JPH05500598A - Solid-phase sequencing method for single-stranded and double-stranded nucleic acids - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Solid-phase sequencing method for single-stranded and double-stranded nucleic acids Background of the invention Advances in nucleic acid sequence analysis in genetic engineering and molecular biology are very rapid. and is expected to continue for years to come. Obtain information about the primary structure of nucleic acids This includes genetic diseases, oncogenes, advances in biology, protein production, and other essential for understanding the field at the molecular level.

Elucidating the entire nucleic acid sequence of a long DNA molecule is difficult and time consuming. Therefore, the nucleus Numerous efforts have been made to make acid sequencing faster and more convenient. in general , sequence analysis of nucleic acids can be done by chemical or enzymatic methods. these two people Comparing the methods, the chemical approach by MaXall and G11bert ( 1) is the enzymatic method of Sanger and co-workers (2) or a modification thereof. has some fundamental drawbacks. Chemical sequencing techniques are very versatile. Analysis of long sequences often has a relatively high error rate. Enzymatic distribution In sequence determination, the DNA is, for example, cloned into a suitable vector and primed. After hybridization with the DNA polymer, this primer molecule extended by using enzymes and nucleotide triphosphates. Ru.

a mixture of dideoxynucleotide triphosphates, the so-called stopping reagents, or complementary strands with known end groups by lowering the concentration of one of the monomers. A shorter homologous piece of is generated. Radiolabeling of primers, radiolabeled monomers Further marking can be achieved by introducing a non-radiolabeled moiety, e.g. a fluorescent substance. If a car is added, gel electrophoresis separation is performed, followed by autoradiography or or by real-time laser spectrophotometry, the newly generated arrays can be observed. .

Reactions in chemical and enzymatic sequencing are typically performed in solution reactions.

Automation of such solution reactions is relatively expensive and requires robotic equipment (3). Such complex equipment is required. Therefore, important advances in sequencing This was the use of a solid-phase material that is commonly used. However, chemical (Maxarn-Gi 1ber t) Regarding the sequencing method, Jiosentani et al. (4) Solid-phase approaches such as those described in This limits its use in sequencing very long nucleic acids.

For enzymatic methods of sequence analysis, the only published approach uses solid phase technology. (S, see 5tahl et al. (5)). In this method, a specific vector This includes the cloning of the DNA to be sequenced within cRrT28, which is Ru. In this method, the target nucleic acid sequence is transferred to the enzyme Bg12 or PstE2. Biotinylated UTP and Klenow DNA polymers were excised with associated sticky ends. Labeling is possible by loading with lase. Next, the double-stranded DNA is Binds to the Vidin-Sepharose support by biotin-avidin interaction.

The DNA is then denatured and after hybridization of appropriate primers Sequencing proceeds. However, the method described by 5tahl et al. It has some basic drawbacks. The first limitation is that a specific vector is required. Is Rukoto.

This vector is a special type of vector that is selected in a cloning experiment. It is different from the vector. This generally involves converting the cloned sequence into a new vector. This involves moving to another location, which requires additional cost and effort. In particular, (b) If it is a single-stranded nucleic acid, firstly, to label this nucleic acid, convert it into double-stranded DNA. the second strand must be discarded by denaturation before sequencing.

Considering the shortcomings of current methods, it is especially arrangement of nucleic acid sequences or nucleic acid fragments for sequence analysis of very long nucleic acid sequences. A suitable solid phase is required for column analysis. The development of such technology is especially genome sequencing (6) and other large-scale sequencing projects of different organisms. Considering the project, important objectives and The present invention is particularly useful for large-scale sequencing projects such as those described above, including enzyme alignment of suitable nucleic acids. Development of new solid phases for column analysis (in accordance with a preferred embodiment of the present invention) , a method for sequence analysis of single-stranded and double-stranded nucleic acids on a solid-phase support, and its Including equipment. This allows nucleic acids for sequencing purposes to be solid and insoluble in water. modified nucleic acid units that act to anchor themselves to a polymeric support of Extend from the end. In a preferred method, a chemical or enzyme-catalyzed reaction is Denatured nucleotides that are attached to nucleic acid strands by reaction include, in particular, 5-bromo-2 deoxyuridylate or generally a halogenated nucleotide, or A nucleotide with appropriate substitutions in its base or sugar moieties. fixed like this Nucleic acids that can be converted into single-stranded DNA or RNA strands or double-stranded DNA or R It is a chain like a part of NA. Solid supports that are insoluble in water include cellulose, Polymers and inorganic supports such as wallose or Sephadex, especially primarily silicon It is a support having a skeleton structure formed by silicon and oxygen atoms.

