EP1005574A2 - Method for producing nucleic acid polymers - Google Patents

Method for producing nucleic acid polymers

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Publication number
EP1005574A2
EP1005574A2 EP98947434A EP98947434A EP1005574A2 EP 1005574 A2 EP1005574 A2 EP 1005574A2 EP 98947434 A EP98947434 A EP 98947434A EP 98947434 A EP98947434 A EP 98947434A EP 1005574 A2 EP1005574 A2 EP 1005574A2
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Prior art keywords
oligonucleotides
linkable
nucleic acid
polymerase
primary strand
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EP98947434A
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German (de)
French (fr)
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Peter Hegemann
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae

Definitions

  • the invention relates to a method for producing nuclear acid polymers
  • Another method for producing long nucleic acid polymers is the so-called fill-in method, in which two single-stranded nucleic acid chains are hybridized with one another, overhanging ends being filled in with the aid of DNA polymerases, so that the double-stranded product is longer than the oligonucleotides used, but also the Fill-m method cannot produce a product that is longer than the sum of the two nuclear acid chains used
  • Khorana developed a process named after him, in which several chemically synthesized oligonucleotides with an average length of 15 nucleotides, which overlap in a suitable arrangement without gaps, were enzymatically linked to a double strand by polynucleotide ligase (Khorana, HG et al, J Mol Biol 27, 209 -17, 1972, and subsequent publications, Khorana, HG et al, J Biol Chem 251, 3 (10), 565-70, 1976 and subsequent publications) by sequentially ligating a few (4-8) oligonucleotides into longer intermediates, purifying the intermediates and subsequent ligation of the intermediates with one another, double-stranded nuclear acid chains with a length of 514 base pairs (Edge, MD et al, Nature 292, 756-62, 1981), were synthesized later up to approximately 1000 bp, which could then be cloned into bacterial expression vectors
  • Khorana The methods described by Khorana are usually radiolabelled in order to identify the band of the desired product in the gel highly radioactive 32 P marker represents a potential hazard that could not be avoided with this method
  • the present invention has for its object to provide an uncomplicated, reliable and inexpensive method for the synthesis of nucleic acid polymers with a length of more than 1000 bases in one step, in which the disadvantages listed above can be overcome
  • linkable oligonucleotides which, when arranged continuously and after linking, can form a primary strand
  • one or more non-linkable oligonucleotides each of the non-linkable oligonucleotides comprising two mutually bordering regions, the first of which is the 3 'end of a linkable Oligonucleotide is complementary and the second of which is complementary to the 5 'end of a further linkable oligonucleotide
  • FIGS. 1 and 2 The method according to the invention is shown schematically in FIGS. 1 and 2.
  • Khorana process it offers the decisive advantage that the synthesis of a single-stranded nucleic acid polymer can be carried out in a single reaction batch. All the linkable and non-linkable oligonucleotides required for the synthesis of the primary strand are used simultaneously; by adding a linking agent, a paris strand of covalently linked oligonucleotides is formed which can reach a length of more than 1000, for example 1500 bases.
  • steps (b) and (c) are repeated several times, the oligonucleotides previously hybridized with one another being separated from each other before each repetition of these two steps, i.e.
  • the double strand previously formed by the hybridization is denatured.
  • the denaturation can be carried out by increasing the temperature or increasing the pH value can be carried out in a manner known to the person skilled in the art.
  • the multiple denaturing and renaturing with subsequent linking leads to a considerable improvement in the yield of P ⁇ marstrang, which is otherwise impaired, for example, by chain breaks which are a consequence of the incorporation of incompletely phosphorylated P ⁇ marstrangoligonucleotide
  • the effect of such chain terminations on the overall yield is minimized by repeating steps (b) and (c)
  • steps (b) and (c) are carried out not only once, but several times. In a preferred embodiment, they are repeated 1 to 8 times, particularly preferably 3 to 5 times
  • the number of oligonucleotides used can vary between 2 linkable oligonucleotides and 150 linkable oligonucleotides.
  • the number of non-linkable oligonucleotides is in each case equal to the number of linkable oligonucleotides, one higher or one lower, at least one, preferably between 5 and 100, particularly preferred 10 and 50 linkable oligonucleotides used
  • the linking of the linkable oligonucleotides of the Pmarmar strand can comprise different reactions.
  • Linking here is understood to mean, for example, a reaction of an enzymatic, chemical or also photochemical nature.
  • T4-DNA ligases are used as linking agents
  • a thermostable ligase for example Pfu ligase (Stratagene)
  • Pfu ligase (Stratagene)
  • thermostable ligase for example Pfu ligase (Stratagene)
  • T4 DNA ligase thermolabile DNA ligases
  • the use of this thermostable ligase allows the steps of hybridizing and ligating the oligonucleotides to be carried out at high temperatures of 45 ° to 80 ° C., preferably 70 ° C.
  • oligonucleotides to be linked must be labeled on the terminal nucleotides with photosensitive molecules which contain carbon-carbon double bonds and which can be subjected to (2 + 2) photocyclodimensation, such as, for example Stilbene, allen, mono-, di- and triendicarboxylic acid derivatives or styrene derivatives
  • the non-linkable oligonucleotides only serve as a template for the formation of the nucleic acid polymer.
  • these oligonucleotides are not phosphorylated at their 5 'end. This means that these oligonucleotides are enzymatically linked, which are only responsible for the attachment of the linkable oligonucleotides in the desired arrangement , not possible
  • non-linkable oligonucleotides in which, for example, the 3 'end is a dideoxynucleotide or a thionucleond. Corresponding modifications are also possible at the 5' end.
  • the method can also be used with non-linkable oligonucleotides which are phosphorylated at the 3 'end This modification likewise prevents the enzymatic linkage of the oligonucleotides.
  • non-linkable oligonucleotides must be designed such that they cannot form a covalent bond to the respectively adjacent non-linkable oligonucleotide under the conditions chosen for linking the oligonucleotides forming the primary strand
  • the oligonucleotides used in the method according to the invention can have a length of 30 to 1500 nucleotides.
  • the length of the nucleotide selected in each case is dependent on several factors, including the probability of the formation of secondary structures and the purity of the selected starting materials. However, it is preferred that the oligonucleotides have a length of 30 to 200, particularly preferably 30 to 100 or 30 to 60 nucleotides
  • the single-stranded nucleic acid polymers already generated in the synthesis can in turn be combined with the method according to the invention into single-stranded nucleic acid polymers of several kilobases in length (FIG. 3).
  • the method is carried out as explained above, with linkable oligonucleotides for the P ⁇ marstrang nucleic acid polymers of, for example, 1500 bases can be used
  • the non-linkable oligonucleotides which are intended to ensure the correct arrangement of the oligonucleotides of the P ⁇ mar strand next to one another, are possibly only 30 to 50 nucleotides long.This order of magnitude allows hybridization with two oligonucleotides of the P ⁇ mar strand to be arranged adjacent to one another
  • the use of non-linkable oligonucleotides with up to 300 nucleotides or more is also possible and, where, for example, otherwise strong secondary structures have been formed in the primary strand, can be very useful
  • Each of the non-linkable oligonucleotides contains two adjoining regions, which have complementate with the 3 'or 5' end of two adjacent oligonucleotides for the primary strand.
  • the regions of complementary between the linkable oligonucleotides for the primary strand and the non-linkable oligonucleotide nucleotides of the opposite strand are each about 15 to 30 base pairs long, preferred
  • a preferred embodiment of the method then consists in an upstream reaction of attaching the terminal oligonucleotides for the P ⁇ mar strand and / or oligonucleotides complementary to the terminal oligonucleotides of the P ⁇ mar strand to the ends of a linearized vector, to link them to the vector and subsequently to carry out the method according to claim 1
  • the vector ends are preferably cohesive.
  • This method leads to a circulated product which can be transfected directly into a suitable host organism, for example bacteria, sucker or insect cells.
  • "transfection” includes various techniques, how to understand eg electroporation microinjection infection transfection supported by eg "Ca-P0 4 ", DEAE, hydrophobic molecules etc.
  • either the 5'-end oligonucleotide of the pmarmar strand at its 5'-end or the 3'-end oligonucleotide of the pmarmar strand at its 3'-end is provided with a hapten or both terminal oligonucleotides with different haptens. This is made possible the fixation of the oligonucleotide coupled to the hapten before or after its linkage with other oligonucleotides of the primary strand to a fixed support. This separates the hapten-coupled strand from all precursors and by-products without hapten z.
  • the chosen hapten-carrier combination must be stable under the intended conditions.
  • a preferred hapten is eg biotin, which is bound by streptavidm coupled to a carrier.
  • the oligonucleotides can be coupled with antigens which are recognized and bound by antibodies fixed to a carrier. The further synthesis of the primary strand or one of the further steps described below can then be carried out on this carrier.
  • the second hapten could be useful, for example, for detection methods to be carried out later with the nucleic acid polymer
  • a polymerase enzyme is added to the reaction mixture of a synthesis reaction carried out in accordance with the present process in order to convert the primary strand, which is a single nucleic acid strand, into a to form a klemsaure double strand or to specifically amplify the complementary strand (ie the opposite strand to the primary strand) (asymmetric PCR)
  • a specific primer is an oligonucleotide (backwinder) which is complementary to the 3 'end of the complete primary strand
  • the reaction mixture of a synthesis reaction carried out according to the present method a polymerase enzyme and additionally terminal forward and backward separators are added in large excess to the synthesis product used.
  • a polymerase enzyme and additionally terminal forward and backward separators are added in large excess to the synthesis product used.
  • the principle of the polymerase chain reaction (Saiki et al, Science 239, 487 -491, 1988) from the single-stranded nucleic acid polymers, double-stranded nucleic acid polymers
  • the polymerase enzyme is selected from the group of DNA polymerases, eg BE coli polymerase I, Klenow polymerase, T4 DNA polymerase and reverse transceptase.
