WO1990001562A1

WO1990001562A1 - Solid-phase sequencing method for single- and double-stranded nucleic acids

Info

Publication number: WO1990001562A1
Application number: PCT/US1989/002185
Authority: WO
Inventors: Jose Flavio Ramalho-Ortigao; Gabriele GRÖGER; Gustav Jirikowski; Hartmut Seliger
Original assignee: Applied Biosystems, Inc.
Priority date: 1988-05-18
Filing date: 1989-05-18
Publication date: 1990-02-22
Also published as: DE3816934A1; EP0417161A4; EP0417161A1

Abstract

The invention includes a method and apparatus for the sequence analysis of single- and double-stranded nucleic acids on solid phase supports whereby nucleic acids for the purpose of sequencing are terminally elongated with modified nucleic acid units, which themselves serve as units for anchoring to a water insoluble and solid polymer support. In the preferred mode, modified nucleotides which are to be attached to a nucleic acid chain by chemical or enzymatically catalyzed reactions are in particular 5-bromo-2'-deoxyuridylate or in general other nucleotides halogenated or otherwise appropriately substituted at the base or sugar moieties, or streptavidin. Nucleic acids which can be immobilized in this way are either single-stranded DNA- or RNA-chains or such chains as parts of the double-stranded DNA or RNA. Water insoluble and solid supports are polymers such as cellulose, sepharose or sephadex as well as inorganic supports, in particular supports which have a backbone structure formed mainly by silicon and oxygen atoms. The water-insoluble solid supports have effector groups, in particular, they may contain immobilized antibodies specific to 5-bromo-2'-deoxyuridylate or other appropriate units capable of selective interaction with other halogenated nucleotides or nucleotides appropriately substituted at the base or sugar moieties. The binding of the nucleic acid strands which are to be sequenced to the polymer support is effected by interactions of the modified nucleotide units with the effector groups immobilized to the support, in particular by receptor ligand interactions. An apparatus is disclosed for carrying out the method.

Description

SOLID-PHASE SEQUENCING METHOD FOR SINGLE- AND DOUBLE-STRANDED NUCLEIC ACIDS

Background of the Invention

The growth of Genetic engineering and molecular biology sequence analysis of nucleic acids has been extremely rapid and is expected to remain so for the next several years. Obtaining information about the primary structure of nucleic acids is the prerequisite for the understanding of the molecular basis of hereditary diseases, oncogenesis, developmental biology, evolution of proteins and other areas.

The elucidation of total nucleic acid sequences of long chain DNA molecules is a difficult and time consuming task. Therefore many efforts are underway to make nucleic acid sequencing more rapid and simpler. Generally the sequence analysis of nucleic acids can be done by chemical or enzymatic methods. When both methods are compared, the chemical approach according to Maxam and Gilbert (1) has some basic disadvantages with respect to enzymatic methods used by Sanger and coworkers (2) or variants thereof. The chemical sequencing procedure is too short ranged and the analysis of longer sequences is often accompanied by a relatively high rate of error. For enzymatic sequencing DNA is e.g. cloned into an appropriate vector; after hybridization with a primer this primer molecule is elongated by the use of a DNA polymerase and nucleotide triphosphates . The admixture of a so- called stop-reagent, a dideoxynucleotidetriphosphate , or by lowering the concentration of one of the mono ers allows one to produce shorter homologous pieces of the complementary strand with known end groups. If furthermore a marker is applied, either by- radioactive labeling of the primer or by incorporation of radioactively labelled monomers or by incorporation of non-radioactively labelled moieties, e.g. fluorescent substances, one can visualize the newly produced sequences by gel electrophoretic separation either by autoradiography or through the use of real time laser spectrophotometry.

Both chemical as well as enzymatic sequencing reactions have so far been done usually as solution reactions. The automation of such solution reactions is relatively expensive and requires a complicated apparatus such as robotic devices (3) . An important advance therefore was the use of solid phase materials for the sequencing reactions. Solid phase approaches, as they have been described by osentani et al . (4) for the chemical (Maxa -Gilbert) sequencing method, however, are only of limited use for the sequencing of very long nucleic acids due to the disadvantages stated above .