The solid support, which is insoluble in water, contains effector groups.

ups), in particular they are specific for 5-bromo-2'-deoxy-uridylate. Differentially immobilized antibodies or other halogenated nucleotides or salts Other groups or sugar moieties that can selectively interact with appropriately substituted nucleotides may include appropriate units. Attachment of the nucleic acid strand to be sequenced to a polymer support In this case, the interaction between the effector group immobilized on the support and the modified nucleotide unit is action, particularly through receptor-ligand interactions. According to a preferred method: Nucleic acids immobilized on a solid phase are subjected to solution reactions or modifications thereof using the enzymatic methods described above. Sequence according to. In particular, sequence analysis has been It can be applied according to the approach described in the invention. This allows the sequenced The nucleic acids or primers to be treated can be attached to a solid phase by the methods described above. Change , this process can be automated, thereby using the support materials and fixation techniques described above. Apply a series of oligonucleotide primers (chromosome walking) using , sequence analysis can be performed continuously.

Five dishes of Yeongseodan I Ching FIG. 1 shows a solid-phase DNA sequencing method according to the method of the present invention.

FIG. 2 shows the method for double-stranded nucleic acids of the invention.

FIG. 3 shows a method for immobilizing primers according to the present invention.

FIG. 4 shows a template immobilization method according to the present invention.

Figure 5 shows chemical induction of 5-bromouridine at the 3' end of a nucleic acid according to the invention. Show how to enter.

FIG. 6 shows the preparation of oligonucleotides by the phosphite-triester method according to the present invention. A method for introducing bromouridine at the 5' end is shown.

FIG. 7 shows a schematic diagram of an apparatus for carrying out the method of the invention.

definition Solid phase J refers to materials such as Sepharose, Sephadex, and other glycodopolymers. solid, water-insoluble polymers, and inorganic supports, especially controlled pore glasses , refers to silica gel, etc.

"Anchoring group is a ligand-receptor complex, antigen-antibody complexes, such as protein A-antibody complexes or other complex proteins; and a biotin-streptavidino combination.

A "nucleic acid (sequence)" is all possible types and structures of nucleic acid sequences. nuclear Acids (sequences) include DNA and RNA, and also include deoxy and ribomonomers. Contains a polynucleotide composed of. Nucleic acids (sequences) can be single-stranded or double-stranded. Can exist as a main chain.

A "nucleic acid fragment" is composed of mononucleotides or 2-100 monomeric units. The components may include nucleotides in oligomers.

"Denatured nucleic acid (sequence)" means a biological nucleic acid sequence or a modified nucleic acid (sequence) fragments containing (partially) substitutions in their bases or sugar residues. Ru.

"-order structure" refers to a sequence of bases.

- Na words H In the solid-phase system for enzymatic sequence analysis of nucleic acids, which is the subject of the present invention, The enzyme-catalyzed polymerization on the surface of the An insoluble polymer is used, which can be used. Sequencing reactions are particularly rapid and Polymeric support capable of retaining excess aqueous solution (buffer solution) in the body cavity site in the desired manner.

Examples of such supporting materials are cellulose, sepharose or inorganic polymers. – especially inorganic polymers composed of a backbone of silicon and oxygen atoms. Can be mentioned. Particularly useful materials for enzyme-catalyzed sequence analysis are Sepharose and It was found that the glass material (7) has a controlled pore size.

The properties of the polymeric support are of some importance for applications in enzymatic sequencing of nucleic acids. There is a need.

