U.S. patent application number 15/589837 was filed with the patent office on 2017-08-24 for programmable oligonucleotide synthesis.
The applicant listed for this patent is Synthetic Genomics, Inc.. Invention is credited to Raphael Carapito, Peer Staehler.
Application Number | 20170240956 15/589837 |
Document ID | / |
Family ID | 38963235 |
Filed Date | 2017-08-24 |
United States Patent
Application |
20170240956 |
Kind Code |
A1 |
Staehler; Peer ; et
al. |
August 24, 2017 |
PROGRAMMABLE OLIGONUCLEOTIDE SYNTHESIS
Abstract
The invention relates to methods and devices for preparing
synthetic nucleic acids.
Inventors: |
Staehler; Peer; (Mannheim,
DE) ; Carapito; Raphael; (Strasbourg, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Synthetic Genomics, Inc. |
La Jolla |
CA |
US |
|
|
Family ID: |
38963235 |
Appl. No.: |
15/589837 |
Filed: |
May 8, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13741231 |
Jan 14, 2013 |
9644225 |
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15589837 |
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13441186 |
Apr 6, 2012 |
8367335 |
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13741231 |
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12438425 |
Feb 23, 2009 |
8173368 |
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PCT/EP2007/007417 |
Aug 23, 2007 |
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13441186 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P 19/34 20130101;
C12Q 1/686 20130101; C07H 21/00 20130101; C12Q 1/6806 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 23, 2006 |
DE |
10 2006 039 479.8 |
Claims
1. A method of preparation of synthetic nucleic acids, in
particular of nucleic acid double strands, comprising the steps:
(a) preparation of a multiplicity of different nucleic acid
fragments by solidphase synthesis, preferably at various positions
of a common solid support, and (b) joining together of at least two
nucleic acid fragments from (a) by binding to one another or by
covalent linkage, characterized in that the method comprises
amplification steps in which the synthesized nucleic acid fragments
or/and optionally double-stranded intermediates formed therefrom
are submitted to amplification, e.g., PCR.
2. A method for isolating an individual nucleic acid having the
desired sequence from a mixture of nucleic acids, comprising: (a)
monoclonizing a mixture of nucleic acids, (b) parallel sequencing
the monoclonized nucleic acids of step (a), (c) localizing a
nucleic acid with the desired sequence in the nucleic acids of step
(b), (d) isolating the nucleic acid from step (c), and (e)
optionally further processing of isolated nucleic acid obtained in
step wherein the isolation takes place by at least one of steps (i)
to (iv): (i) isolating one or more beads on which said nucleic acid
molecule is immobilized, (ii) cleaving off the nucleic acid
molecule from a support on which said nucleic acid molecule is
immobilized, (iii) selectively amplifying the nucleic acid molecule
by spatially-resolved addition of PCR reagents, and (iv) eluting
the nucleic acid molecule by laser capture method.
3. A method for quality control of assembled nucleic acid
molecules, said method comprising: (a) providing a mixture of
assembled nucleic acid molecules suspected of containing sequence
defects, (b) amplifying the mixture of (a) to receive a clonal
population thereof, (c) immobilizing the clonal population of (b)
on a support, (d) parallel sequencing the clonal population
immobilized according to (c), (e) localizing at least one clonal
nucleic acid molecule that meets a predefined criterion, and (f)
obtaining the at least one clonal nucleic acid molecule of (e).
4. The method of claim 3, wherein the assembled sequences provided
in step (a) are built each from one or more oligonucleotides.
5. The method of claim 3, wherein the assembled sequences represent
genes, gene clusters, chromosomes, genomes or fragments thereof
6. The method of claim 3, wherein amplification in step (b)
comprises an emulsion-based PCR.
7. The method of claim 3, wherein immobilizing the clonal
population according to step (c) comprises immobilization on
beads.
8. The method of claim 3, wherein the predetermined criterion in
step (e) is the desired correct sequence.
9. The method of claim 3, wherein the obtaining the clonal nucleic
acid molecule of (f) comprises: (i) detachment of the clonal
nucleic acid molecule from the support, (ii) copying without
disrupting the covalent linkage to the support, (iii) laser
capture, or/and (iv) spatially resolved PCR.
10. Use of a quality controlled DNA target sequence obtained by the
method of claim 1 for assembly of a gene, gene cluster, chromosome,
genome or a fragment thereof
11. A method of assembly of nucleic acid molecules comprising: (a)
providing single-stranded nucleic acid molecules to be assembled
into a target nucleic acid molecule, (b) assembling by
hybridization complementary regions of the single-strands nucleic
acids of (a), and (c) controlling the quality of the mixture of
assembled nucleic acid molecules obtained in step (b) by the method
of claim 3.
12. The method of claim 11, wherein step (b) results in a gene,
gene cluster, chromosome, genome or a fragment thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 13/741,231 filed Jan. 14, 2013, now issued as
U.S. Pat. No. 9,644,225; which is a continuation application of
U.S. application Ser. No. No. 13/441,186 filed Apr. 6, 2012, now
issued as U.S. Pat. No. 8,367,335; which is a divisional
application of U.S. application Ser. No. 12/438,425 filed Feb. 23,
2009, now issued as U.S. Pat. No. 8,173,368; which is a 35 USC
.sctn.371 National Stage application of International Application
No. PCT/EP2007/007417 filed Aug. 23, 2007; which claims the benefit
under 35 USC .sctn.119(a) to Germany Patent Application No. 10 2006
039 479.8 filed Aug. 23, 2006. 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 methods and devices for preparing
synthetic nucleic acids.
[0004] Background Information
[0005] There is a high demand for synthetic nucleic acids in
molecular biology and biomedical research and development.
Synthetic nucleic acids (DNA, RNA or their analogues) are mainly
prepared using column-based synthesizers.
[0006] Particularly important and widespread applications for
synthetic nucleic acid polymers are primers for the polymerase
chain reaction (PCR) (Critical Reviews in Biochemistry and
Molecular Biology 26 (3/4), 301-334, 1991) and the sequencing
method according to Sanger (Proc. Nat. Acad. Sci. 74, 5463-5467,
1977).
[0007] Synthetic DNA also has a role in the preparation of
synthetic genes. Methods of gene synthesis are described for
example in U.S. Pat. No. 6 586 211 B1, in PCT/EP2004/013131, in WO
00/13017 A2, in S. Rayner et al., PCR Methods and Applications 8
(7), 741-747, 1998, in WO 90/00626 A1, in EP 385 410 A2, in WO
94/12632 A1, in WO 95/17413 A1, in EP 316 018 A2, in EP 022 242 A2,
in L. E. Sindelar and J. M. Jaklevic, Nucl. Acids Res. 23 (6),
982-987, 1995, in D. A. Lashkari, Proc. Nat. Acad. Sci. USA 92
(17), 7912-7915, 1995, and in WO 99/14318 A1, which are
incorporated as reference.
[0008] Another two fields of application with increasing demand are
the production of microarrays or biochips from oligonucleotide
probes (1. Nature Genetics, Vol. 21, Supplement (complete), January
1999, 2. Nature Biotechnology, Vol. 16, 981-983, October 1998, 3.
Trends in Biotechnology, Vol. 16, 301-306, Jul. 1998) and the
preparation of interfering RNA (iRNA or RNAi) for the modulation of
gene expression in target cells (PCT/EPO1/13968).
