24

The science of law is identified and relationship is identified

Sequence polymorphism has nothing in common with each other genomic dna.Can analyze the blood or other body fluid or the tissue that obtain from the crime scene like this, and compare with suspect's sample.The pleomorphism site mark of sufficient amount is got off, form the uniqueness sign of sample.Thereby SBH can be very easy to the polymorphism of ground mark mononucleotide and form this sign.

One group of dna fragmentation (10-1000) of sample and suspect can be increased.Will be on one or several inferior array from segmental DNA point of representative of sample and suspect, each inferior array is replicated 4 parts.In 3 circulations, 12 probes can determine whether each DNA site exists allelotrope A or B in every duplicate samples (comprising suspect's).Sample and suspect's pattern is mated, can find the suspect.

Can prove with same process or negative father and mother and child's sibship.From children and adult preparation DNA and amplification polymorphism locus; Can determine A or the allelic pattern of B by hybridization separately.The pattern that is obtained is compared with the positive and negative control, can help to determine family relationship.In this case, only need allelic integral part and father and mother can confirm once side's coupling.The numerous mistake of statistics that can avoid in the program of marker gene seat number, or once more sudden change cover effect.

Embodiment 25

Assessment population or the gene diversity of species and the species diversity of ecologic niche

To a large amount of locus (for example, several genes or whole Mitochondrial DNA) on the allelic variation frequency detect, caused setting up dissimilar conclusions, such as more such conclusions, they relate to environment to genotype, to the historical and evolution of population or to the population susceptibility, to extinction, and other influence.Can carry out these assessments by detecting specific known allelotrope, perhaps by some locus are checked order completely again, order-checking can be determined the sudden change once more of gene again, and the latter can disclose mutagenic compound and the slight change in the environment.

In addition, check order again, just can examine or check the species diversity in the microorganism world by dna sequence dna (as the gene of ribosome-RNA(rRNA) or the proteinic gene of high conservative) to evolution conservative.Can be from environment and prepare DNA with the specific gene of the corresponding primer amplification of conserved sequence.Preferably dna fragmentation is cloned on the plasmid vector (or be diluted to a such level with it: contain 1 molecule in each hole on the porous plate, carry out amplification in vitro then).The preface of can resurveying according to the clone that previously described mode will prepare like this.Thereby obtain two category informations.At first, can obtain catalogue not of the same race and individual density in each.A part of in addition information can be used for detecting ecological factor or pollute Ecosystem System Influence.This will disclose whether pollute the extinction that has caused some species, and perhaps the relative abundance between species is changed.This method can be applied to the dna sequencing in the fossil equally.

Embodiment 26

To the detection of nucleic acid species or quantitatively

Utilize a probe to can detecting and quantitative analysis DNA or RNA thing class, this probe is to comprising that one is fixed on unmarked probe on the matrix and the label probe in solution.Under the situation of underlined probe and ligase enzyme,, these thing classes can detect in the unlabelled probe with quantitative by being exposed to.Especially, the probe by linkage flag probe on the sample nucleic acid main chain and unmarked probe obtain extending forms the thing class that extension probes just indicates existence to detect.Therefore, remove not the label probe that connects after, if there is marker in the special site of the array on the matrix, just show there is a sample thing class that the amount of marker is indicated the expression level of this thing class.

Selectively, earlier with 1 or a plurality of unlabelled probe be arranged on the matrix, and with 1 or a plurality of label probe import in the solution.According to a kind of like this method, promptly utilize and can distinguish fluorescigenic dyestuff under the wavelength, can make the probe variation on the array.Utilize this mode, be added to cDNA mixture on the array, determine whether to exist this cDNA thing class and expression level thereof with being specific to the mark of determinand class and unmarked probe in detecting.According to an embodiment preferred, can measure the partial sequence of cDNA in this way by selecting to contain have the unmarked and label probe of overlap right with tested cDNA.

Special pathogen is genomic to be existed and quantity to detect can to select probe, and this is right by add the probe of selecting in composition, and this probe is to only combination in purpose pathogenic genes group individuality.Just, though it is right not to be specific to certain probe of pathogen gene group, the right combination of probe is special.Equally, in the detection or order-checking of cDNA, this thing happens possibly: particular probe is special for the thing class right and wrong of a cDNA or other type.Yet, can decide the existence and the quantity of Special Thing class by such result, the combination that promptly is positioned at the selection probe on the array position of a uniqueness shows the existence of a Special Thing class.

Without polymerase chain reaction (PCR) or other purpose amplification procedure, only just can detect an infectious medium that has 10kb or bigger DNA with an immobilized detection chip.According to other method,, can analyze the genome of the infectious medium that comprises bacterium and virus by through the single target nucleic acid sequence of pcr amplification and utilize the existence of the special label probe of target sequence being hybridized to come the testing goal gene.Because this analysis only has specificity to single target sequence, therefore must utilize such as these method amplification genes of PCR, provide enough target sequences so that provide the signal that can survey.

According to present embodiment, it provides improving one's methods of a peculiar nucleotide sequence that utilizes Format 3 types to react to detect infectious medium, wherein to prepare a solid phase detection chip, this chip contains the array that is made of multiple different immobilized oligonucleotide probe, and probe has specificity to the infectious medium of being studied.One point comprises the mixture of being made up of with the unmarked probe of target nucleic acid complementary many, and it makes the special marker of something class is concentrated in a position, thereby higher than the susceptibility of diffusion or single probe mark.This multiprobe may be the overlap of target nucleic acid sequence, but also may be non-overlapping sequence, can also be non-conterminous.5-12 Nucleotide of these probe preferably approximatelies is long.

Nucleic acid samples is added probe array, the target sequence in the sample will with a plurality of fixed probe hybridizations.Select a group echo multiprobe, they can be combined on the target sequence adjacent with stationary probe specifically, then they are added on the array of unmarked oligonucleotide probe mixture with sample.Ligase enzyme is joined the adjacent probe that connects in the chip on the sample.Rinsing detection chip then has or not marker to determine whether to exist sample nucleic acid to remove not hybridization and probe that is not connected and sample nucleic acid by detection.This method can provide reliable result, and the volumetric molar concentration of its sample media that uses has reduced by 1000 times.