There are several ways to immobilize a target nucleic acid or nucleic acid fragment on a support material. method. The following immobilization method is used for small nucleic acid fragments and up to 100 kb. have been shown to be particularly advantageous for binding single- or double-stranded nucleic acids in .

In a preferred method, attachment involves attaching the nucleic acid of interest to the support via a spacer. It consists of being combined with. A large number of different substances are available, depending on the conditions of use, in particular: Depending on the stability required in the sequence of sequencing reactions selected, can be used as a server. Particularly good results of enzyme sequencing methods have shown that the common composition ■ Obtained using the support system.

This composition I is a polymeric protein antibody nucleic acid, wherein the polymer is e.g. glass or controlled pore glass in which the protein is e.g. protein A or other suitable protein, the antibody being e.g. a mouse anti-BrdU antibody or a modified protein. Other antibodies against leotide, in which the nucleotides are e.g. Nucleic acids, or e.g. unprotected BrclU strands or denatured nucleos BrdU chain linked to the 3″ or 5″ end (unprotected BrdU chain or Nucleic acid fragment (tailed at the 3” or 5’ end by another modified nucleotide strand) It is. The size of this nucleic acid or nucleotide fragment is one non-stranded short oligonucleotide. Large DNA with thousands of monomer units (single-stranded and double-stranded) It can range up to. Correlated to the corresponding antibody, e.g. 5-promodeo The modified nucleic acid component of the xyuridylate may be present at the 3' or 5' terminal region, or It can be suitably immobilized within the nucleic acid strand.

Attachment of the protein to the support can be accomplished by, for example, direct binding of the antibody to the polymer. Although such reagents are not absolutely necessary, they can be It can be done.

Although it is recommended to use proteins, especially protein A, as spacers, Other proteins can also be applied in a similar manner.

Furthermore, an aspect of the present invention is to immobilize a nucleic acid of interest via ligand receptor binding. For example, the polymer matrix or the protein immobilized on the alcoholic acid itself It is an antibody that In preferred use, bromouridine, halogen-substituted Other uridine derivatives or nucleotides such as adenone, thymidine, guar/sine , 7tidine, i/sine derivatives, or other substituted or modified bases or sugars. It is an antibody against a part. In other embodiments, biotin streptavidin Gin type is used.

Attachment of the desired nucleic acid involves adding modified nucleotides at the 3' or 5' end. More primary extension or introduction of degenerate nucleotides into the nucleic acid strand is necessary. considered essential. For example, nucleic acids are transferred to the nucleus catalyzed by terminal nucleotide transferases According to Ortigao et al. (Reference 8), promodeo It can be extended by xyuridylate. Similarly, other nuclei whose bases have been denatured Cleotides are oligonucleotides or large molecules up to 11 (kb) to be added to the 3' end of the nucleic acid of interest, which can be stranded or double-stranded DNA. Can be used as a substrate.

(Hereafter the clue) Alternatively, the oligonucleotide or polynucleotide is located at the 5' or 3' position. its configuration by chemical reaction with a nucleic acid component that is tailed or modified with May be expanded within the column. In such chemical synthesis, the same method as ■-phosphonate method is used. Like, phosphodiester, phosphate triester, submersible! / that of phosphoric acid triester A phosphoramidite method using each can be used. And the latter three methods is the most frequently used solid-phase chemical synthesis method.

Oligonucleotides or polynucleotides labeled or tiled by one of the methods described above. Any of the nucleotides can be attached to a polymeric support according to the invention. present invention One aspect of this is to use oligonucleotides with appropriate modifications as primers for sequence analysis. Immobilization of leotide and template for sequencing reaction, i.e. Both immobilizing long single-stranded or double-stranded polynucleotides as fixed targets That's the way to go.

Solid-phase sequence analysis is then performed on the immobilized target DNA or or using immobilized primers. Primer strand hybridization Separation may be performed prior to separation. Non-hybrid second stratum The second primer can then be re-immobilized and sequenced using a second primer.