  • the nucleic acid double strands formed in this way can be either DNA-DNA double strands or DNA-RNA double strands be (Kleppe et al, Proc NatI Acad
  • the added polymerase is a temperature-stable polymerase, for example Taq polymerase.
  • the temperature-stable enzyme loses only a little of its activity even after several temperature cycles and can therefore catalyze the polymerase reactions of several successive cycles
  • the polymerase reaction is preferably carried out repeatedly several times in order to amplify the nucleic acid double strand exponentially.
  • each cycle comprises denaturing already existing nuclear acid double strands by conventional measures, for example by increasing the temperature or the pH. the attachment of terminal polymers under suitable conditions and a polymerase-catalyzed nucleic acid synthesis.
  • a temperature-stable polymerase the addition of further polymerase is unnecessary, but where one of the non-temperature-stable polymerases has been used for the polymerization reaction, this is inactivated by the thermal denaturation, in this case, fresh polymerase enzyme must be added in each cycle.
  • the polymerase reaction is usually carried out 5 to 15 times, preferably about 8 to 12 times. The main advantage the repetition of the polymerase reaction is then that due to the position of the primers (final) targeted complete synthesis product can be exponentially amplified
  • the denaturation of the double strands is usually carried out at temperatures of more than 90 ° C.
  • the reaction mixture is then slowly cooled so that the final primers, depending on their composition, can hybridize with the single strands at temperatures between 80 ° C. and 45 ° C.
  • Polymerase-catalyzed DNA synthesis can be carried out using a temperature-stable enzyme in the range from 45 to 70 ° C, which minimizes the formation of u ⁇ specific hybrids
  • the present invention furthermore relates to a single-stranded nucleic acid polymer which has been obtained by the process according to the invention.
  • a single-stranded nucleic acid polymer which has been obtained by the process according to the invention.
  • primary strands must be designed so that they are complementary to one another at their 3 'end in a range of a few base pairs, for example 20 to 60, preferably 30 to 40 base pairs, so that in a subsequent in vitro or in vivo polymerase reaction, the 3 'end of each primary strand can serve as a polymer for a mattress-dependent polymerase
  • Figure 1 describes a preferred embodiment of the method according to the invention
  • Figure 2 shows a detailed view of the overlap area between two adjacent linkable oligonucleotides and a non-linkable oligonucleotide, which comprises two adjacent regions, one of which is complementary to the 3 'end of a first linkable oligonucleotide and the second of which the 5' end of a second linkable oligonucleotide is complementary
  • the overlap area between a region of the non-linkable oligonucleotide with the complementary region of one of the linkable oligonucleotides should be at least 15 bp
  • Figure 3 illustrates the implementation of the method with very long linkable oligonucleotides, which are correctly arranged next to one another by hybridization with much shorter non-linkable oligonucleotides.For this purpose, it is sufficient if the non-linkable oligonucleotides ensure stable hybridization with the two linkable oligonucleotides to be arranged adjacent without having to be complementary to the entire later primary strand
  • Figure 4 shows the principle of the Fill-m method using the example of two long oligonucleotides that are only complementary in their 3 'region. By hybridizing the complementary 3' regions, the 3 'end of each of the oligonucleotides serves as a polymer for a polymerase reaction along the template of the each other oligonucleotides
  • Figure 5 a) Oligonucleotides for the synthesis of the primary strand of the algal-adapted GFP mutant S65T / F64L (FG1 to FG15) b) Oligonucleotides of the opposite strand (not connectable) (FG14rev to FGIrev) c) Ohgon nucleotide FG15rev, polymer for the synthesis of the polymerase-catalyzed catalyst Figure 6 Modified gene (S65T / F64L) for the Green Fluorescent Protein (GFP) from Aquorea victorea.
  • S65T / F64L Green Fluorescent Protein
  • the gene was modified compared to the natural GFP gene in such a way that it only contains codons preferred by green algae, for example Chlamvdomonas
  • the underlined nucleotides were only filled in by the polymerase reaction (matrix FG15rev)
  • the following shows the synthesis of a modified gene for green fluorescent protein (GFP) from the jellyfish Aequorea victorea.
  • the codon usage is modified in the synthetic gene in such a way that the codons of green algae such as Chlamvdomonas are preferred.
  • 30 oligonucleotides with a length of 45 to 50 bases used
  • the oligonucleotides of the primary strand are designated as FG1 to FG15 and are 45 to 47 bases long.
  • the non-linkable oligonucleotides of the complementary strand are designated as FG rev to FGI rev and are 46 bases (FG14rev to FGI rev) long
  • Sequences of the oligonucleotides are shown in Fig. 5a, b
  • the reaction was carried out in a programmable thermal cycler.
  • the oligonucleotides which form the linkable strand were phosphorylated at the 5 'end before the synthesis reaction
  • Step 1 oligonucleotide phosphorylation at the 5 'end (100 ⁇ l mixture)
  • T4 polynucleotide kmase 10 units Biolabs
  • the reaction is carried out at 37 ° C. for 4 h and then terminated by incubation at 95 ° C. for 5 minutes.
  • Step 2 carrier-free nucleotide polymer synthesis (primary strand)
  • the reaction was carried out in a programmable thermal cycler. Recombinant Pfu DNA ligase from Stratagene was used. The buffer conditions were changed to the ligase conditions with KCI, NP-40, MgCI 2 , whereby the ions were taken into account in the kinase batch.
  • the reaction mixture is incubated for 3 minutes at 95 ° C. and for 3 minutes at 80 ° C. During the incubation at 80 ° C., 3 ⁇ l of Pfu DNA ligase (12 units of thermostable ligase from Stratagene) are added and the following temperature cycle is then carried out 3 times:
  • Variant 1 The product is precipitated to separate the Pfu ligase, if necessary amplified and cloned using PCR and Taq polymerase.
  • Variant 2 In order to facilitate the separation, the primer FG1 is used biotinylated. After completion of the synthesis, the reaction mixture is adjusted to pH 13 with NaOH Streptavidin magnetic balls (Dynal) are added and the synthesis product is held in place with a magnet on the wall of the reaction vessel, while all the backwashers, unused forward packers and ligase are removed with the supernatant. The clean synthesis product, now coupled to streptavidm, is placed in 30 ⁇ l of neutral buffer recorded and can, for example, be amplified as in step 3 (see below)
  • Variant 1 1 ⁇ l of the synthesis product are amplified in 1 cycle with 1 U of thermostable Pfu polymerase (Stratagene) in 12 cycles in a standard buffer (It manufacturer) in a 50 ⁇ l batch.
  • the oligonucleotide FG1 serves as the forward spanner, the oligonucleotide FG15rev (50 bases, Fig. 5c) final concentration 25 pmol per 50 ⁇ l.
  • Variant 2 For amplification by means of PCR, two terminal polymers are added which have a total length of 30 or 33 bases, of which only 21 or 24 bp hybridize with the synthesis product. The "overhangs" each contain an interface of the "multiple cloning region" of the vector pBenesc ⁇ pt II KS ' (Stratagene) The amplified product is cut with the enzymes Xhol and Knpl, purified using Quiaex II (Quiagen) and cloned by default

Abstract

The conventional synthesis of nucleic acid polymers from several oligonucleotides consists of several cycles of intermediate product synthesis, cleaning the intermediate product and the synthesis of a full length product (to a maximum length of approx. 1000 nucleotides). The novel method is carried out in a single reaction step and can result in nucleic acid polymers with more than 1000 nucleotides. According to the invention, two or more cross-linkable oligonucleotides are prepared. Said cross-linkable oligonucleotides can form a primary strand with continuous sequencing and after cross-linking. One or more non-cross-linkable oligonucleotides are also prepared. Each of the non-cross-linkable oligonucleotides has two adjacent areas, the first of which is complementary to the 3' end of a cross-linkable oligonucleotide and the second of which is complementary on the 5' end of another cross-linkable oligonucleotide. The cross-linkable oligonucleotides are hybridized with the complementary areas of the non-cross-linkable oligonucleotides and then cross-linked by enzymatic, chemical or photochemical means. The method is suitable for de novo-synthesis of long nucleic acid chains.