For the enzymatic method of sequence analysis only one approach using a solid phase technique has so far been published (See S. Stahl et al . (5)) . That method involves the cloning of the DNA to be sequenced in a special vector, eRIT 28, for which the target nucleic acids are excised with sticky ends relating to the enzymes Bgl 2 or PstE 2, which allows labelling by fill up with biotinylated UTP plus Klenow DNA polymerase. The double stranded DNA is then coupled to an avidin-sepharose support via biotin-avidin interaction. The DNA is then denatured and the sequencing proceeds after hybridization of an appropriate primer. The method described by Stahl et al . , however, has some basic disadvantages. A limitation in the first place is the requirement of a particular vector. This vector is unusual and normally not a vector of choice in cloning experiments. This will generally involve a transfer of a cloned sequence into this new vector which involves additional costs and labor. In particular, if the target nucleic acid is a single stranded nucleic acid, it first has to be transformed into double stranded DNA for the purpose of labelling, the second strand being discarded by denaturation prior to sequencin .

In view of the difficulties of existing methods there is a demand for appropriate solid phases for applications in sequence analysis of nucleic acids or nucleic acid fragments which will in particular serve to allow a rapid automated and inexpensive sequence analysis also of very long chain nucleic acids in large numbers. The development of such techniques is . an important objective in particular in view of the project of sequence analysis of the human genome (6) and other large scale sequencing projects of different organisms .

Summary of the Invention

This invention features the development of new solid phases for the enzymatic sequence analysis of nucleic acids which are particularly suitable for the before-mentioned large scale sequencing projects.

In accordance with preferred embodiments, the invention includes a method and apparatus for the sequence analysis of single- and double-stranded nucleic acids on solid phase supports whereby nucleic acids for the purpose of sequencing are terminally elongated with modified nucleic acid units, which themselves serve as units for anchoring to a water insoluble and solid polymer support. In the preferred mode, modified nucleotides which are to be attached to a nucleic acid chain by chemical or enzymatically catalyzed reactions are in particular 5-bromo-2 ' -deoxyuridylate or in general other nucleotides halogenated or otherwise appropriately substituted at the base or sugar moieties. Nucleic acids which can be immobilized in this way are either single-stranded DNA- or RNA-chains or such chains as parts of the double-stranded DNA or RNA. Water insoluble and solid supports are polymers such as cellulose, sepharose or sephadex as well as inorganic supports, in particular supports which have a backbone structure formed mainly by silicon and oxygen atoms. The water-insoluble solid supports have effector groups, in particular, they may contain immobilized antibodies specific to 5-bromo- ' -deoxy- iridylate or other appropriate units capable of selective interaction with other halogenated nucleotides or nucleotides appropriately substituted at the base or sugar moieties. The binding of the nucleic acid strands which are to be sequenced to the polymer support is effected by interactions of the modified nucleotide units with the effector groups immobilized to the support, in particular by receptor ligand interactions. According to the preferred method, the nucleic acids immobilized to the solid phase are sequenced according to enzymatic procedure previously described for solution reactions or variants thereof. The sequence analysis in particular can be applied according to the procedure described in the invention for both single- and double-stranded nucleic acids, whereby the nucleic acid to be sequenced or alternatively the primer can be bound to the solid phase in the way described above. Also, the process can be automated, whereby a progressive sequence analysis can be done by the use a series of oligonucleotide primers (chromosome walking) applying the support material and immobilization procedure described above.

Brief Description of the Drawings

Fig. 1 shows a solid phase DNA sequencing according to the method ofthe invention.

Fig. 2 shows a method of the invention for double stranded nucleic acids.

Fig. 3 shows a method of immobilization of primer according to the invention.

Fig. 4 shows a method of immobilization of templates according to the invention.

Fig. 5 shows a method for chemical introduction of 5-bromouridine at the 3' end of a nucleic acid according to the invention.

Fig. 6 shows a method according to the invention for the introduction of bromouridine at the 5 ' -end of an oligonucleotide in the phosphite-tricster method.

Fig. 7 shows a schematic diagram of an apparatus for performing the method of the invention.

DEFINITIONS

"Solid Phase" describes a solid water insoluble polymer, such as sepharose, sephadex, other glycosidic polymers as well as inorganic supports, especially controlled pore glass, silicagel and others.

