`
`
`
`
`
`(54) Title) METHOD FOR PRODUCING POLYMERS
`
`(54) Bezeichnung: VERFAHREN ZUR HERSTELLUNG VON POLYMEREN
`
`WELTORGANISATION FUR GEISTIGES EIGENTUM
`‘ PCT
`Internationales Biiro
`INTERNATIONALE ANMELDUNG VEROFFENTLICHT NACH DEM VERTRAG UBER DIE
`INTERNATIONALE ZUSAMMENARBEITAUF DEM GEBIET DES PATENTWESENS(PCT)
`
`
`
`(51) Internationale Patentklassifikation 7 :
`(11) Internationale Verdffentlichungsnummer: WO 00/49142
`
`
`C12N 15/10, C12P 19/34
`
`(43) Internationales
`
`Veréffentlichungsdatum:
`
`
`
`24. August 2000 (24.08.00)
`
`
`
`(21) Internationales Aktenzeichen: PCT/EP00/01356|(81) Bestimmungsstaaten: AU, CA, JP, US, europdisches Patent
`
`
`(AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT,
`
`(22) Internationales Anmeldedatum: 18. Februar 2000 ( 18.02.00)
`
`
`LU, MC,NL, PT, SE).
`
`
`
`
`
`Veréffentlicht
`(30) Priorititsdaten:
`199 07 080.6
`19. Februar 1999 (19.02.99)
`DE
`Mit internationalemRecherchenbericht.
`
`
`
`199 28 843.7
`24. Juni 1999 (24.06.99)
`DE
`Vor Ablaufderfiir Anderungen der Anspriiche zugelassenen
`61 RA G
`
`
`
`199 40 752.5
`27. August 1999 (27.08.99)
`DE
`Frist; Veréffentlichung wird wiederholt falls Anderungen
`SHC
`\rcrYEP99/06316
`27. August 1999 (27.08.99)
`EP
`
`
`eintreffen.
`
`199 57 116.3
`26. November 1999 (26.11.99) DE
`0 104 é
`NAN
`SF
`Nok. a
`&
`<
`
`
`NT 5 Anmelder (fur alle Bestipznuungsstaaten ausser US): BEBYE
`
`[DE/DE];
` Gasselweg 15, D-69469 Weinheim (DE).
`
`(72) Erfinder; und
`
`(75) Erfinder/Anmelder
`(nur fiir US):
`STAHLER, Peer, F.
`
`
`[DE/DE]; Riedfeldstrasse 3, D-68169 Mannheim (DE).
`
`STAHLER, Cord,
`F.
`[DE/DE];
` Siegfriedstrasse
`9,
`
`D-69469 Weinheim (DE). MULLER, Manfred [DE/DE];
`Mannheimerstrasse 11, D-69198 Schriesheim (DE).
`
`
`
` (74) Anwalte: WEICKMANN, H. usw.; Kopemikusstrasse 9,
`D-81679 Munchen (DE).
`
`
`
`
`
`(57) Abstract
` The invention relates to a method for producing
`polymers, especially synthetic nucleic acid double strands
`
`
`of optional sequence, comprising the following steps:
`(a)
`
`providing a support with a surface which contains a plurality
`
`of individual reaction zones, (b) location—resolved synthesis
`of nucleic acid fragments with different base sequences
`respectively in several of the individual reaction areas and
`(c) removing the nucleic acid fragments from the individual
`
`reaction areas.
` (57) Zusammenfassung
` Die Erfindung betrifft ein Verfahren zur Herstellung
`
`
`
`von von_synthetischenPolymeren, insbesondere
`
`
`
`Nukleinsduredoppelstrangen wahlfreier Sequenz, umfassend
`die Schritte:
`(a) Bereitstellen eines Trigers mit einer
`Oberflache, die eine Vielzahl von individuellen Reak-
`tionsbereichen enthilt;
`(b) ortsaufgeléstes Synthetisieren
`von Nukleinsdurefragmenten mit jeweils unterschiedlicher
`
`Basensequenz an mehreren der individuellen Reaktionsbere-
`iche; und (c) Abldsen der Nukleinsaurefragmente von individuellen Reaktionsbereichen.
`
`
`
`
`
`

