U.S. patent application number 11/751604 was filed with the patent office on 2007-12-06 for microfluidic-based gene synthesis.
This patent application is currently assigned to MASSACHUSETTS INSTITUTE OF TECHNOLOGY. Invention is credited to Peter A. Carr, Joseph M. Jacobson, David Kong.
Application Number | 20070281309 11/751604 |
Document ID | / |
Family ID | 38724077 |
Filed Date | 2007-12-06 |
United States Patent
Application |
20070281309 |
Kind Code |
A1 |
Kong; David ; et
al. |
December 6, 2007 |
Microfluidic-based Gene Synthesis
Abstract
A method for synthesizing long DNA constructs from
oligonucleotide precursors directly within a microfluidic device
uses several oligonucleotides at once. A precursor mix containing
at least two oligonucleotide precursors with at least partial base
complementarity is introduced into an input of a microfluidic chip
and at least one cycle of at least one gene synthesis protocol are
applied to fabricate a DNA construct containing the sequence of at
least two oligonucleotide precursors. A method for the synthesis of
a modified DNA construct includes electroporating at least one
oligonucleotide encoding for at least one point mutation and having
homology with at least one DNA region of a target cell into the
target cell and incorporating the oligonucleotide into the target
cell DNA through the action of recombination protein beta or a
recombination protein beta functional homolog.
Inventors: |
Kong; David; (Lexington,
MA) ; Carr; Peter A.; (Medford, MA) ;
Jacobson; Joseph M.; (Newton, MA) |
Correspondence
Address: |
NORMA E HENDERSON;HENDERSON PATENT LAW
13 JEFFERSON DR
LONDONDERRY
NH
03053
US
|
Assignee: |
MASSACHUSETTS INSTITUTE OF
TECHNOLOGY
Cambridge
MA
|
Family ID: |
38724077 |
Appl. No.: |
11/751604 |
Filed: |
May 21, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60801812 |
May 19, 2006 |
|
|
|
Current U.S.
Class: |
435/6.14 ;
435/91.2 |
Current CPC
Class: |
C12N 15/902 20130101;
C12P 19/34 20130101; C12N 15/70 20130101 |
Class at
Publication: |
435/006 ;
435/091.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12P 19/34 20060101 C12P019/34 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with U.S. government support under
Grant Number CCR-0122419, awarded by the National Science
Foundation. The government has certain rights in this invention.
Claims
1. A method for the synthesis of at least one DNA construct from
oligonucleotide precursors, comprising the steps of: introducing a
precursor mix into an input of at least one microfluidic device
having at least one input, the precursor mix containing at least
two oligonucleotide precursors with at least partial base
complementarity; and applying at least one cycle of at least one
gene synthesis protocol to fabricate at least one DNA construct
containing the sequence of at least two of the oligonucleotide
precursors.
2. The method of claim 1, wherein the gene synthesis protocol
comprises at least the step of applying a time varying thermal
field to the precursor mix and at least one enzyme.
3. The method of claim 1, wherein the gene synthesis protocol
comprises at least the step of applying a force to move the
precursor mix relative to a spatial thermal gradient.
4. The method of claim 1, wherein the precursor mix comprises at
least polymerase and nucleosides suitable for polynucleotide
synthesis.
5. The method of claim 1 wherein the precursor mix comprises at
least ligase and the gene synthesis protocol comprises at least the
step of containing the mix for an effective incubation time.
6. The method of claim 1, wherein the oligonucleotide precursors
comprise a DNA tile set and the DNA construct is a DNA tile or a
set of DNA tiles.
7. The method of claim 1, wherein some portion of the
oligonucleotide precursors are derived from an oligonucleotide
array.
8. The method of claim 7, wherein the oligonucletide precursors are
cleaved from the oligonucleotide array.
9. The method of claim 8, wherein the means of oligonucleotide
cleavage is at least one enzyme.
10. The method of claim 8, wherein the means of oligonucleotide
cleavage is at least one chemical.
11. The method of claim 1, wherein the microfluidic device further
comprises means for hierarchical DNA assembly.
12. The method of claim 1, further comprising the steps of:
combining fabricated DNA constructs from multiple microfluidic
devices into at least an additional microfluidic device; and
applying at least one cycle of at least one gene synthesis protocol
to combine at least two of the DNA constructs to fabricate at least
one larger DNA construct.