Another aspect of the present invention, can enlarge the signal that label probe produces by some means, as adding a general tail for free probe, this tail contains a plurality of product pigments, enzymatic or radioactive marker, perhaps itself easily by the another one multiple labelling the combination of Probe medium specificity.Just can carry out partial signal in this way amplifies.Can applying marking or unlabelled probe when amplify the second stage.In amplified this second stage, a length dna sample that has multiple labeling can cause strength of signal to amplify 10-100 doubly, and this can make signal amplify 100,000 times altogether.By utilizing two aspects of present embodiment, needn't use PCR or other amplification step, the strength of signal near 100,000 times just can obtain the positive findings that probe one DNA connects.

According to another aspect of the present invention, can prepare an array or a super array that comprises a whole set of probe (for example probe of 4096 6 base sequences).This array can be used to any nucleic acid species detect or carry out part to completely the order-checking, they are general in this sense.A single point on array may comprise the probe of a thing class or the mixture of probe, for example in a reaction mixture of synthetic N (1-3) B (4-6) N (1-3) type (N represents 4 kinds of all Nucleotide, B represents a kind of special Nucleotide, the scope of relevant numeral base number, as, 1-3 represents " 1 to 3 base ").By the signal of different piece on the molecule of collecting same longer nucleic acid thing class, these mixtures can provide stronger signal for the nucleic acid species of lower concentration.Can the probe groups that this is general be divided into many subgroups, these subgroup points become the array by the separated unit of some barriers, and barrier wherein can prevent that the hybridization buffer that contains sample and label probe from spreading.

When a known nucleic acid species of sequence is detected, select a kind of more sequence of number Nucleotide that contains, comprise the label probe in unlabelled stationary probe and the solution.Label probe can be synthetic or be selected from a whole set of base sequence (as 7 base sequences) of synthetic in advance.Label probe is added in the unit array of corresponding stationary probe.A pair of like this fixing with label probe will be hybridized at close position with target sequence, in case add ligase enzyme, probe is just covalently combined.

If unit array comprises 1 above stationary probe (be in the difference part respectively or be positioned on the same point), described probe is positive in given nucleic acid species, all corresponding label probes can be mixed so to join in this unit array.When what detect is to mix nucleic acid thing time-like, label probe is mixed just seem particularly important.The example of the nucleic acid species mixture of a complexity is a mRNA in the cell or tissue.

According to one embodiment of the invention, the unit array of stationary probe make can each is possible stationary probe use with the mixture of the label probe of comparatively small amt.If implement a multiple labelling scheme, can use the mixture of more complicated label probe.Preferred multiple labelling method can be used different fluorescence dyes or can carry out isolating molecular marked compound with mass spectrograph.

Selectively, according to a preferred embodiment of the present invention, select some short stationary probes, the common and many nucleic acid array hybridizings of these probes.The probe that these are short and the mixture of label probe are used in combination, and preparing these label probe mixtures is to have a label probe at least corresponding to each stationary probe in order to make.Preferred mixture is those wherein mixtures of not corresponding with a plurality of stationary probes label probe.

Embodiment 27

Utilize the fragment of all possible 10 base sequences inquiry HIV virus

In the embodiment of this SBH form III, go up array that combines all possible 5 base sequences (1024 5 possible base sequences) of preparation at nylon membrane (as Gene Screen).Utilize 5 ' end of 5 '-TTTTTT-NNN-3 ' (G, T in this step of synthetic, waits mole to add all 4 kinds of bases for all 4 kinds of base A of N=, C), synthetic bonded 5 base sequence oligonucleotide.The accurate place of these oligonucleotide on nylon membrane, after air-dry, is fixed oligonucleotide with UV treatment with air-dry point.Make the density of oligonucleotide reach 18 oligonucleotide of every square nanometers in this way.After the UV treatment, handle nylon membrane in 60-80 ℃ with the stain remover that contains damping fluid.The oligonucleotide point is divided into the inferior array that 10 row 10 are listed as, and each inferior array has point and 36 control points of 64 5 base sequences.16 inferior arrays have 1024 5 base sequences, and it has comprised all possible 5 base sequences.

Utilize physical barriers, for example hydrophobic band is separated each the inferior array in the array, in the time of can avoiding the hybridization of each inferior array and sample like this, with the crossed contamination of adjacent inferior array.In a preferred embodiment, hydrophobic band is that (this solvent is well known in the art) the silicon cave solution (for example, ordinary silicon cave glue and sealing compound) that is used in the suitable solvent is made.Form line with this silicon cave lipoprotein solution between inferior array, hydrophobic of cell separated in the conduct after solvent evaporation of this line.

In the embodiment of this Format III, 5 base sequences of free or dissolved (uncombined) are to utilize 5 '-NN-3 ' (all 4 kinds of base A of N=, C, G, 3 ' terminal synthetic T).In this embodiment, free 5 base sequences and bonded 5 base sequences are combined generates all possible 10 base sequences, and this 10 base sequence is for the known dna below the 20kb is checked order.The double-stranded DNA sex change of 20kb is formed the single stranded DNA sequence of 40kb.The ssDNA of this 40kb and 4% institute might 10 base sequences hybridization.Feasible pending free or dissolving (uncombined) 5 base sequences can being converged of 10 base sequences and known target sequence bonded low frequency is used to handle each inferior array, and do not lose sequence information.In a preferred embodiment, 16 probes are merged in each inferior array, and all possible 5 base sequences are present in 64 set that contain free 5 base sequences.Like this, utilize 1024 inferior arrays (set of each free 5 base sequence has 16 inferior arrays) just can generate all possible 10 base sequence probes at a DNA sample.