`

`
`
`Method for producing polymers
`
`Description
`
`producing
`for
`a method
`to
`relates
`invention
`The
`polymers,
`in particular synthetic nucleic acid double
`strands of optional sequence.
`
`10
`
`15
`
`20
`
`25
`
`Technical background of the invention
`Manipulation and construction of genetic elements such
`as,
`for
`example,
`gene
`fragments, whole
`genes
`or
`regulatory regions
`through
`the
`development
`of
`DNA
`recombination technology, which is often also referred
`to as genetic engineering,
`led to a particular need for
`genetic
`engineering methods
`and
`further
`development
`thereof
`in
`the
`areas
`of
`gene
`therapy, molecular
`medicine (basic research, vector development, vaccines,
`regeneration, etc.).
`Important areas of application are
`also the development of active substances, production
`of active substances in the context of the development
`of
`pharmaceuticals,
`combinatorial
`biosynthesis
`(antibodies, effectors such as growth factors, neural
`transmitters, etc.), biotechnology (e.g. enzyme design,
`pharming, biological production methods, bioreactors,
`etc.),
`diagnostics
`(BioChips,
`receptors/antibodies,
`enzyme
`design,
`etc.)
`and
`environmental
`technology
`(specialized
`or
`custom microorganisms,
`production
`processes, cleaning-up, sensors, etc.).
`
`30
`
`Prior art
`
`foremost
`and
`first
`Numerous methods,
`methods,
`allow specific manipulation
`different purposes.
`
`enzyme-based
`of
`DNA
`for
`
`35
`
`to use available genetic
`have
`said methods
`All of
`on
`the one hand, well-
`material. Said material
`is,
`defined to a
`large extent but allows,
`on the other
`hand,
`in a kind of “construction kit
`system” only a
`
`

`

`
`
`of
`combinations
`possible
`of
`amount
`limited
`particular available and slightly modified elements.
`
`the
`
`In this connection, completely synthetic DNA has so far
`played only a minor part
`in the form of one of
`these
`combinatorial elements, with the aid of which specific
`modifications of
`the available genetic material
`are
`possible.
`
`amount of work
`large
`the
`share
`known methods
`The
`certain
`duration
`of
`a
`required,
`combined with
`appropriate operations,
`since the stages of molecular
`biological and in particular genetic experiments
`such
`as DNA isolation, manipulation,
`transfer into suitable
`target
`cells,
`propagation,
`renewed
`isolation,
`etc.
`usually have to be repeated several
`times. Many of the
`operations which come
`up can only insufficiently be
`automated and accelerated so that
`the corresponding
`work remains
`time-consuming and labor-intensive.
`For
`the isolation of genes, which must precede functional
`study and characterization of
`the gene product,
`the
`flow of information is in most cases from isolated RNA
`(mRNA) via cDNA and appropriate gene
`libraries via
`complicated screening methods
`to a single clone. The
`desired DNA which has been cloned in said clone is
`frequently
`incomplete,
`so
`that
`further
`screening
`processes follow.
`
`DNA
`of
`recombination
`above-described
`the
`Finally,
`allows,
`only limited flexibility and
`has
`fragments
`together with the described amount of work required,
`only few opportunities for optimization.
`In view of the
`variety and complexity in genetics,
`functional genomics
`and proteomics,
`i.e.
`the study of gene product actions,
`such optimizations in particular are a bottleneck for
`the further development of modern biology.
`
`A common method is recombination by enzymatic methods
`(in vitro): here,
`DNA elements
`(isolated genomic DNA,
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`

`

`plasmids,
`
`amplicons,
`
`viral
`
`or
`
`bacterial
`
`genomes,
`
`vectors) are first cut into fragments with defined ends
`
`by appropriate restriction enzymes. Depending on
`
`the
`
`composition of these ends, it is possible to recombine
`
`the fragments
`
`formed and to link them to form larger
`
`DNA
`
`elements
`
`(likewise
`
`enzymatically).
`
`For
`
`DNA
`
`propagation purposes,
`
`this is frequently carried out
`
`in
`
`a plasmid acting as cloning vector.
`
`10
`
`The
`
`recombinant
`
`DNA normally has
`
`to be propagated
`
`clonally in suitable organisms
`
`(cloning)
`
`and,
`
`after
`
`this time-consuming step and isolation by appropriate
`
`methods,
`
`is again available for manipulations such as,
`
`for example,
`
`recombinations. However,
`
`the restriction
`
`15
`
`enzyme cleavage sites are a
`
`limiting factor
`
`in this
`
`method: each enzyme recognizes a specific sequence on
`
`the (double-stranded) DNA, which is between three and
`
`twelve nucleotide bases
`
`in length,
`
`depending on
`
`the
`
`particular
`
`enzyme,
`
`and
`
`therefore,
`
`according
`
`20
`
`a
`
`particular
`
`number
`
`to
`
`of
`
`Statistical
`
`distribution,
`
`cleavage sites at which
`
`the
`
`DNA
`
`strand is
`
`cut
`
`is
`
`present on each DNA element. Cutting the treated DNA
`
`into defined fragments, which
`
`can
`
`subsequently be
`
`combined to give the desired sequence,
`
`is important for
`
`25
`
`recombination.
`
`Sufficiently
`
`different
`
`and
`
`specific
`
`enzymes are available for
`
`recombination technology up
`
`to a limit of 10 - 30 kilo base pairs (kbp) of the DNA
`
`to be cut.
`
`In addition, preliminary work and commercial
`
`suppliers provide corresponding vectors which take up
`
`30
`
`the
`
`recombinant
`
`DNA
`
`and
`
`allow cloning
`
`(and
`
`thus
`
`propagation
`
`and
`
`selection).
`
`Such
`
`vectors
`
`contain
`
`suitable cleavage sites for efficient recombination and
`
`integration.
`
`35
`
`With increasing length of the manipulated DNA, however,
`
`the rules of statistics give rise to the problem of
`
`multiple and unwanted cleavage sites. The statistical
`
`average
`
`for
`
`an
`
`enzyme
`
`recognition
`
`sequence
`
`of
`
`6
`
`nucleotide bases
`
`is one cleavage site per
`
`4000 base
`
`