"Anchoring groups" are ligand-receptor complexes, e.g. antigen antibody complexes such as protein A antibody conjugate or other conjugated proteins, and biotin-stre avidin combinations .

"Nucleic acids" are all possible types and structures of nucleic acids. This encompasses DNA as well as RNA and also polynucleotides composed of deoxy and ribomono ers . The nucleic acids can be present as single or double strands.

"Nucleic acid fragments" can be constituents ranging from mononucleosides or -nucleotides via oligomers, composed of 2-100 monomer units.

"Modified nucleic acids" are biological nucleic acids or their fragments obtained by enzymatic or chemical methods and (partially) containing substitutions at the base or sugar residues.

"Primary structure" relates to the sequence of bases .

Detailed Description of the Preferred Embodiments

The solid phase system for enzymatic sequence analysis of nucleic acids which is the subject of this invention makes use of an insoluable polymer on the surface of which enzyme catalysed polymerization can be done manually or in an automatic apparatus. The sequencing reactions proceed particularly fast and in the desired way with polymer supports which allow an excess of aqueous solutions (buffer solutions) to sites in carrier cavities. Examples for such support materials are cellulose, sepharose or inorganic polymers, in particular inorganic polymers consisting of a backbone of silicon and oxygen atoms. Particularly useful materials for the enzyme catalyzed sequence analysis were found to be sepharose and controlled pore glass materials (7).

The nature of the polymer support is of some importance for the application in enzymatic sequencing procedures of nucleic acids.

The fixation of the target nucleic acids or nucleic acid fragments to the support material can be done in several ways. The immobilization method described below has been shown to be particularly advantageous for the binding both of small nucleic acid fragments as well as single or double stranded nucleic acids of up to 100 kb .

In a preferred mode, the attachment consists of binding the target nucleic acid to the support via a spacer. A number of different substances can be used as spacers depending on the conditions of the application, in particular depending on the stability required under the set of sequencing reactions chosen. Particularly good results for enzymatic sequencing have been obtained with support systems of the general composition I: polymer protein antibody nucleic acid, in which polymer is for example sepharose or controlled pore glass, the protein is for example protein A or another appropriate protein, antibody is for example mouse anti-BrdU antibody or another antibody against a modified nucleoside, a nucleotide is for example a single-stranded or double-stranded nucleic acid or nucleic acid fragment tailed for example at its 3' or 5' end with an unprotected BrdU chain or chains of other modified nucleotides. This nucleic acid or nucleotide fragment can range in size from a single short-chain oligonucleotide to a large DNA (single-as well as double-stranded) with thousands of monomer units. A modified nucleic acid constituent, for example 5-bromodeoxyuridylate, which correlates to its respective antibody, can be affixed preferentially at the 3' or 5' terminal position, or also within the nucleic acid chain.

The attachment of the protein to the support can be done for instance by carbodiimide coupling, although such reagents arc not absolutely necessary, if for example an antibody will bind directly to the polymer. The use of proteins as spacers is recommended, particularly protein A, but other proteins can alternatively be applied in analogous procedures .

A further aspect of invention is the immobilization of the target nucleic acid via ligand receptor binding, e.g. an antibody to the protein immobilized onto the polymer matrix itself. In preferential use are antibodies to bromouridinε, other halogen-substituted uridine derivatives or derivatives of nucleosides adenosine, thymidine, guanosine, cytidine, inosine et al. or against other substituted or modified bases or sugar moieties. In another embodiment, a biotin strepavidin system is used.

The attachment of the target nucleic acid requires primary elongation with a modified nucleotide at the 3'- or 5'- terminus or incorporation of modified nucleotides within the nucleic acid chain. For example, the nucleic acid can be elongated by bromodeoxyuridylate according to Ortigao et al . (reference 8) by 3 ' -terminal reaction of the nucleic acid catalyzed by terminal nucleotidyl transferase. In a similar way other nucleotides modified at their bases can be used as substrates to be added to the 3'- end of the target nucleic acid, which can be either oligonucleotide or single or double - stranded DNA of up to many kb .