[0009] The aforesaid fields of application of molecular biology
provide valuable contributions in the development of active
compounds, the production of active compounds, combinatorial
biosynthesis (antibodies, effectors such as growth factors,
neurotransmitters etc.), in biotechnology (e.g., enzyme design,
pharming, biological production methods, bioreactors etc.), in
molecular medicine in tissue engineering, in the development and
application of new materials (e.g., materials such as spider silk
and mother of pearl), in the development and use of diagnostic
agents (microarrays, receptors and antibodies, enzyme design etc.)
or in environmental engineering (specialized or tailor-made
microorganisms, production methods, remediation, sensors etc.). The
method according to the invention can thus be employed in all these
areas.
PRIOR ART
[0010] The commonest method for the preparation of synthetic
nucleic acids is based on the fundamental work of Caruthers and is
known as the phosphitamide method (M. H. Caruthers, Methods in
Enzymology 154, 287-313, 1987). The sequence of the resultant
molecules can be controlled by the order of synthesis. Other
methods, such as the H-phosphonate method, serve the same purpose
of successive synthesis of a polymer from its subunits, but have
not found such widespread application as the method according to
Caruthers.
[0011] To make it possible to automate the chemical method of
polymer synthesis from subunits, solid phases are generally
employed, on which the growing molecular chain is anchored. On
completion of synthesis it is split off, which requires a suitable
linker between the actual polymer and the solid phase. For
automation, as a rule the method employs solid phases in the form
of activated particles, which are packed in a column, e.g.,
controlled pore glass (CPG). These solid phases as a rule only
carry one specifically removable type of oligo with a programmed
sequence. The individual synthesis reagents are then added in a
controllable manner in an automatic machine, which mainly provides
the automated addition of the individual reagents to the solid
phase. The quantity of molecules synthesized can be controlled by
the amount of support material and the size of the reaction
batches. For the aforementioned molecular-biological methods, these
amounts are either sufficient or even too high (e.g., in the case
of PCR primers). Some degree of parallel operation for production
of a multiplicity of different sequences is achieved through
arranging several columns in an assembly of apparatus. Thus,
equipment with 96 parallel columns is known by a person skilled in
the art.
[0012] A variant and further development for the production of
synthetic nucleic acids is the in situ synthesis of microarrays
(array arrangement of nucleic acids in a matrix). This is carried
out on a substrate that is loaded with a multiplicity of different
sequences during the synthesis. The great advantage of the in situ
synthesis methods for microarrays is the preparation of a
multiplicity of oligomers of different and defined sequence at
addressable locations on a common support. The synthesis has
recourse to a manageable set of feed materials (in the case of DNA
microarrays, as a rule the 4 bases A, G, T and C) and from these it
builds up any sequences of nucleic acid polymers.
[0013] The individual molecular species can be demarcated on the
one hand by separate fluidic compartments for addition of the
synthesis feed materials, as is the case e.g., in the so-called in
situ spotting method or piezoelectric techniques, based on inkjet
printing technology (A. Blanchard, in Genetic Engineering,
Principles and Methods, Vol. 20, Ed. J. Sedlow, 111-124, Plenum
Press; A. P. Blanchard, R. J. Kaiser, L. E. Hood, High-Density
Oligonucleotide Arrays, Biosens. & Bioelectronics 11, 687,
1996).
[0014] An alternative method is the spatially-resolved activation
of synthesis sites, which is possible through selective
illumination, through selective or spatially-resolved generation of
activation reagents (deprotection reagents) or through selective
addition of activation reagents (deprotection reagents).
[0015] Examples of the methods known to date for the in situ
synthesis of microarrays are [0016] photolithographic light-based
synthesis (McGall, G. et al.; J. Amer. Chem. Soc. 119; 5081-5090;
1997), [0017] projector-based light-based synthesis
(PCT/EP99/06317), [0018] fluidic synthesis by means of physical
separation of the reaction spaces (known by a person skilled in the
art from the work of Prof. E. Southern, Oxford, UK, and of the
company Oxford Gene Technologies, Oxford, UK), [0019] indirect
projector-based light-controlled synthesis by light-activated
photo-acids and suitable reaction chambers or physically separated
reaction spaces in a reaction support, [0020] electronically
induced synthesis by spatially-resolved deprotection on individual
electrodes on the support using proton production induced by the
electrodes (known from, among others, the products of the company
Combimatrix) and [0021] fluidic synthesis by spatially-resolved
deposition of the activated synthesis monomers (known from A.
Blanchard, in Genetic Engineering, Principles and Methods, Vol. 20,
Ed. J. Sedlow, 111-124, Plenum Press; A. P. Blanchard, R. J.
Kaiser, L. E. Hood, High-Density Oligonucleotide Arrays, Biosens.
& Bioelectronics 11, 687, 1996).
[0022] Methods of preparation of synthetic nucleic acids, in
particular nucleic acid double strands on a common solid support,
are also known from WO 00/49142 and WO 2005/051970.
SUMMARY OF THE INVENTION
[0023] An improved method is to be provided for the preparation of
synthetic nucleic acids of optional sequence, in particular nucleic
acid double strands, through the preparation of suitable
solid-phase-supported synthetic libraries. Moreover, an improved
method is to be provided for the preparation of synthetic nucleic
acids of optional sequence, in particular nucleic acid double
strands, through the preparation of suitable solid-phase-supported
synthetic libraries and the subsequent joining together of at least
two nucleic acid fragments from the library through binding or
covalent linkage of these two nucleic acid fragments to one
another, wherein preparation of the library includes control of the
quantitative proportions of the constituents of the library to one
another.
[0024] The nucleic acid fragments are joined together preferably by
a specific hybridization reaction between overlapping regions of
mutually complementary segments of the nucleic acid fragments,
thereby obtaining longer synthetic double-stranded nucleic acids.
The individual sequence segments used for building up longer
nucleic acids preferably have a length of 20-100 or 20-300
nucleotide building blocks, preferably of 25-50 or 25-100
nucleotide building blocks, for example about 30 nucleotide
building blocks. The sequence segments are preferably selected in
such a way that they at least partially overlap a sequence segment
of the antisense strand of the complementary nucleic acid that is
to be synthesized, so that the nucleic acid strand to be
synthesized can be built up by hybridization of individual sequence
segments. In an alternative embodiment, the sequence segments are
preferably selected so that the sequence segments on both strands
of the nucleic acid to be synthesized completely overlap, and
accordingly preparation of a more or less complete double strand
now only requires covalent linkage of the phosphodiester backbone.
The length of the complementary regions or overlaps between
individual fragments is e.g., 10-50 or 10-100 nucleotide building
blocks, preferably 12-25 or 20-80 nucleotide building blocks,
especially preferably about 15-20 nucleotide building blocks and
most preferably about 15 or about 20 nucleotide building blocks. If
the overlapping or complementarity region between two nucleic acid
fragments has a high AT content, e.g., an AT content >50%,
preferably an AT content >60%, especially preferably an AT
content >65%, the binding constant is lower in comparison with
GC-richer sequences. Accordingly, for thermodynamic reasons,
hybridization between these fragments may be of comparatively low
efficiency. This can have an influence on the assembly of 2 or more
fragments. A possible sequence-dependent consequence is a reduced
yield of nucleic acid double strands with the correct target
sequence.
