The method of determining the nucleic acid sequence (options) and set for use in determining the sequence of nucleic acid

 

(57) Abstract:

In the invention described are methods and kit for fast and efficient creation of nucleic acid sequence based on the hybridization of two sets of small samples of oligonucleotides of known sequences. Extremely large molecules of nucleic acids, including chromosomes and undissolved RNA, can be arranged in the sequence without prior cloning and sublimirovanny. Methods according to this invention may also allow to solve the existing problems associated with the technology of creating sequences, such as, for example, a low value of the signal-to-noise and difficulty of selectively attaching many fragments of nucleic acids to the surface, making longer or more complex samples and applying labels to a larger number of substances. 3 C. and 38 C.p. f-crystals, 10 ill.

This application is a partial continuation of concurrently pending application for U.S. patent 08/303.058, filed September 8, 1994, which is a partial continuation of patent application U.S. 08/127.420, filed September 27, 1993: the entire text and drawings of them specifically included Sud is rstv energy LDRD 03235 and contract W-31-109-ENG-38 between the U.S. Department of energy and University of Chicago, representing a National laboratory of argon.

The present invention in General relates to the field of molecular biology. In particular, the invention provides for the creation of new methods and compounds, allowing highly efficient formation of sequences of nucleic acid molecules. Methods according to this invention is suitable for sequences of long nucleic acid molecules, including chromosomes and RNA, with the processes of cloning and sublimirovanny.

Currently, the formation of nucleic acid sequences is an integral part of scientific progress. Sequencing, i.e., the primary structure of molecules and segments of nucleic acids is important in relation to specific projects, exploring a range of specific application areas. Information about the organization of the sequence affects science, medicine, agriculture and all areas of biotechnology. The creation of nucleic acid sequences, of course, vitally important for research on the human genome and other large-scale initiatives, which aim to advance our understanding of the evolution and function of organisms and help in p is the slot evident. For example, the human genome Project (PPP), multi-national initiative dedicated to creating a sequence the entire human genome, is carried out in different centres. However, progress in this area and mostly slow and expensive. Organization of nucleic acid sequences is usually determined on polyacrylamide gels that separate DNA fragments in the range 1 - 500 base-pairs with a difference in length by one nucleotide. The real definition of a sequence, i.e. the order of the individual nucleotides A, G, C and T, can be achieved in two ways. First, using the method of Maxima and Gilbert chemical cleavage of the DNA fragment at specific nucleotides (Maxam and Gilbert, 1977), or, secondly, using the method of creating a sequence ending chain of dideoxy described by Sanger and colleagues (Sanger and others, 1977). Both methods are time consuming and take a lot of time.

Recently been proposed other methods of generating nucleic acid sequences that do not use phase electrophoresis, and these methods can in General be called a Creation sequence by hybridization or LNG (Drmanac and others, 1991; Kantor and others, 1992; Drmanac and Chervenkov, U.S. patent 5.202.231).

Razvi basis, known as chips create a sequence. The usefulness of LNG mainly by the fact that this technology were issued U.S. patents. However, although LNG has the potential to increase the speed with which you can form a sequence of nucleic acids, all existing methods of LNG have several disadvantages.

LNG can be performed in two main ways, which are often called "format 1" and "format 2" (Cantor and others, 1992). In format 1, the oligonucleotides of known sequence, usually about 100 to 1000 nucleotides in length, built on a solid base or filter, so you unknown samples contact (neutralized) (Strezoska and others, 1991; Drmanac and Chervenkov, U.S. patent 5.202.231). Replica series then being "scanned" by the hybridization of the sets of labeled samples with a length of approximately 6-8 radicals. In format 2, the chip formation sequence is formed from a set of oligonucleotides of known sequences with a length of approximately 6-8 radicals (southern, WO 89/10977; hrapko and others, 1991; southern and others, 1992). Nucleic acids of unknown sequence then rushing about and allow them to hybridisierung to associated oligo.

Unfortunately, both of these formats JV is the Isla other significant problems include attaching different parts of the nucleic acid, you need to arrange in sequence to a solid support surface or cooking a large set of longer samples. In format 2, the main problems include labels nucleic acids of unknown sequence, usually the resulting high signal-to-noise ratio that can be determined only short sequences.

Another problem in the format 2 is the formation of secondary structure that prevents access to certain purposes, and different conditions required for samples with different GC content. Therefore, this technology is the obvious way to benefit from the establishment of a new procedure for the organization of nucleic acid sequences and, in particular, a procedure that avoids the tedious process of cloning or/and sublimirovanny.

The present invention is designed to overcome these and other drawbacks inherent in the previous developments, by creating new methods and compounds for the formation of nucleic acid sequences. The latest methods described here are commonly called inventors "format 3" and they represent a significant improvement over existing methods LNG format 1 and teinaava acids is determined by hybridization with two sets of small samples of oligonucleotides of known sequences. The method according to the invention allows to obtain highly selective sequence of extremely large molecules of nucleic acid, including chromosomal material or RNA, without prior cloning, sublimirovanny or gain. In addition, these methods do not require a large number of samples, a comprehensive synthesis of longer sampling or tagging a complex mixture of nucleic acid segments.

In order to determine the sequence of nucleic acid according to the methods of this invention are mainly sequences from a nucleic acid by hybridization with complementary sequences from two sets of small samples of oligonucleotides (oligo) with a certain length and a known sequence, which include most of the combinations of sequences of this length of the sample. It then parses the given sequence to determine the overlapping lengths of the found sequences and reconstructed or going the full sequence of nucleic acid from such overlapping sequences.

How education sequence can be performed with apolonio. Alternatively, you can apply the method described as "holding cycle (alternation)" when two sets of small oligo hybridize simultaneously with unknown sequences. The term "series" is used, since the election of the method arises from the subsequent temperature increase to "melt" those hybrids that complementary. Such methods a series of generally applied in other areas of molecular biology, such as PCR, and its easy to see specialists when reading this description.

This invention is applicable to formation of a sequence of nucleic acid molecules is very large. In practice, the molecule is a nucleic acid sequence which is necessary to form, mainly divided into fragments to obtain fragments of the nucleic acid of low or intermediate length, easy to manipulate.

The notion of a fragment of the nucleic acid, as it is used here, just the usual means a nucleic acid molecule with a length of approximately 10 base pairs (BP) 100 BP. It is believed that the most preferred methods are those in which the nucleic acid molecule, consequently the length, i.e., from about 10 BP to 40 BP. However, it should be emphasized that the present invention is not the way of a complete education sequences of small fragments of nucleic acid, but rather a method of forming sequence of the nucleic acid molecules per se, which involves identifying parts of the sequence from the inside of the molecule is whether the implementation using the whole molecule or, for simplicity, achieved initial fragmenting the molecules at the sites of smaller size from about 4 to 1000 bases.

The sequence of the nucleic acid molecules are determined by hybridization to a small sample of oligonucleotides of known sequence. When people talk about "small samples of oligonucleotides", the term "minor" means a sample with a length of less than 10 BP and preferably samples with a length of about 4 BP and about 9 BP. In one example, the education sequence are considered particularly suitable sample with a length of about 6 bps. For oligo sets, which includes all combinations of sequences for the selected length of the sample, the number will be represented as 4F, where F is the length of the sample. For example, for a 4-measure set will contain 256 samples; 5-measure set will be solartechnik and it is possible to carry out automated synthesis.

In the methods according to this invention, one set of small samples of oligonucleotides of known sequence, which can be called the first set will be attached to a solid support, i.e., contact (neutralized through) on this basis so that he can participate in the hybridization reactions. Another set of small samples of oligonucleotides of known sequence, which can be called the second set of samples that are in solution and labeled with a detectable label. The oligo sets may contain samples of the same length or different lengths.

The process of sequential hybridization indicates that the nucleic acid molecules or fragments of unknown sequence can be hybreed to certain sets of samples of oligonucleotides of known sequences in separate periods of time (Fig. 1). Molecules or fragments of nucleic acid will be mainly are denatured, allowing hybridization, and will be added to the first linked set of samples under conditions of selective hybridization with ensuring that hybridize only fragments with complementary sequences. Fragments with complementary sequences is, set of samples in the solution to the already formed by the combination of fragments and samples. Labeled probes that hybridize next to a fixed breakdown will remain attached to the base and can be detected, which does not happen when there is a gap between the fixed and labeled samples (Fig. 1).

The simultaneous hybridization indicates that the nucleic acid molecule of unknown sequence can contact with certain sets of samples of oligonucleotides of known sequences at the same time.

Hybridization occurs under conditions of selective hybridization. Then the fragments with complementary (not complementary) sequences of "melted", i.e. removed with increasing temperature, and then is the next stage of the election hybridization, allowing hybridisierung any second additional samples. Then labeled samples, which were hybridisierung next to a fixed sample, are detected in the same way.

Nucleic acid sequence, which is "complementary (complementary)", are those that are capable of pairing bases according to standard rules of complementarity Watson-Cu or modified purine always will mate on the basis of the smaller pyrimidines with education only known combinations. These include standard Paris guanine, paired with cytosine (G:C) and adenine, paired with either thymine (A:T), in the case of DNA or with uracil (A:U), in the case of RNA. Also consider the use of modified bases or so-called "universal base (base)" (M. Nichols and others, 1994).

Used herein, the term "complementary (complementary) sequences" means nucleic acid sequence, which in principle are complementary along its entire length and have very few discrepancies between the databases. For example, nucleic acid sequence with 6 bases in length can be called complementary if they hybridize in five of the six positions with only one mismatch. Naturally, the sequence of nucleic acids that are "fully complementary" are those nucleic acid sequence that is fully complementary over its entire length and not have mismatches of bases.

After identification by hybridization to oligo with known sequences of different individual sequences that are part of fragments of nucleic acids, then these individual sequences are analyzed to find peracre the as the end of the 3' sequence or Vice versa, are identified. The full sequence of the molecule or fragment of the nucleic acid can then contributes, i.e. it can be reconstructed from certain thus overlapping sequences.

The process of finding overlapping sequences and reconstructing the full sequence is usually determined by computational analysis. For example, if the labeled probe 5'-TTTTTT-3' 's hybrid to the place that contains a fixed probe 5'-AAAAAA-3', is determined by the 12-dimensional sequence from the inside of the nucleic acid molecules, namely, 5'-AAAAAATTTTTT-3' (th. ID. N. 1), i.e. a sequence of two hybridized samples combined to detect previously unknown sequence. The next question that must be answered is what nucleotide is next only to that specific sequence 5'-AAAAAATTTTTT-3' (th. ID. N. 1). There are four options presented in the fixed-break 5'-AAAAAT-3' and labeled probes 5'-TTTTTA-3' for A; 5'-TTTTTT-3' for T; 5'-TTTTTC-3' for C; and 5'-TTTTTG-3' for G. If, for example, probe 5'-TTTTTC-3' is positive, and the other three are negative, then the collected sequence is extended to the 5'-Athelny in place, containing a fixed sample AAAATT.

This process is repeated until all positive (F+P) the sequence of the oligonucleotides are not used or will not be identified as a false positive.

Thus, this invention creates a very effective way to organize the sequence of fragments and molecules of nucleic acids long. As here defined, large nucleic acid molecules are those molecules that need to be divided into fragments to generate the sequence. Their length is typically at least about 45 or 50 base pairs (BP), and often more. In fact, the methods according to the invention can be used to organize the sequence of nucleic acid molecules with virtually no upper limit, so that we can form a sequence of about 100 BP, 1 kilobase (KB, 100 KB, 1 Megabase (MB) 50 MB or more, to complete chromosomes, inclusive, such as chromosomes of human, having a length of about 100 MB. Such a large number is quite within the framework of the present invention, and the formation of a sequence of such number of databases will require 2 sets of 8-Mer or 9-Mer (so that F+P is approximately equal to the DNA micromachinery chromosomal bands, cosignee DNA or insert YAC, or can be RNA, including mRNA, grnk, t or sn.