Alternatively, oligo- or polynuclootides can be tailed at the 5'- or 3'- position or elongated within their sequence by chemical reaction with a modified nucleic acid constituent. For such chemical syntheses, the phosphodiester , phosphotriester , phosphite triester resp. phosphoramidite procedures can be used as well as the H-phosphonate method, the latter 3 procedures being the most frequently used for solid-phase chemical synthesis.

Any oligo- or polynucleotide labelled or tailed in one of the above described fashions can be coupled to a polymer support according to the invention. An aspect of the invention is both the fixation of oligonuclotides containing an appropriate modification as primers for sequence analysis as well as, on the other hand, a fixation of long single- or double-stranded polynucleotides as templates for the sequencing reactions, thus, as targets for sequencing.

The solid-phase sequence analysis is then done with the immobilized target DNA or the immobilized primer according to Figs. 1-4. Prior to hybridization of a primer a strand, separation can be done. The non-hybridized second strand can subsequently be re-immobilized and sequenced with a second primer. If there is a choice between immobilization of the target nucleic acid or of the sequencing primer, preference is on the immobilization of the sequencing target nucleic acid, since, in that case, the sequence homologs obtained by the polymerase reaction can be easily isolated by denaturation. If the sequencing has been done by immobilization of the primer, the elongation products obtained must first be dentured from the target nucleic acid, and then the newly formed dideoxy-terminated sequences must be cleaved from the polymer support, e.g. by high salt or low pH solutions .

The sequence analysis as such can be done in analogy to the enzymatic sequencing in solution (2) without the requirement of previous cloning of the target nucleic acid. The fragments that are later separated and identified on the sequencing gel are elongated from a primer with use of dideoxynucleotides as stop reagents and a DNA polymerase in a way similar to the normal Sanger reactions. The visualization of the thus synthesized homologs can be done by reading a label either for the primer or for one of the nucleoside triphosphates , such label can be a radiolabel such as ~ - P or *-^,*⁵S or a fluorescent moiety as are known in the art. In particular the latter triphosphate labelling allows a substantial incorporation of label into the newly synthesized nucleotide fragments and, thus permits reading of nucleic acid sequences up to approximately 5000 bases by the solid-phase technique.

The sequencing of longer nucleic acids requires the application of several primers (Fig. 4) . The sequencing is started with the first primer. Then the 3¹- end of the longest newly generated fragments is read and a primer nested near that end is used for the start of a new cycle of sequencing. This cyclic procedure is repeated until the total sequence of the nucleic acid is elucidated.

The process of the invention entails the following steps :

Fixation of a nucleic acid or fragments tagged with modified bases via a suitable receptor onto an insoluble polymer or a similar attachment procedure.

Hybridization of a primer (or sequencing target).

Manual or automated enzyme-catalyzed sequence analysis .

The procedure described above, when there is reversibility of the receptor-1igand binding, can be combined with an affinity-chromatographic purification of a target nucleic acid.

The following examples provide a detailed description of various aspects of the preferred embodiment of the invention, but are not meant to be limiting as to the concept of the invention as embodied in the attached claims.

Examples

Used Abbreviations:

dATP Deoxyadenosinetriphosphate ddATP Dideoxyadenosinetriphosphate ddCTP Dideoxycytidinetriphosphate ddGTP Dideoxyguanosinetriphosphate ddTTP Dideoxythymidinetriphosphatc

BSA Bovine Serum Albumin

BrdU 5-Brom-2 ' -deoxyuridine

DTT Dithiothreitol

PAGE Polyacryla ide Gel

PBS Phosphate Buffered Saline

SB Sequencing Buffer

Example 1 Functionalization of a support material with antibody

500 mg Protein A-sepharose

5 ml IxPBS with 0,9% NaCl and 0.05% NaN₃ 5 ml IxPBS 500 ml mouse-BrdU-antibody-solution

(25 mg lyophilisate in 500 ml bidistilled water)

Protein A-sepharose was swollen in 5 ml IxPBS with NaCl and NaN₃ within 15 min. at room temperature and stored in the refrigerator. A sedimented 250 ml gel-probe was transferred into a column, which was sealed with glass wool and connected to a two way valve. Sepharose was washed five times with 1 ml IxPBS each time. The washing solution was pressed manually with mild pressure through the column. 500 ml of mouse BrdU antibody solution were added to the gel and the mixture was shaken overnight. The antibody-solution was rinsed off and the support material was washed with 20 ml IxPBS.