`

`one
`is
`it
`8 nucleotide bases
`for
`and
`(4°)
`pairs
`65,000
`(48). Recombination using
`cleavage
`site per
`therefore
`is
`not particularly
`restriction enzymes
`Suitable for manipulating relatively large DNA elements
`(e.g. viral genomes, chromosomes, etc.).
`
`recombination in cells igs
`Recombination by homologous
`known,
`too. Here,
`if identical
`sequence sections are
`present
`on
`the
`elements
`to be
`recombined,
`it
`is
`possible to newly assemble and manipulate relatively
`large DNA elements by way of
`the natural process of
`homologous
`recombination. These
`recombination events
`are substantially more indirect than in the case of the
`restriction enzyme method and, moreover, more difficult
`to control. They often give distinctly poorer yields
`than
`the
`above-described
`recombination
`using
`restriction enzymes.
`
`A second substantial disadvantage is restriction to the
`identical sequence sections mentioned which, on the one
`hand, have to be present in the first place and, on the
`other
`hand,
`are
`very
`specific
`for
`the particular
`system.
`The
`specific
`introduction
`of
`appropriate
`sequences itself then causes considerable difficulties.
`
`An additional well-known method is the polymerase chain
`reaction (PCR) which allows
`enzymatic
`DNA
`synthesis
`(including high multiplication)
`due
`to the bordering
`regions of
`the section to be multiplied indicating a
`DNA replication start by means of short,
`completely
`synthetic DNA oligomers
`(“primers”). For
`this purpose,
`however,
`these flanking regions must be known and be
`specific
`for
`the
`region
`lying
`in
`between. When
`replicating the
`strand,
`however,
`polymerases
`also
`incorporate
`wrong
`nucleotides,
`with
`a
`frequency
`depending on the particular enzyme,
`so that. there is
`always
`the danger of
`a certain distortion of
`the
`starting sequence. For
`some applications,
`this gradual _
`distortion can
`be very disturbing. During
`chemical
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`
`
`

`

`the above-
`for example,
`sequences such as,
`synthesis,
`can
`be
`restriction
`cleavage
`sites
`described
`(limited)
`incorporated into the primers. This allows
`manipulation of
`the complete sequence. The multiplied
`region can now be in the region of approx.
`30 kbp, but
`most of this DNA molecule is the copy of a DNA already
`present.
`
`The primers are prepared using automated solid phase
`synthesis
`and
`are
`widely
`available,
`but
`the
`configuration of all automatic synthesizers
`known
`to
`date leads to the production of amounts of primer DNA
`(gmol-range reaction mixtures) which are too large and
`not
`required for PCR, while the variety in variants
`remains limited.
`
`Synthetic DNA elements
`(inter alia in:
`Since the pioneering work of Khorana
`Shabarova: Advanced Organic Chemistry of Nucleic Acids,
`VCH Weinheim;)
`in the 1960s,
`approaches
`in order
`to
`assemble double-stranded DNA with genetic or
`coding
`sequences
`from chemically synthesized DNA molecules
`have repeatedly been described. State of
`the art here
`is genetic elements of up to approx.
`2 kbp in length
`which
`are
`synthesized from nucleic
`acids. Chemical
`solid phase synthesis of nucleic acids and peptides has
`been automated. Appropriate methods
`and devices have
`been
`described,
`for
`example,
`in
`US 4353989
`and
`US 5112575.
`
`short
`from
`synthesized
`is
`DNA
`Double-stranded
`(see
`to
`two methods
`according
`oligonucleotides
`PCR Methods and Applications, Cold
`Holowachuk et al.,
`Spring Harbor Laboratory Press): on the one hand,
`the
`complete double strand is synthesized by synthesizing
`Single-stranded nucleic acids (with suitable sequence),
`attaching complementary regions by hybridization and
`linking the molecular backbone by,
`for example,
`ligase.
`On
`the other hand,
`there is also the possibility of
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`