The process of determining the sequence of a long nucleic acid molecule involves simply finding sequences of length F+P from molecules and combining sequences using a suitable algorithm. In practice, first, most likely divided into fragments of a nucleic acid molecule, the sequence in which you want to create, to obtain smaller fragments, such as fragments of nucleic acids with intermediate length. Then there are sequences of length F+P hybridization, for example, sequential hybridization of the fragments to the complementary sequences of the 2 sets of small samples of oligonucleotides of known sequence, as described above. Thus, it is possible to reconstruct the complete sequence of the nucleic acid is extremely large molecules from the overlapping sequences of length F+P.

Regardless of whether a nucleic acid sequence which you want to organize, itself a fragment with an intermediate length or first processed, the OIC acid by hybridization of two sets of small samples of oligonucleotides of known sequence is the basis for the described methods education sequence. This process usually involves the following stages:

a) conversion of a set or series of attached or connected (neutralized) samples of oligonucleotides into contact with fragments of nucleic acid hybridization conditions effective to allow the fragments with complementary sequence is sufficient to hybridizers in the sample that forms the primary complexes, where the fragment has both hybridized and dehybridization, or "free," sequence;

b) bringing the primary complexes into contact with a set of labeled samples of oligonucleotides in solution in the hybridization conditions effective to allow the samples with complementary sequences to hybridisierung to dehybridization free or sequence fragment that forms the secondary complexes, where the fragment hybridized, and attached (linked) to the sample and labeled probe;

C) removal of secondary complexes of any of labeled samples that were not hybridized next to attached sample, which leaves only the neighboring secondary complexes;

g) detection of neighboring secondary complexes by detecting the presence of label in the labeled sample; and

d) nahoko by combining or compounds known sequences hybridized and labeled samples.

Hybridization or washing conditions chosen for one or two stages of hybridization may be carried out according to certain example education sequence. For example, both conditions are hybridization can be arranged to allow samples of the oligonucleotide to hybridisierung to this fragment of the nucleic acid when they contain complementary sequences, for example, in principle the matching sequence, for example, those sequences that hybridize in 5 of the 6 positions. Preferably the steps of hybridization carried out using a simple robotic device that is typically used in the existing procedures of the education sequence.

Alternatively, the hybridization conditions can be arranged so as to permit hybridization of only those samples and fragments of oligonucleotides, which are fully complementary sequence. These more selective or "strict" conditions can be used for both the individual stages of the process of sequential hybridization or only for each one stage. In such cases, samples of oligonucleotides, whether related or mechelu complementary sequence to the fragment.

The selected hybridization conditions largely dictate the degree of complexity required for the data analysis. Equally computer software available to analyze any data generated, can dictate the conditions of hybridization, which need to be applied in this laboratory. For example, in the electoral process both phase hybridization will be performed under conditions allowing hybreed only oligo and fragments with a fully complementary sequences. Because mismatched bases will not, this method implies that the least sophisticated computational analysis and for this reason, it is presently preferred application of the invention in practice. However, the use of less selective conditions for one or both stages of hybridization is also included in the scope of this invention.

Suitable hybridization conditions for use at one point or both can usually be determined by optimization procedures or the first of a series of studies. Different types of the first studies are usually conducted by experts on the organization of sequences of nucleic acids in establishing operating procedures and adjustment procedures for this laboni stages; used buffers and pH and strength of ions can vary and thereby optimized.

In preferred embodiments, the method of forming nucleic acid sequence according to this invention includes a sampling stage for sampling the secondary hybridization complexes that contain directly adjacent associated and labeled sample, in contrast to those that are not directly adjacent and separated by one, two or multiple databases.

There are many different processes for removal of labeled samples that did not hybridisierung directly next to the attached breakdown, i.e. not hybridisierung rear sides, each of which leaves only the directly adjacent secondary complexes.

Such selective processes can be based solely on the washing steps with controlled rigor, where applicable, the conditions of hybridization are arranged directly adjacent samples remain hybridized due to the increased stability created by the interactions between sets of adjacent nucleotides. Again, such washing conditions, such as temperature, concentration, time, b is directly adjacent.

In preferred embodiments of the invention directly adjacent associated and labeled samples will be connected, i.e., the United covalent bond to perform the washing steps to remove any non-formed connection samples. Education links can be achieved by treatment with a solution containing a chemical binder, for example, water-soluble carbodiimide or CYANOGEN bromide. Preferably, you can use the enzyme ligase, such as T4 ligase DNA from bacteriophage T4, which industrial supplied by many firms (e.g., Biolabs). In any case, you can then delete not directly adjacent labeled sample through a more stringent washing conditions, which may not affect covalently labeled United and fixed samples.

The remaining neighboring secondary complexes will be detected by observation of the presence of the label of the labeled samples present inside the complexes. Samples of oligonucleotides can metalsa chemically detectable label, such as fluorescent dyes, or adequately modified for detection by chemiluminescent procedures manifestations, or radioactive labels, such as the radioactive isotopes, and discovered by mass spectrometry.

Currently considered the most preferred method of implementation in practice of this invention involves the implementation stages of hybridization under conditions designed to allow hybreed only those samples and fragments of oligonucleotides, which are fully complementary sequence, and allow to remain hybridized only directly neighboring samples. This method therefore requires the least complex computational analysis.

If the nucleic acid molecule of unknown sequence longer than about 45 or 50 BP, one effective way to determine its sequence typically involves the processing of molecules for the formation of fragments of the nucleic acid intermediate length and defining sequences of the fragments. The nucleic acid molecule, be it DNA or RNA, can be divided into fragments of any of numerous ways, including, for example, by cutting restrictive enzyme digestion, shear (shearing) by physical means such as supersonic processing, processing NaOH or shift at low pressure.

In some examples, the OS is up to 9 BP can be designed to obtain fragments of nucleic acids in length, from about 10 BP to 40 BP. Naturally, longer samples will mainly be used in conjunction with the formation of the sequence of longer fragments of the nucleic acid and Vice versa. In some preferred embodiments used a small sample of the oligonucleotides will have a length of about 6 BP, and fragments of a nucleic acid sequence which should be organized, will have a length in the main about 20 BP. Optionally, the fragments can be separated according to size in order to obtain fragments of the appropriate length, for example, fragments can be processed on the gel, such as agarose gel, and fragments of approximately the desired length can be cut off.

The method of determining the sequence of nucleic acid molecules can also be explained on the example, using the following conditions. Initially arbitrarily segmented certain amount of a nucleic acid sequence which you want to form to obtain a mixture of fragments of nucleic acids in length T. Prepared a series of related samples of oligonucleotides of known sequence and length F and the set of labeled samples of oligonucleotides in solution with known sequences of length P, where F + P = T, predp contact with a mixture of fragments of nucleic acid hybridization conditions, effective to allow the formation of complexes with hybridized complementary sequences of length F and dehybridization sequences of fragments of length T-F. Preferably hereditarian sequence length F will contain only fully complementary sequence.

Then the primary complexes are brought into contact with a set of labeled samples of oligonucleotides in the hybridization conditions effective to allow the formation of secondary complexes with hybridized complementary sequences of length F and the adjacent hybridized complementary sequences of length P. In the preferred embodiments of the invention only labeled samples with a fully complementary sequences will be allowed to hybreed, and only those samples that hybridizing directly associated with the breakdown will be allowed to remain hybridized. In the most preferred examples at this stage neighboring connected and labeled probe oligonucleotides will form a link.

Then detect the secondary complexes by detecting the presence of the label and is the combination of known sequences hybridized connected and labeled samples. Then the overlapping length sequences of length F+P will be identified, allowing to reconstruct or to collect the full sequence of nucleic acid from certain overlapping sequences.

In the methods according to this invention, the oligonucleotides of the first set can be attached to a solid support, i.e., contact (offset) by any of the methods known in the art. For example, the attachment may be by means of addressable driven by laser photodestructive (Fodor and others, 1991; Pease and others, 1994). One in the General case, the preferred method is the attachment of oligo through the phosphate group with the use of such reagents as nuke phosphoramidite or nucleoside hydrogen Fosforit, as described by Sapernom and Masoom (PCT application WO 90/03382) using the basics of glass, nylon or Teflon.

Another preferred method is a method generated by the light synthesis described Pithom and others (1994). You can buy associated with the basis of a series of oligonucleotides, for example, those that are sold by companies Affymetrix and Beckman.

Linked oligonucleotides can be formed in a number that contains all the samples of the sludge is esto series of linked oligonucleotides, located with the formation of the so-called "chip create sequence". One example of the chip - this is when the hydrophobic segments are used to create separate spatial areas. Chips create a sequence can be constructed for various applications, such as planning, partial create sequence create sequence outlined areas for diagnostic purposes, creating a sequence mPHK and the creation of large-scale genome sequencing. For each application it is possible to construct a concrete chip samples of different sizes or with an incomplete set of samples.

In one embodiment of the invention two sets of samples of oligonucleotides will be samples of length 6 bases, i.e., 6-measures. In this case, each set of oligo contains 4096 individual samples. Samples of the first set are preferably fixed in a row on the microchip, it is most convenient when placed in 64 rows and 64 columns. The second set of 4096 oligo will metalsa detectable label and distributed on a set of separate tubes. In this example, 4096 chips are combined in a large number or in several rows. After hybridization of nucleic acid fragments Nebolchi and only one out of every 4096 nucleotides will be added to each microarray.

Among other objects of this invention are kits of materials to create sequences of nucleotide acids. Basically, these kits include a solid base to which are attached a number of samples of oligonucleotides of known sequences, as shown in Fig. 2A, Fig. 2B and Fig. 2C, where the oligonucleotides are able to participate in hybridization reactions, and a set of tanks containing solutions of labeled samples of oligonucleotides of known sequences. Also considered such schemes as those shown in Fig. 4. It describes the use of "universal base" or a method of attaching or end position to give a new dimension hybridization of the fragments.

In these sets of materials attached sample oligonucleotides and samples in solution can have a length of approximately 4-9 BP, preferably of the sample length of about 6 bps. Oligo can be in the state of chemically detectable or radioactive labels, and in General the preferred sample, labeled 32Pand even more preferred samples, labeled 33P. Sets of materials can also include chemical or other binder, such as EN zymes is trojstva with 96 tips or 96 pins, buffers, reagents for cutting long molecules of nucleic acid and tools for sizing DNA fragments. Sets of materials may also include labeled devices RNA, so that samples can be removed by RNase treatment and re-use chips generate the sequence.

In the drawings, the present invention is illustrated.

In Fig. 1 shows the main stages of the process of hybridization. Step 1: Its target DNA sequence which is necessary to form a (T), 's hybrid electoral conditions attached to a number of samples of oligonucleotides. Described the place with a sample of Fxand Fy. Complementary sequence for Fxand Fyare at different positions So Stage 2: Labeled samples Pi(one sample per chip) hybridize to a number. Described probe having a complementary target at T, which is adjacent to Fxbut not with Fy. Step 3: By applying selective conditions or reagents selectively melted complexes without neighboring samples. A specific example is the education connection labeled samples with a fixed breakdown, when labeled sample it hybridises "back to back" with the attached sample. Positive signals from the beam formed communication samples.

In Fig. 2A, Fig. 2B and Fig. 2C presents the components of a sample set of materials for education sequence.

Fig. 2A. Chips create a sequence that represents the number of 4Pidentical sections, each of which contains identical (or different) numbers of oligonucleotides. Sections can be separated by physical barriers or hydrophobic strips. It is believed that in the series there 4000-16000 of oligotypes.