If there is a choice between immobilization of target nucleic acids or immobilization of sequencing primers, In this case, the target nucleic acid for sequence determination is preferably immobilized, so that Sequence analogs obtained by the reaction can be easily isolated by denaturation. ply By immobilization of the mer, the resulting extension product, when sequenced, first Denatured from the nucleic acid and the newly formed dideoxy terminal sequence e.g. High concentration salt solution or low pi (unless cleaved from the polymer support by solution) No.

Such sequence analysis is a solution-based method that does not require prior cloning of the target nucleic acid. It can be done analogously to in-liquid enzymatic sequencing (2). later isolated and for sequencing Fragments identified on the gel were treated with dideoxynucleotides as stopping reagents, and D from the primers in a manner similar to the conventional San- reaction using NA polymerase. Extended. Analogs synthesized in this way can be used with primers or nucleoside 3 It can be visualized by reading the label for either one of the phosphates.

Such labels may be radiolabels such as 32p or 353, or It can be any fluorescent moiety known in the art. In particular, the latter triphosphate labeling This makes it virtually possible to introduce labels into newly synthesized nucleotide fragments; This makes it possible to read nucleic acid sequences of up to about 5000 bases by solid phase method.

Sequencing of longer nucleic acids requires the application of several types of primers (Figure 4) . Sequencing is initiated using the first primer. And the newly generated The 3' end of the longest fragment was read and inserted near the end (nest ed) Primers are used to start a new sequencing cycle. This circulation method is This process is repeated until the entire sequence of the nucleic acid is solved.

The method of the invention includes the following steps: a nucleic acid labeled with a modified base or transfer the fragments through suitable receptors to insoluble polymers or similar adsorption means. to fix; hybridization of primers (or sequencing targets); and manual or automated enzyme-catalyzed sequence analysis.

If the receptor-ligand binding is reversible, the method described above will It can be combined with purification by identity chromatography.

The following examples provide detailed descriptions of various aspects of preferred embodiments of the invention. However, there is no meaning in limiting the inventive concept as set forth in the appended claims.

1 plate Practicing employee dATP deoxyadenosine triphosphate ddATP dithioquine adenosine triphosphate ddeTP phosphate dideoxycytidine triphosphate ddGTP dideoxyguanosyl ddTTP dideoxythymidine triphosphate BSA rough serum albumin hmm BrdU 5-bromo-2-thioxyuridine DTT dithiothreitol PAGE polyacrylamide gel PBS phosphate buffered saline SB Sequencing buffer Tip 1! mouth.

Functionalization of support materials using antibodies 500mg Protein A Sepharose 5ml O,9% NaCl and O,O 1xPBS 5 ml IXPBS containing S% NaN3 500ml? Us-BrdU-antibody solution (2 in 500 ml double-distilled water) 5mg lyophilizate) Protein A Sepharose containing NaCl and NaN3 The cells were swollen in 5 ml of 1x PBS for 15 minutes at room temperature and stored in the freezer. precipitation Transfer 250ml of gel probe to the column and seal the column with glass wool. and connected to a two-way valve. Wash Sepharose 5 times with 1 ml of 1x PBS each. Purified. The wash solution was gently pressurized by hand through the column. Mouse BrdU antibody 500 ml of molten Ti was added to the gel and the mixture was shaken overnight. Rinse the antibody solution , the support material was washed with 20 ml of 1xPBS.

The Five Lords of Senwa Labeling of nucleic acids using modified bases Alternative A) Terminal debovine 7 nucleotidyl transferase (terminal -deoxynucleotidyl-transferase) Reaction between lomodeoxyuridine and nucleic acid 0.2-10p+aol oligo or polynucleotide z, B, M13 β-Mercaptoethanol 1mM Potassium cacodylate IQQmM BSA 100μg/m1 CoC121mM 5-bromodeoxyuridine 400mM 5-3 phosphate 16U terminal deoxynucleotidyl transferase Incorporation of bromodeoxyuridine is carried out by enzymes, terminal deoxynucleotidyl It is catalyzed by spherase and occurs at the 3' position of the nucleic acid. The reactant is the catalyst After adding the enzyme, incubate for 15 minutes at 37°C in a total volume of 15 μl in the buffer system (described above). Incubated. If radiolabeling of nucleic acids is used, 10-15 pmo Repeat the same reaction by adding a-32P-dATP or a-35S-dATP of 1. You can get this with . Alternatively, the T4-polymer can be used before or after the tail reaction. Nucleotide kinase and either γ-32P-ATP or γ-”5-ATP 5' phosphorylation of nucleic acids catalyzed by molecules can be used.

