U.S. patent application number 15/426860 was filed with the patent office on 2017-05-25 for method for producing polymers.
The applicant listed for this patent is Synthetic Genomics, Inc.. Invention is credited to Manfred Mueller, Cord F. Staehler, Peer F. Staehler.
Application Number | 20170147748 15/426860 |
Document ID | / |
Family ID | 27438924 |
Filed Date | 2017-05-25 |
United States Patent
Application |
20170147748 |
Kind Code |
A1 |
Staehler; Peer F. ; et
al. |
May 25, 2017 |
METHOD FOR PRODUCING POLYMERS
Abstract
The invention relates to a method for producing polymers, in
particular synthetic nucleic acid double strands of optional
sequence, comprising 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) detachment of the nucleic acid
fragments from individual reaction areas.
Inventors: |
Staehler; Peer F.;
(Mannheim, DE) ; Staehler; Cord F.; (Weinheim,
DE) ; Mueller; Manfred; (Schriesheim, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Synthetic Genomics, Inc. |
La Jolla |
CA |
US |
|
|
Family ID: |
27438924 |
Appl. No.: |
15/426860 |
Filed: |
February 7, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12400493 |
Mar 9, 2009 |
9568839 |
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15426860 |
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11785505 |
Apr 18, 2007 |
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12400493 |
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10455369 |
Jun 6, 2003 |
7790369 |
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11785505 |
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09869332 |
Jul 26, 2001 |
6586211 |
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PCT/EP00/01356 |
Feb 18, 2000 |
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10455369 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01J 2219/00596
20130101; B01J 2219/00675 20130101; B01J 2219/00689 20130101; B01J
2219/00317 20130101; B01J 2219/00511 20130101; B01J 19/0093
20130101; B01J 2219/00711 20130101; B01L 2300/0654 20130101; B01L
3/502707 20130101; G03F 7/70216 20130101; B01J 2219/00441 20130101;
B01J 2219/00585 20130101; B01J 2219/00626 20130101; C40B 50/14
20130101; B01J 2219/00722 20130101; C40B 40/06 20130101; B01J
2219/00448 20130101; G16B 50/00 20190201; B82Y 30/00 20130101; B01J
2219/00725 20130101; C12N 15/10 20130101; B01L 2300/069 20130101;
B01J 2219/00529 20130101; B01J 2219/0059 20130101; B01J 2219/00479
20130101; C12N 15/66 20130101; B01J 2219/00702 20130101; B01J
2219/00605 20130101; B01J 2219/00608 20130101; B01J 2219/00704
20130101; B01J 2219/00603 20130101; B01J 2219/00648 20130101; B01J
2219/00439 20130101; C40B 40/10 20130101; C12P 19/34 20130101; B01J
19/0046 20130101; B01J 2219/00436 20130101; B01J 2219/00659
20130101; B01J 2219/00497 20130101; B01L 3/5085 20130101; G16B
30/00 20190201; B01J 2219/00621 20130101; B01J 2219/00432 20130101;
B01L 2300/0864 20130101 |
International
Class: |
G06F 19/22 20060101
G06F019/22; G06F 19/28 20060101 G06F019/28; C12P 19/34 20060101
C12P019/34 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 19, 1999 |
DE |
199 07 080.6 |
Jun 24, 1999 |
DE |
199 28 843.7 |
Aug 27, 1999 |
DE |
199 40 752.5 |
Aug 27, 1999 |
EP |
PCT/EP99/06316 |
Nov 26, 1999 |
DE |
199 57 116.3 |
Claims
1. Method for directly converting digital target sequences into
nucleic acids comprising the steps (a) providing a digital target
sequence, (b) fragmenting the digital target sequence into suitable
oligomeric building blocks, (c) synthesizing said oligomeric
building blocks on at least one support by parallel synthesis
steps, (d) detaching the oligomeric building blocks from the
support, (e) bringing the oligomeric building blocks into contact
with one another, and (f) linking the phosphodiester backbone
enzymatically.