Fig. 2B is a view of the increasing section of the chip containing the 4Fplots, each with a specific breakdown of the oligonucleotide (4000-16000), synthesized or installed in this place. Sites can be as small as several microns, and the section size of approximately 1 to 10 mm

Fig. 2C represents the set of tubes or one or more plates with multiple pockets with a corresponding number of pockets (in this case, 4Ppockets). Each pocket contains a number of specifically labeled oligonucleotide.

More samples can be stored its if tagging is not performed during synthesis; in this case, a set of materials to create a sequence will contain the necessary components for labelling samples. Li, when a certain number of labeled samples are transferred into the section of the chip. The transfer can be carried out by pipette (simple or multi-channel) or a number of pins (studs) transferring the liquid by surface tension. Tools to migrate may also be incorporated into the Toolkit.

Fig. 3A, Fig. 3B and Fig. 3C. Hybridization of DNA fragments obtained by an arbitrary cut a certain number of DNA molecules. In Fig. 3A fragment of T1 DNA that contains the full purpose for fixed and non-fixed labeled samples. Fig. 3B represents the case where the fragment of T-DNA incorrectly cut. In Fig. 3C there is enough space for hybridization of the sample P, but the neighboring sequence not complementary to it. In the case B, and case C signal will be reduced due to saturation of the molecules attached sample F. Simultaneous hybridization of the DNA fragments and labeled samples and the cyclic process of hybridization are some possible ways to increase the output of the right neighboring hybridisable.

Fig. 4. Using "universal base" as a means of communication or in end position for hybridization. Universal base (M-base, Nichols and others , 1994) or all 4 bases can davlatligini number of samples. In addition, the use of universal bases at the free end of the sample creates a gasket that allows you to read the sequence in another frame (frame).

Determining sequences of nucleic acid molecules finds important use in all areas of fundamental and applied biological research (Drmanac and Chervenkov, 1990). The present invention creates a new and effective ways to create sequences and nucleic acid. One of the intended uses of this methodology in combination with other methods of education sequences is the use in the work on the human genome Project (PPP).

Currently there are two methods of creating a sequence hybridization (LNG). In the first, format 1, unknown genomic DNA or oligonucleotides with a length of up to 100-200 built on a solid substrate. Then these DNA "polled" by the hybridization of a set of labeled samples, which are usually 6-8-measures. In the reverse method, format 2, oligomers 6-8 nucleotides bind (neutralized) on a solid basis, and allow them to otjihase to parts cloned and labeled DNA.

In either of the two types of analysis of LNG have included what their methods LNG, it is those that are associated with the synthesis of a large number of trials and difficulties of effective selective hybridization. Full recognition of the match-mismatch is difficult for two main reasons. First, end-discrepancy samples longer than 10 bases very indiscriminately, and, secondly, a complex mixture of labeled segments of DNA, which is obtained by analysis of long DNA fragments generates high background.

The present invention provides an efficient selective hybridization without a large number of samples or samples with increased length and also eliminates many of the steps of applying labels and cloning that are specific disadvantages of the known methods of LNG. Described in highly effective ways to create nucleic acid sequence, called the creation of the sequence in the format of 3 based on hybridization with two sets of small samples of oligonucleotides of known sequences, and thus it is possible to determine at least twice the length of the sequence. These methods allow to organize the sequence of extremely large molecules of nucleic acid, including chromosomes, and resolve sabreena has an extremely high potency, because it can also be used to create RNA sequences and even unaugmented RNA samples.

After the present invention, as described in the application U.S. 08/127.402 and Drmanac (1994), was presented another variant of LNG, called positional LNG (PSPG) (Original and others, 1994). PSPG is essentially a variant of LNG format 2 (in which the oligonucleotides of known sequences bind (neutralized) and used for hybridization to the nucleic acids of unknown sequence that was previously marked). In PSPG related samples, not being a simple, odnokratnye samples, this duplexes containing odnokratnye 3' overhangs. Biotinylated duplex sample bound to streptavidin coated magnetic beads with the formation of some sort associated sample and then mixed with 32P-labeled target nucleic acid sequence to which you want to create. Then add ligase T; DNA binding any hybridized target DNA to the shorter end of the duplex samples.

However, while this is an interesting approach, PSPG (as it is stated by the Original and others, 1994) does not reflect the significant improvements in sravnenie the length of the sequence, which can be defined for one cycle of the method. In PSPG also supported burdensome requirement of applying labels to an unknown target DNA, which is not required in format 3. In General PSPG available for use in comparative studies or planning, but not in the new creation sequence of the genome. Thus, it differs significantly from the format 3, which, though wide is applicable in all areas of creating sequences, is a very powerful tool for the formation of sequences of even the largest genomes.

A nucleic acid sequence which you want to organize, you can first be divided into fragments. This can be achieved by any means, including, for example, cutting restrictive enzyme digestion, especially with Cvi JI, as described by Fitzgerald and others (1992); offset by physical means, such as ultrasonic processing; processing NaOH, etc., If desired fragments of the appropriate length, for example, approximately 10 to 40 BP, can be cut out of the gel. The full nucleic acid sequence of the original molecule such as a chromosome, will be determined by defining posledovatelno the

Therefore, it does not require an intermediate step of determining the sequence of the fragment, rather the sequence of the whole molecule skonstruiruem of contoured sequences F+P.

For purposes of the following discussion in the General case will be made that 4 base comprise nucleic acid sequences that you want to organize. This is A, G, C and T for DNA and A, G, C and U for RNA. However, it may be useful in some embodiments to use modified base in small samples of oligonucleotides. To perform this invention, the first is usually prepared a number of small samples of oligonucleotides of a certain length, which includes all combinations of sequences of this length of the sample. This number represents the 4N(4 to the power of N), where the length of the sample is denoted by N. for Example, there are 4096 possible sequences for the 6-dimensional samples (46= 4096).

One set of these samples of length F (4F) will be fixed in a square layout on the microchip, which can be in the range from 1 mm2up to 1 cm2. In this example they will be arranged in 64 rows and 64 columns. Of course, ensures that the sample oligo were attached or otherwise due the th P, number 4Pwill be also synthesized. Oligo in this set "P" will be metalsa detectable label and will be distributed on the set of tubes (Fig. 2A, Fig. 2B and Fig. 2C).

4Pof chips are combined in a large series (or multiple series of approximately 10 - 100 cm2for convenient sizes), where P corresponds to the length of the oligonucleotides in the second set of oligomer (Fig. 2B and Fig. 2C). Again, as a convenient example, P is chosen as 6 (P = 6).

A nucleic acid sequence which is necessary to organize, will be separated into fragments with getting smaller nucleic acid fragments of unknown sequence. The average length of these fragments, called T, usually must be greater than the combined length F and P^and may be approximately three times greater than the length of F (i.e. F+P T and T is approximately equal to 3F). In this example, the goal will be to obtain fragments of nucleic acids with a length of approximately 20 base pairs. These fragments will be denaturirovannyj and added to the big ranks in the conditions that facilitate hybridization of complementary sequences.

In the simplest and preferred in the present form of the invention will be a choice is selected nucleotides in the fragment of the nucleic acid are complementary to all 6 nucleotides F samples oligonucleotide. Such hybridization conditions will be determined by the usual initial research optimization, which specify conditions such as temperature, concentration of different components, the duration of the stages and used buffers, including the pH of the buffer.

At this stage each microchip contains certain hybridized complexes. They will be in the form of samples: fragment complexes, in which the entire sequence of samples hybridized to the fragment, but the fragment, being longer, has some dehybridization sequence, forming a "tail" or "tails" of the complex. In this example, the hybridized complementary sequences are of length F, and dehybridization sequence will have a total length of T-F.

Complement of the fragment can be located at the corresponding end or towards him, so that it is possible to form one longer dehybridization tail. Alternatively, the complement of the fragment can be located towards the opposite end, so that there are two dehybridization tail (Fig. 3A, Fig. 3B and Fig. 3C).

After washing to remove nedovolnaya fragments nook who is to each microarray for hybridization to the tails of the fragment of the nucleic acids of unknown sequence, issued from samples: fragment complexes. Only one of each of the 4 nucleotides will be added to each microarray. Now preferred hybridization conditions that allow you to have significant education links only if all 6 nucleotides labeled samples are complementary for 6 consecutive nucleotides of the tail fragment of nucleic acid. Conditions of hybridization is determined as described above initial research, which are optimized such elements as temperature, concentration, time, buffers, etc.

At this stage, each chip will contain certain "secondary hybridized complexes. They will be in the form of sample : fragment : complexes of samples in which the entire sequence of each sample was hybridized to the fragment and in which the fragment probably has some dehybridization sequence. In these secondary hybridized complexes associated with the sample and labeled sample can be hybridized to the fragment, so that the two samples are directly adjacent or located to each other back to back.

However, given the fact that the fragments are in General longer, the s, separated by one or more bases.

Then large ranges are handled through a process to remove dehybridization labeled samples. In preferred embodiments used the process will remove some not only dehybridization labeled samples, but not adjacent hybridized labeled samples. The process makes use of the electoral conditions in order to allow these secondary hybridization complexes that contain neighboring connected and labeled samples, most likely of those secondary hybridization complexes, in which the fragment of the nucleic acid hybridized to 2-m samples, but these samples are not adjacent. This is an important aspect of the invention, in the sense that it will allow the final contouring of the partition sequence fragment corresponding to a combined sequence associated sample and labeled samples.

The selectivity process used to remove dehybridization and not adjacent hybridized samples from a number while leaving adjacent hybridized samples attached can be again controlled by the washing process. Adjacent hybridized samples will not be affected by the chosen conditions and preferred embodiments will be processed large numbers, so any neighboring samples will be covalently linked, for example, by treatment with a solution containing a chemical binder or, more preferably, the enzyme ligase, such as T4 ligase DNA (Landegren and others, 1988; Wu and Wallace, 1989).

In any case, the full range will be subjected to stringent washing, so the only remaining label associated with a number will be in the form of double stranded complexes probe-fragment probe hybridized with neighboring parts of length F+P (i.e., 12 nucleotides in this example). Using this two-step reaction, hybridization may be very high selectivity, because it takes into account the 3 or 4 of the independent electoral processes: selective hybridization of the fragment T to the sample length F of databases; selective hybridization of the sample length P of the base fragment T; electoral stability full match (F+T+P) hybrid compared with the P hybrids or even with mismatched hybrids containing not adjacent samples F+P; and electoral education links 2 end bases F and P.

Then discovered the so-called neighboring secondary complexes by monitoring the location of the remaining labels on the series. With the position of the label can almosta related (neutralized) and labeled samples. Then you can reconstruct or to collect the full nucleic acid sequence of the original molecule such as a chromosome, thus determined, the overlapping sequences F+P.

If education links used in the process of creating a sequence, as now preferred, a conventional chip

oligonucleotides cannot be reused. The inventor believes that this will not limit, because there are different methods of disposal. For example, you can create specific easily fissionable relationship between the samples and then split the connection upon detection.

Alternatively, you can use the ribonucleotides of the second sample, sample P, or use ribonucleotide for the database connection in the sample P, so that the sample can then be removed by treatment with RNase or uracil-DNA glycosylation (Craig and others , 1989). Other considered methods must establish communication by chemical education ties, which can be selectively cut off (Valley and others, 1988).

Other variants and improvements of this methodology create sequence are also considered and included in the scope of this invention. This includes apogee described by Hoheisel and Leehom (1990). You can apply and hybridization with conduction cycles to increase the hybridization signal, as it is used in the technology of PRC. In these cases, will be used cycles with different temperatures for the repetition of the hybridization of some of the samples. The invention also provides for the determination of shifts in frames read using ravnomernykh quantities of samples that have different base at an end position. For example, using ravnomernye 7-measures, of which the first 6 bases it's the same specific sequence, and the last position can be A, T, C or G in the alternative.