`

`as
`edges
`the
`overlapping
`regions
`synthesizing
`by
`attachment
`single-stranded
`nucleic
`acids,
`hybridization,
`filling in the single-stranded regions
`via enzymes
`(polymerases) and linking the backbone.
`
`at
`
`the genetic
`length of
`total
`the
`In both methods,
`element is restricted to only a few thousand nucleotide
`bases due
`to,
`on
`the one hand,
`the expenditure and
`production costs of nucleic acids in macroscopic column
`synthesis
`and,
`on
`the other hand,
`the logistics of
`nucleic acids being prepared separately in macroscopic
`column synthesis and then combined. Thus,
`the same size
`range as in DNA recombination technology is covered.
`
`a
`can be described as
`the prior art
`summarize,
`To
`procedure in which,
`in analogy to logical operations,
`the available matter
`(in this case genetic material
`in
`the
`form of nucleic acids)
`is
`studied and combined
`(recombination).
`The
`result
`of
`recombination
`experiments of
`this kind is then studied and allows
`conclusions,
`inter alia about
`the elements employed and
`their combined effect. The procedure may therefore be
`described as
`(selectively)
`analytical
`and
`combina-
`torial.
`
`The prior art
`studies
`of
`modification
`impossible.
`impossible.
`
`thus
`any
`of
`the
`Systematic
`
`systematic
`allow any
`not
`does
`combinations
`whatsoever.
`The
`combined
`elements
`is
`almost
`testing of modifications
`is
`
`Subject of the invention and object achieved therewith
`It
`is
`intended to provide
`a method
`for directly
`converting
`digital
`genetic
`information
`(target
`sequence,
`databases,
`etc.)
`into biochemical genetic
`information
`(nucleic
`acids) without making
`use
`of
`
`10
`
`15
`
`20
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`25
`
`30
`
`35
`
`nucleic acid fragments already present.
`
`

`

`The
`invention
`therefore
`relates
`to
`a method
`for
`producing polymers,
`in which a plurality of oligomeric
`building blocks is synthesized on a support by parallel
`synthesis steps,
`is detached from the support and is
`brought into contact with one another to synthesize the
`polymer. Preference is given to synthesizing double-
`stranded nucleic acid polymers of at
`least 300 bp,
`in
`particular at least 1000 bp in length. The nucleic acid
`polymers
`are preferably selected from genes,
`gene
`clusters,
`chromosomes, viral and bacterial genomes or
`sections thereof. The oligomeric building blocks used
`for
`synthesizing the polymer
`are preferably 5-150,
`particularly preferably 5-30, monomer units in length.
`In successive steps,
`it is possible to detach in each
`case partially complementary oligonucleotide building
`blocks from the support and to bring them into contact
`with one another or with the polymer intermediate under
`hybridization conditions. Further examples of suitable
`polymers are nucleic acid analogs and proteins.
`
`
`
`(c)
`
`the nucleic
`of
`detachment
`individual reaction areas.
`
`acid fragments
`
`from
`
`fragments
`acid
`the nucleic
`of
`sequences
`base
`The
`synthesized in individual reaction areas are preferably
`chosen such that
`they can assemble to form a nucleic
`acid double strand hybrid. The nucleic acid fragments
`can then be detached in step (c)
`in one or more steps
`
`the invention
`In a particularly preferred embodiment,
`relates to a method for producing synthetic DNA of any
`optional
`sequence
`and
`thus
`any
`known
`or
`novel
`functional genetic elements which are contained in said
`
`sequence. This method comprises the steps
`(a)
`provision of a support having a surface area which
`contains a plurality of individual reaction areas,
`location-resolved
`synthesis
`of
`nucleic
`acid
`fragments
`having
`in
`each
`case different
`base
`
`(b)
`
`sequences
`
`in several of
`
`the individual
`
`reaction
`
`areas, and
`
`