Example 2

Labelling of a Nucleic Acid with a Modified Base

Alternative A) Reaction of nucleic acids with 5-Bromodeoxyuridine by the use of terminal- deoxynucleotidyl-tranferase

0.2 - lOpmol Oligo- or polynucleotide , z.B. M13 β-Mercaptoethanol 1 M Potassium-Cacodylate 100 mM BSA 100 ug/ml

Cods-* 1 mM

5-Bromodeoxyuridine 400 mM 5 ' -triphosphate 16 u Terminal-deoxynucleotidyl- transferase

The incorporation of bromodeoxyuridine is catalysed by the enzyme terminal-deoxynucleotidyl- transferase and takes place at the 3'- position of the nucleic acid. The reactants were incubated in the buffer system (see above) after the addition of the catalyzing enzyme for 15 minutes at 37°C in a total volume of 50 ml. If radioactive labelling of the nucleic acid is to be used, it is possible to obtain this in the same reaction by the addition of 10-15 p ol alpha-^3aP-dATP or alpha-^aβS-dATP or alternatively, before or after the tailing reaction, by means of the 5 ' -phosphorylation of the nucleic acid, catalyzed by T4-polynucleotide-kinase and either gamma-^3S2P-ATP or gamma- ^*-^*S-ATP .

After the tailing-reaction, the reaction product is desalted and purified of the excess BrdU through filtration over a 10 cm length, 5 mm diameter sephadex-column, thus freeing it from reactions with unreacted Bromodeoxyuridine triphosphate. The olution-medium is bidistilled water pH 8.

Alternative B) Incorporation of BrdU through chemical oligonucleotide-synthesis

During the chemical oligonucleotide synthesis on a solid support, BrdU can be incorporated at every desired position.

a) If BrdU is to be incorporated at the 3'-terminus of the oligonucleotide, the derivatization of a support material is necessary (demonstrated in Figure 5 ) .

b) If BrdU is to be incorporated at the 5'-position of the oligonucleotide, the incorporation takes place by the utilization of BrdU-phosphoramidite in the chain which is synthesized (demonstrated in Figure 6) .

Due to the relatively high cost for modified bases at the present time, alternative A is preferred.

Example 3

Loading capacity of the solid support

10 ml Antibody-loaded gel

IxPBS 10 pmol BrdU labelled, radioactively labelled oli onucleotide

The binding-capacity of the described support in Example 1 was determined by means of a BrdU-labelled oligonucletode . For easier detection, the oligonucleotide was radioactively labelled after the bromouridinylation step by the addition of alpha-^a*-^*P-ATP . The incorporation rate was 40,000 cpm/pmol. The lyophilysed oligonucleotide was resuspended in a in concentration of 1 pmol/10 ul . A 10 ul gel sample was transferred to a column, and sealcd with glass wool. The sepharose was washed twice with 100 ul IxPBS each time. 1 pmol oligonucleotide is applied to the column and pressed in. After a 15 minute incubation at 37°C the gel was washed five times with 1 ml IxPBS. A capacity of 0.05 pmol oligonucleotide per ul gel could be determined. This process was repeated until no further increase could be found. The limit of the capacity was near 0.2 pmol/ul gel.

35% of the immobilized nucleic acid could be reeluted off the support by five washing-steps with 50 ul 1 M acetic acid, pH 2, each time. 87% of the anchored nucleic acid was recovered after a five time washing with 50 ul 2 M NaCl in IxPBS. A nearly quantitative release (98%) of the nucleic acid from the support was found by elution with glycine buffer pH 3.

After the removal of the nucleic acid from the gel the support material is again ready for use in a new sequencing reaction cycle.

Example 4

Solid phase sequencing

M13 labeled with BrdU and M13-sequencing-primer This procedure includes the following steps:

1. Annealing of the M13-target to the support

4 gel samples of 5 ul volume were washed twice with 100 ul IxPBS each time. 0.2 ug 5 ' BrdU-labelled target in 5 ul SB were loaded onto each gel sample and pre-reaction mixtures were incubated at 37°C for 15 minutes. Each sample was washed with 50 ul SB each time .