Includes the following examples demonstrate preferred embodiments of the invention. Professionals need to understand what is described in the following examples, the methods are methods that open inventor for the normal functioning of the practical use of the invention, and thus they can be considered components of the preferred modes of such practical use.

However, professionals need to understand in the light of this description that you can make a lot of changes in the specific embodiments that are described here, and still get the origin is e related to the basis of oligonucleotides

Oligonucleotides, i.e., small segments of nucleic acid can be easily prepared, for example, by direct synthesis of the oligonucleotide by chemical means, as is usually the case in practice, using an automated synthesizer oligonucleotides.

Related basis nucleotides can be prepared by any method known in the art, using any suitable basis, such as glass, polystyrene or Teflon. One strategy consists in finding the exact location of the oligonucleotides synthesized by standard synths. Binding (neutralization) can be achieved by passive adsorption (Inouye and Hondo, 1990); by using UV-light (Nagata and others, 1985; Dalen and others, 1987; Morry and Collins, 1989) or by the formation of covalent bonds the base-modified DNA (Keller and others, 1988; 1989).

You can apply another strategy is to use the quality of education due to the strong interaction of Biotin-streptavidin. For example, the Original and others (1994) describe the use of biotinylated samples, although this duplex probes that are bound to streptavidin coated magnetic beads. Streptavidin coated beads can be bought in the company Dynal, Oslo. Of course, the same chemistry education in different companies for example, Operon Technologies (Alameda, California).

The firm Nunc Laboratories (high speed Internet access, Illinois) also sell suitable to the use of the material. Nunc Laboratories has developed a method by which DNA can be covalently bind to the surface with microchromosome called CovaLink NH. CovaLink NH is the surface of polystyrene to which is grafted a secondary amino group (> NH), which serve as the bridge position for further education covalent bonds. Modules CovaLink can be purchased in Nunc Laboratories. DNA molecules can be bound to CovaLink exclusively at the 5'-end by connection of phosphoramidate that allows binding (neutralization) of more than 1 pmol of DNA (Rasmussen and others, 1991).

Use strips CovaLink NH for covalent binding of DNA molecules at the 5'-end was described (Rasmussen and others, 1991). This technology uses the relationship of phosphoramidate (Chu et al , 1983). This is useful, as is preferably the binding (neutralization) using only one covalent bond. The relationship of phosphoramidate binds DNA with secondary amino groups CovaLink NH, which are situated at the end of the shoulder pads, covalently grafted to the surface of polystyrene over his shoulder strip length of 2 nm.

To bind the group. Probably, even you can covalently bind Biotin with CovaLink and then use streptavidin for binding samples.

More specifically, the method of forming a bond involves dissolving the DNA in the form (7.5 ng/ál) and denaturing for 10 min at 95oC and cooling on ice for 10 min 0.1 M 1-Mei with ice temperature, pH 7.0 (1-MeIm7) is then added to a final concentration of 10 mM 1-MeIm7. Then a solution of ss DNA is applied to the strips CovaLink (75 ál/pocket), standing on ice.

Carbodiimide 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), dissolved in 10 mM 1-MeIm7, refreshed and 25 μl added to his pocket. The strips are incubated in thermostat 5 hours at 50oC. After holding the strips are washed using, for example, Nunc-Wash Measurement; first strips are washed 3 times, then they promahivaetsya the washing solution for 5 min and finally washed 3 times with washing solution is of 0.4 N NaOH and 0.25% SDS, heated to 50oC).

It is believed that another suitable method for use with the invention described in the application WO 90/03382 (southern and Maskos). This method of preparation of the oligonucleotide associated with basis, involves attaching the nucleoside 3'-reagent through FOSFA the th basis. Then the oligonucleotide is synthesized on supported nucleoside protecting groups are removed from the chain synthetic oligonucleotide under standard conditions, which are not otscheplaut of the oligonucleotide from the Foundation. Among the suitable reagents include the nucleoside phosphoramidite and nucleoside hydrogen Fosforit.

More specifically, in order to use this method, the basis, for example a glass plate, derivatives in contact with a mixture of xylene, glycidoxypropyltrimethoxysilane and a small amount of diisopropylethylamine 90oC for half a day. She then thoroughly washed with methanol, ether and dried in air. Then derivational base is heated with stirring in hexamethyleneimine containing a catalytic amount of concentrated sulfuric acid, for half a day in an argon atmosphere at 80oC to obtain alkyl hydroxyl derivational basis. After washing with methanol and ether base is dried in vacuum and stored under argon at -20oC.

Then the synthesis of the oligonucleotide is performed manually under standard conditions using derivateservlet glass plates as a solid basis. First nucleates the basis of oligonucleotides of high purity.

You can apply the strategy of "chip" for the preparation of a series of DNA samples. For example, addressable driven by the laser photodeposition can be used in the chemical synthesis of oligonucleotides directly on the glass surface, as described by Fodor and others (1991). Samples can also be linked (to neutralize) nylon bases, as described by van ness and others (1991); or be associated with a Teflon using the method of Duncan and Cavalier (1988).

Fodor and others (1991) describe the directed light synthesis of dinucleotides, which is applicable to spatially directed synthesis of complex compounds for use in microprosodic devices. It is based on a method that uses light to the direction of simultaneous synthesis of chemical compounds on a solid basis. Structure exposure to light or other forms of energy through a mask or other spatially addressable means determines which areas of the canvas are activated to create a chemical bond.

Activation of the light comes from the removal photolabile protecting groups from the selected sites. After deprotection (unprotect) the first compound containing photolabile protecting group is exposed to on HSR substrate is illuminated through a second mask, which activates another area for reaction with the second protected building block. The structure of the masks used in this lighting, and the sequence of reagents to determine the final products and their placement. In the method of Fodor's possible a high degree of miniaturization, because the density of places of synthesis involves only physical constraints on the spatial addressability, i.e., the diffraction of light. Each connection is available and its location is precisely known. Therefore, oligo-chip, thus obtained, can be easily used in LNG.

Fodor and others (1991) describe the activated light education dinucleotide as follows. 5'-Nitrouracil thymidine was synthesized from 3'-O-thymidine acetate. After deprotection with a base of 5'-nitrouracil thymidine was attached to zaminirovannoy substrate by contacting the 3'-hydroxyl group. Protecting groups nitrofural removed lighting through 500 μm mask type chessboard. Then, the substrate was treated activated by phosphoramidite 2'-deoxycytidine. To monitor the reaction fluorometrically, deoxycytidine modified by the educational communications protected FMOC-aminohexyl attached to exoticism Amin. Polubotko substrate FITC. Therefore, following this method, it is possible to synthesize related basis oligonucletide.

The bond formation of the oligonucleotide with nylon base, as described by van ness and others (1991), requires the activation of nylon surface through alkylation and selective activation of 5'-amine oligonucleotides cyanur-chloride as follows. Nylon surface atriums using triethyloxonium tetrafluoroborate with the formation of amine-reactive imitat-esters on the surface of nylon and 1-methyl-2-pyrrolidone is used as the solvent. The surface of the nylon is not polished to the maximum possible surface area.

Then activated the surface reacts with poly(ethylenimine) (Mr approximately 10K-70K) with the formation of the polymer coating, which creates an extended surface amine to attach oligo. The oligonucleotide(s) with amine tail selectively reacts with excess cyanur-chloride, exclusively amine tail, giving 4,6-sodium dichloro-1,3,5-triazinyl-oligonucleotide(s) in quantitative yield. The offset of one chlorine environment cyanur-chloride amino group reduces the reactivity of the remaining groups chlorine. This leads to increased gidroliticescoe time in buffer containing aqueous solutions (pH 8,3, 4oC, 1 week) and can be easily isolated and purified by chromatography dimensional elution or by ultrafiltration.

This reaction is specific for amino tail with no apparent reaction to the nucleotide environments. Then coated with PEI surface nylon reacts with activated cyanur-chloride of the oligonucleotide. High concentrations of "exciting" sequence can easily be contacted on the surface and unreacted amines are covered succinoyl anhydride in the final stage of the process of derivatization.

One particular method of preparation of the oligonucleotides associated with the base, is to use the generated light synthesis described Pithom and others (1994). These authors used existing methods of photolithography for forming a series of related samples of oligonucleotides (DNA chips). These methods, in which light is used to direct the synthesis of samples of oligonucleotides in miniaturizing series high-density, use photolabile 5'-protected N-acyl-deoxy nucleoside phosphoramidite, the surface chemistry of the Creator of the links and universal strategy of combinatorial synthesis. The matrix of their 256 spatial certain samples of oligonucleotides can fores described.

Pease and others (1994) presented a strategy suitable for use in the guided light in the synthesis of oligonucleotides. In this way the surface of solid supports modified photolabile protecting groups, is illuminated through a photolithographic mask, yielding reactive gidroksilnye groups in the illuminated areas. Then 3'O-phosphoramidite-activated deoxynucleoside (protected 5'-hydroxyl photolabile group) is on the surface and bonds are formed at the places that were affected by light. After coating the oxidation of polozka washed and the surface is illuminated through the 2nd mask for exposure to additional hydroxyl groups for the formation of ties. The second 5'-protected, 3'O-phosphoramidite-activated deoxynucleoside is applied to the surface. Selective foodarticle and cycles of education relations are repeated until it turns the desired range of products. As used photolithography, the process can be miniaturizing for the formation of a series of high-density samples of oligonucleotides, the sequence of which is known in every place.

Path synthesis for the preparation of the required 5'O-(alpha-methyl-6-nitropiperonyl)-N-fwbk -2'-Alicia N-acyl-2'-deoxynucleoside, which is reacted with 1-(2-nitro-4,5-methylenedioxyphenyl)-ethyl-1-chloroformate obtaining 5'-MeNPoc-N-acyl-2'-deoxynucleoside. In the second step the 3'-hydroxyl is reacted with 2-cyanoethyl N,N'-diisopropylchlorophosphoramidite using standard procedures to obtain 5'-MeNPoc-N-acyl-2'-deoxynucleoside-3'-O-(2-cyanoethyl-N-N - aminobutiramida)phosphoramidites. Svetosavska group is stable under ordinary conditions of synthesis of phosphoramidite and can leave the water base. These reagents can permanently be stored in argon 4SOOC.

It was reported periods polypropelene photolysis 28, 31, 27 and 18 for MeNPoc-dT, MeNPoc-dcibu, MeNPoc-dGPACand MeNPoc-dAPACrespectively (Pease and others, 1994). Therefore, lithographic synthesis recommended lighting time 4.5 min (9 t1/2MeNPoc-dC) to provide > 99% removal MeNPoc protecting groups.

Suitable synthetic base consists of 5.1 x 7.6 cm glass substrate prepared by cleaning in a concentrated NaOH, followed by extensive rinsing in water. Then the surface derivatized 2 hours with a solution of 10% (volume/volume) bis(2-hydroxyethyl) aminopropyltriethoxysilane (firm Petrarch Chemicals, Bristol, PA) in 95% ethanol, washed thoroughly with ethanol and ether, dried in Intesa is attached by a reaction derivatizing substrates with 4,4'-dimethoxytrityl (DMT)-hexamethylene-O-cyanoethyl phosphoramidite.

In summary, to initiate synthesis of a sample of the oligonucleotide, a suitable derivative of deoxynucleoside of phosphoramidite is attached to a synthetic base through educational communication. Then areas of the canvas are activated for the synthesis of lighting through, for example, holes 800 x 12800 micron photolithographic mask. Can be additional cycles of synthesis of phosphoramidite (DMT-protected deoxynucleoside) to form any desired sequence, such as any 4-, 5-, 6-, 7-, 8-, 9- or even 10-dimensional sequence. After removal of phosphate and ekzoticheskih amine-protecting groups concentrated NH4OH for 4 hours a substrate can be installed in a thermostatically controlled chamber hybridization with the water jacket, ready to work.