`

`-g-
`
`under conditions such that a plurality,
`
`i.e. at
`
`least
`
`some of the detached nucleic acid fragments assemble to
`form a nucleic acid double strand hybrid. Subsequently,
`the nucleic acid fragments
`forming one strand of
`the
`
`least
`at
`can
`hybrid
`strand
`double
`acid
`nucleic
`partially be linked covalently to one another. This may
`be carried out by enzymatic
`treatment,
`for
`example
`using ligase, or/and filling in gaps
`in the strands
`using DNA polymerase.
`
`The method comprises within the framework of a modular
`
`system the synthesis of very many individual nucleic
`acid strands which serve as building blocks and, as a
`
`a double-stranded nucleic acid sequence which
`result,
`can be more
`than 100,000 base pairs
`in length is
`generated,
`for
`example
`in
`a microfluidic
`reaction
`
`support.
`
`The
`
`highly
`
`complex
`
`synthetic
`
`nucleic
`
`acid which
`
`preferably consists of DNA is produced according to the
`method and according to the following principle: first,
`relatively short
`DNA
`strands
`are
`synthesized in a
`multiplicity of reaction areas on a reaction support by
`in situ synthesis. This may take place,
`for example,
`using the supports described in the patent applications
`DE
`199 24 327.1,
`DE
`199 40 749.5,
`PCT/EP99/06316
`and
`
`PCT/EP99/06317.
`
`In this connection, each reaction area
`
`the individual and specific synthesis
`is suitable for
`of an individual given DNA sequence of approx.
`10 - 100
`nucleotides
`in length. These
`DNA
`strands
`form the
`building blocks for the specific synthesis of very long
`DNA molecules. The fluidic microprocessor used here may
`carry
`reaction
`spaces
`specially designed
`for
`the
`application.
`
`by
`carried out
`thus
`is
`itself
`synthesis
`DNA
`The
`following the automated solid phase synthesis but with
`
`some novel aspects:
`
`the “solid phase” in this case is
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`an
`
`individual
`
`the
`
`reaction area
`
`on
`
`the
`
`surface of
`
`

`

`
`
`support,
`
`for example the wall of
`
`the reaction space,
`
`i.e.
`
`it is not particles introduced into the reaction
`
`is the case in a conventional synthesizer.
`space as
`Integration of the synthesis in a microfluidic reaction
`support
`(e.g.
`a
`structure with optionally branched
`channels
`and reaction spaces) makes
`it possible to
`
`introduce the reagents
`
`and other components
`
`such as
`
`enzymes.
`
`10
`
`After synthesis,
`
`the synthesized building blocks are
`
`detached from said reaction areas. This
`
`detachment
`
`process may be carried out
`specifically for
`individual,
`
`location- or/and time-
`several
`or
`all
`DNA
`
`strands.
`
`15
`
`In a preferred variant of the method it is provided for
`
`a plurality of
`
`reaction areas to be established and
`
`utilized within a fluidic space or compartment so that
`
`the DNA strands synthesized therein can be detached in
`
`20
`
`one operation step and taken away from the compartment
`
`which fluidically connects the reaction areas.
`
`Subsequently, suitable combinations of the detached DNA
`strands
`are
`formed. Single-stranded or/and double-
`
`25
`
`stranded building blocks
`
`are
`
`then
`
`assembled,
`
`for
`
`example, within a reaction space which may comprise one
`
`or more reaction areas for the synthesis. Expediently,
`
`the
`
`sequence of
`
`the
`
`individual building blocks
`
`is
`
`chosen such that, when bringing the individual building
`
`30
`
`blocks
`
`into
`
`contact with
`
`one
`
`another,
`
`regions
`
`complementary to one another are available at
`
`the two
`
`ends
`
`brought
`
`together,
`
`in order
`
`to make possible
`
`specific
`
`attachment
`
`of
`
`further
`
`DNA
`
`strands
`
`by
`
`hybridizing said regions. As
`
`a
`
`result,
`
`longer
`
`DNA
`
`35
`
`hybrids are formed. The phosphorus diester backbone of
`
`these DNA hybrids may be covalently closed,
`
`for example
`
`by ligases, and possible gaps in the double strand may
`
`be filled in in a known manner enzymatically by means
`
`of polymerases. Single-stranded regions which may be
`
`