After the tail reaction, the reaction product was desalted and made into a 10 cI++ long and 5 mm diameter septum. Purify excess BrdU by filtration on a Addex column. In this way, The reaction product is liberated from the reactant containing bromodeoxyuridine triphosphate in the reaction. let The elution solvent is double distilled water with pF18.

Alternative B) Incorporation of BrdU by chemical oligonucleotide synthesis During the chemical synthesis of oligonucleotides on a solid support, BrdU is can be captured at the desired location.

a) If BrdU is to be incorporated at the 3′ end of the oligonucleotide, Derivatization of the support material is required (as shown in Figure 5).

b) If BrdU is to be incorporated at the 5′ end of the oligonucleotide by using BrdU phosphoramidites in the synthesized nucleotide chain. Uptake thus occurs (as shown in Figure 6).

Alternative A is currently preferred due to the relatively high cost of modified bases. .

Sourachi Goshu Supporting capacity of solid support 10ml Gel carrying antibodies xPBs 10 pmol BrdU labeled and radiolabeled oligonucleotide The binding ability of the support described in Example 1 was demonstrated by using the BrdU-labeled oligonucleotide. It was determined using After the bromouridylation step for easier detection Radiolabel the oligonucleotide by adding α-32P-ATP to did. The uptake rate was 40. It was OOOcpm/pmol. freeze-dried (lyophilysed) oligonucleotide at 1 piol/10μl resuspended at concentration. Transfer 10 μl of gel sample to the column and seal with glass wool. I did a rule. Sepharose was washed twice with 100 μM IXPBS each. 1p mol of oligonucleotide was applied to the column and pressurized.

After incubating for 15 minutes at 37°C, the gel was washed 5 times with 1 ml of 1x PBS. Purified. The loading capacity is 0.05 pmol oligonucleotide per 1 μl gel. It may be determined that the This method is repeated until no further increase is observed. returned. The limit of loading capacity was approximately 0.2 μmol/μm gel.

35% of the immobilized nucleic acids were washed 5 times with 50 μl of 1M acetic acid (pH 2). It could be re-eluted from the support by a cleaning step. 87% of the immobilized nucleic acids were was recovered with 5 washes with 2M NaCl in 1x PBS. pH 13 Guzo Nucleic acids are almost quantitatively (98%) removed from the support when eluted with thin buffer. It was confirmed that it was released.

After removing the nucleic acids from the gel, the support material is again cycled in a new sequencing reaction. It can be used for

BrdU-labeled M13 and M13 sequencing primers This method uses the following steps: two that encompass the process 1. Annealing the M13 target to the support. Washed twice with 0 μm 1×PBS. 0.2μg 5°Brd in 5μl SB The U-labeled target was loaded onto each gel sample and the pre-reaction mixture was incubated at 37°C for 15 minutes. I bet. Each sample was washed with 50 μm SB.

2. Primer annealing 4 μm oligonucleotide primer (concentration: 0.2 μmol/μl) Seed was added to each of the gel samples and incubated for 15 minutes at 37°C.

3. Sequencing reaction For each gel sample, add 1 μl of DTT, 2 μl of diluted GTP labeling mixture, 0.5 μl of a-”’P-dATP (1pa+ol) or 0.5μl α-355-d Add ATP (1 pmol) and 2 μm diluted Seq. Incubated for 0 minutes. The same procedure was performed for other bases. Those skilled in the art are aware of other Labels, e.g. fluorescently labeled primers or fluorescent markers/terminators It will be appreciated that may be used as well. Similarly, other chain exchanges Tender, e.g. DNA polymerase I (Klenow fragment), T7 sequence Polymerase and Taq polymerase may also be used. 4. Termination reaction 2.5 μI ddGTP for C column.

25 μm dclATP for A column.