Of course, you can easily buy the chip DNA, such as the one described above activated light chips, from a commercial source. In this regard, you can contact the firms Affymetrix of Santa Clara, California 95051, and Beckman.

Example II

Modified oligonucleotides for use in samples

Modified oligonucleotides can be used in all procedures of this invention to improve the special is th base. For example, you can use the pyrimidines with halogen at position C5. It is believed that this improves the stability of the duplex by the impact on the installation base layers. You can also apply 2,6-diaminopurine to give the 3rd link halogen in its base pairing with thymine, which thermally stabilizes DNA-duplexes. It was reported that the use of 2,6-diaminopurine leads to a significant improvement of the stability of short duplex oligomers. Its inclusion, as proposed, provides a more stringent annealing conditions triggering substances, thereby improving the specificity of formation of duplex and suppressing background problems or eliminating the use of shorter oligomers.

Synthesis options triphosphate these modified nucleotides described by Hoheisel and Leehom (1990). 5-chloro-2'-deoxyuridine and 2,6-diaminopurine 2'-deoxynucleoside acquired, for example, the company Sigma. The phosphorylation is performed as follows: 50 mg of dry 2-NH2-dAdo taken in 500 μl of dry triethyl phosphate with stirring in argon. Added 25 μl of POCl3and the mixture is maintained in a thermostat at -20oC. Meanwhile, 1 mmol pyrophosphoric acid dissolved in 0.95 ml of tri-n-butylamine and 2 ml of methanol and you the second time is also added to 70 μl of tri-n-butylamine. Finally, it is dissolved in 2 ml dry of dimethyl formamide.

After 90 min at -20oC mixture phosphorylation evaporated to remove excess POCl3and added tri-n-butylammonium pyrophosphate in dimethyl formamide. The curing is carried out for 1.5 min at room temperature. The reaction is stopped by adding 5 ml of 0.2 M triethylammonium bicarbonate (pH 7.6) and the mixture is kept on ice for 4 hours. 5-Cl-dUrd conditions will be identical, but added to 50 μl of POCl3and the phosphorylation is carried out at room temperature for 4 hours.

After hydrolysis, the mixture is evaporated, the pH is set at 7.5 and extracted in 1 volume of diethyl ether. Separation of the products produced, for example, in (2.5 x 20 cm) of Q-Sepharose column using a linear gradient of 0.15 M to 0.8 M triethylammonium the bicarbonate. When storing frozen nucleotides stable for a long time. You can also use indiscriminate similar base or universal base, as it is designed by Nichols and others (1994). This new analogue, 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole (denoted M), was formed for use in samples of oligonucleotides and initiating substances to solve the problems the project is agenturnoj sequence peptides. This analog increases the stacking layers and simultaneously minimizes interactions with the formation of hydrogen bonds, without destroying the spatial duplex DNA. Similar Meters of nucleoside was designed to maximize interaction when laying layers using appreticeship polar substituents associated with heteroaromatic rings, increasing intra - and naradiya interaction when laying to reduce the role of hydrogen bonds in the specificity of base pairing. Nichols and others (1994) prefer 3-nitropyrrole 2'-deoxyribonucleoside because of its structural and electronic similarity with p-nitroaniline, whose derivatives belong to the smallest known interlayers double retinue DNA.

Dimethoxytrityl-protected phosphoramidite nucleoside M can also be included in the nucleosides used as initiator substances for education sequence and the polymerase chain reaction (PCR). Nichols and others (1994) showed that a significant number of nucleotides can be replaced by M without loss of specificity of the triggering substance. Unique property M is its ability to replace long chains of adjacent nucleosides and still give functional initiator substances by the ladder of education sequences, and all PRC with 3 different containing M-initiating substances led to the expansion of the right product (Nichols and others, 1994).

The ability containing 3-nitropyrrole oligonucleotides can act as initiator substances confidently shows that the duplex structure should be formed with the complementary strands. Optical thermal profiles obtained for pairs of oligonucleotides d(5'-C2-T5XT5G2-3') and d(5'-C2A5YA5G2-3') (where X and Y can be A, C, G, T or M), as reported fit the usual sigmoidal pattern observed for the transition from double to single strands (strands) of DNA. The values of Tmoligonucleotides containing base pairs XM (where X was A, C, G or T and Y was M), as reported, all fall in the range of 3oC (Nichols and others, 1994).

Example III

Preparation forming sequences of chips and series

This example describes the physical embodiments of forming a sequence of chips, considered the inventor.

A basic example is the use of 6-mers that are attached to the surfaces 50 microns to obtain a chip with dimensions of 3 x 3 mm, which can be combined to produce a number of 20 x 20 cm is building a 9-dimensional chip with dimensions of 5 x 5 mm. 4000 units of such chips can be used to create a series of 30 x 30, see Fig. 2A, Fig. 2B and Fig. 2C shows another example of the row in which 4000-16000 of oligotypes located on the square. Plate or set of tubes, as also indicated, can be packaged with this range as part of a set of materials to create sequences.

The rows can be separated physically from each other, or with hydrophobic surfaces. One possible way to use the separation of the hydrophobic stripe is to apply this technology as Iso-Grid Microbiology System, manufactured by QA Laboratories, Toronto, Canada.

Hydrophobic grid membrane filter (HGMF) has been used in analytical Microbiology of food for about a decade, where they are uniquely attractive to those that have advanced numerical range and automated counting of colonies. One industrial supply grid is an ISO-GRID(TM) from the company QA Laboratories Ltd. (Toronto, Canada), which consists of a square (60 x 60 cm) polysulfone polymer (Gelman Tuffryn HT-450, a pore size of 0.45 MJ), which is printed in black paint hydrophobic mesh, consisting of 1600 (40 x 40) square cells. HGMF was previously modified by the addition of bacterial suspensions vacuum, f is krabov limited grid cells with a known position and size of the membrane, HGMF operates more as a unit MPN than the usual plate or a membrane filter. Peterkin and others (1987) reported that these HGMF can be used for distribution and storage of genomic libraries when used with the Replicator HGMF. One such tool replicates the growth from each of the 1600 cells ISO-GRID and allows you to make many copies of an exemplary HGMF (Peterkin and others, 1987).

Sharpe and others (1989) also used the ISO-GRID HGMF from QA Laboratories and automated meter HGMF (MI-100 Interpreter) and the Replicator RP-100. They have reported a method of maintaining and sorting the many cultures of microbes.

Peterkin and his colleagues once described the sorting method of DNA samples using hydrophobic grid-membrane filter (Peterkin others, 1989).

These authors reported the effective hybridization of colonies directly on the HGMF. Earlier poor results were obtained due to the low DNA binding ability polysulfone polymer, on which are printed HGMF. However, Peterkin and others (1989) reported that binding of DNA to the surface of the membrane was improved when processing replicated and sustained in thermostat HGMF polyethylenimine, polycation, before contact with DNA. Although this early work was used cellular attachment of DNA and the G

In order to quickly identify useful sequence, Peterkin and others (1989) used radiochannel plasmid DNA from different clones and tested its specificity relative DNA prepared HGMF. Thus, DNA from recombinant plasmids were quickly sorted by hybridization of colonies relative to 100 organisms to replicate the HGMF, which can be easily and reproducibly be prepared.

We need to solve two main problems. Manipulation with small (2-3 mm) chips and parallel execution of thousands of reactions. The solution offered by this invention, is to hold the chips and samples in the respective rows. In one example, the chips containing 250000 9-mers are synthesized on a silicon substrate in the form of plates 8 x 8 mM (15 μm/oligonucleotide Pease and others, 1994), built in format 8 × 12 (96 chips) with a groove between them in 1 mM. Samples are added to either a multichannel pipette or a number of pins, one sample per 1 chip. For calculation of all 4000 6-mers need to apply 42 number of chips, or using different rows, or re-using one set of rows of chips several times.

In this case, when early item of the application; F= 9; P=6; F+P=15.

Chips m is, the AK x=4 to 10 and n= 1-4. In order to achieve more efficient hybridization and to avoid the potential impact of any oligonucleotides basis specified database, you can surround especificamente databases, which seems such a formula as a (N)nBx(N)m (Fig. 4).

Example IV

Preparation of fragments of nucleic acids

A nucleic acid sequence which you want to create, can be obtained from any suitable source, such as a cDNA, genomic DNA, chromosomal DNA, microcassette chromosomal bands, cosignee or YAC inserts and RNA, including mRNA without any expansion stages. For example, Sambrook and others (1989) describe 3 of the Protocol to isolate DNA with high molecular weight from mammalian cells (pages 9.14-9.23).

Then the nucleic acids are fragmented by any of the methods known in the art, including, for example, the use of restrictive enzymes, as described on page 9.24-9.28 Sambrook and others (1989), shear ultrasound and processing of NaOH.

Shift at low pressure is also relevant, as described by Sripuram and others (1990). In this method, the DNA samples are passed through a small French pressure sensor with set pressures from small to intermediate. Richment. The results of these studies indicate that the shift at low pressure is a useful alternative sound and enzymatic methods DNA fragmentation.

One particularly suitable method for separation of DNA fragments, as is, is one that uses two endonuclease recognition database, CviJI, described by Fitzgerald and others (1992). These authors described an approach for the rapid fragmentation and fractionation of DNA on specific dimensions, which they considered suitable for "fractional" cloning and creating sequences. The present inventor provides that it will also be particularly suitable for generating arbitrary but relatively small DNA fragments for use in the present technology education links.

Restrictive endonuclease CviJI usually splits the probability of the sequence PnGCPy between G and C, leaving blunt ends. Atypical reaction conditions, which alter the specificity of this enzyme (CviJI**this gives a quasi-random distribution of DNA fragments from small molecules pUC19 (2688 base pairs). Fitzgerald and others (1992) quantitatively assessed the arbitrariness of this strategy slicing using CviJI**the product of the NGOs associated, without repair all, the cloning vector lacZ minus M13. Sequence analysis of the 76 clones showed that CviJI**limits PyGCPy and PuGCPu in addition to places PuGCPy and that new data on the sequence accumulated at a speed consistent with random fragmentation.

As reported in the literature, the advantages of this approach compared to fractionation by sanicula (sound) and agarose gel include: required fewer DNA (0.2 to 0.5 μg instead of 2-5 µg) and has fewer steps (no need prelink, repair all, chemical extraction or electrophoresis agarose gel and elution). It is suggested that these benefits will also be useful in the preparation of DNA to create sequences in format 3.

Regardless of how you obtained or are fragments of the nucleic acid, it is important denaturing the DNA to get to the hybridization of single curly pieces. This is achieved by keeping the DNA solution for 2-5 minutes at 80-90oC. Then the solution is quickly cooled to 2oC to prevent the re-Naturali DNA fragments before they come into contact with the chip. Phosphate groups also need to be removed from genomeid can be prepared by automated synthesis, what is routine for specialists, for example, using Applied Biosystems. Alternatively, samples can be prepared using methods Biotechnologies Inc. using layers of porous substrates Teflon.

Samples of oligonucleotides can be mentioned, for example, radioactive labels (355, 32P, 33Pand preferably 33Pfor series with areas of 100-200 microns or 100-400 μm; non-radioactive isotopes (Jacobsen and others, 1990); or fluorophores (Brembo and others , 1988). All such methods of applying labels are conventional technique, as shown in the relevant sections of Sambrook and others (1989) and in other literature, for example, Schubert and others (1990), Murakami and others (1991) and Keith and others (1991).