Description

Verfahren zum Herstellen von Nukleinsäurepolymeren Process for the preparation of nucleic acid polymers
Die Erfindung betrifft ein Verfahren zum Herstellen von NuklemsaurenpolymerenThe invention relates to a method for producing nuclear acid polymers
Der zunehmende Einsatz rekombinanter Gene in der Gen- und Biotechnologie sowie der medizinischen Analytik hat einen großen Bedarf an Verfahren zur "de novo"-Synthese von langen Nuklemsaureketten ausgelost Die synthetische Herstellung beliebig gewählter Nukieinsauresequenzen kann in vielen Fallen eine zeitsparende Alternative für aufwendige Klonierungs- und Modifizierungsverfahren sein Eine routinemäßige Synthese langer Nuklemsaureketten kann ebenfalls entscheidende Vorteile beim "drug-design" bieten, z B bei der Herstellung von "maßgeschneiderten" Antikörpern, Inhibitor/Aktiva- tormolekulen, Ribozymen oder in der DNA-Chip-Technologie Desweiteren konnten auf diese Weise gezielt modifizierte Nukleotide, z B markiert durch (Fluoreszenz)Farbstoffe oder Enzyme, in eine Nukleinsaurekette eingebaut werden Neben den hier aufgeführten Beispielen ergeben sich noch eine Vielzahl weiterer Anwendungsmoglichkeiten für gezielt hergestellte NukleinsaurepolymereThe increasing use of recombinant genes in gene and biotechnology as well as medical analysis has a great need for processes for the "de novo" synthesis of long nucleic acid chains. The synthetic production of any chosen nucleic acid sequences can in many cases be a time-saving alternative for complex cloning and Be a modification process A routine synthesis of long nuclear acid chains can also offer decisive advantages in "drug design", eg in the production of "tailor-made" antibodies, inhibitor / activator molecules, ribozymes or in DNA chip technology deliberately modified nucleotides, eg marked by (fluorescence) dyes or enzymes, can be incorporated into a nucleic acid chain. In addition to the examples listed here, there are a number of other possible uses for specifically produced nucleic acid polymers
Bei der einfachsten Variante der herkömmlichen Gensynthese, der direkten Klonierung, werden zwei lange, einander vollständig komplementäre Oligonukleotide miteinander hybridisiert Auf Grund der derzeitigen technischen Limitierung bei der Herstellung der Oligonukleotide auf eine Lange von 150 - 200 Basen ist auch die Große der resultierenden Hybπdisierungsprodukte auf diesen Langenbereich beschranktIn the simplest variant of conventional gene synthesis, direct cloning, two long, completely complementary oligonucleotides are hybridized with one another. Due to the current technical limitation in the production of the oligonucleotides to a length of 150-200 bases, the size of the resulting hybridization products is also on these Limited area
Ein weiteres Verfahren zum Herstellen langer Nukleinsaurepolymere ist die sogenannte Fill-in-Methode, bei der zwei einzelstrangige Nuklemsaureketten miteinander hybridisiert werden, wobei überhangende Enden mit Hilfe von DNA-Polymerasen aufgefüllt werden, so daß das doppelstrangige Produkt langer als die eingesetzten Oligonukleotide ist Aber auch mit der Fill-m-Methode kann kein Produkt erzeugt werden, das langer als die Summe der beiden eingesetzten Nuklemsaureketten istAnother method for producing long nucleic acid polymers is the so-called fill-in method, in which two single-stranded nucleic acid chains are hybridized with one another, overhanging ends being filled in with the aid of DNA polymerases, so that the double-stranded product is longer than the oligonucleotides used, but also the Fill-m method cannot produce a product that is longer than the sum of the two nuclear acid chains used
Bei der "Shot Gun-Gensynthese" werden komplementäre, einzelstrangige Oligonukleotide zusammen mit einem durch Restriktionsenzyme geöffneten Expressionsvektor direkt in Zellen traπsfiziert, wobei ein zirkularisiertes Produkt nur entstehen kann wenn alle Teilsequenzen durch die Enzymmaschineπe des Wirtsorganismus in geeigneter Weise ligiert werden Die Effizienz der erfolgreichen Ligationen bei dieser Methode ist i a sehr gering, insbesondere wenn viele Oligonukleotide für die Gensynthese eingesetzt werdenIn "shot gun gene synthesis", complementary, single-stranded oligonucleotides are transfected directly into cells together with an expression vector opened by restriction enzymes, a circularized product being able to be produced only if all the partial sequences are suitably produced by the enzyme machinery of the host organism The efficiency of the successful ligations with this method is generally very low, especially if many oligonucleotides are used for gene synthesis
1972 entwickelte Khorana ein nach ihm benanntes Verfahren, bei dem mehrere chemisch synthetisierte Oligonukleotide von durchschnittlich 15 Nukleotiden Lange, die sich in geeigneter Anordnung lückenlos überlappen, enzymatisch durch Polynukleotidligase zu einem Doppelstrang verbunden wurden (Khorana, H G et al , J Mol Biol 27, 209-17, 1972, und Folgeveroffentlichungen, Khorana, H G et al , J Biol Chem 251, 3 (10), 565- 70, 1976 und Folgeveroffentlichungen) Durch sequenzielles Ligieren von wenigen (4 - 8) Oligonukleotiden zu längeren Zwischenprodukten, Aufreinigen der Zwischenprodukte und anschließendes Ligieren der Zwischenprodukte miteinander wurden doppelstrangige Nuklemsaureketten mit einer Lange von 514 Basenpaaren (Edge, M D et al , Nature 292, 756-62, 1981 ), spater bis ca 1000 bp synthetisiert, die anschließend in bakterielle Expressionsvektoren kloniert werden konntenIn 1972, Khorana developed a process named after him, in which several chemically synthesized oligonucleotides with an average length of 15 nucleotides, which overlap in a suitable arrangement without gaps, were enzymatically linked to a double strand by polynucleotide ligase (Khorana, HG et al, J Mol Biol 27, 209 -17, 1972, and subsequent publications, Khorana, HG et al, J Biol Chem 251, 3 (10), 565-70, 1976 and subsequent publications) by sequentially ligating a few (4-8) oligonucleotides into longer intermediates, purifying the intermediates and subsequent ligation of the intermediates with one another, double-stranded nuclear acid chains with a length of 514 base pairs (Edge, MD et al, Nature 292, 756-62, 1981), were synthesized later up to approximately 1000 bp, which could then be cloned into bacterial expression vectors
Mit diesen Verfahren sind mehrere entscheidende Nachteile verbundenThere are several key disadvantages associated with these methods
1 ) Nach jedem Ligationsschπtt mußten die Produkte bzw Zwischenprodukte durch1) After each ligation step, the products or intermediate products had to go through
Auftrennung auf einem Polyacrylamid-Gel (PAA-Gel) von unerwünschten Nebenprodukten der Ligationsreaktion gereinigt werden Dieser zeitintensive Arbeitsschritt ist mit einem hohen personellen und finanziellen Aufwand verbundenSeparation on a polyacrylamide gel (PAA gel) from unwanted by-products of the ligation reaction to be cleaned This time-consuming work step is associated with high personnel and financial expenditure
2 ) Bei der Elution der Zwischen- und Endprodukt aus dem PAA-Gel mußten beträchtliche Ausbeuteverluste in Kauf genommen werden2) Considerable losses in yield had to be accepted in the elution of the intermediate and end product from the PAA gel
3 ) Das bisher längste Produkt, das mit Hilfe dieser Technik hergestellt werden konnte, hatte eine Lange von ca 1000 Basenpaaren Da die meisten eu- und prokaryotischen Gene eine kodierende Sequenz von durchschnittlich 300 - 3000 Baseπpaaren haben, ist diese Lange für die meisten Anwendungen ungenügend3) The longest product to date that could be produced using this technique had a length of approx. 1000 base pairs. Since most eu and prokaryotic genes have a coding sequence of 300-3000 base pairs on average, this length is insufficient for most applications
4 ) Für die benotigte Aufreinigung über PAA-Gele wurden die Nukleotide bei dem von4) For the required purification using PAA gels, the nucleotides were removed from the
Khorana beschriebenen Verfahren üblicherweise radioaktiv markiert, um die Bande des gewünschten Produktes im Gel identifizieren zu können Die Verwendung hoch radioaktiver 32P-Marker stellt ein Gefahrdungspotential dar, das bei diesem Verfahren nicht vermieden werden konnteThe methods described by Khorana are usually radiolabelled in order to identify the band of the desired product in the gel highly radioactive 32 P marker represents a potential hazard that could not be avoided with this method
Der vorliegenden Erfindung liegt die Aufgabe zugrunde, ein unkompliziertes, zuverlässiges und preiswertes Verfahren für die Synthese von Nukleinsäurepolymeren mit einer Lange von mehr als 1000 Basen in einem Schritt bereitzustellen, bei dem die oben aufgeführten Nachteile überwunden werden könnenThe present invention has for its object to provide an uncomplicated, reliable and inexpensive method for the synthesis of nucleic acid polymers with a length of more than 1000 bases in one step, in which the disadvantages listed above can be overcome
Diese Aufgabe wird gelost durch ein Verfahren zum Herstellen eines Nukleinsaurepoly- mers, umfassendThis object is achieved by a method for producing a nucleic acid polymer, comprising