In this embodiment, target DNA is represented the segment of 2 600bp of HIV virus.The fragment of representing these 600bp with 60 eclipsed 30 base sequences (each 30 base sequence and adjacent 30 base sequences have the overlapping of 20 Nucleotide).Target DNA of the set of 30 base sequences simulation, this DNA through techniques well known shearing, digestion and/or random PCR handle and generate one very little pulsating with hangar.

As described in the embodiment of the Format III of front, wait 5 base sequences of marked free with radio isotope, vitamin H, fluorescence dye.Free 5 base sequences of mark with hybridize and be connected with target DNA together in conjunction with 5 base sequences.In a preferred embodiment, the ligase enzyme that in reaction system, adds 300-1000 unit.Embodiment according to the front determines hybridization conditions.After connecting and removing target DNA and excessive free probe, (utilizing the technology of describing among the embodiment of front) analyzes the position that array is determined label probe.

Known dna sequence in the target DNA, and known free the reaching in conjunction with 5 base sequences in each inferior array indicating which will be connected on free 5 base sequences of mark in conjunction with 5 base sequences in each inferior array.Along with the variation of each target DNA, will lose from the signal of 20 future positions, and obtain 20 new signals by forecasting sequence.In these 10 new points, determined in each new point that in conjunction with the overlap of 5 base sequences which free mark 5 base sequence is combined.

Utilize aforesaid method, array and the set of free mark 5 base sequences, detect the dna sequence dna of HIV with all possible 10 base sequences.Utilize the method for this Format III, we can correctly identify tested pulsating " wild-type " sequence, can identify those sequences " sudden change " of introducing on these segments equally.

Embodiment 28

The order-checking of reiterated DNA sequences

In one embodiment, in an improved Format III method, the reiterated DNA sequences in the target DNA is checked order with " transcribed spacer oligonucleotide ".The transcribed spacer oligonucleotide of the different lengths on the reiterated DNA sequences (having determined tumor-necrosis factor glycoproteins) and target DNA, first known adjacent oligonucleotide and second at first round SBH known or one group may hybridize by adjacent oligonucleotide (SBH learns from the first round) with the other side of transcribed spacer.When a transcribed spacer that coincide with the repetition DNA fragment length was hybridized with target sequence, two adjacent oligonucleotide can be connected on the transcribed spacer.If first known oligonucleotide sequence is fixed on the matrix, and second known or possible oligonucleotide sequence is labeled, so when the transcribed spacer of a suitable length and target DNA hybridization, will form a bonded and connect product, this product comprises second the known or possible oligonucleotide that is labeled.

Embodiment 29

Utilize FORMAT 3 SBH to check order by tapping point

In one embodiment, utilize the 3rd group of oligonucleotide sequence and improved form III method, the tapping point in the target DNA is checked order.Behind the first round SBH, may identify some tapping points during the layout sequence.This problem can solve like this, hybridize by the oligonucleotide that overlaps with one of known array that causes tapping point, and then hybridize with an other oligonucleotide target sequence, this oligonucleotide be labeled and one of the sequence of stretching out with tapping point corresponding.After suitable oligonucleotide and target DNA hybridization, the oligonucleotide that is labeled can be connected with other oligonucleotide.In a preferred embodiment, select first kind of oligonucleotide, it is formed branch by the Nucleotide at one or several tapping point place (can discern a branched sequence like this), and second kind of oligonucleotide also is selected, and it is initial and read in the tapping point sequence from first kind of oligonucleotide.Select one group of the third oligonucleotide, the corresponding all possible branched sequence of this oligonucleotide, and have one or several Nucleotide (corresponding) overlapping with first oligonucleotide with the tapping point sequence.These oligonucleotide and target DNA are hybridized, have only the third oligonucleotide that has suitable branched sequence (it is complementary with the tapping point of first oligonucleotide) to produce and be connected product with first, second oligonucleotide.

Embodiment 30

Be used to analyze the multiple probe of target nucleic acid

In the present embodiment, with different marker label probe groups, therefore, each probe in the group can both differentiate with other probe.Like this, this group probe can contact in same hybridization with nucleic acid and can not lose any detecting probe information.In a preferred embodiment, different markers is different radio isotope, or different fluorescent marks, or different EMLs.These probe series can be used for form I, II or the III of SBH.

In Format I SBH, the probe of one group of distinctive mark is hybridized with the target nucleic acid that is fixed on the medium, and used hybridization conditions can be distinguished the mispairing of mating fully and having only a base pair.The specific probe that is connected with target nucleic acid can be identified by they different marks, and has determined coupling fully at least in part by this link information.

In Format II SBH, hybridize with the different probe tagged target nucleic acid and with probe array.Discern and probe bonded specific target nucleic acid by its different markers, and determine the coupling fully of nucleic acid at least in part by these combining informations.

In FormatIII SBH, the probe of one group of distinctive mark and stationary probe and target nucleic acid are hybridized, and hybridization conditions can be distinguished the mispairing of mating fully with a base pair.Label probe at a contiguous stationary probe on the target nucleic acid is incorporated on the stationary probe, detects and distinguish this product by their not isolabeling.

In a preferred embodiment, the distinctive mark thing is EMLs, can utilize electron capture mass spectrograph (EC-MS) that it is detected.Can be by multiple backbone molecule, certain preferred some fragrant chain prepares EMLs, as referring to Xu etc., J.Chromatog.764:95-102 (1997).EML reversibly and stably is connected on the probe, after the hybridization of probe and target nucleic acid, removes EML from probe, and the EC-MS that utilizes standard to its identify (as, can detect EC-MS by Gas Chromatography-mass Spectrometer (GCMS)).

Embodiment 31

Detect the low frequency target nucleic acid

SBH Format III has enough resolving abilities that such Sequence Identification is come out, and this sequence and it the only similar sequence of the difference of a Nucleotide exist with 1 to 99 part in sample.Therefore, can identify the extremely low nucleic acid of concentration in the nucleic acid samples, for example a sample that derives from blood with Format III.