[0025] One aim of the method according to the invention is to
influence the thermodynamic relations during assembly from 2 or
more fragments by controlling or by controlling and regulating the
quantitative proportions of the fragments in a reaction batch, in
order to improve the yield of correct nucleic acid double strands.
In particular, the thermodynamic parameters are modulated in a
reaction for binding at least 2 nucleic acid fragments to one
another. Modulation of the thermodynamic parameters means, in
particular, that the binding of the two nucleic acid fragments to
one another, which is subject to the law of mass action, is
improved. It is especially preferable for the modulation of the
thermodynamic parameters in the reaction to comprise control of the
quantitative proportions of individual nucleic acid fragments; in
particular through the use of larger amounts of nucleic acid
fragments that have a high proportion of AT. If at least some
nucleic acid fragments, for modulation of their thermodynamic
parameters in the reaction of at least 2 nucleic acid fragments,
are used in an increased amount relative to other fragments, this
can be achieved for example in that at least some nucleic acid
fragments, which have a high AT content, are used in an increased
amount relative to other fragments.
[0026] By controlling the quantitative proportions of individual
nucleic acid fragments, in particular by using larger amounts of
nucleic acid fragments that have a high proportion of AT, the yield
of correct hybridization products and therefore also the yield of
correct nucleic acid double strands can be improved. The quantity
of the population of the corresponding nucleic acid fragments can
thus be improved by preferably .gtoreq.10%, especially preferably
.gtoreq.50% or even more, e.g., by up to a factor of 100 or 1000
relative to other nucleic acid fragments without a high proportion
of AT.
[0027] Apart from the proportion of AT, there are also other
parameters that have an influence on the yield of target sequences.
These include the varying synthesis efficiency of the individual
fragments or oligonucleotides during extension in the synthesis
process. A person skilled in the art knows, for example, that
building block G in phosphoramidite methods couples at lower yield
to the polymer strand that is to be extended than the other
nucleotide building blocks. Moreover, a person skilled in the art
is aware of dependences of the synthesis efficiency on the complete
sequence of the polymer strand that are empirically evident, but
not in every case already predictable. This includes for example
the synthesis of several G building blocks in succession.
[0028] These deviations in the availability or kinetics of
individual fragments for the assembly of 2 or more fragments into a
target sequence can also be influenced by controlling or
controlling and regulating the quantitative proportions. The
deviation may be well known and it may be possible to calculate it,
or it may only be known empirically and observed in experiments.
Accordingly, the method according to the invention can be optimized
e.g., iteratively with measurement of the result, for example the
yield of target sequence. One embodiment of the invention is the
use of a stored-program device, in order to control the predicted
optimal composition of the quantitative proportions on the basis of
known regularities for new fragment sequences and target sequences.
During reaction this can take place by means of a computer or
similar control equipment. The influencing factors and settings can
be recorded in a database, which in one embodiment is contained in
a stored-program device and is used directly or indirectly in the
control of the synthesis.
[0029] The reaction products of a library synthesis are
characterized by considerable variety of the sequences, programming
of which is freely selectable during the synthesis operation. A
numerical example will illustrate the great variety of such a
library. A microarray from the GENIOM.RTM. system, for which the
nucleic acid molecule populations are synthesized on individual
synthesis locations in a special microfluidic support, can for
example (status in the year 2006) synthesize up to 60,000 freely
selectable oligonucleotides with a sequence of up to 60
nucleotides. The equipment provides spatially-resolved synthesis of
the nucleic acids using a projector-based method (see e.g., WO
00/13018 or WO 00/13017).
[0030] The aim of the improved method is to provide nucleic acids
with high and rationally programmable diversity of the sequences
and controllable quantitative proportions of the individual
sequence representatives or fragments (constituents of the library)
for subsequent processes in a next step.
[0031] Examples of subsequent processes, for which the invention
can be used, are: [0032] production of nucleic acid fragments as
primers for primer extension methods, strand displacement
amplification, polymerase chain reaction, site directed mutagenesis
or rolling circle amplification, [0033] production of synthetic
genes, gene fragments, gene clusters, gene transfers, gene vectors,
chromosomes, genomes, optimized genomes, minimal gene clusters,
minimal genomes, completely synthetic genomes or of mixtures with
directed or randomized variants of synthetic genes, gene fragments,
gene clusters, gene transfers, gene vectors, chromosomes, genomes,
optimized genomes, minimal gene clusters, minimal genomes,
completely synthetic genomes, [0034] modulation of gene expression
by means of RNAi or antisense methods, in which one more copy of
the nucleic acid or nucleic acids produced can be provided by an
RNA polymerase, [0035] production, extraction, purification,
isolation or preparation of analytes (sample preparation) for the
logically subordinate analysis by microarrays, by sequencing
methods, by parallel sequencing methods, by amplification methods
(strand displacement amplification, polymerase chain reaction or
rolling circle amplification) or analysis in gel electrophoresis,
[0036] RNA libraries with e.g., 2 or more sequences for translation
in vitro or in vivo, [0037] cloning of the nucleic acids produced
alone or in combination with further sequences by means of vectors
or plasmids, [0038] production of minimal genomes, optimized
genomes, reduced genomes, mixed genomes of various species, wherein
the whole planned stock of the genome or a portion thereof can be
encoded by the synthetic nucleic acids, [0039] production of target
organisms with permanently or temporarily integrated, transformed,
transfected or otherwise inserted synthetic genes, gene fragments,
gene clusters, gene transfers, gene vectors, chromosomes, genomes,
optimized genomes, minimal gene clusters, minimal genomes,
completely synthetic genomes or of mixtures with directed or
randomized variants of synthetic genes, gene fragments, gene
clusters, gene transfers, gene vectors, chromosomes, genomes,
optimized genomes, minimal gene clusters, minimal genomes,
completely synthetic genomes, [0040] production of the target
organisms described in the preceding item for the improvement,
change or reduction of a native substance occurring in the target
organism, e.g., an amino acid chain, a protein, an organic
substance, a hydrocarbon, a drug or a precursor thereof, a nucleic
acid, a pheromone, or some other substance for the provision of an
article used by humans or for production of a substance that is
then put to further use, [0041] ligation of the nucleic acids in
vectors, YACs, BACs, chromosomes or plasmids, [0042] validation or
testing of hybridization assays and associated reagents and kits by
means of the nucleic acid polymers produced, in the areas of
microarrays, biochips, dot blots, Southern or Northern blots, bead
arrays, serial analysis of gene expression (SAGE), PCR, real-time
PCR, [0043] reference or calibration methods or steps within assays
from the areas of microarrays, dot blots, Southern or Northern
blots, bead arrays, serial analysis of gene expression (SAGE), PCR,
real-time PCR, [0044] production of binding agents such as aptamers
and ribozymes and indirect production via the translation in vivo
or in vitro of peptides, proteins, antibodies, antibody fragments,
peptides acting analogously to antibodies, proteins acting
analogously to antibodies, [0045] production, via translation in
vivo or in vitro of glycoproteins, proteoglycans or complexes with
optionally peptide fraction, protein fraction, RNA fraction or DNA
fraction, such as ribosomes or proteasomes.
DETAILED DESCRIPTION OF THE INVENTION
[0046] Preferred methods of production of synthetic nucleic acids
from a solid support are known from WO 00/49142 and WO 2005/051970.
Reference is made expressly to the contents of these documents, and
they are incorporated herein in their entirety.
