`
`
`
`24. August 2000 (24.08.00)
`
`C12N 15/10, ClZP 19/34
`
`(21) Internationales Aktenzeichen:
`
`PCT/EPOO/01356
`
`DE
`19. Februar 1999 (19.02.99)
`DE
`24. Juni 1999 (24.06.99)
`DE
`27. August 1999 (27.08.99)
`EP
`27. August 1999 (27.08.99)
`26. November 1999 (26.11.99) DE
`
`WELTORGANISATION EUR GEISTIGES EIGENTUM
`‘ PCT
`lmemationales Biiro
`INTERNATIONALE ANMELDUNG VEROFFENTLICHT NACH DEM VERTRAG UBER DIE
`INTERNATIONALE ZUSAMMENARBEIT‘AUF DEM GEBIET DES PATENTWESENS (PCT)
`
`
`(51) Internationale Patentklassifikation 7 :
`
`
`
`
`(11) Internationale Veriitt’entlichungsnummer: W0 (IO/49142
`
`
`(43) Internationales
`
`Veriiffentlichungsdatum:
`
`
`
`
`
`
`(81) Bestimmungsstaaten: AU, CA, JP, US, européiisches 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).
`
`
`
`
`(30) Prioritiitsdaten:
`Vert’lfl‘entlicht
`
`199 07 080.6
`Mit internationalem Recherchenbericht.
`
`
`
`199 28 843.7
`Var Ablaufderfiir Anderungen der Ampriiche zugelassenen
`61 R . (/
`
`199 40 752.5
`Frist; Verbfientlichung wird wiederholt falls Anderungen
`2 Q
`
`
`
`PCT/EP99/06316
`eintrefi'en.
`’ V S EC
`
`
`199 57 116.3
`‘0
`1 '4
`
`.7)
`
`
`Sled c.
`67w- Q
`
`Anmelder (fu‘r alle Best
`ungsstaaren ausser US): FEB}?
`[DE/DE];
`
`Gasselweg 15, D—69469 Weinheim (DE).
`(72) Erfinder; und
`
`(75) Erfinder/Anmelder
`(nur fi‘ir US):
`STAHLER, Peer, F.
`
`
`[DE/DE]; Riedfeldstrasse 3, D—68169 Mannheim (DE).
`
`STAHLER, Cord,
`F.
`[DE/DE];
`Siegfriedstrasse
`9,
`
`D—69469 Weinheirn (DE). MULLER, Manfred [DE/DE];
`Mannheimerstrasse 11, D—69198 Schriesheim (DE).
`
`
` (74) Anwfilte: WEICKMANN, H. usw.; Kopemikusstrasse 9,
`D—81679 Milnchen (DE).
`
`
`(54) Title: METHOD FOR PRODUCING POLYMERS
`
`(54) Bezeichnung: VERFAHREN ZUR HERSTELLUNG VON POLYMEREN
`
`
`
`
`
`
`
`(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.
`
` (S7) Zusammenfassung
` Die Erfindung betrifft ein Verfahren zur Herstellung
`synthetischen
`von
`insbesondere
`Polymeren,
`von
`Nukleinsauredoppelstrangen wahlfreier Sequcnz, umfassend
`
`die Schritte:
`(a) Bereitstellen eines Tragers mit einer
`
`Oberflache, die eine Vielzahl von individuellen Reak-
`tionsbereichen enthalt;
`(b) ortsaufgelostes Synthetisieren
`
`von Nukleinsaurefragmenten mit jeweils unterschiedlicher
`
`
`Basensequenz an mehreren der individuellen Reaktionsbere-
`
`
`iche; und (c) Ablbsen der Nukleinsaurefragmente von individuellen Reaktionsbereichen.
`
`
`
`

`

`Method for producing polymers
`
`Description
`
`5
`
`10
`
`15
`
`20
`
`25
`
`producing
`for
`a method
`to
`relates
`invention
`The
`polymers,
`in particular synthetic nucleic acid double
`
`strands of optional sequence.
`
`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
`thereof
`
`engineering methods
`in
`the
`areas
`of
`
`and
`gene
`
`development
`further
`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,
`
`design,
`enzyme
`(specialized
`or
`
`environmental
`and
`etc.)
`custom microorganisms,
`
`technology
`production
`
`processes, cleaning—up, sensors, etc.).
`
`3O
`
`Prior art
`
`Numerous methods,
`
`first
`
`and
`
`foremost
`
`enzyme—based
`
`methods,
`
`allow specific manipulation
`
`of
`
`DNA
`
`for
`
`different purposes.
`
`35
`
`All of
`
`said methods
`
`have
`
`to use available genetic
`
`material. Said material
`
`is,
`
`on
`
`the one hand, well—
`
`defined to a
`
`large extent but allows,
`
`on
`
`the other
`
`hand,
`
`in a kind of “construction kit
`
`system" only a
`
`
`
`