In one embodiment, these two sequences are sequences of decision cystic fibrosis, and difference is that one of them has lacked 3 Nucleotide.The probe of these two sequences is to be fixed on the matrix, can distinguish the probe of absence type and wild-type, and the two common be labeled in abutting connection with probe.Utilize these target sequences and probe, can detect a deletion mutantion that is present in 99 wild-types with SBH FormatIII.

Embodiment 32

Be used to analyze the polarizer device and the method for target nucleic acid

Can the device that the blended material comes tectonic analysis nucleic acid take place with two nucleic acid arrays and optional a kind of nucleic acid of two arrays that can stop before needs.Can be with the array in a series of matrix supportive devices, matrix includes but not limited to nylon membrane, nitrocellulose filter or other above disclosed material.In preferred embodiments, a kind of matrix is a kind ofly to be divided into the film of sub-district by hydrophobic band, or another foraminate upholder, can clog gel or sponge in the hole.In the present embodiment, probe is placed in the sub-district or aperture of film, is added to gel or sponge and a kind of solution (having or do not have target nucleic acid) on the film or in the hole, can dissolves probe like this.This solution that is dissolved with probe is contacted with the nucleic acid of second array.Nucleic acid can be, but to be not limited to be oligonucleotide probe or target nucleic acid, can be with probe or target nucleic acid mark.Can include but not limited to radio isotope with any this area marker commonly used, fluorescent mark or electrophoresis mass labels come labeling nucleic acid.

To stop the mixed material of nucleic acid to be placed between two arrays, its modes of emplacement guarantees that the nucleic acid of two arrays will be mixed in together after this material is removed.The form of material can be sheet, film or other barrier form, and this material can be made of any nucleic acid blended material that can stop.

Can use this device like this in SBH Format I: first array of device contains the target nucleic acid that is fixed on the matrix, second array of device has nucleic acid probe, these probes are labeled, thereby and can remove the target nucleic acid on first array is inquired about.Optional these two arrays are separated by layer of substance, this material can stop connecing of probe and target nucleic acid to be melted, and after removing this layer material, probe just can interact with target nucleic acid.After suitable cultivation and (choosing wantonly) rinse step, can " read " to go out the target nucleic acid array and can mate fully with target nucleic acid to measure which probe.Thisly recognize that to read can be automatization, also can be artificial (as, by naked eyes identification autoradiogram(ARGM)).In SBH Format II, the similar previously described process of process, just target nucleic acid is labeled and probe is fixed.

Selectively, can this device of following use in SBH FormatIII: form two nucleic acid probe array,, and one of them array can be fixed on the matrix with two arrays or the labeled nucleic acid probe of one of them.Can stop the mixed material of probe that two arrays are separated with one deck.By adding target nucleic acid and removing the blocking layer so that two kinds of probes and target nucleic acid mix mutually and start Format II and react.The probe that is attached to the consecutive position on the target nucleic acid is connected in together (for example by the base stacking interaction or by being connected with the covalent linkage of main chain), reads the result and links to each other with target nucleic acid in contiguous site to determine which probe.After one group of probe is fixed on the matrix, can reads that array probe of fixed and determine which probe and stationary probe in another array link together.Identical with top method, this reading method can be automatically (as, utilize the ELISA counter) or artificial (as utilizing the visual inspection autoradiogram(ARGM)).

Embodiment 33

Three-dimensional probe array

In a preferred embodiment, oligonucleotide probe is fixed in the cubical array.Cubical array comprises many layers, and each layer can independently and break away from other layer to be analyzed, and perhaps all layers of cubical array are analyzed simultaneously.Cubical array comprises that for example, a kind of array that is placed on the matrix has a plurality of depressions on the matrix, and probe is positioned at the different depths (each aspect is made of the probe that is positioned at the similar degree of depth of depression) of these depressions; Perhaps be placed in the array on such matrix, described matrix has the depression of different depths, and probe is positioned at the bottom of depression, the peak of separating depression or the joint portion (each aspect is made up of all probes that are in certain depth) of peak and depression; Perhaps be placed in the array on the matrix of being made up of many platy layers, these platy layers form cubical array.

The material that is used for synthetic these cubical arraies is known in the field, comprising previously mentioned some materials that also are fit to do the upholder of probe array of this specification sheets.In addition, other some can be supported the suitable material of oligonucleotide probe, and preferably flexible material also can be used as matrix.

Embodiment 34

CDNA clone's bunch signal processing

Utilize Standard PC R, the analysis of SBH sequence signal and mulberry lattice sequencing technologies, obtain many different nucleotide sequences by the cDNA library.By the insertion sequence in pcr amplification library, used the special primer of the carrier sequence of insertion sequence both sides in the amplification procedure.These samples are put on nylon membrane and with the oligonucleotide probe of proper amt to be inquired about, and measures the concentration of positive bonding probes, thereby sequence signal is provided.With the clone be gathered into have similar or identical sequence signal bunch, from each bunch, select a representational clone and carry out gel order-checking.In a typical mulberry lattice order-checking flow process, utilize counter-rotating M13 sequencing primer to infer the pulsating 5 ' terminal sequence of the insertion of increasing.With the PCR product purification, and carry out fluorescence dye termination cycle sequencing.Do the order-checking of single passage gel with 377Applied Biosystems (ABI) sequenator.Great majority clone through this method selection and order-checking has the sequence that has nothing in common with each other, and seldom has identical sequence.

Embodiment 35

High yield is produced chip

In a preferred embodiment, a kind of device that is used for the mass production probe array may comprise the drum or the dish of a rotation, and it is connected with an inkjet deposited device, (as droplet amount head); With a suitable automation system, anorad gantry for example.A particularly preferred embodiment about this device will cooperate Fig. 1-3 to be described.

Device comprises a cylinder (1), is combined with suitable matrix on it.This matrix can be any matrix that is applicable to probe array described above.In a preferred embodiment, matrix is a kind of flexible material, and array directly generates on matrix.In selectable embodiment, flexible matrix is combined on the cylinder, and one chip is fixed on the matrix.On each independent chip, form array then.