a) das Bereitstellen von 2 oder mehr verknupfbaren Oligonukleotiden die bei kontinuierlicher Anordnung und nach Verknüpfen einen Pπmarstrang bilden können, und einem oder mehreren nicht verknupfbaren Oligonukleotiden, wobei jedes der nicht verknupfbaren Oliognukleotide zwei anemandergrenzende Bereiche umfaßt, deren erster dem 3'-Ende eines verknupfbaren Oligonukleotids komplementär ist und deren zweiter dem 5'-Ende eines weiteren verknupfbaren Oligonukleotids komplementär ista) the provision of 2 or more linkable oligonucleotides which, when arranged continuously and after linking, can form a primary strand, and one or more non-linkable oligonucleotides, each of the non-linkable oligonucleotides comprising two mutually bordering regions, the first of which is the 3 'end of a linkable Oligonucleotide is complementary and the second of which is complementary to the 5 'end of a further linkable oligonucleotide
b) das Hybridisieren von Oligonukleotiden für den Pπmarstrang mit den komplementären Bereichen der nicht verknupfbaren Oligonukleotide undb) the hybridization of oligonucleotides for the primary strand with the complementary regions of the non-linkable oligonucleotides and
c) das Verknüpfen der Oligonukleotide des Pπmarstrangesc) linking the oligonucleotides of the Pmarmar strand
Das erfindungsgemaße Verfahren ist in den Abb 1 und 2 schematisch dargestellt. Es bietet im Unterschied zum Khorana-Verfahren den entscheidenden Vorteil, daß die Synthese eines einzelstrangigen Nuklemsaurepolymers in einem einzigen Reaktionsansatz durchgeführt werden kann Dabei werden alle zur Synthese des Pπmarstrangs benotigten verknupfbaren und nicht verknupfbaren Oligonukleotide gleichzeitig eingesetzt; durch Zugeben eines Verknupfungsmittels entsteht ein Pπmarstrang kovalent verknüpfter Oligonukleotide, der eine Lange von mehr als 1000, z B 1500 Basen erreichen kann In bevorzugten Ausfuhrungsformen werden die Schritte (b) und (c) mehrfach wiederholt, wobei vor jeder Wiederholung dieser beiden Schritte die zuvor miteinander hybridisierten Oligonukleotide voneinander getrennt werden, d h der durch die Hybridisierung zuvor gebildete Doppelstrang denaturiert wird Die Denaturierung kann durch Temperaturerhöhung oder Erhöhung des pH-Wertes auf dem Fachmann bekannte Weise durchgeführt werden Das mehrfache Denaturieren und Renaturieren mit anschließendem Verknüpfen fuhrt zu einer erheblichen Verbesserung der Ausbeute an Pπmarstrang, die sonst z B durch Kettenabbruche, die eine Folge des Einbaus unvollständig phosphory- lierter Pπmarstrangoligonukleotide sind, beeinträchtigt wird Der Einfluß solcher Kettenabbruche auf die Gesamtausbeute wird durch die Wiederholung der Schritte (b) und (c) minimiertThe method according to the invention is shown schematically in FIGS. 1 and 2. In contrast to the Khorana process, it offers the decisive advantage that the synthesis of a single-stranded nucleic acid polymer can be carried out in a single reaction batch. All the linkable and non-linkable oligonucleotides required for the synthesis of the primary strand are used simultaneously; by adding a linking agent, a paris strand of covalently linked oligonucleotides is formed which can reach a length of more than 1000, for example 1500 bases In preferred embodiments, steps (b) and (c) are repeated several times, the oligonucleotides previously hybridized with one another being separated from each other before each repetition of these two steps, i.e. the double strand previously formed by the hybridization is denatured. The denaturation can be carried out by increasing the temperature or increasing the pH value can be carried out in a manner known to the person skilled in the art. The multiple denaturing and renaturing with subsequent linking leads to a considerable improvement in the yield of Pπmarstrang, which is otherwise impaired, for example, by chain breaks which are a consequence of the incorporation of incompletely phosphorylated Pπmarstrangoligonucleotide The effect of such chain terminations on the overall yield is minimized by repeating steps (b) and (c)
Die Schritte (b) und (c) werden erfindungsgemaß nicht nur 1 Mal , sondern mehrfach durchgeführt In einer bevorzugten Ausfuhrungsform werden sie 1 bis 8 Mal besonders bevorzugt 3 bis 5 Mal wiederholtAccording to the invention, steps (b) and (c) are carried out not only once, but several times. In a preferred embodiment, they are repeated 1 to 8 times, particularly preferably 3 to 5 times
Die Anzahl der eingesetzten Oligonukleotide kann zwischen 2 verknupfbaren Oligonukleotiden und 150 verknupfbaren Oligonukleotiden schwanken Die Anzahl der nicht verknupfbaren Oligonukleotide ist jeweils gleich der Anzahl der verknupfbaren Oligonukleotide, um eines hoher oder um eines niedriger, mindestens also eins Bevorzugt werden zwischen 5 und 100, besonders bevorzugt 10 und 50 verknupfbare Oligonukleotide eingesetztThe number of oligonucleotides used can vary between 2 linkable oligonucleotides and 150 linkable oligonucleotides. The number of non-linkable oligonucleotides is in each case equal to the number of linkable oligonucleotides, one higher or one lower, at least one, preferably between 5 and 100, particularly preferred 10 and 50 linkable oligonucleotides used
Das Verknüpfen der verknupfbaren Oligonukleotide des Pπmarstranges kann verschiedene Reaktionen umfassen Unter Verknüpfen wird hier z B eine Reaktion enzymati- scher, chemischer oder auch photochemischer Natur verstanden So wird im Fall der enzymatischen Verknüpfung (Ligation) als Verknupfungsmittel z B T4-DNA-Lιgase verwendet In der bevorzugten Ausfuhrungsform wird eine thermostabile Ligase, z B Pfu- Ligase (Stratagene), eingesetzt, die den Vorteil bietet, daß bei wiederholten Zyklen des Denatuπerens durch Temperaturerhöhung , des Hybπdisierens und Ligierens der Oligonukleotide kein neues Enzym zugesetzt werden muß wahrend dies bei Verwendung thermolabiler DNA-Ligasen, wie z B T4-DNA-Lιgase, der Fall ist Desweiteren gestattet die Verwendung dieser thermostabilen Ligase, die Schritte des Hybndisierens und des Ligierens der Oligonukleotide bei hohen Temperaturen von 45° bis 80°C, bevorzugt 70°C, durchzufuhren Diese stπngenten Temperaturbedingungen bedeuten, daß der Schritt des Hybndisierens der Oligonukleotide in komplementärer Anordnung mit hoher Spezifitat erfolgt und im Vergleich zum Khorana-Verfahren erheblich weniger Nebenprodukte anfallen Ligationen mit herkömmlicher Ligase werden üblicherweise in einem Temperaturbereich von 40°C bis 4°C durchgeführt und können daher zu einem höheren Anteil unspezifischer Hybridisierungen durch Fehlpaarung oder Sekunda rstrukturen fuhrenThe linking of the linkable oligonucleotides of the Pmarmar strand can comprise different reactions. Linking here is understood to mean, for example, a reaction of an enzymatic, chemical or also photochemical nature. In the case of enzymatic linking (ligation), for example, T4-DNA ligases are used as linking agents In In the preferred embodiment, a thermostable ligase, for example Pfu ligase (Stratagene), is used, which has the advantage that no repeated enzyme has to be added during repeated cycles of denaturing by increasing the temperature, hybridizing and ligating the oligonucleotides, while using more thermolabile DNA ligases, such as T4 DNA ligase, is the case Furthermore, the use of this thermostable ligase allows the steps of hybridizing and ligating the oligonucleotides to be carried out at high temperatures of 45 ° to 80 ° C., preferably 70 ° C. These constant temperature conditions mean that the step of hybridizing the oligonucleotides in a complementary arrangement with High specificity occurs and, compared to the Khorana process, significantly fewer by-products are obtained. Ligations with conventional ligase are usually carried out in a temperature range from 40 ° C to 4 ° C and can therefore lead to a higher proportion of non-specific hybridizations due to mismatch or secondary structures
Eine chemische Verknüpfung der Oligonukleotide, die von Goeddel und Kollegen beschrieben worden ist (Goeddel D V et al PNAS 76 1979), ist ebenfalls möglichA chemical linkage of the oligonucleotides, which has been described by Goeddel and colleagues (Goeddel D V et al PNAS 76 1979), is also possible
Eine weitere Möglichkeit, Oligonukleotide miteinander verknüpfen, bietet die photochemische Verknüpfung Dabei müssen die zu verknüpfenden Oligonukleotide an den end- standigen Nukleotiden mit photosensitiven Molekülen markiert sein die Kohlenstoff- Kohlenstoff-Doppelbmdungen enthalten und einer (2+2) Photocyclodimensation unterliegen können, wie z B Stilben-, Allen-, Mono-, Di- und Triendicarbonsauredeπvate oder StyroldeπvateAnother possibility of linking oligonucleotides with one another is provided by the photochemical linkage. The oligonucleotides to be linked must be labeled on the terminal nucleotides with photosensitive molecules which contain carbon-carbon double bonds and which can be subjected to (2 + 2) photocyclodimensation, such as, for example Stilbene, allen, mono-, di- and triendicarboxylic acid derivatives or styrene derivatives
Erfindungsgemaß dienen die nicht verknupfbaren Oligonukleotide lediglich als Matrize zur Bildung des Nuklemsaurepolymers In einer bevorzugten Ausfuhrungsform sind diese Oligonukleotide an ihrem 5'-Ende nicht phosphoryliert Dadurch ist eine enzymatische Verknüpfung dieser Oligonukleotide, die nur für die Anlagerung der verknupfbaren Oligonukleotide in der gewünschten Anordnung verantwortlich sind, nicht möglichAccording to the invention, the non-linkable oligonucleotides only serve as a template for the formation of the nucleic acid polymer. In a preferred embodiment, these oligonucleotides are not phosphorylated at their 5 'end. This means that these oligonucleotides are enzymatically linked, which are only responsible for the attachment of the linkable oligonucleotides in the desired arrangement , not possible