`

`
`
`limited
`
`amount
`
`of
`
`possible
`
`combinations
`
`of
`
`the
`
`particular available and slightly modified elements.
`
`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.
`
`The
`
`known methods
`
`share
`
`the
`
`large
`
`amount of work
`
`required,
`
`combined with
`
`a
`
`certain
`
`duration
`
`of
`
`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
`
`incomplete,
`frequently
`processes follow.
`
`so
`
`that
`
`further
`
`screening
`
`Finally,
`
`the
`
`above—described
`
`recombination
`
`of
`
`DNA
`
`fragments
`
`has
`
`only limited flexibility and
`
`allows,
`
`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
`
`3O
`
`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
`
`15
`
`20
`
`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
`
`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
`
`a
`
`particular
`
`number
`
`to
`
`of
`
`25
`
`3O
`
`35
`
`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
`
`recombination.
`
`Sufficiently
`
`different
`
`and
`
`specific
`
`enzymes are available for
`
`recombination technology up
`
`to a limit of 10 — 3O kilo base pairs (kbp) of the DNA
`
`to be cut.
`
`In addition, preliminary work and commercial
`
`suppliers provide corresponding vectors which take up
`
`the
`
`recombinant
`
`DNA
`
`and
`
`allow cloning
`
`(and
`
`thus
`
`propagation
`
`and
`
`selection).
`
`Such
`
`vectors
`
`contain
`
`suitable cleavage sites for efficient recombination and
`
`integration.
`
`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
`
`

`

`pairs
`
`(46)
`
`and
`
`for
`
`8 nucleotide bases
`
`it
`
`is
`
`one
`
`(48). Recombination using
`65,000
`site per
`cleavage
`therefore
`is
`not particularly
`restriction enzymes
`suitable for manipulating relatively large DNA elements
`
`(e.g. viral genomes, chromosomes, etc.).
`
`Recombination by homologous
`
`recombination in cells is
`
`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
`
`to the bordering
`due
`(including high multiplication)
`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
`
`a
`with
`nucleotides,
`wrong
`incorporate
`depending on the particular enzyme,
`so that
`
`frequency
`there is
`
`always
`
`the danger of
`
`a certain distortion of
`
`the
`
`this gradual a
`some applications,
`starting sequence. For
`distortion can
`be very disturbing. During
`chemical
`
`10
`
`15
`
`20
`
`25
`
`3O
`
`35
`
`
`
`

`

`synthesis,
`
`sequences such as,
`
`for example,
`
`the above—
`
`described
`
`restriction
`
`cleavage
`
`sites
`
`can
`
`be
`
`incorporated into the primers. This allows
`
`(limited)
`
`manipulation (If
`
`the complete sequence. The nmltiplied
`
`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.
`
`10
`
`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
`
`(pmol—range reaction mixtures) which are too large and
`
`15
`
`required for PCR, while the variety in variants
`not
`remains limited.
`
`20
`
`25
`
`3O
`
`35
`
`Synthetic DNA elements
`
`Since the pioneering work of Khorana
`
`(inter alia in:
`
`Shabarova: Advanced Organic Chemistry of Nucleic Acids,
`
`VCH Weinheinu)
`
`in the 19605,
`
`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
`
`described,
`been
`US 5112575.
`
`for
`
`example,
`
`in
`
`US 4353989
`
`and
`
`Double—stranded
`
`DNA
`
`is
`
`synthesized
`
`from
`
`short
`
`oligonucleotides
`
`according
`
`to
`
`two methods
`
`(see
`
`Holowachuk et al.,
`
`PCR Methods and Applications, Cold
`
`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
`
`