[0047] Preferably, in the method according to the invention, the
synthetic nucleic acids are prepared by synthesizing a multiplicity
of different nucleic acid fragments at various positions of a
common solid support. Preferably the synthesis of the nucleic acid
fragments comprises construction from nucleotide building blocks by
wet-chemical and/or photochemical methods on the support,
subsequent detachment of the nucleic acid fragments and assembling
of the fragments to the desired nucleic acid double strand.
Furthermore, the synthesis can include amplification steps, in
which the synthesized nucleic acid fragments and/or optionally
double-stranded intermediates formed from them are submitted to
amplification, e.g., PCR. For this purpose, nucleotide building
blocks and an enzyme that brings about amplification can be added.
Amplifications can take place on the support, i.e., before and/or
after detaching the nucleic acid fragments, and/or after elution
from the support.
[0048] The support can be selected from flat supports, porous
supports, reaction supports with electrodes, reaction supports with
particles or beads, microfluidic reaction supports, which
optionally have surface modifications such as gels, linkers,
spacers, polymers, amorphous layers and/or 3D matrices, and
combinations of the aforesaid supports. Preferably the support is a
microfluidic support.
[0049] The nucleic acid fragments are preferably produced by
spatially and/or time-resolved in situ synthesis on the support,
for example by spatially and/or time-resolved illumination by a
programmable light source matrix. The spatially and/or
time-resolved synthesis can take place in a microfluidic support
with one or more fluidic reaction spaces and one or more reaction
regions within a fluidic reaction space.
[0050] Different amounts of nucleic acid fragment species used for
assembly can be produced by using several regions and/or larger
regions for the synthesis of the particular nucleic acid fragments
on the support. An appropriately modified support is also an object
of the invention.
[0051] A further--optionally independent--object of the invention
comprises carrying out the assembly of nucleic acid fragments to
nucleic acid double strands in several steps. In a first step, the
nucleic acid fragments synthesized on the support are provided at
the 5'- and/or 3'-end with one or more generic primer sequences of
preferably 10-20 or 10-100 bases, especially preferably of 10-30
bases, even more preferably about 15 bases, with the primer
sequences being selected so that amplification is possible directly
for the individual fragment, for a proportion of all fragments in a
mixture, for all fragments in a mixture or after hybridization of
two or more nucleic acid fragments with partial complementary
sequence. After cleaving off the nucleic acid fragments provided
with primers from the support and optionally after elution from the
support and optionally a hybridization of fragment pairs with
complementary sequence, a subsequent amplification takes place,
e.g., by PCR, by adding corresponding primers. In the amplification
reaction there is formation of nucleic acid fragments that contain
the generic primer sequence at their ends. After cleaving-off the
primer sequence, e.g., by means of restriction endonucleases, the
resultant nucleic acid fragments can be submitted to further
amplification cycles, in order to produce a nucleic acid double
strand.
[0052] In one embodiment, the nucleic acid double strand produced
by synthesis of fragments and their subsequent assembly is inserted
into a vector, e.g., a plasmid, and transferred into a suitable
host cell, e.g., a bacterial cell.
[0053] The preparation of the nucleic acid polymers offers, at
several points of the method, the possibility of introducing
modifications or labeling into the reaction products by known
methods. This includes labeled nucleotides, which are modified
e.g., with haptens or optical markers, such as fluorophores and
luminescence markers, labeled primers or nucleic acid analogues
with special properties, such as special melting point or
accessibility for enzymes. Embodiments of the invention can
therefore include the following functions and methods and can make
the associated laboratory processes possible: [0054] labeling of
the oligonucleotides, of the fragments, of covalently or
noncovalently bound hybrid strands constructed therefrom and of the
target sequences, for direct or indirect detection with a measuring
instrument, e.g., an optical, magnetic, electric or
chemiluminescent measuring instrument or method of measurement,
[0055] labeling of the oligonucleotides, of the fragments, of
covalently or noncovalently bound hybrid strands constructed
therefrom and of the target sequences, for direct or indirect
detection by other molecular structures such as enzymes, receptors,
proteins, [0056] isolation or extraction of the oligonucleotides,
of the fragments, of covalently or noncovalently bound hybrid
strands constructed therefrom and of the target sequences, [0057]
binding of the oligonucleotides, of the fragments, of covalently or
noncovalently bound hybrid strands constructed therefrom and of the
target sequences by the haptens or other building blocks that can
be used in a molecular recognition reaction, of a functional group
or of a modification, [0058] detection of the oligonucleotides, of
the fragments, of covalently or noncovalently bound hybrid strands
constructed therefrom and of the target sequences, [0059]
decomposition, selective separation, opening, degradation or
enzymatic digestion of the oligonucleotides, of the fragments, of
covalently or noncovalently bound hybrid strands constructed
therefrom and of the target sequences, [0060] attachment of the
oligonucleotides, of the fragments, of covalently or noncovalently
bound hybrid strands constructed therefrom and of the target
sequences to a target structure or to target structures, e.g., in
or on a reaction support, on a glass slide, in a reaction vessel,
to a target organelle, to a target cell, to a target organ, to an
organism, to a surface, to a biological surface, to a molecular
complex such as a chromosome, virus particle or protein
complex.
[0061] An example of application of the invention and the course of
the method using the GENIOM.RTM. platform are presented below:
[0062] 1. Design of a microarray from 6000 different 30-40mer, in
particular 30mer or 40mer nucleic acid fragments in the GENIOM.RTM.
equipment. In designing the microarray, the number of synthesis
spots per sequence is chosen between 1 and 100 (in single steps),
with nucleic acid fragments (oligos) with tested or predicted
weaker binding relative to other sequences that participate in
assembly later, being represented with a number of synthesis spots
that is preferably greater by 50% or more, than at least one other
oligo-sequence, in order to increase the percentage in the mixture
and thus promote the corresponding hybrids versus the other,
stronger-binding sequences.
[0063] 2. Adding-on of a generic primer-sequence of 10-15, in
particular 10 or 15 bases to all 6000 oligos, in particular at both
ends in each case, so that all sequences comprise e.g., 60 bases.
The primer sequence can be selected so that a PCR reaction is
possible by hybridization of in each case two oligos with
complementary sequence.
[0064] 3. Production of the microarray based on the design from 1.
and 2. by synthesis in the GENIOM.RTM. equipment.
[0065] 4. Cleaving-off and elution of the oligos from the reaction
support.
[0066] 5. PCR of the library with addition of a primer pair
suitable for the primer sequences added in 2. In the PCR there is
formation of e.g., 60mers, each of which can carry an insert of
e.g., 40 bases with optional sequence and uniform sequences at both
ends.
[0067] 6. Cleaving-off of the primer sequence.
[0068] 7. Incubation of the resultant 30-40mer library or of the
30mer or 40mer library with new PCR reagents with addition of a
primer pair, which e.g., only binds to sequences in the hybrid that
are at least 2 fragments apart and therefore leads to a "nested
PCR" of a longer fragment (direct assembly and PCR amplification of
a synthetic gene is known by a person skilled in the art from
publications).
[0069] 8. Further processing or storage of the resultant
amplification product (amplicon). In one embodiment the amplicon is
cloned in bacteria using a plasmid and after growth of clones a
number of 10 clones or 10 inserts in the clones is sequenced. The
sequence-tested synthetic gene is ready.