In relation to the radiolabelling of the usual ways - this phase ends using T4 polynucleotide kinase or labeling with high specific activity using Maple or even T7 polymerase. This is described as follows.

Synthetic oligonucleotides are synthesized without a phosphate group at their ends 5' and can therefore be easily marked by means of the transfer of the gamma-32Por gamma-33Pfrom (gamma-32P)ATPor (gamma-33)ATPusing the enzyme of bacteriophage T4 which is high, as the specific activity of gamma-32P)ATPor (gamma-33P)ATP. The following reaction is to mark 10 pmoles of the oligonucleotide to high specific activity. Labeling on different amounts of the oligonucleotide can be easily achieved by increasing or decreasing the size of the reaction while maintaining constant concentrations of all components.

The reaction mixture will be created with the use of 1.0 μl of the oligonucleotide (10 pmoles/µl); 2,0 ál 10x bacteriophage T4 polynucleotide kinase buffer; 5,0 ál (gamma-32P)ATPor (gamma-33P)ATP(Sp. act. 5000 Ci/mmol; 10 mCi/ml in aqueous solution) (10 pmoles); and 11.4 μl of water. 8 units (about 1 µl) of the bacteriophage T4 polynucleotide kinase are added to the reaction mixture, well mixed and held for 45 minutes at 37oC. the Reaction mixture is heated for 10 minutes at 68oC for inactivation of bacteriophage T4 polynucleotide kinase.

Then determine the transfer efficiency 32Por 33Pto the oligonucleotide and its specific activity. If the specific activity of the sample is acceptable, it is cleared. If the specific activity is too low, add another 8 units 68oC to inactivate the enzyme.

Cleaning radiometric oligonucleotides can be achieved by precipitation with ethanol; deposition with pyridinium bromide; chromatography through a Biogel P-60; or by chromatography on a column Sep-Pak C18.

Samples with higher specific activities can be obtained by using the fragment maple E. coli DNA polymerase I for synthesizing strands of DNA dopolnyaya to the synthetic oligonucleotide. Short-initiating substance it hybridises to the template oligonucleotide, whose sequence is complementary to the desired radiochannel samples. Then initiating substance expands using fragment maple E. coli DNA polymerase I for inclusion (alpha-32P)dNTP or (gamma-33P)dNTR in a template-directed manner. After the reaction, the template and the product separated by denaturing, followed by electrophoresis through a polyacrylamide gel under conditions of denaturation. With this method, if desired, to form samples of oligonucleotides containing some radioactive atoms in a molecule of the oligonucleotide.

To use this method, you need to mix in pipe microfuge the calculated amount (alpha-32P)dNTP or (alpha-33P)dNTP, it is Rada template. The dNTP concentration should not be less than 1 micron at any stage during the reaction. Then the trumpet adds the appropriate amount of initiator substances and template DNA and initiating substance in a molar ratio of 3-10 times the template.

Then add 0.1 volume of 10 x buffer maple and well mixed. Then add 2-4 unit fragment maple E. coli DNA polymerase I, 3 μl reaction volume, mixed and held for 2-3 hours at 4oC. If desired, the course of the reaction can be monitored by removing small (0.1 µl) multiples and measuring the fraction of radioactivity, which was able to precipitate, 10% trichlorosucrose acid (TCA).

The reaction mixture is diluted with equal volumes of the filling gel buffer, heated to 80oC for 3 minutes and then the whole sample was loaded on a denaturing polyacrylamide gel. After electrophoresis the gel autoradiographed that allows you to find the sample and to remove it from the gel. There are also the following different ways fluorophores mark. Brembo and others (1988) describe the synthesis of fluorescently labeled initiating substances. Similar deoxyuridine with primary aminoven shoulder formers communication" of the 12 atoms, PR is ical intermediate substance with obtaining 5'(methyl propanol)-2'-deoxyuridine. Reaction with dimethoxytrityl-chloride gives the corresponding actuator 5'-dimethoxytrityl. Ester methyl hydrolyzed, is activated and reacts with appropriate manuallyand alkyl-diamine. After purification, the resulting nucleosides shoulder formers communication is converted to the nucleoside analogues capable for chemical synthesis of the oligonucleotide.

Then get oligonucleotides that contain one or two bases with the shoulder formers connection with the use of a modified chemistry of fosforito. In a solution of 50 nmoles of the oligonucleotide with the shoulder formers links in 25 μl of sodium bicarbonate 500 mM (pH 9,4) are added to 20 μl of FITC 300 mM in dimethyl-sulfoxide. The mixture was stirred at room temperature for 6 hours. The oligonucleotide is separated from free FITC by elution from columns of Sephadex G-25 1 x 30 cm with 20 vV ammonium acetate (pH 6) with a combination of fractions of the first peak of UV absorption.

In the General case, the fluorescent labeling of the oligonucleotide at the 5'-end in the beginning assumed 2 stages. First N-protected derivative of aminoalkyl of phosphoramidite is added to the 5'-end of the oligonucleotide during automated DNA synthesis. After removal of all the protecting group is m followed by purification of labeled oligonucleotide from excess dye using reverse phase HPLC or PAGE.

Schubert and others (1990) described the synthesis of phosphoramidite, which allows you to get oligonucleotides labeled with fluorescein during automated DNA synthesis. Methyl ester of fluorescein alkiliruya 4-chloro(4,4'-dimethoxytrityl)butanol-1 in the presence of K2CO3and KI in DMF for 17 hours. After removing groups of trityl 1% TFAb in chloroform product hospitilised standard procedures by bis (diisopropylamino)methoxypropene. Phosphorylation of the specified derivative obtained fluorescein leads to sufficient output H-phosphonate. The resulting amidic (solution 0.1 M in dry acetonitrile) is used for the automated synthesis of different initiating substances using beta cyanoethyl phosphoramidite chemistry and DNA synthesizer. Cleavage from the basics and deprotection are made using 25% aqueous ammonia solution for 36 hours at room temperature. The raw product is purified by PAGE, and labeled initiating substance apparently as pale-green fluorescent band at 310 nm. Elution and the diesel plant with cartridges RP 18 give the desired product.

Fluorescent labeling 5'-end of the sample in the method Schubert, directly the conventional phosphoramidite. After deprotection and removal of ammonia by liofilizirovanny using the speed vacuum or precipitation with ethanol fluorescently labeled oligonucleotides can directly be used for the formation of DNA sequences in the format of 3 LNG.

Murakami and others have also described the preparation labeled with fluorescein oligonucleotides. This synthesis is based on the method supported by the polymer phosphoramidite and hydrogen phosphonate. The Ethylenediamine or hexamethylenediamine were used as a leash. They are put through phosphoramidate the relationship that was formed by oxidation of the hydrogen-phosphonate intermediate substance in solution CCl4. Modified oligonucleotides are subjected to staining using primary amine-orienting reagent FITC, on the balls. The resulting modified oligonucleotide is cleaved from the beads and then clears RPLC.

Keith and others (1991) describe the use of samples of oligonucleotides that are directly attributable to exude alkaline phosphatase in combination with a direct chemiluminescent substrate (AMPPD), to allow detection of the samples. Phosphatase of alkaline can covalently to connect with a modified base of the oligonucleotide. After hybridization EOE gives fluorescence without excitation, i.e. the laser is not needed. It is believed that a strong signal can be formed with the use of such technology.

Labeled samples can be easily purchased in various industrial sources, including GENsET, not to synthesize.

Example VI

The removal of a phosphate group

As exude alkaline phosphatase bacteria (BAP), and Escherichia exude alkaline phosphatase calves (CIP) catalyze the removal of radicals 5'-phosphate of DNA and RNA. Therefore, they are appropriate for the removal of 5'phosphates from DNA and/or RNA to prevent the formation of links and incorrect hybridization. The removal of phosphate, as described by Sambruna and others (1989), is carried out after cutting or other shear genomic DNA.

BAP more active of the 2 exude alkaline phosphatases, but he is also much more resistant to heat and cleaning substances. Therefore, it is difficult to slow down BAP fully at the end of dephosphorylation reactions. Proteinase K is used for digestion CIP, which should be removed entirely, if the subsequent communication should be effective. An alternative method is to inactivate CIP by heating to 65oC for 1 hour (or 75oC for 10 minutes) in the presence of 5 mM EDTA (pH 8.0), followed by purification dephosphorylating DNA by extraction with phenol: x the organization

Below are some examples to describe the performance of the educational methodology of the sequences considered by the inventor. First, the entire chip's hybrid mixture of DNA complexity 100 million BP (chromosome 1 person). The guidelines for conducting the hybridization can be found in articles such as Drmanac and others (1990); hrapko and others (1991) and the Original and others (1994). In these articles specified temperature ranges hybridization, buffers and wash steps, which are suitable for use at the initial stage of format 3 LNG.

This inventor especially considering conducting hybridization for up to several hours at high salt concentrations and at low temperatures (-2 to 5oC) because of the relatively low concentration of the resulting target DNA. For this purpose, applied SSC buffer instead of buffer phosphate (Drmanac and others , 1990), which is deposited at 10oC. Washing does not necessarily have to be extensive (several minutes) because of the second stage and can be completely eliminated, if the conduction cycles of hybridization is used to create a sequence highly complex DNA samples. The same buffer is used for hybridization and washing, so that you is simple robotic devices on each row, for example, the number of 8 x 8 mm (example III), added one labeled probe, for example, 6-Mer. Used 96-Konakovo or 96-pin device, doing this for 42 operations. Again, the range of electoral conditions, as previously described in the scientific literature.

This inventor in particular considering the following conditions. First, after adding the labeled samples and extracts for only a few minutes (due to the high concentration of added oligonucleotides) at low temperature (0-5oC) the temperature is increased to 3 to 10oC depending on the length of F+P and is added to the washing buffer. At this time, used the washing buffer is compatible with any response education connections (for example, in the range of salt concentration of 100 mM). After adding ligase temperature again rises to 15-37oC to allow the rapid formation of ties (less than 30 min) and further izbiranii hybrids with a full match and mismatches.

The use of cationic detergents (detergents) substances is also being considered for use in the format of 3 LNG, as described by Ponticum and Berg (1991). These authors describe the use of two simple cationic cleaners, determinado aminotrimethylene bromide (TMAB), in which one of the methyl groups replaced by either a 12-carbon (DTAB), or 16-carbon (CTAB) alkyl group. TMAB is the salt of bromide ion Tetramethylammonium, the reagent used in the experiments re-natureloving nucleic acids to reduce the shift of the content of G-C temperature melting. DTAB and CTAB is similar to the structure of sodium dodecyl sulfate (SDS) with the substitution of the negatively charged sulfate SDS positively charged Quaternary amine. Although SDS is typically used in hybridization buffers to reduce non-specific education links and to slow nucleases, it does not greatly affect the rate of re-naturebase.

During the process of education of links you can add the enzyme labeled samples or after the step of washing to reduce the background.

Although it has not previously been proposed for use with any method of LNG technology ligase well established in the field of molecular biology. For example, hood and colleagues have described a method of detecting a gene through ligase (Landegren and others, 1988), a methodology which can easily be adapted for use in format 3 of LNG. Landegren describes the analysis of the presence of these DNA sequences on the basis of Ecole DNA.