`

`synthesizing
`
`regions
`
`overlapping
`
`at
`
`the
`
`edges
`
`single-stranded
`
`nucleic
`
`acids,
`
`attachment
`
`as
`
`by
`
`hybridization,
`
`filling'
`
`in the single-stranded.
`
`regions
`
`via enzymes
`
`(polymerases) and linking the backbone.
`
`10
`
`15
`
`In both methods,
`
`the
`
`total
`
`length of
`
`the genetic
`
`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.
`
`To
`
`summarize,
`
`the prior art
`
`can be described as
`
`a
`
`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
`
`20
`
`(recombination).
`experiments sf
`
`recombination
`of
`result
`The
`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
`torial.
`
`25
`
`(selectively)
`
`analytical
`
`and
`
`combina—
`
`The prior art
`
`thus
`
`does
`
`not
`
`allow any
`
`systematic
`
`studies
`
`of
`
`any
`
`combinations
`
`whatsoever.
`
`The
`
`modification
`
`of
`
`the
`
`combined
`
`elements
`
`is
`
`almost
`
`impossible.
`
`Systematic
`
`testing of modifications
`
`is
`
`3O
`
`impossible.
`
`Subject of the invention and object achieved therewith
`
`It
`
`is
`
`intended to provide
`
`a method
`
`for directly
`
`converting
`
`digital
`
`genetic
`
`information
`
`(target
`
`35
`
`sequence,
`
`databases,
`
`etc.)
`
`into biochemical genetic
`
`information
`
`(nucleic
`
`acids) without making
`
`use
`
`of
`
`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.
`
`In a particularly preferred embodiment,
`
`the invention
`
`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,
`
`(b)
`
`location—resolved
`
`synthesis
`
`of
`
`nucleic
`
`acid
`
`fragments
`
`having
`
`in
`
`each
`
`case different
`
`base
`
`sequences
`
`in several of
`
`the individual
`
`reaction
`
`areas, and
`
`(c)
`
`the nucleic
`of
`detachment
`individual reaction areas.
`
`acid fragments
`
`from
`
`The
`
`base
`
`sequences
`
`of
`
`the nucleic
`
`acid
`
`fragments
`
`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
`
`

`

`_ 8 _
`
`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
`
`nucleic
`
`acid
`
`double
`
`strand
`
`hybrid
`
`can
`
`at
`
`least
`
`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
`
`systen1 the synthesis of very' many individual nucleic
`
`acid strands which serve as building blocks and, as a
`
`result,
`
`a double—stranded nucleic acid sequence which
`
`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,
`
`strands
`
`are
`
`synthesized in a
`
`10
`
`15
`
`20
`
`relatively short
`
`DNA
`
`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/O63l6
`
`and
`
`PCT/EP99/O63l7.
`
`In this connection, each reaction area
`
`is suitable for
`
`the individual and specific synthesis
`
`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.
`
`25
`
`3O
`
`35
`
`The
`
`DNA
`
`synthesis
`
`itself
`
`is
`
`thus
`
`carried out
`
`by
`
`following the automated solid phase synthesis but with
`
`some novel aspects:
`
`the “solid phase” in this case is
`
`an
`
`individual
`
`reaction area
`
`on
`
`the
`
`surface of
`
`the
`
`
`
`

`

`
`
`support,
`
`for example the wall of
`
`the reaction space,
`
`i.e.
`
`it is not particles introduced into the reaction
`
`space as
`
`is the case in. a conventional synthesizer.
`
`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 .
`
`After synthesis,
`
`the synthesized. building blocks are
`
`detached from said reaction areas. This
`
`detachment
`
`process may be carried out
`
`location— or/and time-
`
`specifically for
`
`individual,
`
`several
`
`or
`
`all
`
`DNA
`
`strands.
`
`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
`
`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—
`
`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
`
`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
`
`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
`
`15
`
`20
`
`25
`
`3O
`
`35
`
`

`

`‘10—
`
`present may be
`
`filled in by
`
`enzymes
`
`(e.g. Klenow
`
`fragment) with the addition. of suitable nucleotides.
`
`By bringing
`formed.
`are
`DNA molecules
`longer
`Thus
`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
`
`10
`
`In this way it is possible to generate very
`molecules.
`long DNA sequences as completely synthetic molecules of
`
`more than 100,000 base pairs in length.
`
`15
`
`20
`
`25
`
`3O
`
`35
`
`The
`
`amount
`
`of
`
`individual building blocks which
`
`is
`
`required for
`
`a
`
`long synthetic DNA molecule is dealt
`
`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/O63l6 and PCT/EP99/06317.
`
`The miniaturized
`
`reaction
`
`support
`
`here
`
`causes
`
`a
`
`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.
`
`
`
`