`

`- 10 -
`
`(e.g. Klenow
`enzymes
`filled in by
`present may be
`fragment) with the addition of suitable nucleotides.
`Thus
`longer
`DNA molecules
`are
`formed.
`By bringing
`together clusters of DNA strands synthesized in this
`way within reaction spaces it is in turn possible to
`generate
`longer part
`sequences
`of
`the
`final
`DNA
`molecule. This may be done
`in stages,
`and the part
`sequences are put
`together
`to give ever
`longer DNA
`molecules.
`In this way it is possible to generate very
`long DNA sequences as completely synthetic molecules of
`more than 100,000 base pairs in length.
`
`is
`individual building blocks which
`of
`amount
`The
`long synthetic DNA molecule is dealt
`a
`required for
`with in the reaction support by parallel synthesis of
`the building blocks in a location- or/and time-resolved
`synthesis process.
`In the preferred embodiment,
`this
`parallel
`synthesis
`is carried out by light -dependent
`location-
`or/and time-resolved DNA
`synthesis
`in a
`fluidic microprocessor which is also described in the
`patent applications DE 199 24 327.1, DE 199 40 749.5,
`PCT/EP99/06316 and PCT/EP99/06317.
`
`a
`causes
`here
`support
`reaction
`The miniaturized
`reduction in the amount of starting substances by at
`least a factor of 1000 compared with a conventional DNA
`synthesizer. At
`the same time, an extremely high number
`of nucleic acid double strands of defined sequence is
`produced. Only in this way is it possible to generate a
`very large variety of individual building blocks, which
`is required for the synthesis of long DNA molecules, by
`using an economically sensible amount of resources. The
`synthesis of a sequence of 100,000 base pairs, composed
`of overlapping building blocks of
`20 nucleotides
`in
`length,
`requires
`10,000
`individual building blocks.
`This can be achieved using appropriately miniaturized
`equipment
`in a highly parallel synthesis process.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`
`
`

`

`- 11 -
`
`and
`For efficient processing of genetic molecules
`is
`systematic inclusion of all possible variants
`it
`necessary to produce
`the
`individual building block
`sequences
`in a
`flexible and economic way. This
`is
`achieved
`by
`the method
`preferably
`by
`using
`a
`programmable
`light
`source matrix
`for
`the
`light-
`in
`dependent
`location-
`or/and
`time-resolved
`situ
`Synthesis of the DNA strands, which in turn can be used
`as building blocks
`for
`the synthesis of
`longer DNA
`strands.
`This
`flexible
`synthesis
`allows
`free
`programming of the individual building block sequences
`and thus also generation of any variants of
`the part
`sequences or
`the final sequence, without
`the need for
`substantial modifications
`of
`system
`components
`(hardware). This programmed synthesis of
`the building
`blocks and thus the final synthesis products makes it
`possible
`to
`systematically process
`the variety of
`genetic
`elements. At
`the
`same
`time,
`the
`use
`of
`computer-controlled
`programmable
`synthesis
`allows
`automation
`of
`the
`entire
`process
`including
`communication with appropriate databases.
`
`the
`sequence of
`the
`sequence,
`a given target
`With
`individual building blocks can be selected efficiently,
`taking
`into
`account
`biochemical
`and
`functional
`parameters. After putting in the target sequence (e.g.
`from a database),
`an
`algorithm makes
`out
`suitable
`overlapping regions. Depending on the task, different
`amounts of
`target
`sequences
`can be produced, either
`within one reaction support or spread over a plurality
`of reaction supports. The hybridization conditions for
`formation
`of
`the
`hybrids,
`such
`as,
`for
`example,
`temperature, salt concentrations, etc., are adjusted to
`the
`available
`overlap
`regions
`by
`an
`appropriate
`algorithm.
`Thus, maximum attachment
`specificity is
`ensured.
`In a
`fully automatic version,
`it
`is also
`possible to take target
`sequence data directly from
`public or private databases
`and
`convert
`them into
`appropriate target
`sequences. The products generated
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`
`
`

`

`-12-
`
`may in turn be introduced optionally into appropriately
`automated processes,
`for
`example
`into
`cloning
`in
`suitable target cells.
`
`Synthesis in stages by synthesizing the individual DNA
`strands
`in reaction areas within enclosed reaction
`spaces
`also
`allows
`the
`synthesis
`of
`difficult
`sequences,
`for example those with internal
`repeats of
`sequence
`sections, which
`occur,
`for
`example,
`in
`retroviruses and corresponding retroviral vectors. The
`controlled detachment of building blocks within the
`fluidic
`reaction spaces makes
`a
`synthesis
`of
`any
`sequence possible, without problems being generated by
`assigning the overlapping regions
`on
`the individual
`building blocks.
`
`for
`necessary
`requirements
`quality
`high
`The
`inter
`Synthesizing very long DNA molecules can be met
`alia by using real-time quality control. This comprises
`monitoring
`the
`location-resolved
`building
`block
`synthesis,
`likewise
`detachment
`and
`assembly
`up
`to
`production of
`the final
`sequence. Then all processes
`take place
`in
`a
`transparent
`reaction support.
`In
`addition,
`the possibility to
`follow reactions
`and
`fluidic
`processes
`in
`transmitted
`light mode,
`for
`example by CCD detection,
`is created.
`
`preferably
`is
`support
`reaction
`The miniaturized
`designed such that a detachment process is possible in
`the individual reaction spaces and thus the DNA strands
`synthesized on the reaction areas located within these
`reaction
`spaces
`are
`detached
`individually
`or
`in
`clusters.
`In a
`suitable embodiment of
`the reaction
`Support it is possible to assemble the building blocks
`in reaction spaces ina process in stages and also to
`remove building blocks, part
`sequences or
`the final
`product or else to sort or fractionate the molecules.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`