13. The method of claim 1, wherein the DNA construct is a gene and
further comprising the step of expressing the gene as a protein
gene product.
14. The method of claim 1, further comprising the step of applying
at least one form of error correction.
15. The method of claim 14, wherein the error correction comprises
at least one cycle of hybridization by selection.
16. A method for the synthesis of a modified DNA construct
comprising the steps of: electroporating, into at least one target
cell, at least one oligonucleotide having homology with at least
one DNA region of the target cell and encoding for at least one
point mutation; and incorporating the oligonucleotide into the
target cell DNA through the action of recombination protein beta or
a recombination protein beta functional homolog.
17. The method of claim 16, wherein a plurality of oligonucleotides
is employed and the step of electroporating is repeated a plurality
of times.
18. The method of claim 16, wherein the step of electroporating is
carried out using an electroporation means comprised within a
microfluidic system.
19. A method for the synthesis of a modified DNA construct,
comprising the steps of: introducing, into at least one target
cell, at least one polynucleotide construct having homology with at
least one DNA region of the target cell and encoding for at least
one point mutation; generating, using the polynucleotide construct,
at least one oligonucleotide within the target cell having homology
with at least one DNA region of the target cell and encoding for at
least one point mutation; and incorporating the oligonucleotide
into the target cell DNA through the action of recombination
protein beta or a recombination protein beta functional homolog.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 60/801,812, filed May 19, 2006, the entire
disclosure of which is herein incorporated by reference.
FIELD OF THE TECHNOLOGY
[0003] The present invention relates to gene synthesis and, in
particular, to methods for the direct synthesis of a gene, genes,
gene systems, or long DNA constructs from oligonucleotide
precursors within a microfluidic device.
BACKGROUND
[0004] A great proportion of the activity and expenditure in the
field of molecular biology is devoted to the isolation,
modification, and expression of genes (sequences of nucleotides
ranging from .about.400 bases to several kilobases) and DNA
constructs (e.g. gene systems, such as metabolic pathways and other
long DNA constructs, ranging from several Kb to .about.300 Kb) for
a range of applications, including the production of protein-based
drugs, the production of chemicals and biofuels, and the creation
of protein libraries, gene knockouts, and other mutations and
modifications aimed at garnering a fundamental understanding of
molecular biology.
[0005] Although it has been feasible for some time to synthesize
small genes (<1 Kb) ab initio from synthetic oligonucleotides
(i.e. sequences of DNA up to .about.100 nucleotide bases) [e.g.
Gupta et al., Proc Nal Acad Sci, 57, 148 (1968); Stemmer et al.,
Gene, 164, 49 (1995)], the cost and error rate associated with this
procedure has been limiting in terms of the size gene or the size
of the gene library that can economically be synthesized. Recent
advances in the use of DNA oligonucleotide microarray-based methods
for synthesizing the synthetic oligonucleotide precursors [e.g.
Tian et. al., Nature, 432, 1050 (2004); Zhou et al., Nucleic Acids
Res, 32, 5409 (2004); Richmond et al., Nucleic Acids Res, 32,
5011], as well as error correcting methods [e.g. Carr et. al.,
Nucleic Acids Res, 32, e162 (2004); Tian et. al., Nature, 432, 1050
(2004)] for assembling such oligonucleotides into genes or larger
DNA constructs, coupled with the vast sequence knowledge that has
been accumulated through genome sequencing projects, have opened up
the new possibility of `bit to gene`, in which a gene or other DNA
construct can be downloaded or designed in silico and then directly
synthesized. However, such procedures still involve macroscopic
liquid volumes (typically greater than 1 microliter and as much as
milliliters) and macroscopic fluid handling, which typically
requires costly robotic handlers. In the case of microarray-derived
oligonucleotide precursors, these precursors also require
amplification, which introduces errors. Thus, the overall cost and
time currently associated with synthesizing gene-length and longer
DNA constructs (e.g. bacterial genomes) are prohibitive to
widespread use and availability.
[0006] Court et al. have previously disclosed methods for inducing
homologous combination using single-stranded nucleic acids [U.S.