2. Annealing of the primer

1 ul of the oligonucleotide-primer (cone: 0.2 pmol/ul) was added to each of the four gel samples and. hybridized at 37°C during 15 minutes.

3. Sequencing-reaction

To each gel sample 1 ul DTT , 2 ul diluted GTP-label-mix, 0.5 ul alpha-³*^**P-dATP (1 pmol) or alternatively 0.5 ul alpha-^3*-^*S-dATP (1 pmol) and 2 ul diluted sequenase were added and incubated for 10 minutes at room temperature, and similarly for other bases. Those skilled in the art will appreciate that other labels could be used as well , for example fluorescently labelled primers, or fluorescent dideoxy terminators. Similarly other chain extenders could also be used, for example DNA polymerase I (Klinow Fragment), T7-sequenase, and Taq-polymerase.

4. Stop-reaction

To the G-column 2.5 ul ddGTP to the A-column 2.5 ul ddATP to the C-column 2.5 ul ddCTP and to the T-column 2.5 ul ddTTP were added and the reaction mixture was incubated for 30 minutes at 37°C. Thereafter the support was washed twice with 50 ul IxPBS each time.

5. Elution

After the synthesis, the newly hybridized nucleic acids were denatured by the addition of a formamide bluemarker solution. Per 40,000 cpm 1 ul of the eluent is used. 6. Gel-electrophoresis

The eluents were immediately loaded on a 0.2 mm denaturing PAGE and separated. Per well 40,000 cpm were loaded. After the gel run an autoradiograph was made .

EXAMPLE 5

Solid Phase Sequencing Using Biotin-Streptavidin

This approach involves use of biotin-streptavidin interaction rather than antigen-antibody interaction, and represents another example of the general concept of the invention using a different class of 1igand-receptor binding. The procedure is as follows: 1. Labelling of nucleic acids with 11-Biotin-dUTP 0.2-10 pmol nucleic acid or oligonucleotide β-Mercaptoethanol 1 mM

Potassium-Cacodylate 100 mM BSA 100 ug/ml

CoClj 1 mM

11-Biotin-dUTP 200 uM

16 u Terminal-deoxynucleotidyl- transferase

The incorporation of Biotin-dUTP is catalysed by the enzyme terminal-deoxynucleotidyl-transferase and takes place at the 3'-position of the nucleic acid. The reactants are dissolved in the tailing buffer (composition as above) and incubated for 30 min. at 37°C after addition of terminal- deoxynucleotidyl- transferase. The total volume of the reaction is 50 ul . The nucleic acid can be radioactively labelled during biotinylation through the addition of 10-15 pmol alpha-³²P-dNTP or alpha-^3**-S-dNTP (see Example 2). Alternatively the nucleic acid can be radioactively labelled before or after the tailing reaction by the phosphorylation reaction with radioactive triphosphate.

The product is desalted by loading on a sephadex column and eluted with distilled water (pH 8, and apply trace of NH₃) .

A second way to incorporate biotinylated monomers is during the chemical synthesis of oligonucleotides . This chemical labelling is described in Example 2, Alternative B. Chemical labeling is particularly important for the case of the sequence analysis with annealing of the primer to the support (Figure 3) .

2. Annealing of the biotinylated nucleic acid to the solid support

40 ul Streptavidin-agarose (commercially available)

10 pmol biotinylated nucleic acid or oligonucleotide

IxPBS

The stεptavidin-agarose is washed twice with IxPBS (200 ul each time). The biotinylated nucleic acid is loaded onto the support in a total volume of 20 ul and the suspension is incubated for 30 min. at 37°C. The supernatant is taken off and the support is washed three times with 200 ul IxPBS. Tests with radioactively labelled oligonucleotides gave an incorporation rate of 75-85% into the support material .

3. The sequence analysis is carried out as described for the BrdU-antibody immobilized templates when autoradiography is used for detection of the fragments .