2. Method according to claim 1, wherein the digital target
sequence/nucleic acid is selected from the group consisting of
genes or sections thereof, gene clusters or sections thereof,
chromosomes or sections thereof, or viral or bacterial genomes or
sections thereof
3. Method according to claim 1, wherein the digital target sequence
of step (a) describes functional genetic elements out of the group
consisting of genes or sections thereof, gene clusters or sections
thereof, chromosomes or sections thereof, or viral or bacterial
genomes or sections thereof; or variations thereof
4. Method according to claim 1, wherein the conversion of the
digital target sequence into oligomeric building blocks includes
variation of the nucleic acids.
5. Method according to claim 1, wherein the digital target
sequence/nucleic acid is a nucleic acid double strand.
6. Method according to claim 5, wherein the digital target
sequence/nucleic acid is a double-stranded nucleic acid of at least
300 bp in length.
7. Method according to claim 1, wherein the digital target sequence
derives from a database.
8. Method according to claim 1, wherein suitable oligomeric
building blocks are generated taking into account biochemical and
functional parameters in the fragmenting step (b).
9. Method according to claim 1, wherein the building blocks are
chosen such that they can assemble to form a nucleic acid double
strand hybrid.
10. Method according to claim 1, wherein an algorithm makes out
suitable overlapping regions in the fragmenting step (b).
11. Method according to claim 1, wherein the oligomeric building
blocks are from 5-150 monomer units in length.
12. Method according to claim 1, wherein each oligomeric building
block is synthesized on a different area of a common support (step
(c)).
13. Method according to claim 1, wherein the synthesis of the
building blocks (step (c)) is carried out in a location or/and
time-resolved synthesis process.
14. Method according to claim 1, wherein the synthesis step (c),
the detachment step (d) and the contacting step (e) is carried out
within one compartment.
15. Method according to claim 1, wherein step (c) is carried out in
a microfluidic reaction support having one or more fluidic reaction
compartments and one or more reaction areas within a fluidic
reaction compartment.
16. Method according to claim 1, wherein in step (d) in each ease
partially complementary building blocks are detached from the
support and are brought into contact with one another or with the
polymer intermediate under hybridization conditions.
17. Method according to claim 1, wherein the oligomeric building
blocks are detached in one or more steps under conditions such that
a plurality of detached nucleic acid fragments assemble to form a
nucleic acid double strand hybrid.
18. Method according to claim 1, wherein step (f) includes
treatment with ligase.
19. Method according to claim 1, wherein possible gaps in the
strands after hybridization are filled using polymerase.
20. Method for generating functionally integrated DNA molecules,
comprising the steps (a) providing digital nucleic acid sequences
of several functional elements, (b) integrating the functional
elements into a digital DNA molecule, (c) fragmenting the digital
DNA molecule into suitable oligomeric building blocks, (d)
synthesizing said oligomeric building blocks on at least one
support by parallel synthesis steps, (e) detaching the oligomeric
building blocks from the support, (f) bringing the oligomeric
building blocks into contact with one another, and (g) linking the
phosphodiester backbone enzymatically to form the integrated DNA
molecule.
21. Method of claim 20, wherein the functional element is selected
from the group consisting of genes, part of genes, regulatory
elements, viral packaging signals or viral vectors.
22. Method according to claim 20, wherein the digital DNA molecule
derives from a database.
23. Method according to claim 20, wherein suitable oligomeric
building blocks are generated taking into account biochemical and
functional parameters in the fragmenting step (c).
24. Method according to claim 20, wherein the building blocks are
chosen such that they can assemble to form a nucleic acid double
strand hybrid.
25. Method according to claim 20, wherein an algorithm makes out
suitable overlapping regions in the fragmenting step (b).
26. Method according to claim 20, wherein the oligomeric building
blocks are from 5-150 monomer units in length.
27. Method according to claim 20, wherein each oligomeric building
block is synthesized on a different area of a common support (step
(d)).