`

`- 13 -
`
`be
`its completion, may
`after
`sequence,
`target
`The
`introduced as integrated genetic element
`into cells by
`transfer
`and
`thereby
`be
`cloned
`and
`studied
`in
`functional studies. Another possibility is to firstly
`further purify or
`analyze
`the
`synthesis product,
`a
`possible example of said analysis being sequencing. The
`sequencing process may also be
`initiated by direct
`coupling using an appropriate apparatus,
`for example
`using a device described in the patent applications DE
`199 24 327.1,
`DE
`199 40 749.5,
`PCT/EP99/06316
`and
`PCT/EP99/06317
`for
`the
`integrated
`synthesis
`and
`analysis of polymers.
`It
`is likewise conceivable to
`isolate and
`analyze
`the generated target
`sequences
`after cloning.
`
`The method of the invention provides via the integrated
`genetic elements generated therewith a tool which,
`for
`the further development of molecular biology,
`includes
`biological
`variety
`in
`a
`systematic
`process.
`The
`generation
`of
`DNA molecules with
`desired
`genetic
`information
`is
`thus
`no
`longer
`the bottleneck of
`molecular biological work,
`since all molecules,
`from
`small plasmids via complex vectors to mini chromosomes,
`can be generated synthetically and are available for
`further work.
`
`numerous
`The production method allows generation of
`different nucleic acids and thus a systematic approach
`for
`questions
`concerning
`regulatory
`elements,
`DNA
`binding
`sites
`for
`regulators,
`signal
`cascades,
`receptors, effect and interactions of growth factors,
`etc.
`
`fully
`into a
`elements
`integration of genetic
`The
`it possible to
`Synthetic complete nucleic acid makes
`further utilize known genetic tools such as piasmids
`and
`vectors
`and
`thus
`to
`build
`on
`the
`relevant
`experience. On
`the other hand,
`this experience will
`change rapidly as a result of the intended optimization
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`

`

`- 14 -
`
`for
`of available vectors, etc. The mechanisms which,
`example, make a plasmid suitable for propagation in a
`particular cell type can be studied efficiently for the
`first time on the basis of the method of the invention.
`
`This efficient study of large numbers of variants makes
`it possible to detect
`the entire combination space of
`genetic elements. Thus,
`in addition to the at
`the
`moment
`rapidly developing highly parallel
`analysis
`(inter alia on DNA arrays or DNA chips),
`the programmed
`synthesis of integrated genetic elements is created as
`a second important element. Only both elements together
`can
`form the
`foundation of
`an efficient molecular
`biology.
`
`The programmed synthesis of appropriate DNA molecules
`makes possible not only random composition of
`the
`coding
`sequences
`and
`functional
`elements
`but
`also
`adaptation
`of
`the
`intermediate
`regions.
`This may
`rapidly lead to minimal vectors and minimal genomes,
`whose
`small
`size in turn generates advantages. As
`a
`result,
`transfer vehicles such as,
`for example, viral
`vectors can be made more efficient,
`for example when
`using retroviral or adenoviral vectors.
`
`genetic
`known
`of
`combination
`the
`to
`addition
`In
`is possible to develop novel genetic
`sequences,
`it
`elements which can build on the function of available
`elements. Especially for
`such developmental work,
`the
`flexibility of the system is of enormous value.
`
`the
`The synthetic DNA molecules are in each stage of
`development
`of
`the method
`described
`here
`fully
`compatible with the available recombination technology.
`For “traditional” molecular biological applications it
`is
`also
`possible
`to
`provide
`integrated
`genetic
`elements,
`for
`example
`by
`appropriate
`vectors.
`Incorporation of appropriate cleavage sites even of
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`