Then two of the oligonucleotide covalently connected under the action of a DNA ligase, provided that the nucleotides at the junctions correctly paired bases. Although it was not addressed previously, this situation now occurs in education consistency in format 3. Wu and Wallace also describe the use of the bacteriophage T4 DNA ligase to join two adjacent short synthetic oligonucleotides. Their reactions are the formation of ties oligo was carried out in 50 mM Tris HCl pH to 7.6, 10 mM MgCl2, 1 mM ATP, 1 mM DTT and 5% PEG. The formation of the communication was carried out by heating to 100oC for 5-10 min, followed by cooling to 0oC before adding T4 DNA ligase (1 unit; Bethesda Research Laboratory). Most of the reactions of formation of linkages was carried out at 30oC and ended by heating to 100oC for 5 minutes

Then there is the final washing, suitable for selective detection of hybridized adjacent to, or formed a relationship, oligonucleotides with a length of (F+P). This washing step is carried out in water for a few minutes at 40-60oC to flush all neobrazovanix connection labeled samples and all other compounds in order to reduce the background. Thanks covalently bound IU).

Depending on the label receiving images of the chips is done on different machines. For radioactive labels, you can use the storage phosphor screen and PhosphorImager scanner (firm Molecular Dynamics, Sunnyvale, CA). The chips are placed in the cassette and covered with a phosphor screen. After 1-4 hours of exposure, the screen is scanned and the image file is stored on the hard drive of the computer.

For the detection of fluorescent labels are used with a CCD camera and EPI-fluorescence or confocal microscopy. For chips formed directly on the elements of the image with a CCD camera, the detection can be carried out as described by Eggers and others (1994).

Detectors with charge-coupled devices (CCD) are used as the active solid foundations, which quantitatively detect and form the image of the distribution of labeled target molecules in the analyses based on samples. These devices are inherent in microelectronics characteristics that combine highly parallel analyses, ultrasensitive detection, high performance (throughput), integrated data collection and calculation. Eggers and others (1994) describe the use of the quantitative assessment with an accuracy of seconds due to the high sensitivity and applied direct connection.

The approach of the integral detection with CCD allows the detection of events of the formation of molecular bonds on the chips. The detector quickly forms a two-dimensional picture, which uniquely characterizes the sample. In particular the work of molecular detector CCD separate biological sample contacted (neutralized) directly on the picture elements of the CCD and can be attached to a disposable strip-cover, placed on the surface of the CCD. The molecules of the sample can be marked with a radioisotope, chemiluminescent or fluorescent labels.

After exposure to the sample number of samples based on a CCD photons or products of the decay of the radioisotope are emitted at the location of image elements, where the sample formed a connection, in the case of 3 format, with two complementary samples.

In turn, a pair of electron-hole are generated in silicon, when charged particles or radiation from labeled samples

fall on the control electrodes of the CCD. Then the electrons are collected under the adjacent control electrodes of the CCD and sequentially read on the module display. The number of photoelectrons generated at each picture element, is directly proportional to the share of the different relationships can be quantified (Eggers and others, 1994).

As recently reported, the CCD-based silicon has advantages such as semiconductor detectors and imaging primarily due to the high sensitivity of the devices in a wide wavelength range (from 1 to 10000 angstroms). Silicon is very sensitive to electromagnetic radiation from the visible spectrum to soft x-rays. For visible light one photon incident on a control electrode of the CCD, leads to one of the charged electron package under the electrode. One beta particle soft x-ray radiation (typically in the range of Kev to MeV) generates from thousands to tens of thousands of electrons. In addition to high sensitivity CCD, described by Eggers and others (1994), provide a wide dynamic range (4-5 orders of magnitude), to detect these because the service charge may include from a few to 105the electrons. Feature detection is linear over a wide dynamic range.

By placing create an image number in the vicinity of the sample collection efficiency is improved at least 10 times compared with the methods based on lenses, such as are used in conventional cameras on the CCD. That is, the sample (emic is tick imaging, such as lenses and mirrors.

When the radioisotope is attached as telling the group to the target molecules, the energy of the particles detected. Several reporting groups that emit particles with different energy, has been used successfully with microinhomogeneity detectors, such as 32P, 33P, 35S, 14C, 125L. Particles with higher energy, such as from 32Pprovide the high sensitivity molecular detection, while particles with lower energy, such as from 35Sprovide the best resolution. Therefore, the choice of reporting the radioisotope can be made on request. When a particular radioisotope label is selected, the detection performance can be predicted by calculating the signal-to-noise ratio, as described by Eggers and others (1994).

An alternative procedure fluorescent detection involves the use of fluorescent or chemiluminescent reporting groups attached to the target molecules. The fluorescent label can be attached covalently or through interaction. Fluorescent dyes, such as ethidium bromide with strong absorption bands in the near UV range (300-350 nm) and the main quantum efficiency by several orders of magnitude lower than at the wavelength of excitation, than the wavelength of the fluorescent signal.

From the point of view of the detection of luminescence mnogokratnaya control electrodes (schema) of the CCD have a built-in ability to filter out the contribution of the incident light in the UV range, but they are very sensitive to the visible luminescence generated by the fluorescent reporting groups.

This is characterized by a selectivity relative to the UV excitation allows to achieve a high signal to noise ratio (more than 100) using the CCD, as mentioned in article Eggers and others (1994).

To link the sample to the detector can be obtained matrix hybridization on inexpensive substrates of SiO2which are then placed on the surface of the CCD after hybridization and drying. This format is cost-effective because the hybridization of DNA is carried out on inexpensive disposable substrates of SiO2that allows you to reuse more expensive on the CCD detector. Alternatively, samples can be linked directly on the CCD with the creation of a specialized matrix samples.

To associate sample cover SiO2homogeneous epoxy layer associated with the surface of the film using an epoxy-silane reagent and standard Hio2through secondary education amines with epoxy ring. The resulting relationship generates 17 can rotate relations division between the 3' base of the oligonucleotide and the surface of SiO2. To ensure full deprotonation amines and to minimize the formation of secondary structure during the creation of a connection, the reaction is carried out in 0.1 M KOH and incubated at 37oC 6 hours.

In format 3 LNG in General, the signals are calculated for each of the billion points. You will need to hybreed all series, for example, 4000 5 x 5 mm for 1 time and possible subsequent use of a smaller number of rows.

Hybridization with the conduction cycles is one possible way to increase the hybridization signals. For 1 cycle the majority of the samples hybridizing with fragments of DNA from tail sequences, complementary for labeled samples. With increasing temperature, these hybrids will melt (Fig. 3). For the next cycle some of them (about 0.1%) hybridize with the corresponding DNA fragment and additional labeled sample form of communication. In this case, the electoral fusion hybrids DNA mismatches at the same time for both sets of samples.

Hybridization is mperature for thermostable ligase. Then the temperature is reduced to 15-37oC and the chip is aged for up to 10 minutes and then the temperature rises to 37oC or higher for several minutes, and then again decreases. Cycles can be repeated up to 10 times. In one embodiment, the optimal a higher temperature (10-50oC) can be used without cycles and you can spend a longer reaction education communication (1-3 hours).

The procedure described here allows to produce complex chips using standard synthesis and precise determination of the locations of oligonucletides, because you have a relatively small number of oligonucleotides. For example, if all 7-dimensional oligo synthesized (16384 samples), you can define lists 256 million 14-mers.

One important variant of the invented method is to use more than 1 different labeled samples on the base line. This can be done by referring to 2 goals; multiplexing to reduce the number of separately hybridized series; or the determination of the list even longer oligo sequences, such as 3 x 6 or 3 x 7. In this case, if you use 2 labels, specificity 3 consecutive oligonucleotides can be almost absolute, because a positive place lesofat chips, contains samples BxNy, where y is from 1 to 4. These chips make it possible to read the sequence in different frames. This can also be achieved by use of appropriate sets of labeled samples, or both samples F and P may have some unspecified end position (i.e., an element with a terminal degeneration). You can also use a universal base as part of the educational communication for the connection of samples in a certain sequence with a solid Foundation. This makes the sample more than ready for hybridization and makes the structure more stable. If the sample has 5 bases, for example, you might use 3 universal database as educational communication (Fig. 4).

Example VIII

Analysis of the obtained data

The image files are analyzed by the image analysis program, such as the DOTS program (Drmanac and others, 1993), and caliroots and evaluated included statistical functions, for example, the program SCORES (Drmanac and others , 1994). The distribution of the signals is to determine the optimal threshold for conversion of a signal in the +/- output.

From the position of the detected label is determined by F+P nucleotide sequences of fragments by combining known consistently is the notes or the sub-segments of the sequence of the original molecule, such as a chromosome, then assembled from the overlapping sequences F+P, certain computational subtraction.

One option is to convert the signals of hybridization, such as labels, + / - output during the build process sequence. In this case, the Assembly starts with the sequence F+P with a very large account, for example, with F+P sequence AAAAAATTTTTT (PEFC. ID. N. 1). All 4 possible overlapping samples AAAAATTTTTTA (PEFC. ID. N. 3), AAAAATTTTTTT (PEFC. ID. N. 4), AAAAATTTTTTC (PEFC. ID. N. 5) and AAAAATTTTTTG (PEFC. ID. N. 6) and 3 additional samples, which are excellent in the beginning (TAAAAATTTTTT, th. ID. N. 7; CAAAAATTTTTT, th. ID. N. 8; GAAAAATTTTTT, th. ID. N. 9) are compared and determined 3 result: (1) only the initial sample and only 1 of 4 overlapping samples have accounts that are significant and positive in relation to the other 6 samples, in this case, the sequence AAAAAAATTTTTT (PEFC. ID. N. 1) will be extended by 1 nucleotide to the right; (2) none of the sample, except for the initial sample has a significantly positive score, the build will stop, for example, the sequence AAAAAATTTTT (PEFC. ID. N. 10) is at the end of the DNA molecule, the sequence of which is created; (3) found more than 1 significantly polojitelnaa and others, 1989).

Processes computational subtraction use computer programs using existing algorithms (see, for example, Pevzner, 1989; Drmanac and others, 1991; Labat, Drmanac, 1993).

If in addition F+P F(space 1)P, F(space 2)P, F(space 3)P or F(space 4)F is determined, the algorithms will be used to combine all the data sets to correct potential errors or to resolve the situation, when there is a problem of branching (see, for example, Drmanac and others, 1989; Baines and others, 1988).

Example IX

Reuse chips create sequence

If education links used in the process of creating a sequence, then the usual chip oligonucleotides cannot be immediately reused. The inventor considers that this can be solved in different ways.

You can use the ribonucleotides of the second sample, sample P, so that the sample can then be removed by treatment with RNase. In the processing by RNase can be used Mcasa A, endoribonuclease, which specifically corrodes single-stranded RNA 3' to the pyrimidine radicals and breaks down communication phosphate to the adjacent nucleotide. The end products are Piri and divalent cations.

To use the RNase usually withstand the chip in any suitable containing RNase buffer, as described by Sambruna and others (1989). It is appropriate to use 30-50 µl containing RNase buffer 8 x 8 mm or 9 x 9 mm matrix at 37oC from 10 to 60 minutes. Then washed with hybridization buffer.

Although it is not widely used, you can also use the base uracil as described by Craig and others (1989), in specific embodiments. The destruction of combinations related samples to obtain a reusable chip can be achieved by digestion of the repair enzyme of E. coli, Ural-DNA glycosylase, which removes uracil from DNA.

You can also create a specific split the relationship between samples and then split the connection upon detection. For example, this can be achieved by chemical bond formation, as described by Shabarova and others (1991) and Valley and others (1988).

Shabarova and others (1991) describe the condensation of oligodactyly nucleotides CYANOGEN bromide as a condensing agent. In their one-step chemical reaction the formation of ties oligonucleotides were heated to 97oC, slowly cooled to 0oC, then added 1 μl of 10 M Enrichment in acetonitrile.

Valley and others (1988) shows the od of chemical bonding for modification of the sugar-phosphate DNA bases with with water-soluble carbodiimide (CDI) as the binder. Selective cleavage phosphoamide relations involves contact with 15% CH3COOH for 5 min at 95oC. Selective cleavage of communication pyrophosphate involves contact with a mixture of pyridine-water (9: 1) and with just distilled (CF3CO)2O.