28. Method according to claim 20, wherein the synthesis of the
building blocks (step (d)) is carried out in a location or/and
time-resolved synthesis process.
29. Method according to claim 20, wherein step (c) is carried out
in a microfluidic reaction support having one or more fluidic
reaction compartments and one or more reaction areas within a
fluidic reaction compartment.
30. Method according to claim 20, wherein in step (e) in each case
partially complementary building blocks are detached from the
support and are brought into contact with one another or with the
polymer intermediate under hybridization conditions.
31. Method according to claim 20, wherein the oligomeric building
blocks are detached in one or more steps under conditions such that
a plurality of detached nucleic acid fragments assemble to form a
nucleic acid double strand hybrid.
32. Method according to claim 20, wherein step (g) includes
treatment with ligase.
33. Method according to claim 20, wherein possible gaps in the
strands after hybridization are filled using polymerase.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 12/400,493 filed Mar. 9, 2009, now issued as
U.S. Pat. No. 9,568,839; which is a continuation application of
U.S. application Ser. No. 11/785,505 filed Apr. 18, 2007, now
abandoned; which is a divisional application of U.S. application
Ser. No. 7,790,369 filed Jun. 6, 2003, now issued as U.S. Pat. No.
7,790,369; which is a divisional application of U.S. application
Ser. No. 09/869,332 filed Jul. 26, 2001, now issued as U.S. Pat.
No. 6,586,211; which is a 35 USC .sctn.371 National Stage
application of International Application No. PCT/EP00/01356 filed
Feb. 28, 2000, now expired; which claims the benefit under 35 USC
.sctn.119(a) to Germany Patent Application No. 199 57 116.3 filed
Nov. 26, 1999, International Application No. PCT/EP99/06316 filed
Aug. 27, 1999, Germany Patent Application No. 199 40 752.5 filed
Aug. 27, 1999, Germany Patent Application No. 199 28 843.7 filed
June 24, 1999 and Germany Patent Application No. 199 07 080.6 filed
Feb. 19, 1999, all now expired. The disclosure of each of the prior
applications is considered part of and is incorporated by reference
in the disclosure of this application.
BACKGROUND OF THE INVENTION
[0002] Field of the Invention
[0003] The invention relates to a method for producing polymers, in
particular synthetic nucleic acid double strands of optional
sequence.
[0004] Background Information
[0005] 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.).
Prior Art
[0006] Numerous methods, first and foremost enzyme-based methods,
allow specific manipulation of DNA for different purposes.
[0007] 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.
[0008] 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.
[0009] 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 frequently incomplete, so that further screening processes
follow.
[0010] 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.
[0011] A common method is recombination by enzymatic methods (in
vitro): here, DNA elements (isolated genomic DNA, 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.
[0012] 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 to statistical distribution, a particular number of
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
10-30 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.
[0013] 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 cleavage
site per 65,000 (4.sup.1). Recombination using restriction enzymes
therefore is not particularly suitable for manipulating relatively
large DNA elements (e.g., viral genomes, chromosomes, etc.). .sup.1
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.
[0014] 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.
[0015] 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.
[0016] 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 synthesis, sequences such as, for example, the
above-described restriction cleavage sites can be incorporated into
the primers. This allows (limited) 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.
[0017] 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 (.mu.mol-range reaction mixtures) which are
too large and not required for PCR, while the variety in variants
remains limited.
Synthetic DNA Elements
[0018] Since the pioneering work of Khorana (inter alia in:
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 U.S. Pat. No. 4,353,989 and U.S. Pat.
No. 5,112,575.
[0019] 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 as single-stranded nucleic acids,
attachment by hybridization, filling in the single-stranded regions
via enzymes (polymerases) and linking the backbone.
[0020] 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.
[0021] 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 (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 combinatorial.
[0022] 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 impossible.
SUMMARY OF THE INVENTION
[0023] 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 nucleic acid fragments already present.