Those skilled in the art will understand that the use of fluorescent-primers and terminators is also possible as before. The biotin-stroptavidin system has one distinct advantage over the BrdU-sequencing technique in that one can use higher temperatures for a) the annealing of the primer to the template (hence, higher specificity) and, b) the recovering of the synthesized fragments.

The use of higher temperatures permits one to take full advantage of the use of Taq-polymerase for the sequencing reactions.

Automated Apparatus for Solid Phase Sequencing

An apparatus according to the invention is illustrated in Fig. 7. It contains Columns 1-4, such as chromatography columns, each for reactions leading to identification of one of the four nucleobases. The columns are thermostated . Reservoirs are provided to contain washing solution, template solution, primer solution, enzyme mix, ddATP, ddCTP , ddGTP, ddTTP- solutions, and elution buffer. Valves 1-9 are arranged so that washing solution, template, primer and enzyme mix as well as elution buffer can be applied to any of the four columns or to all four columns at the same time, whereas ddATP, ddCTP , ddGTP, ddTTP , are each applied to only one of the four columns. The effluent of each column passes through a two-way valve which directs it either to waste or to a collector (or onto of a sequencing gel) . Air, argon, or nitrogen pressure is applied to move the solution from the reservoirs to the columns. The valves are time controlled through a programmable computer controller .

According to the method of the invention, the nucleic acid fragments (as sequencing template or primer) for the purpose of the sequencing are to be anchored to a water-insoluble solid support. The columns are filled with the support material. The reagents for sequencing are then applied to the columns as required under the computer controller for the different reactions constituting the sequencing cycle. In the case of immobilized sequencing targets the dideoxy-terminated elongation products are hybridized to the immobilized template DNA as described in Example 4. They are subsequently eluted with a formamide dye buffer mixture as described and directly applied to a sequencing gel.

Polyacrylamide-gelectrophoresis and identification of the sequence of the immobilized DNA, is then done by known techniques.

The advantage of the apparatus and the solid phase procedure is that the template through all reaction and washing steps remains anchored to the solid phase support and thus is available for further sequencing cycles. Thus using a set of appropriate primers, it is possible to analyze the sequence of large fragments of e.g. a genomic DNA in a simple and relatively inexpensive fashion. Another advantage is having the option that enzymes, excess monomers etc. can be removed by washing prior to dehybridisation of the dideoxy-terminated polymerisation products. The apparatus features the possibility of full mechanization of all steps included in the Sanger dideoxy sequencing. It can be directly coupled to existing "sequencers", i.e. machines that automate the gel separation and reading process.

A possible alternative application of this apparatus is by filling the columns with immobilized sequencing primer. In that case a preliminary hybridisation step allows one to isolate the DNA which is to be the target of sequencing from a mixture or from a biological material. Sequencing proceeds then essentially as described in Example 4 except that the dehybridisation step removes the sequencing template and the dideoxy terminated fragments subsequently have to be released from the columns as described previously by high salt or low pH- solutions.

Preparations for sequencing:

1. Filling of the columns with the support material.

2. Equilibration for about 2 min. with buffer solution (washing solution from washing solution reservoir) .

3. The columns containing e.g. immobilized anti-BrdU antibody, are then loaded with the nucleic acid template to be sequenced, which has previously been tiled with bromodeoxyridylate as described in Example 2 (i.e. from the Template reservoir).

4. Binding is effected by incubation at 37°C (thermostat) .

Note: Steps 3 and 4 take about 30 min.

The sequencing cycle:

5. Application of primer solution from the primer reservoir into the four columns for 15 min. The primer can be labelled e.g. by radioisotopos , fluoresecent markers etc.

6. "Enzyme mix" containing the polymerase plus deoxynucleotide triphosphates plus stabilizing reagcnts in an appropriate buffer are applied from enzyme mix reservoir to each column.

7. A preincubation time of about 10 min. is allowed to start the polymerization.

8. Each of the four "stop mixes" containing ddATP or ddCTP or ddGTP or ddTTP plus polymerizing enzyme and stabilizing reagents are applied to the appropriate column from their respective reservoirs.

9. Further incubation for 10 min. results in termination of the polymerization.

10. The buffer wash is done for 30 sec. by applying the buffer solution from the buffer reservoir to remove excess of triposphates and enzyme.