Pat. App. Pub. No. 2005/0079618; Court et al., Apr. 14, 2005] and,
in Ellis et al., Proc Natl. Acad. Sci. USA 98:6742-6, 2001, single
oligonucleotides have been employed to modify a genome, but these
procedures do not solve these problems. What has been needed,
therefore, is the ability to make several changes at once, so that
gene-length and longer DNA constructs may be fabricated in a time
and cost-effective manner.
SUMMARY
[0007] The present invention is a method for synthesizing genes,
systems of genes, and other long DNA constructs from
oligonucleotide precursors directly within a microfluidic device.
It permits several changes to be made at once by using several
oligonucleotides at once. Use of the present invention keeps fluid
volumes small (typically less than 1 uL), obviates the need for
most robotics, and permits gene synthesis from oligonucleotide
precursors without requiring an initial amplification of such
nucleotides.
[0008] In one aspect, the present invention is a method to
fabricate genes (.about.400 bases to several kilobases), longer DNA
constructs (from several kilobases to several hundred kilobases),
and gene libraries directly within a microfluidic device. In
another aspect, the present invention provides methods for the
sequential assembly of genes that scale in assembly time
proportional to gene length, as well as hierarchical assembly
methods that scale logarithmically in gene length. In still another
aspect, the present invention provides means for directly coupling
a DNA oligonucleotide microarray to the gene synthesis microfluidic
device, which are capable in combination of going directly from
bits (design of the gene on computer) to physical DNA constructs
without any macroscopic sample handling and without requiring the
intervening amplification of oligonucleotides.
[0009] In one aspect of the present invention, a precursor mix
containing at least two oligonucleotide precursors with at least
partial base complementarity is introduced into an input of a
microfluidic chip and at least one means of oligonucleotide
cleavage and at least one cycle of at least one gene synthesis
protocol are applied to fabricate a DNA construct containing the
sequence of at least two oligonucleotide precursors. In a preferred
embodiment of this aspect, the means of oligonucleotide cleavage
comprises at least polymerase and gene synthesis protocol comprises
applying a time varying thermal field to the precursor mix to
fabricate the DNA construct. Alternatively, a force is applied to
move the precursor mix relative to a spatial thermal gradient. In
another embodiment, the precursor mix contains at least ligase and
the gene synthesis protocol comprises containing the mix for an
effective incubation time.
[0010] In another aspect of the present invention, a method for the
synthesis of a modified DNA construct includes electroporating at
least one oligonucleotide encoding for at least one point mutation
and having homology with at least one DNA region of a target cell
into the target cell and incorporating the oligonucleotide into the
target cell DNA through the action of recombination protein beta or
a recombination protein beta functional homolog.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Other aspects, advantages and novel features of the
invention will become more apparent from the following detailed
description of the invention when considered in conjunction with
the accompanying drawings wherein:
[0012] FIG. 1A is a schematic representation of an example
embodiment of a fluidic chamber enclosing a set of spots of
oligonucleotides that are utilized as building blocks for
synthesizing a single gene or other arbitrary DNA construct
according to one aspect of the present invention;
[0013] FIG. 1B is a schematic representation of an example
embodiment of a fluidic chamber enclosing a set of spots of
oligonucleotides that are utilized as building blocks for
synthesizing multiple genes or arbitrary DNA constructs
simultaneously according to another aspect of the present
invention;
[0014] FIG. 2A is a schematic representation of an example
embodiment of a fluidic architecture for single-stage hierarchical
gene synthesis according to the present invention;
[0015] FIG. 2B is a schematic representation of an example
embodiment of a fluidic architecture whereby n DNA fragments are
assembled into a larger DNA construct in a single stage according
to the present invention;
[0016] FIG. 2C is a schematic representation of an example
embodiment of a fluidic architecture employing multiple stages of
hierarchical gene synthesis according to the present invention;
[0017] FIG. 3 is a schematic representation of an example
embodiment of a fluidic architecture according to the present
invention wherein, upon completion of parallel gene synthesis, the
synthesized genes are subsequently expressed in vitro;
[0018] FIG. 4 is a graphical representation of the probability
fabricating clones containing 95% of N inserts as a function of
insertion probability for an example embodiment of a system,
according to an aspect of the present invention, undergoing 20
insertion cycles;
[0019] FIG. 5 is a schematic representation of an example
embodiment of on-chip hybridization by selection according to one
aspect of the present invention; and
[0020] FIG. 6 is a polyacrylamide gel electrophoresis (PAGE)
showing successful parallel synthesis of genes along with negative
controls in multiple chambers, according to an aspect of the
present invention.