In a preferred embodiment, add that on matrix or chip physical barriers limits a hole array.Can this physical barriers be added on matrix or the chip use device, or alternately, not be fixed on the last physical barriers that just added before of cylinder (1) as yet at chip or matrix.Then single oligonucleotide probe point is placed in each aperture, all probes of being placed in each hole may all have identical sequence, perhaps have different sequences.In a preferred embodiment, the probe of array mid point in each independent hole is with to put in array the probe in other hole different.The sequence testing chip that just can amalgamation contains poly array by these arrays.

Matrix or matrix and chip are fixed to after cylinder (1) goes up, and an engine (not shown) is with rotary cylinder.By the known technology of this area, for example comprise, utilize a fixed sight sensor to reach the light source that rotates with cylinder, can accurately measure the velocity of rotation of cylinder.The precise rotation that calculates more than the utilization, a skimmer (3) moves along arm (2), probe or other reagent can be added to exact position on matrix or the chip by separatory rifle head (8).Skimmer adds pipe (7) by stream and receive probe or reactant from memory (6).The necessary probe of manufacturing array and other reagent are housed in the memory (6).

Described skimmer among Fig. 3.Skimmer can have 1 or a plurality of separatory rifle head (14 and 8).Sample well (13) on the corresponding main body of each separatory rifle head (12) receives probe or other reagent by sample hose (10).Penstock (11) pressurizes to reach 1 pound/inch to cell (9) ², so that probe or reagent flow through separatory rifle head (14 and 8).When each conversion probe or reagent, all necessary lavage specimens QC (10), hole (13) and separatory rifle head (14 and 8).Supply suitable rinsing damping fluid in sample well (13) by sample hose (10) or optional special rinsing tube (not shown), perhaps choose wantonly the rinsing damping fluid is filled it up with in a part or the whole space of chamber.When needing, the rinsing damping fluid is shifted out from sample well and chamber by a vent pipe (not shown) or by sample hose (10) and separatory rifle head (14 and 8).

After utilizing the separatory method that probe is added to all suitable sites on each array or the chip, remove in the cylinder matrix (having or do not have chip) and in conjunction with last new matrix.

Embodiment 36

Utilize and discrete particles compound probe analysis target nucleic acid

In this embodiment, inquire about target nucleic acid with probe, these probes compound (covalently or non-covalently) many discrete particles.Can these particles dispersed differences be come based on the difference of its physics character (or a plurality of physics character), the particle with different physics character is mutually compound with different probes.In a preferred embodiment, probe is a sequence and the known oligonucleotide of length.Therefore, utilize each particulate different physical properties just probe can be identified out.The probe that is applicable to this embodiment is included in described all probes of previous section, comprises probe shorter than total length probe those are on the meaning that information is provided.

The physical property of discrete particles can be anyly can make the characteristic that they are divided into group, and these characteristics are widely known by the people in this area.For example, can be based on their size, fluorescence, absorbancy, electromagnetism electric charge or weight, perhaps whether particle can be labeled dyestuff, radionuclide or EML they are divided into group.Some other suitable mark comprises the part that can be used as specific junction mixture, but these binding substances bonding mark antibody, chemoluminescence agent, enzyme, can and the antibody of tagged ligand specific combination, or the like.In the immunoassay that many markers are applied to easily adopting already.Other some marks comprise antigen, the group and the detectable part of electrochemistry of particular reactive are arranged.Also have some other mark, be included in any mark that previous section is mentioned.These marks and characteristic can be carried out detection by quantitative to it with the common method in this area, comprise for example described method of previous section, and can distinguish particle (for example can use different dye densities or different dye type as a mark) based on detection signal strength or signal type for same particle.In a preferred embodiment, several physical propertys are combined, thus utilize different property combination come distinguishing particles (as, 10 kinds of sizes and 10 kinds of colors combine can distinguish 100 kinds of particle colonies).

Utilize particle-probe can develop the standard combination method, therefore, for example can utilize about 2000 reaction vessels to synthesize all possible 10 base sequences.Carry out first group 1024 and be reflected at synthetic all possible 5 base sequences on 1024 different marking particles.Probe-the particle that produces is mixed, and divide equally in other one group of 1024 reaction vessel.Carry out second group reaction with these samples, the extension products of synthetic all possible 5 base sequences on the probe in the particle set.Utilize physics character to identify preceding 5 Nucleotide of each probe, utilize reactor to identify the feature of 5 Nucleotide in addition of each probe.Thus, utilize 2048 reactors to synthesize all possible 10 base sequence probes.Be easy to this method is improved so that synthesizing all possible n base sequence in the probe length on a large scale.

In a preferred embodiment, according to the particulate fluorescence intensity they are divided in groups.Fluorescent mark with different concns prepares every group of particle, and particle has different fluorescence intensities thus.The fluorescence intensity of fluorescein and its concentration were at 1: 300 to 1: 300, relevant in the scope of 000 (Lockhart etc., 1986), at 1: 3000 to 1: 300, in 000 the concentration range, linear (so fluorescein intensity is linear in the scope of about 1-300).In linear detection range, the usefulness fluorescein (as, 3-259) 256 groups of particles of mark.256 groups of particles can make all possible 4 base sequences be connected to not on the same group the particle.By particle is compiled, possessed 4 contain might 4 base sequences set, utilize A then, G, C, or T extension probes in each set just can form all possible 5 base sequences.Similarly, obtain 16 might 4 base sequences set, by each set being carried out two base (A, G, one of 16 kinds of arrangements of two bases of C and T) extension, (7 base sequences need 64 set can to obtain all possible 6 base sequences, 8 base sequences need 256 set, or the like).