`

`- 15 -
`
`enzymes little used so far is not a limiting factor for
`integrated genetic elements.
`
`Improvements in comparison with prior art
`This method makes it possible to integrate all desired
`functional elements as “genetic modules” such as,
`for
`example, genes, parts of genes,
`regulatory elements,
`viral packaging signals,
`etc.
`into the
`synthesized
`nucleic
`acid molecule
`as
`carrier
`of
`genetic
`information. This
`integration leads to inter alia the
`following advantages:
`
`extremely
`therewith
`develop
`to
`possible
`is
`It
`functionally integrated DNA molecules, unnecessary DNA
`regions being removed (minimal genes, minimal genomes) .
`
`the genetic elements and also
`free combination of
`The
`modifications of the sequence such as,
`for example,
`for
`adaptation to the expressing organism or cell
`type
`(codon
`usage)
`are
`made
`possible
`as well
`as
`modifications of the sequence for optimizing functional
`genetic
`parameters
`such
`as,
`for
`example,
`gene
`regulation.
`
`10
`
`15
`
`20
`
`25
`
`Modifications of the sequence for optimizing functional
`parameters of
`the
`transcript,
`for
`example
`splicing,
`
`
`
`
`regulation mRNA_level,at the regulation at the
`
`
`translation level,
`and, moreover,
`the optimization of
`functional parameters of the gene product,
`such as,
`for
`example,
`the
`amino
`acid sequence
`(e.g.
`antibodies,
`growth
`factors,
`receptors,
`channels,
`pores,
`transporters, etc.) are likewise made possible.
`
`30
`
`35
`
`the system created by the method is
`the whole,
`On
`extremely flexible and allows
`in a manner previously
`not available
`the
`programmed production of genetic
`material
`under
`greatly
`reduced
`amounts
`of
`time,
`
`materials and work needed.
`
`

`

`- 16 -
`
`Using
`the
`available methods,
`it
`has
`been
`almost
`impossible to specifically manipulate relatively large
`DNA molecules
`of
`several
`hundred
`kbp,
`such
`as
`chromosomes
`for
`example.
`Even more
`complex
`(i.e.
`larger)
`viral
`genomes
`of more
`than
`30 kbp
`(e.g.
`adenoviruses) are difficult to handle and to manipulate
`using the classical methods of gene technology.
`
`10
`
`15
`
`20
`
`25
`
`The method of
`
`the invention leads
`
`to a considerable
`
`the
`shortening up to the last stage of cloning a gene:
`gene or the genes are synthesized as DNA molecule and
`then (after suitable preparation such as purification,
`etc.)
`introduced directly into target cells and the
`result
`is
`studied.
`The multi-stage
`cloning process
`which is mostly carried out
`in microorganisms such as
`E. coli
`(e.g.
`DNA isolation, purification,
`analysis,
`recombination,
`cloning
`in
`bacteria,
`isolation,
`analysis, etc.) is thus reduced to the last transfer of
`
`the DNA molecule into the final effector cells. For
`
`synthetically produced genes or gene fragments clonal
`propagation in an intermediate host
`(usually E. coli)
`is no longer required. This avoids the danger of
`the
`gene product destined for
`the target cell exerting a
`toxic
`action
`on
`the
`intermediate
`host.
`This
`is
`distinctly different
`from the toxicity of
`some gene
`products, which, when using classical plasmid vectors,
`frequently leads to considerable problems
`for cloning
`of the appropriate nucleic acid fragments.
`
`30
`
`is the reduction in
`Another considerable improvement
`time and the reduction in operational steps to after
`the
`sequencing of genetic material, with potential
`
`genes and_cloned.found being verified as such
`
`
`
`Normally, after finding interesting patterns, which are
`possible open reading frames
`(ORF), probes are used
`(e.g. by means of PCR)
`to search in cDNA libraries for
`appropriate clones which, however, need not contain the
`whole
`sequence of
`the mRNA originally used in their
`production.
`In other methods,
`an
`expression gene
`
`35
`
`

`

`-17 -
`
`by means
`searched
`is
`library
`Both methods
`can
`(screening).
`substantially using the method of
`
`of
`
`antibody
`an
`shortened
`very
`be
`the invention:
`if a
`
`gene sequence determined “in silico” is present
`after detection of
`an appropriate pattern in a
`
`(i.e.
`DNA
`
`sequence by the computer) or after decoding a protein
`
`sequence,
`
`an appropriate vector with the sequence or
`
`variants
`
`thereof
`
`can
`
`be
`
`generated
`
`directly
`
`via
`
`programmed synthesis of an integrated genetic element
`and introduced into suitable target cells.
`
`10
`
`15
`
`The synthesis taking place in this way of DNA molecules
`
`the direct complete
`100 kbp allows
`of up to several
`synthesis of viral genomes,
`for example adenoviruses.
`These are an important
`tool
`in basic research (inter
`alia gene therapy) but, due to the size of their genome
`(approx.
`40 kbp),
`are difficult
`to
`handle
`using
`cl