2.5 μJ ddeTP for C column and 2.5 μM for T column of ddTTP was added and the reaction mixture was incubated at 37°C for 30 minutes. So Afterwards, the support was washed twice with 50μ each of 1×PBS.

5. Elution After synthesis, add formamide blue marker solution to the newly hybridized nucleic acid. and denatured it. 40. Use 1 μm of eluent per OOO cpm.

6. Gel electrophoresis The eluent was directly supported on 0.2 mm denaturing PAGE for separation. Well half, 4 0. OOOcpm was supported. After running the gel, create an autoradiograph did.

K Kogoji Solid-phase conjugation using biotin-streptavidin This method uses an antigen-antibody phase. This includes using biotin-streptavidin interactions rather than interactions. Contains. The present invention also uses another class of ligand-receptor binding. Here is another example of this concept. The method is as follows: 1. Labeling of nucleic acids using 11-biotin-dUTP 0.2-10 pmol of nucleic acids Or oligonucleotide β-mercaptoethanol 1mM Potassium cacodylate 100mM BSA 100μg/m1 CoC121mM ll-biotin-dUTP 200μM 16U terminal deoxynucleotidyl Transfer of biotin-dUTP is carried out by enzymes, terminal deoxynucleotides, It is catalyzed by Dyltransferase and occurs at the 3° position of the nucleic acid. The reactant is The terminal deoxynucleotidyl trans is dissolved in tail buffer (composition above). After addition of spherase, it was incubated for 30 minutes at 37°C. Complete list of reactants The volume is 50 μl. During biotinylation, the nucleic acid has 10-15 pmo of α- Radiolabeling by adding 32P-dNTPs or α-35S-dNTPs (see Example 2). Alternatively, the nucleic acid can be released before or after the tail reaction. It can also be radiolabeled by a phosphorylation reaction with a radioactive triphosphate.

The product was desalted by loading on a Sephadex column and washed with distilled water ( It is eluted at pH f8 (supplying a trace amount of NH).

A second method of incorporating biotinylated monomers is through chemical synthesis of oligonucleotides. It is between. This chemical label is Alternative B, described in Example 2. chemical label This is particularly important for sequence analysis where primers are annealed to a support. (Figure 3).

2. Annealing of biotinylated nucleic acid to solid support 40 μm strept Avidin-agarose (commercial product) 10 pmol Biotinylated nucleic acid or Ligonucleotide 1χPBS Wash streptavidin-agarose twice with 1x PBS (200 μl each) do. Biotinylated nucleic acid was supported on the support in a total volume of 20 μl, and the suspension was incubated at 37°C. Incubate for 30 minutes. Remove the supernatant and add the support to 200 μl of IXPBS. Wash 3 times with Studies using radiolabeled oligonucleotides have shown that A 75-85% incorporation into the carrier material was obtained.

3. If autoradiography is used to detect fragments, BrdU antibody immobilization Sequence analysis is performed as described for the template.

Those skilled in the art will appreciate that fluorescent primers and terminators can also be used as described above. One thing will be understood. The biotin-streptavidin system is Brd It has clear advantages over U-sequencing. That is, a) the primer is used as a template. b) to recover the synthesized fragments (with higher specificity). The advantage is that higher temperatures can be used.

By using higher temperatures, Taq polymerase can be used in sequencing reactions. There are sufficient benefits to be gained by using

Aiida f゛'s outfit A device according to the invention is illustrated in FIG. The device is a chromatography column columns 1-4, each containing four types of nucleobases (nucleobases). ) is for a reaction that identifies one of the following. The column is temperature controlled.

Washing solution, template solution, primer solution, enzyme mixture, ddATP, d Reservoir containing dCTP, ddGTP, ddTTP solution, and elution buffer Equipped with a bar. Wash solutions, templates, primers and enzyme mixtures, and elution buffer to any or all four columns simultaneously. Valves 1 to 9 are arranged so that ddATP, d dCTP, ddGTP, ddTTP are each placed in only one of the four columns. Supplied.