DETAILED DESCRIPTION
[0021] The present invention is a set of preferred methods and
microfluidic designs wherein microfluidic chips are employed for
the synthesis of genes and other DNA constructs. Embodiments
include the use of DNA oligonucleotide microarrays in conjunction
with a microfluidic device for use in the synthesis of genes and
other DNA constructs. Additional on-chip processes according to the
present invention include on-chip error correction and
transcription and translation of the DNA gene into a protein gene
product.
[0022] As used herein, the following terms expressly include, but
are not to be limited to:
[0023] "DNA tile set" means a group of polynucleotide components
arranged into a specified spatial pattern.
[0024] "DNA oligonucleotide microarray" means a solid substrate
that is typically composed of glass, but could be composed of
silicon, PDMS, or a variety of other materials, which has arrayed
on its surface a multitude of oligonucleotide sequences, and
wherein each sequence is confined to specific, user-designed areas.
For each given area, or `oligonucleotide spot` which is associated
with a given oligonucleotide sequence, a large ensemble of DNA
molecules of that sequence (typically 10.sup.6-1.sup.10 molecules)
is present. A microarray can be composed of as many as hundreds of
thousands of discrete oligonucleotide spots. The terms "DNA
oligonucleotide microarray", "microarray", "array", and "microarray
chip" are used interchangeably.
[0025] "Electroporation" means the use of time-varying electric
fields to allow molecules outside a cell to penetrate the interior
of a cell. In the context of the invention, electroporation is used
to cause oligonucleotides to enter cells.
[0026] "Error correction" means removal of flawed, corrupt, or
otherwise undesired components from a larger set. In the context of
the invention, DNA error correction refers to the reduction of
undesired polynucleotide components, such as by removing them from
a pool of molecules or by altering the flawed molecules to produce
the desired ones.
[0027] "Microfluidic chip" means a device for manipulating
nanoliter to microliter volumes of liquid. Such devices frequently
contain features such as channels, chambers, and/or valves, and can
be fabricated from a variety of different materials, including, but
not limited to, glass and polydimethylsiloxane (PDMS). More complex
versions of these devices are sometimes referred to as a "lab on a
chip" reflecting the integration of several functions in one
device. The terms `microfluidic chip` and `microfluidic device` are
used interchangeably.
[0028] "Oligonucleotide precursor" means a polynucleotide used as a
component to produce a larger polynucleotide. An example would be a
50-mer deoxyribonucleotide used as a component to produce a 500
base pair DNA construct.
[0029] "Precursor mix" means a combination of two or more
oligonucleotide precursors.
[0030] "Recombination protein beta" means the beta protein from
bacteriophage lambda, or its functional equivalents/homologs (for
example, E. coli protein RecT). These proteins are capable of
mediating various types of recombination processes.
[0031] "Shotgun gene (or genome) modification" means the process of
using multiple oligonucleotides to direct the in vivo modification
of DNA molecules. For example, two oligonucleotides can be
electroporated into a cell, and each oligonucleotide can direct the
modification of a different region of the chromosomal DNA. This
usage is distinct from multisite site-directed mutagenesis, which
in the art refers to making multiple modifications to a DNA
molecule in vitro, typically in a highly purified system.
[0032] Single Chamber Microfluidic Gene Synthesis Systems. In a
first preferred embodiment, a microfluidic gene synthesis system
according to the present invention comprises a single microfluidic
chamber. FIG. 1A is a schematic representation of an example
embodiment of a fluidic chamber enclosing a set of spots of
oligonucleotides that are utilized as building blocks for
synthesizing a single gene or other arbitrary DNA construct. In
FIG. 1A, fluidic chamber 102 is aligned to enclose oligonucleotide
spots 104, 106, 108, 110, 112, 114, 116, 118, 120. Each of these
oligonucleotide spots, typically deployed on an oligonucleotide or
DNA array chip 121, is typically composed of 10.sup.6-10.sup.10
individual oligonucleotides of a given sequence. Each sequence is
designed such that the ensemble of molecules, represented in FIG.