Es können ebenfalls nicht verknupfbare Oligonukleotide eingesetzt werden bei denen z B das 3'-Ende ein Didesoxynukleotid oder ein Thionukleond ist Entsprechende Modifizierungen sind auch am 5'-Ende möglich Weiterhin kann das Verfahren mit nicht verknupfbaren Oligonukleotiden, die am 3'-Ende phosphoryliert sind, durchgeführt werden Diese Modifikation verhindert ebenfalls die enzymatische Verknüpfung der Oligonukleotide Sofern das erfindungsgemaße Verfahren nicht mittels enzymatischer Ligation, sondern chemischer oder photochemischer Verknüpfung durchgeführt wird, müssen die nicht verknupfbaren Oligonukleotide dementsprechend so konzipiert sein, daß sie unter den zur Verknüpfung der den Pπmarstrang bildenden Oligonukleotide gewählten Bedingungen keine kovalente Bindung zum jeweils benachbarten nicht verknupfbaren Oligo- nukleotid bilden könnenIt is also possible to use non-linkable oligonucleotides in which, for example, the 3 'end is a dideoxynucleotide or a thionucleond. Corresponding modifications are also possible at the 5' end. The method can also be used with non-linkable oligonucleotides which are phosphorylated at the 3 'end This modification likewise prevents the enzymatic linkage of the oligonucleotides. If the method according to the invention is not carried out by means of enzymatic ligation, but rather chemical or photochemical linkage, the Accordingly, non-linkable oligonucleotides must be designed such that they cannot form a covalent bond to the respectively adjacent non-linkable oligonucleotide under the conditions chosen for linking the oligonucleotides forming the primary strand
Die in dem erfindungsgemaßen Verfahren eingesetzten Oligonukleotide können eine Lange von 30 bis 1500 Nukleotiden haben Die Lange des jeweils gewählten Nukleotides ist von mehreren Faktoren einschließlich der Wahrscheinlichkeit der Ausbildung von Se- kundarstrukturen und der Reinheit der gewählten Ausgangsmateπalien abhangig Es ist jedoch bevorzugt, daß die Oligonukleotide des Pπmarstranges eine Lange von 30 bis 200, besonders bevorzugt von 30 bis 100 oder 30 bis 60 Nukleotiden habenThe oligonucleotides used in the method according to the invention can have a length of 30 to 1500 nucleotides. The length of the nucleotide selected in each case is dependent on several factors, including the probability of the formation of secondary structures and the purity of the selected starting materials. However, it is preferred that the oligonucleotides have a length of 30 to 200, particularly preferably 30 to 100 or 30 to 60 nucleotides
Bei der Synthese bereits erzeugte einzelstrangige Nukleinsaurepolymere können in einem weiteren Schritt wiederum mit dem erfindungsgemaßen Verfahren in einzelstrangige Nukleinsaurepolymere von mehreren Kilobasen Lange zusammengeführt werden (Abb 3) Dazu wird das Verfahren so, wie oben ausgeführt, durchgeführt, wobei als ver- knupfbare Oligonukleotide für den Pπmarstrang Nukleinsaurepolymere von beispielsweise 1500 Basen eingesetzt werden könnenIn a further step, the single-stranded nucleic acid polymers already generated in the synthesis can in turn be combined with the method according to the invention into single-stranded nucleic acid polymers of several kilobases in length (FIG. 3). For this purpose, the method is carried out as explained above, with linkable oligonucleotides for the Pπmarstrang nucleic acid polymers of, for example, 1500 bases can be used
Die nicht verknupfbaren Oligonukleotide, die die korrekte Anordnung der Oligonukleotide des Pπmarstranges nebeneinander gewährleisten sollen, sind gegebenenfalls nur 30 bis 50 Nukleotide lang Diese Größenordnung erlaubt die Hybridisierung mit zwei benachbart anzuordnenden Oligonukleotiden des Pπmarstranges Es besteht keine Notwendigkeit, den ganzen Pπmarstrang abdeckende nicht verknupfbare Oligonukleotide zur Verfugung zu stellen Selbstverständlich ist jedoch die Verwendung von nicht verknupfbaren Oligonukleotiden mit bis zu 300 Nukleotiden oder mehr ebenfalls möglich und kann, wo z B im Pπmarstrang ansonsten starke Sekundarstrukturen ausgebildet werden wurden, durchaus sinnvoll seinThe non-linkable oligonucleotides, which are intended to ensure the correct arrangement of the oligonucleotides of the Pπmar strand next to one another, are possibly only 30 to 50 nucleotides long.This order of magnitude allows hybridization with two oligonucleotides of the Pπmar strand to be arranged adjacent to one another To make available, of course, the use of non-linkable oligonucleotides with up to 300 nucleotides or more is also possible and, where, for example, otherwise strong secondary structures have been formed in the primary strand, can be very useful
Jedes der nicht verknupfbaren Oligonukleotide enthalt zwei aneinandergrenzende Bereiche, die Komplementaπtat mit dem 3'- bzw 5'-Ende zweier benachbarter Oligonukleotide für den Pπmarstrang aufweisen Die Bereiche der Komplementaπtat zwischen den verknupfbaren Oligonukleotiden für den Pπmarstrang und den nicht verknupfbaren Oligo- nukleotiden des Gegenstranges sind jeweils ca 15 bis 30 Basenpaare lang, bevorzugt Each of the non-linkable oligonucleotides contains two adjoining regions, which have complementate with the 3 'or 5' end of two adjacent oligonucleotides for the primary strand. The regions of complementary between the linkable oligonucleotides for the primary strand and the non-linkable oligonucleotide nucleotides of the opposite strand are each about 15 to 30 base pairs long, preferred
Eine bevorzugte Ausfuhrungsform des Verfahrens besteht dann, in einer vorgeschalteten Reaktion die endstandigen Oligonukleotide für den Pπmarstrang und/oder zu den end- standigen Oligonukleotiden des Pπmarstranges ganz oder teilweise komplementäre Oligonukleotide an die Enden eines lineaπsierten Vektors anzulagern, sie mit dem Vektor zu verknüpfen und nachfolgend das Verfahren gemäß Anspruch 1 durchzufuhren Die Vektorenden sind dabei bevorzugt kohasiv Dieses Verfahren fuhrt zu einem zirkulaπsierten Produkt, das direkt in einen geeigneten Wirtsorganismus, z B Bakterien, Sauger- oder Insektenzellen, transfiziert werden kann Unter "Transfektion" sind in diesem Fall verschiedene Techniken, wie z B Elektroporation Mikroinjektion Infektion Transfektion unterstutzt durch z B "Ca-P04", DEAE, hydrophobe Moleküle etc , zu verstehenA preferred embodiment of the method then consists in an upstream reaction of attaching the terminal oligonucleotides for the Pπmar strand and / or oligonucleotides complementary to the terminal oligonucleotides of the Pπmar strand to the ends of a linearized vector, to link them to the vector and subsequently to carry out the method according to claim 1 The vector ends are preferably cohesive. This method leads to a circulated product which can be transfected directly into a suitable host organism, for example bacteria, sucker or insect cells. In this case, "transfection" includes various techniques, how to understand eg electroporation microinjection infection transfection supported by eg "Ca-P0 4 ", DEAE, hydrophobic molecules etc.
In einer bevorzugten Ausfuhrungsform ist entweder das 5'-endstandιge Oligonukleotid des Pπmarstrangs an seinem 5'-Ende oder das 3'-endstandιge Oligonukleotid des Pn- marstrangs an seinem 3'-Ende mit einem Hapten oder beide endstandigen Oligonukleotide mit verschiedenen Haptenen versehen Dies ermöglicht die Fixierung des mit dem Hapten gekoppelten Oligonukleotids vor oder nach seiner Verknüpfung mit weiteren Oligonukleotiden des Primarstranges an einen festen Trager Damit wird eine Abtrennung des Hapten-gekoppelten Stranges von allen Vorstufen und Nebenprodukten ohne Hapten z. B unter denaturierenden Bedingungen (z B pH 13) möglich Die jeweils gewählte Hapten-Trager-Kombination muß unter den vorgesehenen Bedingungen stabil sein Ein bevorzugtes Hapten ist z B Biotin, das durch an einen Trager gekoppeltes Streptavidm gebunden wird Weiter können die Oligonukleotide mit Antigenen gekoppelt werden, die von an einen Trager fixierten Antikörpern erkannt und gebunden werden Die weitere Synthese des Primarstranges oder einer der im folgenden beschriebenen weiteren Schritte kann dann an diesem Trager durchgeführt werden Das zweite Hapten konnte beispielsweise für spater mit dem Nukleinsaurepolymer durchzuführende Detektionsver- fahren nützlich seinIn a preferred embodiment, either the 5'-end oligonucleotide of the pmarmar strand at its 5'-end or the 3'-end oligonucleotide of the pmarmar strand at its 3'-end is provided with a hapten or both terminal oligonucleotides with different haptens. This is made possible the fixation of the oligonucleotide coupled to the hapten before or after its linkage with other oligonucleotides of the primary strand to a fixed support. This separates the hapten-coupled strand from all precursors and by-products without hapten z. B possible under denaturing conditions (eg pH 13) The chosen hapten-carrier combination must be stable under the intended conditions. A preferred hapten is eg biotin, which is bound by streptavidm coupled to a carrier. Furthermore, the oligonucleotides can be coupled with antigens which are recognized and bound by antibodies fixed to a carrier. The further synthesis of the primary strand or one of the further steps described below can then be carried out on this carrier. The second hapten could be useful, for example, for detection methods to be carried out later with the nucleic acid polymer