[0024] 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 shows a vertical section of a reaction support 30
which is orthogonal to the microchannels 33 present thereon, which
are separated from one another by walls 32.
[0026] The bottom 31 of the reaction support is transparent.
Furthermore, a single-stranded nucleic acid 10 with the designation
of the 5' and 3' ends according to convention is depicted
diagrammatically. These are depicted as 10a with the 3' end
covalently bound to the reaction support 30 by solid-phase
synthesis. A light source matrix 20 with a light source and a
controllable illumination exit facing the reaction support 30 is
likewise depicted.
[0027] FIG. 2 shows a top view of reaction support 30 with reaction
areas 12 and the walls 32 between the microchannels 33. The arrows
indicate the direction of flow.
[0028] FIG. 3 shows, similar to FIG. 1, a vertical section through
the reaction support 30, with the single-stranded nucleic acids in
the microchannel 33 being detached.
[0029] FIG. 4 again depicts a top view of the reaction support 30,
with the single-stranded nucleic acids in the microchannel 33 being
detached.
[0030] FIG. 5 shows a top view of the arrangement of microchannels
with fluidic reaction spaces 50, which contain the individual
reaction areas, and reaction chambers, where a part sequence is
assembled. In the reaction space 54 all microchannels within a
reaction support are brought together. The final synthesis product
is assembled there, too, and is removed through exit 55. The
reference numbers 51a and 51b indicate the representations of a
reaction chamber which are shown in enlarged form in FIG. 6 and
FIG. 7 and FIG. 8. The arrows again signal the direction of
flow.
[0031] FIG. 6 shows an enlarged representation of a reaction
chamber 51a after a microchannel with detached single-stranded
nucleic acids.
[0032] FIG. 7 shows an enlarged representation of a reaction
chamber 51a after a microchannel with a double-stranded hybrid 60
composed of two attached complementary nucleic acid single
strands.
[0033] FIG. 8 shows an enlarged representation of a reaction
chamber 51b after bringing together two microchannels with an
assembled double-stranded nucleic acid hybrid 62, enzyme 63 (e.g.,
ligases) for the covalent linkage of the building blocks of the
nucleic acid hybrid 85, a linear covalently linked nucleic acid
double strand 65 and a circular closed nucleic acid double strand
66 (e.g., vector).
[0034] The reference number 64 represents a reaction of the enzymes
with the nucleic acid hybrid.
DETAILED DESCRIPTION OF THE INVENTION
[0035] 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: [0036] (a) provision of a support having a surface area
which contains a plurality of individual reaction areas, [0037] (b)
location-resolved synthesis of nucleic acid fragments having in
each case different base sequences in several of the individual
reaction areas, and [0038] (c) detachment of the nucleic acid
fragments from individual reaction areas.
[0039] 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 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.
[0040] 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 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.
[0041] 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 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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 a known manner enzymatically by means of
polymerases. Single-stranded regions which may be present may be
filled in by enzymes (e.g., Klenow 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.
[0046] 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
and/or 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.
[0047] 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.
[0048] 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 is achieved by the method preferably by using a programmable
light source matrix for the light-dependent location- or/and
time-resolved in 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.
[0049] With a given target sequence, the sequence of the 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 may in turn be introduced
optionally into appropriately automated processes, for example into
cloning in suitable target cells.
[0050] 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.
[0051] The high quality requirements necessary 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.
[0052] The miniaturized reaction support is preferably 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 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.
[0053] The target sequence, after its completion, may be 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.
[0054] 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.
[0055] 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.
[0056] The integration of genetic elements into a fully synthetic
complete nucleic acid makes it possible to further utilize known
genetic tools such as plasmids 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 of available
vectors, etc. The mechanisms which, for 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.
[0057] 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.
[0058] 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.
[0059] In addition to the combination of known genetic sequences,
it is possible to develop novel genetic elements which can build on
the function of available elements. Especially for such
developmental work, the flexibility of the system is of enormous
value.