The effluent of each column can be drained or placed into a collector (or sequencing gel). Assign either one to enter through a two-way valve. air, argon, or nitrogen Apply atmospheric pressure to move the solution from the reservoir to the column. The valve is programmed time controlled by a programmable computer controller.

According to the method of the present invention, fragments of nucleic acids for sequencing (sequencing templates or or as a primer) is immobilized on a water-insoluble solid support. The column is Filled with support material. The reagents for sequencing are then processed under computer control. Under the control, the columns are fed as needed for the various reactions involved in the sequencing cycle. be done. In the case of immobilized sequencing targets, dideoxy terminal extension products are 4. Hybridized to immobilized template DNA as described in Section 4.

They are subsequently eluted with formamide dye buffer and directly distributed as described above. Run on column determination gel. Then polyacrylamide gel electrophoresis and fixation Identification of the sequence of the conjugated DNA is carried out using known methods.

The advantage of the equipment and solid-phase method is that the template remains intact throughout all reaction and washing steps. can be kept immobilized on a solid support and thus further sequencing cycles. It can be used for In this way, by using an appropriate set of primers, Therefore, it is possible to sequence large fragments such as genome DIJA in a simple and relatively inexpensive manner. can be analyzed. Other advantages include eliminating enzymes, excess monomer, etc. The end extension products can be removed by washing before dehybridization. It's about having options. The device is included in Sanger dideoxy sequencing. The feature is that all processes involved can be completely mechanized. It is directly coupled to a machine that automates the gel separation and reading process. can be done.

The device is activated by packing the column with immobilized sequencing primers. Other uses are also possible. In that case, perform the hybridization step in advance. extract DNA targets for sequencing from mixtures or biological materials by Can be isolated. A dehybridization step is then performed on the sequencing template. The plates were removed and the dideoxy-terminated fragments were then washed with the aforementioned high concentration salt solution or low p As described in Example 4, except that it should be released from the column by the H solution. Sequencing proceeds in substantially the same manner as before.

Preliminary for determining the number 1: 1. Pack the column with support material.

2. Equilibrate for approximately 2 minutes with buffer solution (wash solution from wash solution reservoir).

3. For example, place the nucleic acid template to be sequenced on a column containing immobilized anti-BrdU antibody. Hold the plate. As mentioned in Example 2, this template is Treatment with bromodeoxyridylate is being written. (i.e. from the template reservoir) 4. Binding occurs by incubation at 37°C (temperature control).

Note: Steps 3 and 4 require approximately 30 minutes.

Da■゛ Cycle: 5. Supply the primer solution from the primer reservoir to the four columns for 15 minutes. Ru. Primers may be labeled with, for example, radioactive isotopes or fluorescent markers. obtain.

6. Combine the polymerase, deoxynucleotide triphosphate, and sterile reagents in an appropriate The enzyme mixture contained in the buffer is supplied to each column from the enzyme mixture reservoir. Ru.

7. Polymerization begins with a break incubation time of approximately 10 minutes.

8. Polymerization with ddATP or ddCTP or ddGTP or ddTTP Each of the four "stop mixes" containing enzymes and sterile reagents were added to each glue bottle. feed the appropriate column from the server.

9. Incubating for an additional 10 minutes stops polymerization.

10. Wash with buffer by supplying buffer solution from buffer reservoir. Clean for 30 seconds to remove excess triphosphate and enzyme.

11. Deliver elution buffer from its reservoir for 1 minute to denature and remove hybrid The dideoxy-terminated primer extension product is eluted.

Repeat steps 5-11 using new primers for “chromosome walking”. Go back. For sequencing using immobilized primers, steps 5-7 are reversed (r ever) is required.

In addition, the collection of isolates and immobilized dideoxy-terminated extensions prior to gel electrophoresis were A twelfth step is included, consisting of processing the long product.