`

`_ 11 _
`
`For efficient processing of genetic molecules
`
`and
`
`systematic inclusion of all possible variants
`
`it
`
`is
`
`necessary to produce
`
`the
`
`individual building block
`
`sequences
`
`in a
`
`flexible and economic way. This
`
`achieved
`
`by
`
`the method
`
`preferably
`
`by
`
`using
`
`is
`
`a
`
`programmable
`
`light
`
`source matrix
`
`for
`
`the
`
`light—
`
`dependent
`
`location-
`
`or/and
`
`time—resolved
`
`in
`
`situ
`
`10
`
`15
`
`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
`
`components
`system
`of
`substantial modifications
`the building
`(hardware). This programmed synthesis of
`blocks and thus the final synthesis products makes it
`
`possible
`
`to
`
`systematically process
`
`the variety of
`
`the
`
`use
`
`of
`
`genetic
`
`elements. At
`
`the
`
`same
`
`time,
`
`20
`
`computer—controlled
`
`programmable
`
`synthesis
`
`allows
`
`automation
`
`of
`
`the
`
`entire
`
`process
`
`including
`
`communication with appropriate databases.
`
`With
`
`a given target
`
`sequence,
`
`the
`
`sequence of
`
`the
`
`25
`
`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
`
`3O
`
`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
`
`35
`
`by
`regions
`overlap
`available
`the
`algorithm.
`Thus, maximum attachment
`
`appropriate
`an
`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
`
`
`
`

`

`_ 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
`
`for example those with internal
`sequences,
`repeats of
`sections, which
`sequence
`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.
`
`necessary
`requirements
`quality
`high
`The
`for
`synthesizing very long DNA molecules can be met
`inter
`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
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`support it is possible to assemble the building blocks
`in reaction spaces in a process in stages and also to
`remove building blocks, part
`sequences or
`the final
`
`product or else to sort or fractionate the molecules.
`
`

`

`_ 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
`
`for example
`coupling using an appropriate apparatus,
`using a device described in the patent applications DE
`199 24 327.1,
`DE
`199 40 749.5,
`PCT/EP99/O6316
`and
`PCT/EP99/O63l7
`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
`information
`
`of
`is
`
`genetic
`desired
`DNA molecules with
`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.
`
`The production method allows generation of
`numerous
`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 plasmids
`and
`vectors
`and
`thus
`to
`build
`on
`the
`relevant
`
`this experience will
`the other hand,
`experience. On
`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
`
`analysis
`rapidly developing highly parallel
`moment
`(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 nunimal 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
`
`3O
`
`35
`
`

`

`- 15 _
`
`enzymes little used so far is not a limiting factor for
`
`integrated genetic elements.
`
`5
`
`10
`
`15
`
`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
`
`free combination of
`
`the genetic elements and also
`
`modifications of the sequence such as,
`
`for example,
`
`for
`
`20
`
`adaptation to the expressing organism or cell
`(codon
`usage)
`are
`made
`possible
`as well
`
`type
`as
`
`modifications of the sequence for optimizing functional
`
`parameters
`genetic
`regulation.
`
`such
`
`as,
`
`for
`
`example,
`
`gene
`
`25
`
`30
`
`35
`
`Modifications of the sequence for optimizing functional
`
`parameters of
`
`the
`
`transcript,
`
`for
`
`example
`
`splicing,
`
`regulation
`
`at
`
`the
`
`mRNA
`
`level,
`
`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.
`
`On
`
`the whole,
`
`the system created by the method is
`
`in a Inanner previously
`extremely flexible and allows
`not available
`the
`programmed production of genetic
`
`greatly
`under
`material
`materials and work needed.
`
`reduced
`
`amounts
`
`of
`
`time,
`
`
`
`

`

`_ 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
`
`The method of
`
`the invention leads
`
`to a considerable
`
`shortening up to the last stage of cloning a gene:
`
`the
`
`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,
`
`cloning
`
`in
`
`bacteria,
`
`isolation,
`
`20
`
`25
`
`3O
`
`35
`
`recombination,
`
`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.
`
`Another considerable improvement
`
`is the reduction.
`
`in
`
`time and the reduction.
`
`in. operational steps to after
`
`the
`
`sequencing of genetic material, with potential
`
`genes
`
`found
`
`being verified as
`
`such
`
`and
`
`cloned.
`
`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 m