Although the compounds and methods according to this invention has been described in terms of preferred examples of its implementation, for professionals it will be obvious that you can change the connection methods and the steps or sequence of steps described herein ways, without departing from the concept, spirit and scope of the invention. More specifically it is obvious that some substances that are related chemically and physiologically, can be replaced substances described here, and will be received the same or similar results. All such similar substitutes and modifications apparent to specialists, are considered to be within the spirit, scope and concept of the invention defined by the attached claims. All reported substances and methods can be obtained and executed without undue experimentation.

1. The method of determining the sequence of nucleic acid molecules, characterized in that the conducting hybridization of molecules of known sequence, the first set of probes attached to a solid substrate, and the second set of probe is a labeled probe in the solution, create a covalent bond between hybridized with the oligonucleotide from the specified first set of probes and hybridized with the oligonucleotide from the specified second set of probes, identify covalently linked regions of the sequence and determine the sequence of nucleic acid molecules using these identified areas.

2. The method according to p. 1, characterized in that the specified hybridization exercise cycles.

3. The method according to p. 1, wherein the pre-spend fragmentation of a molecule of nucleic acid receiving the nucleic acid fragments of intermediate length.

4. The method according to p. 3, characterized in that the fragments sequentially hybridizing with complementary sequences of these two sets of small oligonucleotide probes of known sequence.

5. The method according to p. 3, characterized in that the obtained fragments simultaneously hybridizing with complementary sequences of the two sets of small oligonucleotides nucleic acid, length which is from about 10 to about 40 nucleotides, using small oligonucleotide probes, the length of which is approximately from 4 to 9 nucleotides.

7. The method according to p. 3, wherein conducting the hybridization of the indicated oligonucleotide probes with fully complementary sequences of these fragments.

8. The method according to p. 3, characterized in that the conducting hybridization oligonucleotide probes with directly neighboring sequences of these fragments.

9. The method according to p. 8, wherein conducting the hybridization of oligonucleotide probes with fully complementary to and directly adjacent sequences of these fragments.

10. The method according to p. 3, characterized in that use oligonucleotide probes covalently linked enzyme bond.

11. The method according to p. 3, characterized in that use oligonucleotide probes are covalently linked through chemical digirolamo agent.

12. The method according to p. 3, characterized in that when hybridization carry out the contacting of the specified first set of small attached oligonucleotide probes with the specified fragmentational sequence hybridisierung probe with the formation of primary complexes, in which the fragment is hybridized and available sequence spend contacting said source complexes with the specified second set of small labeled oligonucleotide probes under conditions that allow only such probes with fully complementary sequences to hybridisierung with a free sequence of the fragment with the formation of secondary complexes, in which the fragment hybridized with the attached probe and labeled probe form a covalent bond between the specified attached probe and the specified labeled probe, remove from the specified secondary complexes labeled probes that are not linked covalently attached to the probe, with the formation of covalently linked complexes, identify covalently bound complexes by detecting the presence of label, identify sequence fragments of the nucleic acids in these covalently linked complexes by combining known sequences hybridized attached and labeled probes.

13. The method of determining the sequence of nucleic acid molecules, characterized in that conduct fragmentation of nucleic acid molecules with getting fragmento and length F and the set of labeled oligonucleotide probes in solution with known sequences with length R, moreover, F + P < T, make contacting a specified number of immobilized oligonucleotide probes with these fragments of nucleic acid hybridization conditions, allowing to form primary complexes with hybridized, fully complementary sequences of length F and dehybridization sequences of fragments with length T - F, produce contact of these complexes with the specified set of labeled oligonucleotide probes in hybridization conditions, allowing to form secondary hybridized complexes with fully complementary sequences of length F and directly adjacent fully hybridized complementary sequences of length P, form a covalent bond between the specified labeled oligonucleotide probes and specified directly adjacent immobilized oligonucleotide probes that detect these secondary complexes by the presence of the label, identify sequences of length F + P fragments of nucleic acid molecules in these complexes by combining the known sequences hybridized immobilized what they determine the complete sequence of the nucleic acid molecule.

14. The method according to p. 13, characterized in that the length of T is approximately three times the length F.

15. The method according to p. 13, characterized in that the length of T is about 10 to 100 nucleotides, the length of F is from about 4 to 9 nucleotides, and the length of P is from about 4 to 9 nucleotides.

16. The method according to p. 15, characterized in that the length of T is about 20 nucleotides, the length F is about 6 nucleotides, and the length of P is about 6 nucleotides.

17. The method according to p. 13, characterized in that the covalent binding between directly neighboring immobilized and labeled oligonucleotide probes carry out enzymatic connection.

18. The method according to p. 13, characterized in that the covalent bond between directly neighboring immobilized and labeled oligonucleotide probes form using chemical digirolamo agent.

19. The method according to p. 13, characterized in that the use of nucleic acid representing the cloned DNA or chromosomal DNA.

20. The method according to p. 13, characterized in that the use of nucleic acid representing mRNA.

21. The method according to p. 13, characterized in that the fragment of the second sodium hydroxide or hydrodynamic fragmentation at low pressure.

22. The method according to p. 13, characterized in that the use of immobilized oligonucleotides, attached to glass, polystyrene or Teflon solid substrate.

23. The method according to p. 13, characterized in that the use of immobilized oligonucleotides attached to a solid substrate through fosfodiesterazu connection.

24. The method according to p. 13, characterized in that the use of immobilized oligonucleotides attached to a solid substrate through lightactive mechanism.

25. The method according to p. 13, characterized in that use labeled non-radioactive isotope or a fluorescent dye oligonucleotide probes.

26. The method according to p. 13, characterized in that use labeled isotopes35S32P or33P oligonucleotide probes.

27. The method according to p. 13, characterized in that the fragment of the nucleic acid molecule or nucleotide probes contain a modified or universal basis.

28. The method according to p. 13, wherein the labeled probes that are not covalently linked to the immobilized probe is removed from the secondary complexes by washing under strict conditions.

29. The method according to p. 13, otlichiya.smotret chip.

30. Kit for use in determining the sequence of nucleic acid molecules, characterized in that it contains a chip with a solid substrate having attached a set of oligonucleotide probes of known sequences, and these oligonucleotides are able to participate in hybridization reactions, as well as a set of containers with solutions labeled oligonucleotide probes of known sequences and vigirously agent.

31. Set on p. 30, characterized in that it used a lot of chips immobilized oligonucleotides installed in the form of a ranked list.

32. Set on p. 30, characterized in that the oligonucleotide probes have a length from about 4 to 9 nucleotides.

33. Set on p. 32, characterized in that the oligonucleotide probes have a length of about 6 nucleotides.

34. Set on p. 30, characterized in that the oligonucleotide probes attached to glass, polystyrene or Teflon solid substrate.

35. Set on p. 30, wherein the oligonucleotide probes attached to a solid substrate through fosfodiesterazu connection.

36. Set on p. 30 of the mechanism.

37. Set on p. 30, wherein the oligonucleotide probes in the state of non-radioactive isotope or a fluorescent dye.

38. Set on p. 30, wherein one of the oligonucleotides contains a modified or universal basis.

39. Set on p. 30, wherein the oligonucleotide probes in the state of isotopes35S32P or33P.

40. Set on p. 30, characterized in that as digirolamo agent used chemically ligiously agent.

41. Set on p. 30, characterized in that as digirolamo agent used the enzyme DNA ligase.

 

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21 cl, 92 dwg, 14 tbl, 24 ex

FIELD: medicine, biology, molecular biology.

SUBSTANCE: invention proposes a new method for differential diagnosis of representatives of family Chlamydiaceae. Method involves isolation of DNA of pathogen, amplification using real-time polymerase chain reaction (PCR) and primers CM1 and CM2 exhibiting specificity to 5'-terminal fragment of gene omp1 followed by post-amplification analysis of curves of PCR-products melting in the presence of nonspecific fluorescent dye SYBR Green I for separation of Chlamydia species and electrophoretic separation of PCR-products. Identification of species is carried out on the basis of differences in PCR-products melting point wherein melting point curves of all fragments of omp1 are characterized by the presence of two peaks reflecting two-stage dissociation of DNA chains in sites with different A/T-saturation degree. Proposed method provides carrying out the differentiation of all species of Chlamydia that are pathogenic for humans. Except for, method provides carrying out the differentiation of Chlamydiaceae causing diseases in animals and also method is simple, rapid and can be used for direct diagnosis of clinical material samples. Invention can be used in medicine, veterinary science and virology for differential diagnosis of representatives of family Chlamydia.

EFFECT: improved method for diagnosis.

2 ex

FIELD: medicine, biology, molecular biology.

SUBSTANCE: invention proposes a new method for differential diagnosis of representatives of family Chlamydiaceae. Method involves isolation of DNA from pathogen, amplification of target using primers CM1 and CM2 showing specificity to 5'-terminal fragment of gene omp1 and electrophoretic separation of polymerase chain reaction (PCR) products. Electrophoresis is carried out in agarose gel with addition of sequence-specific DNA-ligand - bis-benzimide-PEG. The species belonging of PCR-products is determined by comparison of the migration rate of PCR-products in gel with electrophoretic mobility of control. Proposed method provides carrying out the differentiation of all species of family Chlamydiaceae and method is simple and rapid and can be used for direct diagnosis of clinical material samples also. Invention can be used in medicine and virology for differential diagnosis of representatives of family Chlamydiaceae.

EFFECT: improved method for diagnosis.

2 dwg, 2 ex

FIELD: molecular biology, medicine, biochemistry.

SUBSTANCE: invention proposes a method for assay of mononucleotide changes in the known sequences of nucleic acids. Method involves hybridization with PCR-amplified matrix DNA and the following ligation a tandem on its consisting of tetranucleotide that comprises the diagnosed change and two oligonucleotides of the size 8-10 nucleotides being one of that is immobilized on surface of a solid-phase carrier through a 5'-phosphate linker, and the second oligonucleotide is labeled by 3'-end with biotin label. Then tetranucleotide is hybridized with the matrix DNA chain between two oligonucleotides directly that can be ligated with immobilized oligonucleotide through the 5'-end and with non-immobilized oligonucleotide through the 3'-end. The ligation product is detected by its transformation to enzyme label through the complex with high-affinity enzymatic catalyst followed by development of enzyme label in the presence of chromogenic, luminogenic or fluorogenic substrates. Applying a method provides preparing the simple and highly selective agent used for detection of known changes in gene structure.

EFFECT: improved assay method.

8 cl, 3 dwg, 30 ex

FIELD: genetic engineering, biotechnology, biochemistry, agriculture, food industry, medicine.

SUBSTANCE: invention relates to the transformation of plant with nucleic acid encoding enzyme Δ6-desaturase in C. elegans that results to preparing a plant with enhanced content of gamma-linolenic acid and resistance to cold. Desaturase extracted from the plant can be used for preparing a drug used for treatment of disorder in body associated with deficiency of gamma-linolenic acid in it.

EFFECT: valuable biological properties of genes and desaturases.

36 cl, 9 dwg, 2 ex

FIELD: medicine, hematology.

SUBSTANCE: one should isolate DNA out of peripheral blood lymphocytes due to polymerase chain reaction (PCR) technique of DNA synthesis, carry out genotyping for polymorphism of promoter area of TNF-alpha and TNF-beta gene. While detecting genotype LT*22 being characterized by availability of gene TNF-beta mutation in homozygous state, detect persons predisposed to the development of chronic lympholeukosis, and at certain combinations of genotypes TNF*22/LT*22 or TNF*12/LT*11 in patients with chronic lympholeukosis on should predict an aggressive flow of this disease.

EFFECT: higher accuracy of prediction.

3 ex, 3 tbl

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