Probe (in 4 set) with 5 base sequences is inquired about a target nucleic acid.This target nucleic acid carries out mark with another fluorescence dye or other different markers (as mentioned above).Target nucleic acid and 4 set of being labeled are mixed, and complementary probe and target nucleic acid in each set are hybridized.Utilize method well known in the art to detect these hybridization mixtures, identify positive hybridization probe by detecting each particulate fluorescence intensity then.In a preferred embodiment, the mixture that makes probe-particle and target nucleic acid is by in the flow cytometer or in other the separate apparatus, every next particle, thus detect the particulate mark and which probe target nucleic acid determines be and the target nucleic acid complementary.

In a selectable embodiment, with another kind of fluorescence dye or one group of free probe of other mark (as mentioned above) mark, the free probe that each is independent mixes with each 5 base sequence probes set (4 set), and mixture and target nucleic acid are hybridized.When free probe is attached to a site on the target nucleic acid, during the binding site of this site and 5 base sequences adjacent (site of free probe must with the 5 base sequence probes that can be connected terminal adjacent), add a kind of medium and make dissociate probe and 5 base sequence probe covalent attachment (with reference to the description of the relevant suitable media of previous section).Utilize the known method of this area that particle is analyzed, detect which particle (particle that promptly has free probe mark) with free probe covalent attachment, and utilize the particulate fluorescence intensity to identify 5 base sequence probes.In a highly preferred embodiment, the mixture that makes probe-particle, free probe and target nucleic acid is by a flow cytometer, every next particle, and the mark that detects particulate mark and free probe is determined which probe and target nucleic acid complementation.

In preferred embodiments, analyze target nucleic acid with probe-particle composites, all or most operating in the independent device just can be finished.This device has one or more reagent chamber, with the target nucleic acid of damping fluid and mark thorough mixing (can manually add or add automatically target nucleic acid) therein.Mixture is diverted to a plurality of reaction chambers from reagent chamber, and each reaction chamber has a probe particle composites collection.Probe particle and target nucleic acid react under certain conditions, and this conditions permit complementary probe combines with target nucleic acid.From reaction chamber, remove (as by rinsing) excessive target nucleic acid, promptly unconjugated nucleic acid, and utilize mark and particulate on the target nucleic acid to get in touch evaluation to be combined in particle on the target nucleic acid.Utilize the particulate physics characteristic to identify probe.In a preferred embodiment, remove excessive target nucleic acid after, particle passes through duct and arrives detector with single-row from reaction chamber.When one particle passes through detecting instrument, these instruments will detect the mark and the particulate physics character of target nucleic acid.In a selectable preferred embodiment, before or after removing excessive target nucleic acid, for example by size (as exclusion chromatography), electric charge (as, ion exchange chromatography), a kind of and/or in these physics character of density-weight or all particle is divided in groups.Utilize detecting instrument to analyze the particle that these are distributed then.

In a selectable embodiment, damping fluid, target nucleic acid, probe-particle composites collection are arranged in the reagent chamber, with a chemistry or enzymatic be connected reagent.These compositions of thorough mixing are being distributed to them many reaction chambers from reagent chamber then.Each reaction chamber all has the free probe of a mark.Selectively, this probe-particle composites set and the probe that dissociates are placed in the reaction chamber together, and will they add reagent chamber.In addition, the probe that dissociates can be added reagent chamber, probe-particle set can join reaction chamber.Probe-particle, target nucleic acid and free probe react under certain condition, and free probe of this conditions permit and particle probe combine with adjacent site on the target nucleic acid, and therefore, free probe is connected on probe-particle.From reaction chamber, remove (as, by rinsing) excessive free probe (that is, not connecting) and target nucleic acid.Utilize free probe mark to get in touch the probe that detection has been connected, and utilize particulate physics character to identify and particle compound probe with particulate.In a preferred embodiment, remove excessive probes and target nucleic acid after, particle passes passage and arrives detecting instrument from reaction chamber is single-row.When individual particle when the detecting instrument, this instrument will detect with the covalency form and be associated in free probe mark and particulate physics character on the particle.In a selectable preferred embodiment, remove before or after excess probe and the target nucleic acid, utilize the particulate physics characteristic, as size (as exclusion chromatography) by them, electric charge (ion exchange chromatography), and/or density/weight is with its grouping.Utilize the good particle of detecting instrument analysis of allocated.

In a preferred embodiment, one group of second reaction chamber arranged in the instrument, the set of probe particle is placed in second reaction chamber.Target nucleic acid and damping fluid is mixed in reagent chamber, inject first reaction chamber that contains the marked free probe then.Probe and target nucleic acid are mixed, and optional probe and target nucleic acid are hybridized.The mixture inflow of label probe and target nucleic acid is contained in second reaction chamber of probe particle collection.In second reaction chamber, free probe and probe-particle and target nucleic acid are hybridized, and suitable probe is joined together.Can join in the reagent chamber connecting reagent, or add in any one reaction chamber, preferably add in second reaction chamber.Analyze the probe-particulate hybridization product in second reaction chamber as stated above.

In one embodiment, (by PCR or utilize a carrier, for example λ library) target nucleic acid needn't increase before analysis.Because the complicacy of sample sequence increases, the preferred use in the present embodiment than long free probe and particle probe (promptly cross background and tell positive reaction).

Any application of describing before described in this embodiment probe-particle embodiment is applicable to, diagnosis of describing before including but not limited to and order-checking are used.In addition, can these probes-particle scheme be improved by variation or the change that reaches noted earlier.