[0070] It is known in the prior art that the use of different
quantitative proportions of the individual oligonucleotides for
assembly of synthetic genes increases the rate of correct sequences
(Gao, X et al., Nucleic Acids Research, 2003, Vol 31; No.22;
p.143). The stoichiometry of the oligonucleotides has an influence
on the thermodynamic parameters on the basis of the law of mass
action.
[0071] In the preferred embodiment and in a number of the methods
described at the beginning for the production of microarrays in
situ, the design, i.e., the actual loading and allocation of area
and location on the reaction support for an individual
oligonucleotide species, can be selected flexibly and hence also
the quantity of the individual oligonucleotides. Basically this is
possible with all the in situ methods of synthesis known by a
person skilled in the art and enumerated at the beginning. Methods
that can be programmed flexibly and do not require any change of
physical parts in the production setup are particularly preferred
for the embodiments of the invention. For example, the ink-jet
spotting methods, the projection methods and the Combimatrix
electrochemical method are particularly advantageous.
[0072] The quantity of individual oligonucleotides is
correspondingly, after detachment and elution of the oligos, also
controllable in the pool for various oligo-species in solution.
When using projection technology for synthesis on the reaction
support, the quantitative distribution can be set via the number of
micromirrors or projection elements (illumination pixels). A person
skilled in the art can see from this example that also analogously
to other methods, the design determines the quantities. For
example, in a photolithographic method the quantity is determined
by the area of the synthesis locations, and in an indirect method
based on the use of photo-acids it is determined by the number of
physically separated reaction locations that are used for a
sequence.
[0073] The quantitative proportions can be set and controlled by
the user or by software. With suitable software it is possible to
program predetermined values for setting the quantitative
proportions. In this way the process can be automated in certain
places. The inputs can be derived from theoretical models,
bioinformatics, sequence comparison, empirical data or information
in databases.
[0074] For empirical determination and for finding the optimal
number of synthesis units per sequence, in a preferred embodiment
with micromirrors in a projection unit, a hybridization reaction
can be used on a microarray. For this, the selected oligos in
solution are hybridized on a microarray that contains sequences
that are provided for assembly as complementary counterparts on the
respective, corresponding oligos. This analysis simulates the
assembly reaction. The result can, in one embodiment, be determined
with fluorescence markers, which are secured directly or indirectly
to the oligos in solution. The signals from individual analysis
spots can then be evaluated relative to one another or quantified.
As a result of this analysis, the quantitative proportions can be
adjusted by altering the synthesis design.
[0075] Adjustment of the quantitative proportions improves the
binding conditions for oligos with increased amount in the mixture,
and inherently comparatively lower binding strengths are
compensated. The probability of correct incorporation in the full
set of oligos that take part in the reaction is equilibrated for
all oligos.
[0076] A comparatively lower binding strength of an oligo with a
suitable sequence on a second oligo can be caused, apart from other
parameters, by the base composition, mainly by the AT fraction (if
only natural bases are used), by the tendency to secondary
structures or by further interactions with other oligos in the
mixture.
[0077] In one embodiment, the amplification of the oligonucleotides
that are detached is a component part of the method.
[0078] The oligonucleotides or a portion thereof can be synthesized
with generic 5'- and/or 3'-sequences, added onto the sequence of
the nucleic acid that is to be prepared, so that an amplification
of the oligonucleotides or of a portion thereof can then take
place. The amplification or primer sequences are complementary to
corresponding amplification primers and can contain one or more
cleavage sites, preferably Type II cleavage sites. These cleavage
sites enable splitting-off of the primer sequences after
amplification, e.g., by PCR. Several pairs of amplification primer
sequences can be used on one support, to permit a multiplex
amplification, e.g., PCR, in an oligonucleotide library. This means
that subpopulations of the oligonucleotide fragments can contain
specific, but different amplification sequences or pairs of
amplification sequences. The amplicons can be purified or can be
used directly for the amplification reaction, e.g., for PCR.
[0079] In this embodiment, the method can be used for selective
amplification of a subpopulation of the sequences derived from the
support. It also becomes possible to complete nucleic acid
fragments that were not in full-length form after the synthesis.
The amplifications can take place directly in the microchannels of
the support and/or separately from the support in a suitable
reaction vessel. In this way the quantity of nucleic acid fragments
available for gene synthesis can be increased significantly.
Furthermore, the shortened fragments that formed during the
synthesis, and that may hamper the assembly reaction, are diluted
and are present at negligible concentrations compared with the
amplified full-length fragments. Therefore increasing the
proportion of full-length fragments in a mixture intended for gene
synthesis by prior amplification and hence dilution of shortened
synthesis products can also be a component part of this
embodiment.
[0080] In yet another--optionally independent--embodiment, nucleic
acid fragments with the desired correct sequence can be isolated
from a mixture of nucleic acids. For this it is possible for
example to use known techniques, such as emulsion-based PCR or
individual molecular arrays, in which clonal molecular populations
or individual molecules can be isolated and sequenced from a
mixture of DNA fragments. By using such techniques during the gene
synthesis method, the assembled gene can be "monoclonalized" and
each of the individual fragments can be sequenced separately. After
sequence verification, the fragment with the desired sequence can
be identified, isolated and processed further, e.g., by cloning,
DNA based assays, in vitro protein expression etc. The mixture of
DNA fragments can be a PCR product, a ligation product or an
oligonucleotide library.
[0081] Methods in which an emulsion-based PCR simultaneously still
contains preferably in each case a bead or particle in the micelles
or aqueous compartments, are known by a person skilled in the art.
In variants that are particularly optimized and are preferred for
the invention, these are smaller than 1 mm in diameter. For
example, the method of the company 454, which permits the
sequencing of segments in the range 200 to 300 bases (as at 2006),
is known. In this, DNA is fragmented into smaller segments and
these are then supplemented with uniform linker or adapter
sequences by ligation. This mixture is incubated with the beads
described in an emulsion PCR. The primers for the PCR reaction are
present as solid phase on the beads. The reaction result is a
multiplicity of beads, each of which carries clonally only one
fragment from the previously fragmented DNA material covalently on
the surface. In the next step the beads are immobilized in a
reaction support, which contains cavities suitable for the beads
and their size, and then detects the sequences on each of the beads
in parallel by a so-called sequencing by synthesis reaction that is
known by a person skilled in the art.
[0082] In the method according to the invention or in an optionally
independent embodiment, first one or more assembly reactions can be
combined from the oligos from the parallel synthesis. In one
embodiment using emulsion-bead-PCR, the target sequences are to be
selected according to the reading widths of the sequencing reaction
and are therefore preferably 10 to 1000 nucleotides long,
especially preferably 40 to 500. The advantage of this embodiment
is that a mixture of assembled sequences, each built up from 2 or
more oligos and possibly containing defects, is, in the method
presented above, amplified on the beads to clonal populations and
these are then sequenced. Therefore, in a preferred embodiment,
cloning and quality control of the target sequences can be combined
in one step. Localization is effected by immobilization in the
support during sequencing. Those DNA target sequences with
sequences that meet a predefined criterion can be removed in a next
step. In a further preferred embodiment, labeling is carried out by
spatially-resolved addition of a marker, e.g., a specifically or
nonspecifically binding optical marker, such as an intercalator
(Sybergreen).