11. The elution buffer is applied from its reservoir for 1 min. effecting denaturation and elution of the hybridized dideoxy terminated primer elongation products .

Stops No. 5 to 11 are repeated with a new primer for "chromosome walking". Sequencing with immobilized primer requires reversal of steps 5 to 7. Furthermore a 12th step is included consisting of the detachment collection and treatment of the immobilized dideoxyterminated elongation product prior to gel electrophorcsis .

References

1. Maxam, A.M. , and Gilbert, W. (1980). Sequencing end-labeled DNA with base-specific chemical cleavage. In: Methods in Enzymology 65, Grossman, L. and Moldave, L. (eds. ) Academic Press, N.York, pp. 499-560 2. Sanger , F. , Niklen, S. , and Coulson, A.R. (1977) . DNA sequencing with chain terminating inhibitors. Proc.Natl. Acad.Sci.USA 74,5463-5467

3. Wada, A. , Yamamoto, M. , and Soeda, E. (1983) . Automatic DNA sequencer: Computer-programmmed microchemical manipulator for the Maxam-Gilbert sequencing method. Rev. Sci . Instrum . 54,1569-1572

4. Rosenthal, A. , Schwertner, S. , Hahn, V. , and Hunger, H . -D . (1985). Solid-phase methods for sequencing of nucleic acids I. Simulataneous sequencing of different oligodeoxyribonucleotides using a new, mechanically stable anion-exchange paper. Nucleic Acids Res. 13, 1173-1184.

5. Stahl, S. , Hultman, T. , Olsson, A. , Moks , T. , and Uhlen, M. (1988). Solid Phase DNA sequencing suing the biotin-avidin system. Nucleic Acids Res. 16, 3025-3038.

6. Smith, L. , and Hood, L. (1987) . Mapping and sequencing the human genome: how to proceed. Biotechnology 5, 933-939.

7. Groger, G. , and Seliger, H. (1988) A polymeric support for chemical and exnzymatic nucleic acid synthesis. Nucleotides and Nucleosides. In press .

8. Remalho-Ortigao , J.F. , Jirikowski , G.F. , Lindl , T. , and Seliger, H. (1988) Terminal labeling of synthetic oligonucleotides with

5-bromo-2 ' -deoxyuridine for hybridization analysis of DNA and RNA. Nucleic Acids Res. In Press.

Claims

What is Claimed is:

1. In a method for the sequence analysis of single- and double-stranded nucleic acids on solid phase supports, the step of: terminally elongating nucleic acids with modified nucleic acid units, which themselves act as units for anchoring to said solid phase support; whereby said solid phase support is a water insoluble polymer such as cellulose, sepharose or sephadex or an inorganic support, for example a support which has a backbone structure formed mainly of silicon and oxygen atoms, said solid phase support having effector groups, for example such as immobilized antibodies specific to 5-bromo- 2 ' -deoxyuridylate or other appropriate units capable of selective interaction with other halogenated nucleotides or nucleotides appropriately substituted at the base or sugar moieties, or stretavidin; and whereby the nucleic acid strands which are to be sequenced to the polymer support is effected by interactions of the modified nucleic acid units with the effector groups immobilized to the support, in particular by ligand-receptor binding interactions.

2. The method of claim 1 whereby the nucleic acids immobilized to the solid phase are sequenced according to enzymatic procedures used for solution reactions or variants thereof, and whereby the nucleic acid to be sequenced or alternatively the primer used in the sequencing is be bound to the solid phase with said effector groups.

3. The method of claim 2 whereby said step is performed automatically whereby a progressive sequence analysis is performed by the use of a series of oligonucleotide primers for chromosome walking, applying immobilization 1igand-receptor binding.

4. Apparatus for sequencing of single and double stranded nucleic acid fragments by ezymatic sequencing methods, comprising: four columns for holding a solid phase support having an effector group; input means for providing ddATP to one of said columns, ddCTP to another of said columns, ddGTP to yet another of said columns, and ddTTP to the fourth of said columns ; input means for providing primers, templates, and enzymes to each of said four columns, at least one of said primers and templates having affinity for said effector groups ; control means for automatically controlling each of said input means according to a programmed schedule for performing a sequencing cycle.