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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6869—Methods for sequencing
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

• nucleic acids 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.
• 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.
• 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 .
• 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.
• 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.
• 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.
• 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.
• 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.
• 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. 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.
• 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.
• 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 .
• 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.
• 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 .
• the attachment consists of binding the target nucleic acid to the support via a spacer.
• spacers 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.
• ligand receptor binding e.g. an antibody to the protein immobilized onto the polymer matrix itself.
• 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.
• 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.
• 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 .
• 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.
• 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.
• separation can be done prior to hybridization of a primer a strand.
• 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.
• 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 *- , * 5 S or a fluorescent moiety as are known in the art.
• 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 3 1 - 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 :
• 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.
• 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.
• BrdU can be incorporated at every desired position.
• the binding-capacity of the described support in Example 1 was determined by means of a BrdU-labelled oligonucletode .
• 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.
• the support material After the removal of the nucleic acid from the gel the support material is again ready for use in a new sequencing reaction cycle.
• oligonucleotide-primer (cone: 0.2 pmol/ul) was added to each of the four gel samples and. hybridized at 37°C during 15 minutes.
• 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 .
• 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- 32 P-dNTP or alpha- 3** -S-dNTP (see Example 2).
• the nucleic acid can be radioactively labelled before or after the tailing reaction by the phosphorylation reaction with radioactive triphosphate.
• the st ⁇ ptavidin-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 .
• 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.
• FIG. 7 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.
• 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,
• 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 .
• 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.
• 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.
• 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.
• 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.
• 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).
• Binding is effected by incubation at 37°C (thermostat) .
• Steps 3 and 4 take about 30 min.
• the primer can be labelled e.g. by radioisotopos , fluoresecent markers etc.
• Enzyme mix containing the polymerase plus deoxynucleotide triphosphates plus stabilizing reagcnts in an appropriate buffer are applied from enzyme mix reservoir to each column.
• a preincubation time of about 10 min. is allowed to start the polymerization.
• 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.
• 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 .

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