Embodiment 37

Under the situation that the bonded reagent that changes between polynucleotide exists, the interaction of complementary polynucleotide

In this embodiment, by adding one or more reagent, can adjust the coupling and the value of distinguishing of mispairing fully to producing in the complementary polynucleotide cohesive process.In a preferred embodiment, the complementary polynucleotide are a polynucleotide of interest and a polynucleotide probes.Can adjust differentiation and mate ability with mispairing fully by adding reagent, described reagent can be salt, as trialkyl ammonium salts (for example TMAC, Ricelli etc., nucleic acids research 21:3785-3788 (1993)), sodium-chlor, phosphoric acid salt and borate; Can be organic solvent, as methane amide, ethylene glycol, dimethyl sulfoxide (DMSO) and dimethyl formamide, urea, Guanidinium, amino acid analogue such as trimethyl-glycine (Henke etc., nucleic acids research 19:3957-3958 (1997); Rees etc., biological chemistry 32:137-144 (1993)), polyamines such as spermidine and spermine (Thomas etc., nucleic acids research 25:2396-2402 (1997)), or positively charged molecule that can neutralising phosphoric acid main chain negative charge; Can be stain remover, as sodium laurylsulfonate, sarcosyl, little/the major groove binding reagents, positive charge polypeptide and insert reagent such as acridine, ethidium bromide, anthracin.In a preferred embodiment, in hybridization, add the reagent that mixes, so that adjust the ability of distinguishing correct coupling and mispairing.Thereby some of them reagent can be by reducing entropy of fusion influence value of distinguishing of two complementary strands.

In a preferred embodiment, utilize some reagent to improve the ability of the correct coupling of difference from erroneous matching.For example, a kind of denaturing agent methane amide commonly used in the reaction of Format III, is compared with correct coupling, and it has preferential destabilization to erroneous matching.As previously mentioned, open beginning FormatIII reaction, add the methane amide (0%, 10%, 20%, 30%, 40% and 50%) of different amounts then.0%, can detect a correct matched signal, background (erroneous matching) is very high simultaneously.In 10% methane amide, correct preferably matched signal is arranged, background/erroneous matching signal reduces simultaneously.In 20% methane amide, the signal of correct coupling reduces (but can survey), and background/signal error is eliminated simultaneously.In the methane amide of 30%-50%, there is not the signal of correct coupling or background/erroneous matching.

In a selectable embodiment, reduce or increase the Tm of a pair of complementary polynucleotide with a kind of reagent.In a preferred embodiment, utilize the mixture of some reagent to reduce or increase the Tm of a pair of complementary polynucleotide.Reagent can change Tm in many ways, here for two examples (and not meaning that restriction the present invention), (1) destroys reagent (Goodman, the Proc. Natl. Acad. Sci.USA 94:10493-10495 (1997) that the hydrogen bond between two complementary polynucleotide base pairs connects; Moran etc., Proc. Natl. Acad. Sci.USA 94:10506-10511 (1997); Nguyen etc., nucleic acids research 25:3059-3065 (1997)), (2) can neutralize or cover the reagent of the phosphoric acid negative charge in the sugared phosphate backbone of polynucleotide.(Thomas etc., nucleic acids research 25:2396-2402 (1997)).By strengthening or weakening (1) and/or (2), just can regulate the right Tm value of complementary polynucleotide.

In a highly preferred embodiment, add the bound energy that one or more reagent reduce the GC base pair, perhaps increase the bound energy of AT base pair, or both carry out simultaneously.In a preferred embodiment, add one or more reagent, make the bound energy of AT base pair be approximately equal to the bound energy of GC base pair.Like this, the bound energy of two complementary polynucleotide only depends on its length.By adding a kind of reagent of the negative charge that can neutralize or cover the phosphate group in the polynucleotide main chain, can increase the bound energy of these complementary polynucleotide.

Scope of the present invention not only is confined to cited embodiment, and these embodiments only are used for setting forth invention in a certain respect, has the composition of identical function and method also in scope of invention.In fact, considered described preferred embodiment after, those skilled in the art can carry out when of the present invention variously repairing into and changing using.Therefore, the unique restriction to the scope of the invention is to enumerate in the claims.

All documents of quoting in this specification sheets are in this hereby incorporated by reference.

Claims (64)

1. the array of a large amount of oligonucleotide probes comprises:

A kind of matrix;

A kind of material that forms physical barriers, wherein said material are placed on and form apertured grills on the matrix;

Wherein a large amount of oligonucleotide probes are arranged on and form an array in the porous, and wherein a probe spot that is fixed on the matrix is contained in each hole.

2. the array of claim 1, wherein the sequence of the probe that has of each independent spot is different from other probes at other spot places in the array.

3. the array of claim 2, the probe that wherein is arranged in each independent spot has identical sequence.

4. the array of claim 1, the distance between the center of one of them spot and the center of adjacent spots is at least 325 μ m.

5. sequence testing chip contains the array of a large amount of claims 1.

6. sequence testing chip contains the array of a large amount of claims 2.

7. sequence testing chip contains the array of a large amount of claims 3.

8. the array of claim 1, wherein oligonucleotide probe comprises a message part and a reactive group, and this reactive group is used for probe attached to matrix.

9. the array of claim 8, wherein oligonucleotide probe further contains at least one randomization site.

10. the array of claim 9, wherein oligonucleotide probe further contains a spacer.

11. the array of a large amount of oligonucleotide probes comprises the matrix with a plurality of aspects, wherein a large amount of probes are fixed on a plurality of aspects in the matrix.

12. the array of claim 11, wherein each aspect can be analyzed separately.

13. the array of claim 11, wherein multiple aspect can be analyzed simultaneously.

14. the array of claim 11, wherein oligonucleotide probe comprises a message part and a reactive group, and this reactive group is used for probe attached to matrix.

15. the array of claim 14, wherein oligonucleotide probe further contains at least one randomization site.

16. the array of claim 15, wherein oligonucleotide probe further contains a spacer.

17. a method of analyzing target nucleic acid comprises the following steps:

Target nucleic acid is contacted with a large amount of oligonucleotide probes, and wherein said probe is compound with a large amount of different discrete particles, and these particles can be distinguished mutually based on a kind of physical properties, and a different probe is mutually compound with the each type discrete particles;

Detect those and target nucleic acid complementary probe; And

Analyze target nucleic acid from one group of complementary probe.

18. the method for claim 17 further comprises discrete particles is separated into fraction, wherein discrete particles is based on physical properties and is separated.

19. the method for claim 18, wherein the complementary probe group has at least two overlapping probes.

20. the method for claim 18, wherein target nucleic acid sequence is edited in analytical procedure.

21. the method for claim 18 is wherein discerned complementary probe by the physical properties of discrete particles.