1A by 9 distinct oligonucleotides, will assemble into a desired
gene or other arbitrary DNA construct in the presence of the
appropriate enzyme (for example, but not limited to, DNA
polymerase) and other reagents operating under the appropriate gene
synthesis protocols (for example, but not limited to, polymerase
assembly multiplexing), as is well known in the art of de novo gene
synthesis.
[0033] The device shown in FIG. 1A is operated as follows. First, a
reaction mixture, R1, containing reagents, such as DNA polymerase,
MlyI restriction enzyme (for enzymatic cleavage), dNTPs, primers,
Polymerase Chain Reaction (PCR) buffer, and 0.1%
n-dodecyl-beta-D-maltoside, or other suitable reagent mixtures
known in the art, is loaded into chamber 102 with valves 122 open.
Once loaded, valves 122 are closed, thus enclosing the reaction
mixture and oligonucleotide spots in a single volume. Typical
volumes are in the tens and hundreds of nL, though smaller and
larger fluid volumes are possible. Next, the temperature of the
fluid volume is elevated so that the restriction enzyme is active,
thus enabling cleavage of oligonucleotides 104, 106, 108, 110, 112,
114, 116, 118, 120 from microarray surface 121. Upon achieving
oligonucleotide release, the fluid volume is then thermocycled
according to standard PCR protocols (such as, but not limited to,
94.degree. C. for 30 seconds for dehybridization, 55.degree. C. for
30 seconds for annealing, 72.degree. C. for 60 seconds for
annealing, for a total of 45 cycles) to achieve full synthesis of
the desired double-stranded DNA molecules with sequence information
defined by oligonucleotides 104, 106, 108, 110, 112, 114, 116, 118,
120.
[0034] The discussion of the operation of the embodiment of FIG. 1A
assumes that oligonucleotides from the oligonucleotide spots are
cleaved enzymatically from the surface, as is well known in that
art of gene synthesis from oligonucleotide arrays. Examples of such
enzymatic cleavage include, but are not limited to, the use of
restriction enzymes such as MlyI, or other enzymes or combinations
of enzymes capable of cleaving single or double-stranded DNA such
as, but not limited to, Uracil DNA glycosylase (UDG) and DNA
Endonuclease IV. Other means of cleavage known in the art may also
be advantageously employed in the present invention, including, but
not limited to, chemical (base labile) cleavage of DNA molecules or
optical (photolabile) cleavage from the surface. PCR can also be
employed to generate building material for gene synthesis by
copying the oligonucleotides while they are still anchored to the
microarray.
[0035] FIG. 1B is a schematic representation of an example
embodiment of a fluidic chamber enclosing a set of spots of
oligonucleotides that are utilized as building blocks for
synthesizing multiple genes or arbitrary DNA constructs
simultaneously. In FIG. 1B, single chamber 102 encloses four sets
of oligonucleotide spots 124, 126, 128, 130, each of which
comprises oligonucleotides capable of assembling into four distinct
genes or other arbitrary DNA molecules. In this embodiment,
reaction mixture R2, containing similar reagents to mixture R1 of
FIG. 1A but with the exception of having different primers, is
introduced into chamber 102, and valves 122 are closed to isolate a
single reaction volume containing R2 and the various
oligonucleotide spots. A temperature regimen similar to that
employed in conjunction with the embodiment of FIG. 1A is employed
to cleave the oligonucleotides and synthesize multiple genes or
other arbitrary DNA molecules in a single chamber.
[0036] Multiple Chamber (Hierarchical) Microfluidic Gene Synthesis
Systems. A second preferred embodiment of the present invention
comprises multiple chamber (hierarchical) microfluidic gene
synthesis systems. Although single chambers are a good choice for
short DNA constructs, limits exist in terms of the maximum number
of oligonucleotides that can be assembled within a single reaction
chamber system, which consequently limits the size of the largest
possible DNA construct capable of being synthesized in a single
chamber. One solution to this problem is to utilize a hierarchical
approach for DNA synthesis wherein, in order to synthesize a
"large" desired construct, multiple "small" DNA segments are
synthesized in parallel in separate chambers and then introduced
into a subsequent chamber where, after mixing with fresh reagents
to replace those consumed in the initial reactions, such as, but
not limited to, polymerase and dNTPs, the larger desired DNA
molecule is then assembled.