[0083] An optionally independent embodiment comprises a method of
in particular parallel sequencing of at least one nucleic acid in a
mixture comprising assembled nucleic acids, which possibly contain
defective nucleotides, comprising the steps: [0084] (a)
amplification of a mixture comprising assembled nucleic acids,
which are in each case built up from 2 or more nucleic acid
fragments, to clonal populations, [0085] (b) sequencing of at least
one clonal, population from step (a) and [0086] (c) optionally
isolation of at least one nucleic acid, which contains defects,
and/or at least one nucleic acid, which is correct.
[0087] In a preferred embodiment isolation takes place by isolation
of one or more beads. In an alternative embodiment isolation takes
place by selective amplification by spatially-resolved addition of
PCR reagents. In an alternative embodiment labeling takes place by
spatially-resolved addition of a marker, e.g., a specifically or
nonspecifically binding optical marker, such as an intercalator
(Sybergreen), and subsequent elution by the laser capture method,
which is known by a person skilled in the art from the isolation of
individual cells.
[0088] Clones (beads) that are undesirable or are recognized as
defective can be eliminated physically. In one embodiment this can
take place by selective treatment with a strong light source such
as a laser. Alternatively a further immobilization or
derivatization can be carried out, e.g., in a light-dependent
reaction, e.g., crosslinking, covalent modification or the
adding-on of a molecule that facilitates extraction or elimination.
Thus, beads with an undesirable sequence can be selectively
excluded during further exploitation of the sequenced product or
the plurality of products.
[0089] Having been eluted, isolated or otherwise made available for
further steps, the desired target sequences can be used for
building up even longer target sequences. They can also be used as
a mixture for subsequent process steps.
[0090] In one embodiment, all constituents of a genome that are
regarded as necessary are produced in this method. In a preferred
embodiment these DNA segments are, in a subsequent step, inserted
in a target organism, which constructs an assembled genome from
them in vivo. An especially preferred target organism is
Deinococcus radiodurans (also known as Micrococcus radiodurans)r
which can assemble its own genome in vivo after fragmentation,
e.g., ionizing radiation, into fragments smaller than 10,000
bases.
[0091] The beads can be isolated or stored in the sequencing
reaction support and used again at a later time.
[0092] The clonal sequences can be obtained by detachment or
copying without disrupting the covalent linkage to the bead. With
copying without disrupting the covalent linkage to the bead, a bead
is available later for the clonal sequences to be obtained
again.
[0093] Parallel sequencing methods like those described above are
suitable for the verification of mixtures of oligonucleotides, as
described as part of the invention and as starting material for
gene synthesis. In a further--optionally independent--embodiment,
the composition of a library of oligos from a parallel synthesis
method such as the method according to the invention is verified in
a parallel sequencing method with at least 100, preferably with
1,000 to 10,000, especially preferably with 10,000 to 100,000 and
in particular with 100,000 to 100 million parallel sequencing
reactions.
[0094] A person skilled in the art knows other sequencing methods
that can be used in the present invention, for example the
preparation of so-called polonies as clonal DNA on a reaction
support and subsequent sequencing by "sequencing by synthesis
reaction" or by "sequencing by ligation". Products that use these
methods are obtainable from, among others, the company Applied
Biosystems (ABI, USA) under the name Solid and from the company
Solexa/Illumina (USA). A special embodiment with especially
sensitive detection is the "true single molecule sequencing" (tSMS)
from the company Helicos (USA), which also takes place in parallel
and therefore can also be used for the invention.
[0095] In combination with the selection of the quantity of
synthesis capacity, e.g., illumination pixels, mentioned above, a
control or regulating logic system can be used as part of the
invention. Because the sequencing methods included here are highly
parallel, it is possible to record large amounts of data and
therefore provide rational adjustment of synthesis parameters, so
as to base the proportion of usable target sequences on defined
criteria, e.g., proportion of correct target sequences.
[0096] An--optionally independent--further embodiment of the
invention relates to the production of libraries that contain a
multiplicity of variants of a gene, through synthesis of multiple
variants of one or more of the nucleic acid fragments on the
support before gene assembly.
[0097] Yet another--optionally independent--embodiment of the
invention relates to the enzymatic cleaving-off of the nucleic acid
fragments from the support.
[0098] Yet another--optionally independent--embodiment of the
invention relates to the purification of a library of nucleic acid
fragments through rehybridization on a support that contains the
complementary sequences.
[0099] Yet another--optionally independent--embodiment of the
invention relates to the adding-on of primer-specific DNA sequences
to the nucleic acid fragments that form the 5'-end and the 3'-end
of the nucleic acid double strand that is to be synthesized. In
this way several successive reactions can be carried out with the
same primer pair. Moreover, different genes from different supports
can be amplified simultaneously with the same primers, whereby the
method is automated.
[0100] Yet another--optionally independent--embodiment of the
invention relates to the production of synthetic target nucleic
acids for standard microarray analysis methods, e.g., of probes
that are immobilized on standard microarrays.
[0101] The aforesaid embodiments can of course be combined with one
another.
[0102] In general, all reaction supports and solid phases, for
which synthesis of a matrix of nucleic acid polymers is possible,
can be used for the method according to the invention.
[0103] These include, as typical representatives, the following
reaction support formats and solid phases that are known by a
person skilled in the art: [0104] flat reaction support, also
called "chip", [0105] porous supports, [0106] reaction supports
with electrodes, [0107] reaction support with temporarily or
permanently immobilized solid phase of particles or beads, [0108]
microfluidic reaction support, [0109] surface modification: gels,
linkers, spacers, polymers, amorphous layers, 3D matrices.
[0110] Some of these reaction supports can be used in combination,
e.g., a microfluidic reaction support with porous surfaces.
[0111] The DNA probes are preferably constructed by
light-controlled in situ synthesis on a microfluidic support, e.g.,
in a GENIOM.RTM. one instrument (febit biotech GmbH) using suitable
protecting-group chemistry in a three-dimensional microstructure.
In a cyclic synthesis process, illuminations and condensations of
the nucleotides alternate until the desired DNA sequence has been
built up completely in the microchannels at each position of the
array. In this way e.g., up to 48,000 oligonucleotides with a
length of e.g., up to 60 individual building blocks can be
prepared. The oligonucleotides can bind covalently to a spacer
molecule, a chemical spacer on the glass surface of the reaction
support. Synthesis takes place under software control and permits
high flexibility in the construction of the array, which the user
can therefore configure individually according to his needs. For
example, the length of the oligonucleotides, the number of nucleic
acid probes produced or internal controls can be optimized for the
particular experiment.
[0112] In one embodiment, high-quality nucleic acids with a freely
programmable sequence are prepared in the form of oligonucleotides
with a length of 10-200 bases, inexpensively and efficiently in a
plurality of 10 or more different sequences, in order to produce
synthetic coding double-stranded DNA (synthetic genes).
[0113] The construction of double-stranded DNA from
oligonucleotides has been known since the 1960s (works of Khorana
and others; see "Shabarova: Advanced Organic Chemistry of Nucleic
Acids", VCH Weinheim). In the majority of cases it is carried out
by one of two methods (see Holowachuk et al., PCR Methods and
Applications, Cold Spring Harbor Laboratory Press).
[0114] In one case synthesis of the complete double strand is
carried out by synthesis of single-stranded nucleic acids (of
suitable sequence), assembly by hybridization of complementary
regions of these single strands and ligation of the molecular
backbone by enzymes, generally ligase.
[0115] Conversely, there is also the possibility of synthesis of
regions that overlap at the edges as single-stranded nucleic acids,
assembly by hybridization, filling-up of the single-stranded
regions by enzymes (polymerases) and then ligation of the backbone
by enzymes, generally ligase.