22. the method for claim 18, wherein physical properties is associated with the molecule that is selected from dyestuff, radioactive nuleus thuja acid, EML and fluorescence molecule.

23. the method for claim 18, wherein physical properties is selected from size, electric charge, absorbancy and weight.

24. the method for claim 23, wherein physical properties is associated with the intensity of physical properties.

25. the method for claim 23, wherein physical properties is associated with a large amount of differing moleculars.

26. the method for claim 18, the message part of its middle probe is shorter than probe total length.

27. the method for claim 18, wherein target nucleic acid contains a mark and by the marker detection complementary probe on the target nucleic acid.

28. the method for claim 17, particle is independently carrying out on the discrete particles through a detector by inciting somebody to action independently wherein to detect step.

29. the method for claim 28, wherein the complementary probe group has at least two overlapping probes.

30. the method for claim 28, wherein target nucleic acid sequence is edited in analytical procedure.

31. the method for claim 28 is wherein discerned complementary probe by the physical properties of discrete particles.

32. the method for claim 28, wherein physical properties is associated with the molecule that is selected from dyestuff, radioactive nuleus thuja acid, EML and fluorescence molecule.

33. the method for claim 28, wherein physical properties is selected from size, electric charge, absorbancy and weight.

34. the method for claim 33, wherein physical properties is associated with the intensity of physical properties.

35. the method for claim 33, wherein physical properties is associated with a large amount of differing moleculars.

36. the method for claim 28, the message part of its middle probe is shorter than probe total length.

37. the method for claim 28, wherein target nucleic acid contains a mark and by the marker detection complementary probe on the target nucleic acid.

38. the method for claim 28 further comprises target nucleic acid is contacted with a large amount of free oligonucleotide,

The free probe of complementation that is bonded on site of target nucleic acid is covalently bound with discrete particles compound complementary probe with one, and described complementary probe is bonded on the site of target nucleic acid, and this site is adjacent to the site of free probe bonding, reaches

Wherein detect step identification and the covalently bound free probe of discrete particles probe.

39. the method for claim 38, wherein the covalently bound probe groups of complementary contains at least two covalently bound probes of eclipsed.

40. the method for claim 38, wherein target nucleic acid sequence is edited in analytical procedure.

41. the method for claim 38 is wherein discerned and discrete particles compound probe by the physical properties of discrete particles.

42. the method for claim 38 further comprises discrete particles is separated into fraction, wherein discrete particles is based on physical properties and is separated.

43. the method for claim 42 wherein uses flow-cytometer that discrete particles is separated into fraction.

44. the method for claim 38, wherein physical properties is associated with the molecule that is selected from dyestuff, radioactive nuleus thuja acid, EML and fluorescence molecule.

45. the method for claim 38, wherein physical properties is selected from size, electric charge, absorbancy and weight.

46. the method for claim 45, wherein physical properties is associated with the intensity of physical properties.

47. the method for claim 45, wherein physical properties is associated with a large amount of differing moleculars.

48. the method for claim 38, wherein the message part of free probe is shorter than probe total length.

49. the method for claim 38 is wherein short than probe total length with the message part of discrete particles compound probe.

50. the method for claim 38, the wherein message part of free probe and shorter than probe total length with the message part of discrete particles compound probe.

51. a method of analyzing target nucleic acid comprises the following steps:

Under the coupling and other condition of mispairing phase region target nucleic acid is contacted with probe fully making, add wherein that a kind of increase is mated fully and mispairing between the reagent of difference; And

Whether detection probes and target nucleic acid be complementary.

52. the method for claim 51, wherein reagent is selected from the positively charged molecule, stain remover of salt, organic solvent, urea, Guanidinium salt, amino acid analogue, polyamines, other neutralising phosphoric acid skeleton negative charges, little/the major groove wedding agent, positively charged polypeptide and intercalating agent.

53. the method for claim 52, wherein salt is selected from trialkyl ammonium salts, sodium-chlor, phosphoric acid salt and borate.

54. the method for claim 52, wherein organic solvent is selected from methane amide, ethylene glycol, dimethyl sulfoxide (DMSO) and dimethyl formamide.

55. the method for claim 52, wherein amino acid analogue is a trimethyl-glycine.

56. the method for claim 52, wherein polyamines is selected from spermidine and spermine.

57. the method for claim 52, wherein stain remover is selected from sodium laurylsulfonate and sarcosyl.

58. the method for claim 52, wherein intercalating agent is selected from acridine, ethidium bromide and anthracin.

59. the method for claim 51 wherein adds plurality of reagents.

60. the method for claim 59, wherein reagent is selected from the positively charged molecule, stain remover of salt, organic solvent, urea, Guanidinium salt, amino acid analogue, polyamines, other neutralising phosphoric acid skeleton negative charges, little/the major groove wedding agent, positively charged polypeptide and intercalating agent.

61. a method of analyzing target nucleic acid comprises the following steps:

The array of a large amount of immobilized oligonucleotide probes is provided;

The oligonucleotide probe of a large amount of marks is provided;

Form under the condition that the probe that mates fully is different from the probe that a base mispairing is arranged when combining with target nucleic acid with target nucleic acid making, target nucleic acid is contacted with label probe with the immobilization probe, wherein add increase mate fully and base-pair mismatch between the reagent of difference;

The immobilization probe and the label probe in a site that is bonded to target nucleic acid is covalently bound, and described label probe hybridization is in a site of target nucleic acid, and this site is adjacent to the site of immobilization probe bonding; And

Discern covalently bound immobilization probe and label probe.

62. the method for claim 61, wherein reagent is selected from the positively charged molecule, stain remover of salt, organic solvent, urea, Guanidinium salt, amino acid analogue, polyamines, other neutralising phosphoric acid skeleton negative charges, little/the major groove wedding agent, positively charged polypeptide and intercalating agent.

63. the method for claim 61, wherein reagent is methane amide.

64. the method for claim 61 wherein adds plurality of reagents.

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