[0060] The synthetic DNA molecules are in each stage of the
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 enzymes little used so far is
not a limiting factor for integrated genetic elements.
Improvements in Comparison with Prior Art
[0061] 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:
[0062] It is possible to develop therewith extremely functionally
integrated DNA molecules, unnecessary DNA regions being removed
(minimal genes, minimal genomes).
[0063] The free combination of the genetic elements and also
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.
[0064] 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.
[0065] On the whole, the system created by the method is 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.
[0066] 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.
[0067] 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, 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.
[0068] 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 mRNA originally
used in their production. In other methods, an expression gene
library is searched by means of an antibody (screening). Both
methods can be shortened very substantially using the method of the
invention: if a gene sequence determined "in silico" is present
(i.e., after detection of an appropriate pattern in a 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.
[0069] The synthesis taking place in this way of DNA molecules of
up to several 100 kbp allows the direct complete 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
classical genetic engineering methods. As a result, the rapid and
economic generation of variants for optimization in particular is
greatly limited. This limitation is removed by the method of the
invention.
[0070] The method leads to integration of the synthesis, detachment
of synthesis products and assembly to a DNA molecule being carried
out in one system. Using production methods of microsystem
technology, it is possible to integrate all necessary functions and
process steps up to the purification of the final product in a
miniaturized reaction support. These may be synthesis areas,
detachment areas (clusters), reaction spaces, feeding channels,
valves, pumps, concentrators, fractionation areas, etc.
[0071] Plasmids and expression vectors may be prepared directly for
sequenced proteins or corresponding part sequences and the products
may be analyzed biochemically and functionally, for example by
using suitable regulatory elements. This omits the search for
clones in a gene library. Correspondingly, ORFs from sequencing
work (e.g., Human Genome Project) can be programmed directly into
appropriate vectors and be combined with desired genetic elements.
An identification of clones, for example by complicated screening
of cDNA libraries, is removed. Thus, the flow of information from
sequence analysis to function analysis has been greatly reduced,
because on the same day on which an ORF is present in the computer
due to analysis of primary data, an appropriate vector including
the putative gene can be synthesized and made available.
[0072] Compared with conventional solid-phase synthesis for
obtaining synthetic DNA, the method according to the invention is
distinguished by a small amount of material needed. In order to
produce thousands of different building blocks for generating a
complex integrated genetic element of several 100,000 kbp in
length, in an appropriately parallelized format and with
appropriate miniaturization (see exemplary embodiments), a
microfluidic system needs markedly fewer starting substances for an
individual DNA oligomer than a conventional solid-phase synthesis
apparatus (when using a single column). Here, microliters compare
with the consumption of milliliters, i.e., a factor of 1000.
[0073] Taking into account the newest findings in immunology, the
presented method allows an extremely efficient and rapid vaccine
design (DNA vaccines).
Exemplary Embodiments
[0074] To carry out the method, the present invention requires the
provision of a large number of nucleic acid molecules, usually DNA,
whose sequence can be freely determined. These building blocks must
have virtually 100% identical sequences within one building block
species (analogously to the synthesis performance of conventional
synthesizers). Only highly parallel synthesis methods are suitable
for generating the required variance. In order for the system to be
able to work flexibly and, despite the necessary multiplicity of
different building blocks to be synthesized, to require as little
space and as few reagents as possible, the method is preferably
carried out in a microfluidic system within which the individual
sequences are produced in a determinable form. Two types of
programmed synthesis are suitable for systems of this kind, which
are also described in the patent applications DE 199 24 327.1, DE
199 40 749.5, PCT/EP99/06316 and PCT/EP99/06317: these are first
the synthesis by programmable fluidic individualization of the
reaction areas and, secondly, the synthesis by programmable
light-dependent individualization of the reaction areas.