In einer weiteren bevorzugten Ausfuhrungsform wird dem Reaktionsansatz einer nach dem vorliegenden Verfahren durchgeführten Synthesereaktion ein Polymeraseenzym zugesetzt, um aus dem Pπmarstrang, der ein Nuklemsaure-Einzelstrang ist, einen Nu- klemsaure-Doppelstrang zu bilden oder gezielt den komplementären Strang (d h den Gegenstrang zum Primarstrang) zu amplifizieren (asymmetrische PCR) Als spezifischer Primer dient ein zum 3'-Ende des vollständigen Primarstranges komplementäres Oligo - nukleotid (Ruckwartspπmer)In a further preferred embodiment, a polymerase enzyme is added to the reaction mixture of a synthesis reaction carried out in accordance with the present process in order to convert the primary strand, which is a single nucleic acid strand, into a to form a klemsaure double strand or to specifically amplify the complementary strand (ie the opposite strand to the primary strand) (asymmetric PCR) A specific primer is an oligonucleotide (backwinder) which is complementary to the 3 'end of the complete primary strand
In einer bevorzugten Ausfuhrungsform werden dem Reaktionsansatz einer nach dem vorliegenden Verfahren durchgeführten Synthesereaktion ein Polymeraseenzym und zusätzlich endstandige Vorwärts- und Rύckwartspπmer im großen Überschuß zum eingesetzten Syntheseprodukt zugesetzt Auf diese Weise werden nach dem Prinzip der Polymerase-Kettenreaktion (Saiki et al , Science 239, 487-491 , 1988) aus den einzel- straπgigen Nukleinsäurepolymeren doppelstrangige Nukleinsaurepolymere gebildetIn a preferred embodiment, the reaction mixture of a synthesis reaction carried out according to the present method, a polymerase enzyme and additionally terminal forward and backward separators are added in large excess to the synthesis product used. In this way, the principle of the polymerase chain reaction (Saiki et al, Science 239, 487 -491, 1988) from the single-stranded nucleic acid polymers, double-stranded nucleic acid polymers
Das Polymeraseenzym wird dabei aus der Gruppe der DNA-Polymerasen, z B E coli Polymerase I, Klenow-Polymerase, T4 DNA-Polymerase und Reverse Transkπptase, ausgewählt Die dabei gebildeten Nuklemsaure-Doppelstrange können entweder DNA- DNA-Doppelstrange oder DNA-RNA-Doppelstrange sein (Kleppe et al , Proc NatI Acad The polymerase enzyme is selected from the group of DNA polymerases, eg BE coli polymerase I, Klenow polymerase, T4 DNA polymerase and reverse transceptase. The nucleic acid double strands formed in this way can be either DNA-DNA double strands or DNA-RNA double strands be (Kleppe et al, Proc NatI Acad
In einer besonders bevorzugten Ausfuhrungsform ist die zugesetzte Polymerase eine temperaturstabile Polymerase, z B Taq-Polymerase Das temperaturstabile Enzym verliert auch nach mehreren Temperaturzyklen nur wenig von seiner Aktivität und kann deshalb die Polymerasereaktionen mehrerer aufeinander folgender Zyklen katalysierenIn a particularly preferred embodiment, the added polymerase is a temperature-stable polymerase, for example Taq polymerase. The temperature-stable enzyme loses only a little of its activity even after several temperature cycles and can therefore catalyze the polymerase reactions of several successive cycles
Die Polymerasereaktion wird bevorzugt mehrfach wiederholt durchgeführt, um den Nu- kleinsaure-Doppelstrang exponentiell zu amplifizieren Nach dem Prinzip der Polyme- rasekettenreaktion umfaßt dabei jeder Zyklus eine Denaturierung bereits bestehender Nuklemsaure-Doppelstrange durch übliche Maßnahmen, z B durch Erhohen der Temperatur oder des pH, das Anlagern endstandiger Pπmer unter geeigneten Bedingungen und eine Polymerase-katalysierte Nukle sauresynthese Im Fall einer temperaturstabi- len Polymerase erübrigt sich die Zugabe weiterer Polymerase, wo jedoch eine der nicht temperaturstabilen Polymerasen für die Polymerisationsreaktion verwendet worden ist, wird diese durch die thermische Denaturierung inaktiviert, in diesem Fall muß in jedem Zyklus frisches Polymeraseenzym zugefugt werden Üblicherweise wird die Polymerasereaktion 5 bis 15 Mal durchgeführt, bevorzugt etwa 8 bis 12 Mal Der wesentliche Vorteil der Wiederholung der Polymerasereaktion liegt dann, daß aufgrund der Position der Primer (endstandig) gezielt vollständiges Syntheseprodukt exponentiell ampiifiziert werden kannThe polymerase reaction is preferably carried out repeatedly several times in order to amplify the nucleic acid double strand exponentially. According to the principle of the polymer chain reaction, each cycle comprises denaturing already existing nuclear acid double strands by conventional measures, for example by increasing the temperature or the pH. the attachment of terminal polymers under suitable conditions and a polymerase-catalyzed nucleic acid synthesis. In the case of a temperature-stable polymerase, the addition of further polymerase is unnecessary, but where one of the non-temperature-stable polymerases has been used for the polymerization reaction, this is inactivated by the thermal denaturation, in this case, fresh polymerase enzyme must be added in each cycle. The polymerase reaction is usually carried out 5 to 15 times, preferably about 8 to 12 times. The main advantage the repetition of the polymerase reaction is then that due to the position of the primers (final) targeted complete synthesis product can be exponentially amplified
Die Denaturierung der Doppelstrange wird üblicherweise bei Temperaturen von mehr als 90°C durchgeführt Der Reaktionsansatz wird sodann langsam abgekühlt, so daß die endstandigen Primer in Abhängigkeit von ihrer Zusammensetzung bei Temperaturen zwischen 80°C und 45°C mit den dann vorliegenden Einzelstrangen hybridisieren können Die Polymerase-katalysierte DNA-Synthese kann bei Verwendung eines tempera- turstabilen Enzymes im Bereich von 45 bis 70°C durchgeführt werden, wodurch die Bildung uπspezifischer Hybride minimiert wirdThe denaturation of the double strands is usually carried out at temperatures of more than 90 ° C. The reaction mixture is then slowly cooled so that the final primers, depending on their composition, can hybridize with the single strands at temperatures between 80 ° C. and 45 ° C. Polymerase-catalyzed DNA synthesis can be carried out using a temperature-stable enzyme in the range from 45 to 70 ° C, which minimizes the formation of uπspecific hybrids
Gegenstand der vorliegenden Erfindung ist weiter ein einzelstrangiges Nukle saurepo- lymer, das mit dem erfindungsgemaßen Verfahren erhalten worden ist Durch geschickte Kopplung des erfindungsgemaßen Verfahrens, mit dem zunächst einzelstrangige Pn- marstrange hergestellt werden, mit der Fill-m-Methode lassen sich ohne weiteres doppelstrangige Nukleinsaurepolymere von mehreren 1000 Basen erzeugen (Abb 4) Dazu müssen z B Primarstrange so konzipiert sein, daß sie an ihrem 3'-Ende in einem Bereich von wenigen Basenpaaren, z B 20 bis 60, bevorzugt 30 bis 40 Basenpaaren, einander komplementär sind, so daß bei einer nachfolgend in vitro oder in vivo ablaufenden Polymerasereaktion das 3'-Ende jedes Primarstranges als Pπmer fur eine matπzenab- hangige Polymerase dienen kannThe present invention furthermore relates to a single-stranded nucleic acid polymer which has been obtained by the process according to the invention. By skillful coupling of the process according to the invention, with which single-stranded primary strands are initially produced, using the Fill-m method, double-stranded ones can be readily obtained Generate nucleic acid polymers of several 1000 bases (Fig. 4) For this purpose, for example, primary strands must be designed so that they are complementary to one another at their 3 'end in a range of a few base pairs, for example 20 to 60, preferably 30 to 40 base pairs, so that in a subsequent in vitro or in vivo polymerase reaction, the 3 'end of each primary strand can serve as a polymer for a mattress-dependent polymerase
Die folgenden Abbildungen und das Beispiel erläutern die ErfindungThe following figures and the example illustrate the invention
Abbildung 1 beschreibt eine bevorzugte Ausfuhrungsform des erfindungsgemaßen VerfahrensFigure 1 describes a preferred embodiment of the method according to the invention
Es werden zunächst phosphorylierte Oligonukleotide für den Primarstrang und nicht ver- knupfbare Oligonukleotide für den Gegenstrang, die zu den Oligonukleotiden des Pπ- marstrangs komplementär sind und mit jeweils zwei der phosphorylierten Oligonukleotide überlappen, bereitgestellt (a) Anschließendes Ligieren fuhrt zu einem emzelstrangigen Nuklemsaurepolymer (b), dem PπmarstrangFirst there are phosphorylated oligonucleotides for the primary strand and non-linkable oligonucleotides for the opposite strand, which are complementary to the oligonucleotides of the primary strand and each with two of the phosphorylated oligonucleotides overlap, provided (a) Subsequent ligation leads to a single-stranded nuclear acid polymer (b), the pmarmar strand
Abbildung 2 zeigt eine Detailaufnahme des Uberlappungsbereiches zwischen zwei benachbarten verknupfbaren Oligonukleotiden und einem nicht verknupfbaren Oligonukleotid, das zwei aneinandergrenzende Bereiche umfaßt, deren einer dem 3'-Ende eines ersten verknupfbaren Oligonukleotides komplementär ist, und deren zweiter dem 5'- Ende eines zweiten verknupfbaren Oligonukleotides komplementär ist Der Uberlap- pungsbereich zwischen einem Bereich des nicht verknupfbaren Oligonukleotids mit dem komplementären Bereich eines der verknupfbaren Oligonukleotide sollte mindestens 15 bp betragenFigure 2 shows a detailed view of the overlap area between two adjacent linkable oligonucleotides and a non-linkable oligonucleotide, which comprises two adjacent regions, one of which is complementary to the 3 'end of a first linkable oligonucleotide and the second of which the 5' end of a second linkable oligonucleotide is complementary The overlap area between a region of the non-linkable oligonucleotide with the complementary region of one of the linkable oligonucleotides should be at least 15 bp