References 1. Max, A, M,, and G11bert! , (1980), r base Sequencing of end-labeled DNA using specific chemical cleavage J, Methods in Enzymology 65. Grossman, L., and Moldave, Edited by L. (Academic Press, New York) pp, 49 9-560゜2, Sanger, F, , N1klen, S, and Coulson, A. R. (1977), DNA alignment using r-chain termination inhibitors. Column determination J, Proc, Natl, Acad, Sci, USA 74.546 3-5467゜3, Wada, A., Yamamoto, M., and 5oe da, E. (1983), r-movement DNA sequencer: Maxam-Gi computer programmed microchemistry for lbert sequencing Nipicolator”, Rev, sci, Instrum, 54.1569-157 2゜4, Rosenthal, A., 5chvertner, S., Hahn , V., and Hunger, I.

D. (1985), solid-phase methods for nucleic acid sequencing: a novel, mechanically stable Simultaneous sequencing of different oligodeoxyribonucleotides using anion exchange paper Fixed J, Nucleic Ac1ds Res,.

13.1173-1184° 5.5tah1. S., Hultman, T., 01sson, A., Moks , T., and Uhlen.

M. (1988) Solid-phase DNA sequencing using the biotin-avidin system J. , Nucleic Ac1ds Res, , 16, 3025-3038゜6, Sm1th, L., and Hood, L. (19g?), r of the human genome. ? Sobbing and sequencing: How to proceed J, Biotechnology 5, 933-9397, Roger, G., and Seliger, H. (198 8), Polymer support J for chemical or enzymatic nucleic acid synthesis, Nucl Eotides and Nucleosides, in print.

8. Remalho-Ortjgao, J.F., Jjrikovski, G. , F., Lindl, T., and Seliger, H. (1988), [ 5-Bromo-2 for hybridization analysis of DN A and RNA - Terminal labeling of synthetic oligonucleotides using deoxyuridine J, Nuc leic Ac1ds Res,, now in print.

Engraving (no changes to the content) Fig, 1 Solid-phase DNA sequencing using Sanger's dideogen method I separate the pieces Fig, 2 Immobilized target duplex AGE Fig, 3 immobilized primer AGE Fl(!, 4 Immobilization of templates longer than 500 bases 1, denaturation, elution and sequencing on gels Read 2 Synthesis of new primers E! c.

Fig, 5 Chemical nucleic acid synthesis by known methods and isolation of sequences FTg, 6 2. Removal from protecting group and cleavage of immobilized fragmentsFig. 7

Claims (4)

[Claims] 1. In a method for sequence analysis of single-stranded and double-stranded nucleic acids on a solid support, A step of extending the end of a nucleic acid sequence with a modified nucleic acid unit, the step comprising: The nucleic acid unit functions as a unit for immobilizing itself to the solid support, The solid support is aqueous, such as cellulose, Sepharose, or Sephadex. It is an insoluble polymer, or it is composed mainly of silicon and oxygen atoms, for example. an inorganic support such as a support having a formed backbone structure; , such as 5-promo-2'-deoxyuridylate or other halogen nucleotides or nucleotides in which the base or sugar moiety is appropriately substituted or other suitable units capable of selectively interacting with streptapidine (st effector groups such as immobilized antibodies specific for retavidin). have; and Nucleic acid strands to be sequenced on a polymeric support are immobilized on the support. Interactions between defined effector groups and modified nucleic acid units, especially ligands - A process that is influenced by receptor binding interactions. 2. 2. The method according to claim 1, wherein the nucleic acid immobilized on the solid phase is in a solution reaction. sequenced and sequenced by enzymatic methods used for reaction or modification thereof. The nucleic acid or the primer used for sequencing is attached to a solid phase with the effector group. The method is combined with the method. 3. 3. The method of claim 2, wherein said steps are performed automatically and in a continuous manner. Column analysis uses immobilized ligand-receptor binding to investigate chromosome walking. A method carried out using a series of oligonucleotide primers for. 4. For sequencing single-stranded or double-stranded nucleic acid fragments by enzymatic sequencing A device, The device is four columns for holding solid phase supports with effector groups; ddATP in one of the columns, ddCTP in the other one of the columns, and ddTTP to the fourth column. means of input; Inputs that provide primers, templates, and enzymes to each of the four columns means, wherein at least one of the primer and the template is linked to the effector; has an affinity for the ter group; and According to the programmed schedule for performing sequencing cycles, It includes control means for automatically controlling each of the input means.