[0075] In both variants, synthesis is carried out in a microfluidic
reaction support. The design of this reaction support may provide
in the system for the bringing together in stages the detached
synthesis products, i.e., building blocks, by collecting the
nucleic acid strands, after detaching them, in appropriate reaction
areas and the assembly taking place there. Groups of such assembly
areas may then for their part be brought into contact again with
one another so that during the course of a more or less long
cascade the final synthesis products are produced: genetic
information carriers in the form of DNA molecules. The following
variants are suitable here:
[0076] Either synthesis, detachment and assembly are carried out
chronologically but spatially integrated in a microfluidic reaction
support or synthesis, detachment and assembly are carried out
partially in parallel in one or more microfluidic reaction
supports. It is furthermore possible that the microfluidic reaction
support contains only reaction areas for the programmed synthesis
and that subsequently detachment and elution into a reaction vessel
for the assembly are carried out.
[0077] In the case of very large DNA molecules, synthesis,
detachment and assembly can be supplemented by condensation
strategies which prevent break-up of the molecules. This includes,
for example, the use of histones (nuclear proteins which make
condensation of the chromosomes in the nucleus possible in
eukaryotes), the use of topoisomerases (enzymes for twisting DNA in
eukaryotes and prokaryotes) or the addition of other DNA-binding,
stabilizing and condensing agents or proteins. Depending on the
design of the reaction support, this may take place by integrating
the condensation reaction in another reaction chamber provided
therefor or by addition during the combination and assembly in
stages of the building blocks.
[0078] The free choice of sequence is of essential importance for
the controlled and efficient building block assembly in stages to
the final product. For the choice of overlapping complementary ends
influences the specificity of the assembly and the overall
biochemical conditions (salt concentration, temperature, etc.).
When providing a sequence for the gene of interest and after
automatic or manual selection of the other genetic elements
(regulatory regions, resistance genes for cloning, propagation
signals, etc.) for determination of the final product (e.g., a
plasmid vector), the provided sequence is fragmented into suitable
building blocks which are then synthesized in the required number
of reaction supports. The fragments or their overlap regions to be
hybridized are chosen such that the conditions for hybridizing are
as similar as possible (inter alia GC:AT ratio, melting points,
etc.).
[0079] Further extension of the system provides for elements for
purification and isolation of the product forming, which are
likewise designed by microfluidics or microsystem technology. Said
elements may be, for example, methods in which the final
double-stranded DNA after its synthesis using fluorescent synthons
must have a particular total fluorescence. When using proteins with
condensing action, these proteins, where appropriate, may also
carry a fluorescent label which is preferably detectable separately
(reference signal). It is then possible to sort the mixture of
final reaction product in the reaction support structures according
to fluorescence (see Chou et al., Proceedings of the National
Academy of Science PNAS 96:11-13, 1999). Thus a sufficient quality
is achieved in order to directly provide a product for further
work.
[0080] Information from sequencing projects, which is present in
databases, may be studied for genes fully automatically
(computer-assisted). Identified or putative genes (ORFs) are
converted into completely synthetic DNA which may contain, where
appropriate, regulatory and other genetic elements which seem
suitable, so that, for example, one or more vectors are generated.
The product is either made available (e.g., as pure DNA) or
directly introduced to functional studies, inter alia by transfer
into suitable target cells. The information may come from public
databases, from work of decentralized users or from other sources,
for example the method described in the patent applications DE 199
24 327.1 and DE 199 40 749.5.
[0081] It may be of interest that a variance of randomized sequence
occurs at a particular site or sites of the target sequence. An
example is the testing of variants of a binding site into which,
for example over an area of 20 amino acids, i.e., 60 nucleotides,
random variations of nucleotides were incorporated. This may take
place in an embodiment in that during the synthesis process, after
activating a reaction area, a mixture of synthons is added so that
all added synthons can hybridize in a statistically distributed
manner. A modification of this process may provide for DNA building
blocks of different length to be used at a particular position of
the target sequence, for example by producing different building
blocks on different reaction areas, which show the same sequence
for overlapping and hybridization.
* * * * *