Abbildung 3 veranschaulicht die Durchfuhrung des Verfahrens mit sehr langen verknupfbaren Oligonukleotiden, die jeweils durch Hybndisierung mit weitaus kürzeren nicht verknupfbaren Oligonukleotiden korrekt nebeneinander angeordnet werden Für diesen Zweck ist es ausreichend, wenn die nicht verknupfbaren Oligonukleotide eine stabile Hybndisierung mit den beiden benachbart anzuordnenden verknupfbaren Oligonukleotiden gewährleisten, ohne daß eine Komplementaπtat zum gesamten spateren Primarstrang gegeben sein mußFigure 3 illustrates the implementation of the method with very long linkable oligonucleotides, which are correctly arranged next to one another by hybridization with much shorter non-linkable oligonucleotides.For this purpose, it is sufficient if the non-linkable oligonucleotides ensure stable hybridization with the two linkable oligonucleotides to be arranged adjacent without having to be complementary to the entire later primary strand
Abbildung 4 zeigt das Prinzip der Fill-m-Methode am Beispiel zweier nur in ihrem 3'- Bereich komplementärer langer Oligonukleotide Durch Hybndisierung der komplementären 3'-Bereιche dient das 3'-Ende jedes der Oligonukleotide als Pπmer fur eine Polymerasereaktion entlang der Matrize des jeweils anderen OligonukleotidesFigure 4 shows the principle of the Fill-m method using the example of two long oligonucleotides that are only complementary in their 3 'region. By hybridizing the complementary 3' regions, the 3 'end of each of the oligonucleotides serves as a polymer for a polymerase reaction along the template of the each other oligonucleotides
Abbildung 5 a) Oligonukleotide zur Synthese des Primarstranges der algenadaptierten GFP-Mutante S65T/F64L (FG1 bis FG15) b) Oligonukleotide des Gegenstranges (nicht verknupfbar) (FG14rev bis FGIrev) c) Ohgonnukleotid FG15rev, Pπmer fur die Polymerase-katalysierte Synthese des Gegenstranges Abbildung 6 Modifiziertes Gen (S65T/F64L) für das Grüne Fluoreszierende Protein (Green Fluorescent Protein - GFP) aus Aquorea victorea Das Gen wurde gegenüber dem natürlichen GFP-Gen so modifiziert, daß es nur Kodons enthalt, die von Grünalgen, beispielsweise Chlamvdomonas, bevorzugt werden Die unterstrichenen Nukleotide wurden erst durch die Polymerasereaktion aufgefüllt (Matrize FG15rev)Figure 5 a) Oligonucleotides for the synthesis of the primary strand of the algal-adapted GFP mutant S65T / F64L (FG1 to FG15) b) Oligonucleotides of the opposite strand (not connectable) (FG14rev to FGIrev) c) Ohgon nucleotide FG15rev, polymer for the synthesis of the polymerase-catalyzed catalyst Figure 6 Modified gene (S65T / F64L) for the Green Fluorescent Protein (GFP) from Aquorea victorea. The gene was modified compared to the natural GFP gene in such a way that it only contains codons preferred by green algae, for example Chlamvdomonas The underlined nucleotides were only filled in by the polymerase reaction (matrix FG15rev)
Beispiel Synthese eines alqenadaptierten Genes, das für Grünes Fluoreszierendes Protein (GFP) aus der Qualle Aequorea victorea kodiertExample Synthesis of an algal-adapted gene which codes for green fluorescent protein (GFP) from the jellyfish Aequorea victorea
Im folgenden wird die Synthese eines modifizierten Genes für Grünes Fluoreszierendes Protein (GFP) aus der Qualle Aequorea victorea gezeigt Der Kodongebrauch wird im synthetiscnen Gen dahingehend modifiziert daß die Kodons von Grünalgen wie beispielsweise Chlamvdomonas, bevorzugt werden Für die Synthese wurden 30 Oligonukleotide mit einer Lange von 45 bis 50 Basen eingesetzt Die Oligonukleotide des Primarstranges werden dabei als FG1 bis FG15 bezeichnet und sind 45 bis 47 Basen lang Die nicht verknupfbaren Oligonukleotide des komplementären Stranges werden als FG rev bis FGI rev bezeichnet und sind 46 Basen (FG14rev bis FGI rev) lang Die Sequenzen der Oligonukleotide sind in Abb 5a, b gezeigtThe following shows the synthesis of a modified gene for green fluorescent protein (GFP) from the jellyfish Aequorea victorea. The codon usage is modified in the synthetic gene in such a way that the codons of green algae such as Chlamvdomonas are preferred. For the synthesis, 30 oligonucleotides with a length of 45 to 50 bases used The oligonucleotides of the primary strand are designated as FG1 to FG15 and are 45 to 47 bases long. The non-linkable oligonucleotides of the complementary strand are designated as FG rev to FGI rev and are 46 bases (FG14rev to FGI rev) long Sequences of the oligonucleotides are shown in Fig. 5a, b
Die Reaktion wurde in einem programmierbaren Thermocycler durchgeführt Die Oligonukleotide die den verknupfbaren Strang bilden, wurden vor der Synthesereaktion am 5'-Ende phosphoryliertThe reaction was carried out in a programmable thermal cycler. The oligonucleotides which form the linkable strand were phosphorylated at the 5 'end before the synthesis reaction
Schritt 1 Oliqonukleotid-Phosphoryiierunq am 5'-Ende (100 μl-Ansatz)Step 1 oligonucleotide phosphorylation at the 5 'end (100 μl mixture)
10 μl Oligonukleotide FG1 bis FG15 (500 pMol total, gleiche molare Konzentration jedes Oligonukleotides)10 μl oligonucleotides FG1 to FG15 (500 pmol total, same molar concentration of each oligonucleotide)
10 μl Kmasepuffer (10-fach konzentriert, Biolabs)10 μl chamomile buffer (10-fold concentrated, Biolabs)
10 μl ATP 2 mM10 ul ATP 2mM
69 μl H2069 ul H 2 0
1 μl T4 Polynukleotidkmase (10 Einheiten Biolabs) Die Reaktion wird für 4 h bei 37°C durchgeführt und anschließend durch 5-minütige Inkubation bei 95°C beendet.1 μl T4 polynucleotide kmase (10 units Biolabs) The reaction is carried out at 37 ° C. for 4 h and then terminated by incubation at 95 ° C. for 5 minutes.
Schritt 2: Trägerfreie Nukleotidpolymersynthese (Primärstrang)Step 2: carrier-free nucleotide polymer synthesis (primary strand)
Die Reaktion wurde in einem programmierbaren Thermocycler durchgeführt. Es wurde rekombinante Pfu-DNA-Ligase der Firma Stratagene verwendet. Die Pufferbedingungen wurden mit KCI, NP-40, MgCI2 auf die Ligasebedingungen umgestellt, wobei die Ionen im Kinaseansatz berücksichtigt wurden.The reaction was carried out in a programmable thermal cycler. Recombinant Pfu DNA ligase from Stratagene was used. The buffer conditions were changed to the ligase conditions with KCI, NP-40, MgCI 2 , whereby the ions were taken into account in the kinase batch.
10 μl 5'-phosphorylierte Oligonukleotide (50 pMol total) aus Schritt 1 (FG1-FG15)10 ul 5'-phosphorylated oligonucleotides (50 pmol total) from step 1 (FG1-FG15)
1 μl nicht-phosphorylierte Oligonukleotide (50 pMol total) (FGI rev - FG14rev)1 μl non-phosphorylated oligonucleotides (50 pmol total) (FGI rev - FG14rev)
6 μi KCI (100 mM)6 μi KCI (100 mM)
3 μl NP-40 (0,5%)3 μl NP-40 (0.5%)
4 μl MgCI2 (50 mM) 3 μl ATP (10 mM)4 μl MgCI 2 (50 mM) 3 μl ATP (10 mM)
Der Reaktionsansatz wird 3 Min. bei 95°C und 3 Min. bei 80°C inkubiert. Während der Inkubation bei 80°C werden 3 μl Pfu-DNA-Ligase (12 Einheiten thermostabile Ligase der Firma Stratagene) zugesetzt und anschließend 3mal der folgende Temperaturzyklus durchgeführt:The reaction mixture is incubated for 3 minutes at 95 ° C. and for 3 minutes at 80 ° C. During the incubation at 80 ° C., 3 μl of Pfu DNA ligase (12 units of thermostable ligase from Stratagene) are added and the following temperature cycle is then carried out 3 times:
a) 1 Min. 95°C b) in 1 Min. von 95°C auf 70°C abkühlen c) über den Zeitraum von 1 Std. linear von 70°C auf 55°C abkühlen d) 2 Std. bei 55°C inkubierena) 1 min. 95 ° C b) cooling from 95 ° C to 70 ° C in 1 min. c) cooling linearly from 70 ° C to 55 ° C over a period of 1 hour d) 2 hours at 55 ° Incubate C.
Variante 1: Das Produkt wird zur Abtrennung der Pfu-Ligase ausgefällt, gegebenenfalls über PCR und Taq-Polymerase amplifiziert und kloniert.Variant 1: The product is precipitated to separate the Pfu ligase, if necessary amplified and cloned using PCR and Taq polymerase.
Variante 2: Um die Abtrennung zu erleichtern, wird der Primer FG1 biotinyliert eingesetzt. Nach Beendigung der Synthese wird der Reaktionsansatz mit NaOH auf pH13 gebracht Es werden Streptavidin-Magnetkugeln (Dynal) zugesetzt und das Syntheseprodukt mit einem Magneten an der Wand des Reaktionsgefaßes festgehalten, wahrend alle Ruckwartspπmer, unverbrauchte Vorwartspπmer sowie Ligase mit dem Überstand abgenommen werden Das saubere, nun an Streptavidm gekoppelte Syntheseprodukt wird in 30 μl neutralem Puffer aufgenommen und kann z B wie im Schritt 3 (s unten) amplifiziert werdenVariant 2: In order to facilitate the separation, the primer FG1 is used biotinylated. After completion of the synthesis, the reaction mixture is adjusted to pH 13 with NaOH Streptavidin magnetic balls (Dynal) are added and the synthesis product is held in place with a magnet on the wall of the reaction vessel, while all the backwashers, unused forward packers and ligase are removed with the supernatant. The clean synthesis product, now coupled to streptavidm, is placed in 30 μl of neutral buffer recorded and can, for example, be amplified as in step 3 (see below)
Schntt 3 Amplifizierung des Syntheseproduktes mit Hilfe der Polymerasekettenreaktion (PCR)Schntt 3 Amplification of the synthesis product using the polymerase chain reaction (PCR)
Variante 1 1 μl des Syntheseproduktes werden mit 1 E thermostabiler Pfu-Polymerase (Stratagene) in 12 Zyklen im Standardpuffer (It Hersteller) in einem 50 μl-Ansatz amplifiziert Als Vorwartspπmer dient das Oligonukleotid FG1 , als Ruckwartspπmer dient das Oligonukleotid FG15rev (50 Basen, Abb 5c) Endkonzentration 25 pMol pro 50 μl.Variant 1 1 μl of the synthesis product are amplified in 1 cycle with 1 U of thermostable Pfu polymerase (Stratagene) in 12 cycles in a standard buffer (It manufacturer) in a 50 μl batch. The oligonucleotide FG1 serves as the forward spanner, the oligonucleotide FG15rev (50 bases, Fig. 5c) final concentration 25 pmol per 50 μl.