[0116] A preferred course of gene synthesis according to the
invention is as follows: generally, within the scope of a modular
system, many individual nucleic acid strands are synthesized using
the method according to the invention for highly parallel
matrix-based DNA synthesis. The reaction products are sets of
nucleic acids, which serve as building blocks in a subsequent
process. As a result, a sequence matrix is produced that can
contain more than 100,000 different sequences. The nucleic acids
are in single-stranded form and can be eluted from the support or
can be reacted directly in the reaction support. By repeated
copying in one or more operations, multiple copies of the matrix
can be produced without destroying it, and multiplication of the
particular sequences encoded in the matrix is achieved at the same
time. As described in more detail elsewhere, by copying from distal
to proximal it is also possible to cut down the proportion of
shortened nucleic acid polymers on the solid phase, if the copying
initiation site is located distally. An example is a distally
attached promoter sequence.
[0117] The support with the matrix of molecules bound to the solid
phase can be stored for later reuse. The plurality of sequences
produced in one reaction support by an in situ synthesis can thus
be made available for further process steps. At the same time,
through the design of the copying reaction, high quality of the
copied sequences can be achieved.
[0118] Then suitable combinations of the detached DNA strands are
formed. Assembly of the single-stranded building blocks to
double-stranded building blocks takes place within a reaction
space, which in a simple setup can be an ordinary reaction vessel,
e.g., a plastic tube. In another preferred embodiment the reaction
space is part of the reaction support, which in one variant can be
a microfluidic reaction support, in which the necessary reactions
take place. A further advantage of an integrated microfluidic
reaction support is the possibility of integration of further
process steps, such as quality control by optical analysis. In one
embodiment the matrix has already been synthesized in a
microfluidic support, which can then be used simultaneously as
reaction space for the subsequent assembly.
[0119] The sequence of the individual building blocks is selected
so that on bringing the individual building blocks in contact with
one another, complementary regions at the two ends brought together
are available to permit specific assembly of DNA strands through
hybridization of these regions. This results in longer DNA hybrids.
The phosphodiester backbone of the DNA molecule is closed by
ligases. If the sequences are selected in such a way that there are
single-stranded gaps in these hybrids, these gaps are filled
enzymatically by polymerases in a known procedure (e.g., Klenow
fragment or Sequenase). This results in longer double-stranded DNA
molecules. If further use requires these elongated DNA strands to
be available in the form of single strands, this can be achieved by
the methods that are known to a person skilled in the art for
denaturing DNA double strands, such as temperature or alkali.
[0120] By bringing together clusters of DNA strands synthesized in
this way within reaction spaces, it is once again possible to
produce longer partial sequences of the final DNA molecule. This
can be carried out in stages, and the partial sequences are then
assembled to longer and longer DNA molecules. In this way it is
possible to produce very long DNA sequences as a completely
synthetic molecule with a length of more than 100,000 base pairs.
This already corresponds to the order of magnitude of a bacterial
artificial chromosome (BAC). Construction of a sequence of 100,000
base pairs from overlapping building blocks of 20 nucleotides
length requires 10,000 individual building blocks.
[0121] This can be accomplished with most of the highly parallel
methods of synthesis described at the beginning. Those technologies
that produce the array of nucleic acid polymers in a largely freely
programmable manner, and do not rely on the setting-up of technical
components, such as photolithographic masks, are especially
preferred for the method according to the invention. Accordingly,
especially preferred embodiments employ projector-based light-based
synthesis, indirect projector-based light-controlled synthesis by
means of photo-acids and reaction chambers in a microfluidic
reaction support, electronically induced synthesis by
spatially-resolved deprotection on individual electrodes on the
support and fluidic synthesis by spatially-resolved deposition of
the activated synthesis monomers.
[0122] For the rational processing of genetic molecules and the
systematic acquisition of all possible variants, the building
blocks must be produced flexibly and economically in their
individual sequence. The method accomplishes this through the use
of a programmable light source matrix for the light-dependent
spatially-resolved in situ synthesis of the DNA strands that are
used as building blocks. This flexible synthesis permits the free
programming of the individual sequences of the building blocks and
hence also the production of any variants of the partial sequences
or of the final sequence, without any associated substantial
modifications of system components (hardware). Only this programmed
synthesis of the building blocks and hence of the final synthesis
products can provide systematic processing of the plurality of
genetic elements. At the same time, the use of computer-controlled
programmable synthesis makes it possible to automate the entire
process including communication with corresponding databases.
[0123] When the target sequence is specified, the sequence of the
individual building blocks can be selected rationally taking into
account biochemical and functional parameters. Following input of
the target sequence (e.g., from a database) an algorithm searches
for the suitable overlapping regions. Depending on the definition
of the task, varying numbers of partial sequences can be set up,
namely within one reaction support that is to be illuminated or
distributed on several reaction supports. The attachment conditions
for formation of the hybrids, such as temperature, salt
concentration etc., are matched by a corresponding algorithm to the
available overlapping regions. This ensures maximal specificity of
assembly. The data for the target sequence can, in a
fully-automatic version, also be taken directly from public or
private databases and converted to corresponding target sequences.
The resultant products can, once again optionally, be fed into
appropriately automated processes, e.g., into cloning in suitable
target cells.
[0124] Construction in stages by synthesis of the individual DNA
strands in reaction regions within circumscribed reaction spaces
also makes it possible to construct difficult sequences, e.g.,
those with internal repeats of sequence segments, such as are found
e.g., in retroviruses and corresponding retroviral vectors. By
detaching the building blocks within the fluidic reaction spaces,
any sequence can be synthesized, without problems arising from
allocation of the overlapping regions on the individual building
blocks.
[0125] The high quality requirements that are necessary when
constructing very long DNA molecules are fulfilled inter alia
through the use of real-time quality control. The
spatially-resolved synthesis of the building blocks is monitored,
as is the detachment and assembly as far as construction of the
final sequence. Then all processes take place in a transparent
reaction support. It is, moreover, possible to monitor reactions
and fluidic processes in transmitted light, e.g., using CCD
detection.
[0126] The miniaturized reaction support is designed so that a
detachment process is possible in the individual reaction spaces,
and therefore the DNA strands synthesized on the reaction regions
within these reaction spaces are detached in clusters. With
suitable design of the reaction support, assembly of the building
blocks can take place in stages in reaction spaces, as well as the
removal of building blocks, partial sequences or of the final
product, or also sorting or separation of the molecules.
[0127] Once it is completed as an integrated genetic element, the
target sequence can be inserted in cells by transfer and can thus
be cloned and can be investigated in the course of functional
studies. Another possibility is first to carry out further
purification or analysis of the synthesis product, whereas said
analysis can for example be sequencing. The sequencing process can
also begin by direct coupling with suitable equipment, e.g., with a
device operating according to patent application DE 199 24 327 for
integrated synthesis and analysis of polymers. It is also
conceivable for the target sequences produced to be isolated and
analyzed after cloning.
[0128] The method according to the invention provides, with the
integrated genetic elements that it produces, a tool that
encompasses the biological variety in a systematic process for the
further development of molecular biology. The production of DNA
molecules with desired genetic information is therefore no longer
the bottleneck in molecular biological work, because from small
plasmids via complex vectors and as far as mini-chromosomes, all
molecules can be produced synthetically and made available for
further work.