Variante 2: Zur Amplifizierung mittels PCR werden zwei endstandige Pπmer zugegeben, die zwar eine Gesamtlange von 30 bzw 33 Basen aufweisen, wovon aber nur 21 bzw 24 bp mit dem Syntheseprodukt hybridisieren Die "Überhange" enthalten jeweils eine Schnittstelle der "Multiplen Klonierungsregion" des Vektors pBenescπpt II KS' (Stratagene) Das amplifizierte Produkt wird mit den Enzymen Xhol und Knpl geschnitten, über Quiaex II (Quiagen) gereinigt und standardmäßig kloniertVariant 2: For amplification by means of PCR, two terminal polymers are added which have a total length of 30 or 33 bases, of which only 21 or 24 bp hybridize with the synthesis product. The "overhangs" each contain an interface of the "multiple cloning region" of the vector pBenescπpt II KS ' (Stratagene) The amplified product is cut with the enzymes Xhol and Knpl, purified using Quiaex II (Quiagen) and cloned by default
Vorwartspπmer mit Xhol-SchnittstelleForward spanner with Xhol interface
5' CCG CTC GAG ATG GCC AAG GGC GAG GAG CTG5 'CCG CTC GAG ATG GCC AAG GGC GAG GAG CTG
Rückwartspπmer mit Kpnl-SchnittstelleReverse spammer with Kpnl interface
5' CGC GGA TCC TTA CTT GTA CAG CTC GTC CAT GCC 5 'CGC GGA TCC TTA CTT GTA CAG CTC GTC CAT GCC

Claims

WO 99/10358 Λ A PCT/EP98/05219,14Patentansprüche WO 99/10358 Λ A PCT / EP98 / 05219,14 patent claims
1 Verfahren zum Herstellen eines Nukleinsaurepolymeres, umfassend1. A method for producing a nucleic acid polymer comprising
a) das Bereitstellen von 2 oder mehr verknupfbaren Oligonukleotiden, die bei kontinuierlicher Anordnung und nach Verknüpfen einen Primarstrang bilden können, und einem oder mehr nicht verknupfbaren Oligonukleotiden, wobei jedes der nicht verknupfbaren Oligonukleotide zwei aneinandergrenzende Bereiche umfaßt, deren erster dem 3'-Ende eines verknupfbaren Oligonukleotids komplementär ist und deren zweiter dem 5'-Ende eines weiteren verknupfbaren Oligonukleotids komplementär ista) the provision of 2 or more linkable oligonucleotides which, when arranged continuously and after linking, can form a primary strand, and one or more non-linkable oligonucleotides, each of the non-linkable oligonucleotides comprising two adjacent regions, the first of which is the 3 'end of one linkable oligonucleotide is complementary and the second of which is complementary to the 5 'end of a further linkable oligonucleotide
b) das Hybridisieren von Oligonukleotiden für den Primarstrang mit den komplementäre Bereichen der nicht verknupfbaren Oligonukleotide undb) the hybridization of oligonucleotides for the primary strand with the complementary regions of the non-linkable oligonucleotides and
c) das Verknüpfen der Oligonukleotide des Primarstrangesc) linking the oligonucleotides of the primary strand
2 Verfahren nach Anspruch 1 , umfassend eine oder mehrere Wiederholungen der Schπtte (b) und (c), wobei vor jeder Wiederholung eine Denaturierung durchgeführt2 The method according to claim 1, comprising one or more repetitions of the sections (b) and (c), a denaturation being carried out before each repetition
3 Verfahren nach Anspruch 2 wobei die Schritte (b) und (c) 1 bis 8 Mal bevorzugt 3 bis 5 Mal wiederholt werden3 The method of claim 2 wherein steps (b) and (c) are repeated 1 to 8 times, preferably 3 to 5 times
4 Verfahren nach mindestens einem der Ansprüche 1 bis 3 wobei die Oligonukleotide des Pπmarstranges enzymatisch verknüpft werden4 The method according to at least one of claims 1 to 3, wherein the oligonucleotides of the Pmarmar strand are linked enzymatically
5 Verfahren nach Anspruch 4 wobei zur enzymatischen Verknüpfung thermostabile Ligase zugesetzt wird5 The method according to claim 4, wherein thermostable ligase is added for the enzymatic linkage
6 Verfahren nach einem der Ansprüche 4 oder 5 wobei das Hybridisieren und Verknüpfen zwischen 45βC und 80βC, bevorzugt bei 70°C durchgeführt wird 6. The method according to any one of claims 4 or 5 wherein the hybridizing and linking between 45 β C and 80 β C, preferably at 70 ° C is carried out
Verfahren nach mindestens einem der Ansprüche 1 bis 3 wobei die Oligonukleotide des Primarstranges chemisch verknüpft werden Method according to at least one of claims 1 to 3, wherein the oligonucleotides of the primary strand are chemically linked
Verfahren nach mindestens einem der Ansprüche 1 bis 3, wobei die Oligonukleotide des Primarstranges photochemisch verknüpft werdenMethod according to at least one of claims 1 to 3, wherein the oligonucleotides of the primary strand are linked photochemically
Verfahren nach mindestens einem der Ansprüche 1 bis 8, wobei die nicht verknupfbaren Oligonukleotide nicht phosphoryliert sindMethod according to at least one of claims 1 to 8, wherein the non-linkable oligonucleotides are not phosphorylated
Verfahren nach mindestens einem der Ansprüche 1 bis 9, wobei das 3'-Ende oder das 5'-Ende der nicht verknupfbaren Oligonukleotide modifiziert istMethod according to at least one of claims 1 to 9, wherein the 3 'end or the 5' end of the non-linkable oligonucleotides is modified
Verfahren nach mindestens einem der Ansprüche 1 bis 10, wobei die verknupfbaren und nicht verknupfbaren Oligonukleotide jeweils 30 bis 1500 Nukleotide umfassenThe method according to at least one of claims 1 to 10, wherein the linkable and non-linkable oligonucleotides each comprise 30 to 1500 nucleotides
Verfahren nach Anspruch 11 , wobei die verknupfbaren Oligonukleotide jeweils 30 bis 200 Nukleotide, bevorzugt 30 bis 60 Nukleotide, umfassenThe method of claim 11, wherein the linkable oligonucleotides each comprise 30 to 200 nucleotides, preferably 30 to 60 nucleotides
Verfahren nach Anspruch 12, wobei die nicht verknupfbaren Oligonukleotide 30 bis 50 Nukleotide umfassenThe method of claim 12, wherein the non-linkable oligonucleotides comprise 30 to 50 nucleotides
Verfahren nach mindestens einem der Ansprüche 1 bis 13, wobei die Bereiche der Komplementaπtat zwischen verknupfbaren Oligonukleotiden des Primarstranges und nicht verknupfbaren Oligonukleotiden jeweils ca 15 bis 30 bp, bevorzugt 20 bis Method according to at least one of claims 1 to 13, wherein the regions of the complementate between linkable oligonucleotides of the primary strand and non-linkable oligonucleotides each about 15 to 30 bp, preferably 20 to
Verfahren nach mindestens einem der Ansprüche 1 bis 14, wobei vor den Schritten (a) bis (c) gemäß Anspruch 1 die endstandigen Oligonukleotide für den Primarstrang und/oder zu den endstandigen Oligonukleotiden für den Primarstrang ganz oder teilweise komplementäre Oligonukleotide an die Enden eines lineaπsierten Vektors angelagert und mit diesen verknüpft werden The method according to at least one of claims 1 to 14, wherein prior to steps (a) to (c) according to claim 1, the terminal oligonucleotides for the primary strand and / or the terminal oligonucleotides for the primary strand are completely or partially complementary oligonucleotides at the ends of a linearized Vectors are attached and linked to them
16. Verfahren nach einem der Ansprüche 1 bis 15, wobei das 3'-endständige Oligonukleotid für den Primärstrang ein Hapten am 3'-Ende trägt und/oder das 5'- endständige Oligonukleotid für den Primärstrang ein Hapten am 5'-Ende trägt.16. The method according to any one of claims 1 to 15, wherein the 3'-terminal oligonucleotide for the primary strand carries a hapten at the 3'-end and / or the 5'-terminal oligonucleotide for the primary strand carries a hapten at the 5'-end.
17. Verfahren nach mindestens einem der Ansprüche 1 bis 16, umfassend weiter17. The method according to at least one of claims 1 to 16, further comprising
d) das Zusetzen einer Polymerase, um ein Nukleinsäure-Doppelstrangpolymer zu bilden, undd) adding a polymerase to form a double stranded nucleic acid polymer, and
e) das Durchführen einer Polymerasereaktion.e) performing a polymerase reaction.
18. Verfahren nach Anspruch 14, wobei vor oder nach dem Zusatz von Polymerase im Schritt d) zusätzlich endständige Rückwärtsprimer oder endständige Vorwärts- und Rückwärtsprimer zugesetzt werden.18. The method according to claim 14, wherein before or after the addition of polymerase in step d), terminal reverse primers or terminal forward and reverse primers are additionally added.
19. Verfahren nach mindestens einem der Ansprüche 14 bis 17, wobei die zugesetzte Polymerase eine temperaturstabile Polymerase ist.19. The method according to at least one of claims 14 to 17, wherein the added polymerase is a temperature-stable polymerase.
20. Verfahren nach einem der Ansprüche 1 bis 19, umfassend eine oder mehrere Wiederholungen des Schrittes (e), wobei vor jeder Wiederholung eine Denaturierung durchgeführt wird.20. The method according to any one of claims 1 to 19, comprising one or more repetitions of step (e), wherein denaturation is carried out before each repetition.
21. Verfahren nach Anspruch 20, wobei die Denaturierung bei Temperaturen von mehr als 90°C und die Anlagerung endständiger Primer sowie die Polymerase-katalysierte DNA-Synthese bei 45°C bis 70°C durchgeführt werden.21. The method according to claim 20, wherein the denaturation at temperatures of more than 90 ° C and the attachment of terminal primers and the polymerase-catalyzed DNA synthesis are carried out at 45 ° C to 70 ° C.
22. Einzelsträngiges Nukleinsäurepolymer, erhalten nach dem Verfahren gemäß einem der Ansprüche 1 bis 21.22. Single-stranded nucleic acid polymer obtained by the method according to one of claims 1 to 21.
23. Verwendung eines einzelsträngigen Nukleinsäurepolymers nach Anspruch 22 zum Erzeugen doppelsträngiger Nukleinsaurepolymere von mehr als 1000 bp, bevorzugt mehr als 1500 bp. 23. Use of a single-stranded nucleic acid polymer according to claim 22 for producing double-stranded nucleic acid polymers of more than 1000 bp, preferably more than 1500 bp.
EP98947434A 1997-08-22 1998-08-17 Method for producing nucleic acid polymers Withdrawn EP1005574A2 (en)

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DE19736591A1 (en) 1999-02-25
US6472184B1 (en) 2002-10-29
WO1999010358A3 (en) 1999-08-05
DE29823795U1 (en) 2000-03-02

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