[0129] The method of preparation permits the parallel production of
numerous nucleic acid molecules and therefore a systematic approach
for questions concerning regulatory elements, DNA binding sites for
regulators, signal cascades, receptors, action and interactions of
growth factors etc.
[0130] Through integration of genetic elements in a completely
synthetic total nucleic acid, the known genetic tools, such as
plasmids and vectors, can continue to be used, building on relevant
experience. On the other hand this experience will change rapidly
through the endeavors to optimize the existing vectors etc. The
mechanisms that for example make a plasmid suitable for propagation
in a particular cell type can be investigated rationally for the
first time on the basis of the method according to the
invention.
[0131] Through this rational investigation of large numbers of
variants, the entire combination space of genetic elements can be
opened up. Along with highly parallel analysis (including on DNA
arrays or DNA chips) that is currently undergoing rapid
development, the programmed synthesis of integrated genetic
elements is created as a second important element. Only both
elements together can form the foundation of rational molecular
biology.
[0132] With the programmed synthesis of corresponding DNA
molecules, it is not only possible to have any desired composition
of coding sequences and functional elements, but also the regions
in-between can be adapted. This should quickly lead to minimal
vectors and minimal genomes, once again producing advantages
through the smaller size. Transmission vehicles, such as viral
vectors, can thus be made more efficiently, e.g., using retroviral
or adenoviral vectors.
[0133] Beyond the combination of known genetic sequences, the
development of new genetic elements is possible, which can build on
the function of existing elements. It is precisely for such
development work that the flexibility of the system is of enormous
value.
[0134] The synthetic DNA molecules are fully compatible, at every
stage of development of the method described here, with existing
recombinant technology. Integrated genetic elements can also be
provided for "traditional" molecular biological applications, e.g.,
by means of appropriate vectors. The incorporation of corresponding
cleavage sites even for enzymes that have so far found little
application is not a limiting factor for integrated genetic
elements.
[0135] This method makes it possible to integrate all desired
functional elements as "genetic modules", such as genes, parts of
genes, regulatory elements, viral packaging signals etc., into the
synthesized nucleic acid molecule as carrier of genetic
information. This integration offers the following advantages,
among others:
[0136] High-grade functionally integrated DNA molecules can be
developed, omitting unnecessary DNA regions (minimal genes, minimal
genomes).
[0137] Free combination of the genetic elements and the changes to
the sequence, e.g., for adaptation to the expressing organism/cell
type (codon usage), are also made possible, as are changes to the
sequence for optimizing functional genetic parameters, for example
gene regulation.
[0138] Changes to the sequence for optimizing functional parameters
of the transcript also become possible, e.g., splicing, regulation
at the mRNA level, regulation at the translation level, and
furthermore the optimization of functional parameters of the gene
product, for example the amino acid sequence (e.g., antibodies,
growth factors, receptors, channels, pores, transporters etc.).
[0139] Furthermore, it is possible to devise constructs that
interfere with gene expression by the RNAi mechanism. When such
constructs encode more than one RNAi species, several genes can be
inhibited simultaneously in a multiplex system.
[0140] Overall, the system achieved with the method is extremely
flexible and permits, in a manner not previously available, the
programmed construction of genetic material at greatly reduced
expenditure of time, materials and work.
[0141] Directed manipulation of larger DNA molecules, such as
chromosomes of several hundred kbp, was practically impossible with
the existing methods. In fact, more complex (i.e., larger) viral
genomes of more than 30 kbp (e.g., adenoviruses) are difficult to
handle and manipulate with the classical methods of genetic
engineering.
[0142] There is considerable shortening as far as the last stage of
cloning of a gene: the gene or genes are synthesized as a DNA
molecule and then (after suitable preparation, such as purification
etc.) are inserted directly into target cells and the result is
investigated. The multistage cloning process, generally taking
place via microorganisms such as E. coli (e.g., DNA isolation,
purification, analysis, recombination, cloning in bacteria,
isolation, analysis etc.), is therefore reduced to the last
transfer of the DNA molecule into the final effector cells. With
synthetically produced genes or gene fragments, clonal
multiplication in an intermediate host (generally E. coli) is no
longer necessary. We thus avoid the risk that the gene product
intended for the target cell has a toxic action on the intermediate
host. This is a clear contrast from the toxicity of some gene
products, which when using classical plasmid vectors often leads to
considerable problems in cloning the corresponding nucleic acid
fragments.
[0143] Another appreciable improvement is the shorter time and the
reduction in process steps, until, after sequencing of the genetic
material, the resultant potential genes are verified as such and
are cloned. Normally after discovering interesting patterns, which
may be considered as ORF, corresponding clones are sought with
probes (e.g., by PCR) in cDNA libraries, though they need not
contain the entire sequence of the messenger RNA (mRNA) used
originally in their production (the problem of "full length
clones"). In other methods, an antibody is used for searching in an
expression gene library (screening). Both methods can be shortened
considerably with the method according to the invention: if we have
a gene sequence that has been determined "in silico" (i.e., after
recognition of a corresponding pattern in a DNA sequence by the
computer), or after decoding a protein sequence, a corresponding
vector with the sequence or variants thereof can be produced
directly via programmed synthesis of an integrated genetic element
and inserted in suitable target cells.
[0144] The synthesis of DNA molecules in this way up to several 100
kbp permits the direct complete synthesis of viral genomes, e.g.,
adenoviruses. These are an important tool in basic research
(including gene therapy), but because of the size of their genome
(approx. 40 kbp) they are difficult to manipulate with classical
methods of genetic engineering. In particular this greatly limits
the rapid and economical production of variants for optimization.
This limitation is removed by the method according to the
invention.
[0145] With the method, synthesis, detachment of the synthesis
products and assembly to a DNA molecule are integrated in one
system. With the production methods of microsystem engineering, all
necessary functions and process steps up to purification of the
final product can be integrated in a miniaturized reaction support.
These can comprise synthesis regions, detachment regions
(clusters), reaction spaces, feed channels, valves, pumps,
concentrators, separation regions etc.
[0146] Plasmids and expression vectors can be produced directly for
sequenced proteins or corresponding partial sequences and the
products analyzed biochemically and functionally, e.g., using
suitable regulatory elements. The search for clones in a gene
library is therefore omitted. Correspondingly, open reading frames
(ORF) from sequencing work (e.g., the human genome, project) can be
programmed directly in corresponding vectors and combined with
desired genetic elements. Identification of clones, e.g., by costly
screening of cDNA libraries, is unnecessary. The flow of
information from sequence analysis to function analysis is
therefore much shorter, because on the same day that an ORF is in
the computer from analysis of primary data, a corresponding vector
including the presumed gene can be synthesized and made
available.
[0147] Relative to conventional solid-phase synthesis for obtaining
synthetic DNA, the method according to the invention is
characterized by lower expenditure of materials. For the production
of thousands of different building blocks for the production of a
complex integrated genetic element with length of several 100,000
kbp, in correspondingly parallelized format and with corresponding
miniaturization (see Examples of application), a microfluidic
system uses far less feed material than a conventional automatic
machine for solid-phase synthesis for an individual DNA oligomer
(when using a single column). Here, microliters are contrasted with
the consumption of milliliters, i.e., a factor of 1000.
[0148] Taking into account the latest findings in immunology, the
method described permits extremely efficient and rapid vaccine
design (DNA vaccines).
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