[0037] An exemplary device capable of synthesizing a desired gene
from two gene segments 1 and 2, respectively, is shown in FIG. 2A,
which is is a schematic representation of an exemplary fluidic
architecture for single-stage hierarchical gene synthesis. In the
embodiment of FIG. 2A, reaction mix R3, containing the necessary
reagents for oligonucleotide cleavage and gene synthesis of gene
segment 1, is introduced into inlet 202 into a reaction volume
defined by valves 204, 206, 208, 210, which are closed, with R3
exiting through outlet port 212. Similarly, mix R4, containing the
necessary reagents for oligonucleotide cleavage and gene synthesis
of gene segment 2, is introduced into inlet 214 into a volume
defined by valves 216, 218, 220, 210, which are also closed, with
R4 exiting through outlet port 222. Next, valves 224 are closed
shut to seal reaction mixtures R3 and R4 into isolated volumes,
whereupon the device temperature is controlled to activate
enzymatic cleavage of the sets of oligonucleotide spots 225,
227.
[0038] Once cleaved, the reaction mixtures are thermocycled,
preferably in parallel, in order to achieve synthesis of gene
segments 1 and 2. The thermocycled reaction mixes R3 and R4, now
containing assembled gene segments 1 and 2, can then be collected
through outlet ports 226, 228, respectively, by opening valves 206,
218 and introducing pressurized air, water, or other collection
buffer through inlet 230 with valve 232 open and valve 234 closed.
Next, reaction mix R5, containing a fresh supply of polymerase,
dNTPs, primers, and other necessary reagents, is introduced through
inlet 236, with valve 234 still closed and valves 238, 240 open,
with R5 exiting through outlet 242. Once R5 has been introduced
into the fluidic, valves 208, 210, 220 are opened with valves 240,
244, 224, 234 being closed, thus bringing R3 and R4, both
containing gene segments 1 and 2, into contact with R5. Valves 208,
210, 220 are then actuated and employed as a peristaltic pump,
pushing the three fluid segments through rectangular loop 246 until
a homogeneous mixture is achieved. Upon completion of mixing, this
new mixture is then thermocycled, thus assembling the desired
full-length gene. The solution containing this gene can be
retrieved by introducing pressurized air, water, or other buffer
through inlet 230 with valve 232 closed, valve 234 open, and valve
238 closed, with the remaining valves maintaining their previous
configurations.
[0039] Many variations upon the method of hierarchical gene
synthesis of the present invention are possible and are within the
scope of the present invention. For instance, for single-stage
assemblies, such as the one shown in FIG. 2A, instead of combining
two gene segments, multiple chambers synthesizing n gene segments
can be combined in a similar fashion to assemble a large DNA
construct. This is depicted in FIG. 2B, which is a schematic
representation of an embodiment of a fluidic architecture wherein n
DNA fragments are assembled into a larger DNA construct in a single
stage. In FIG. 2B, chambers 252, 254, 256, 258, 260, up to chamber
no contain distinct DNA segments that, when introduced into chamber
262, are combined to yield the desired construct comprised of the
various gene segments in the previous hierarchy level.
[0040] Furthermore, multiple stages of assembly are possible. This
is illustrated in FIG. 2C, which is a schematic representation of
an exemplary embodiment of a fluidic architecture employing
multiple stages of hierarchical gene synthesis. As depicted in FIG.
2C, seven successive thermocycling reactions are conducted to
assemble 64 gene segments in chambers 262 to ultimately produce a
final desired product in chamber 274. Specifically, upon completion
of the 64 individual segments in chambers 262, the products are
then fed to chambers 264, whereupon they are combined into 32 gene
segments, whose products are then fed into chambers 266, and so on,
through chambers 268, 270, 272, until the final full-length product
is synthesized in chamber 274.
[0041] In the preceding discussion for both single chamber and
multiple chamber microfluidic gene synthesis systems, in a
preferred embodiment the oligonucleotide spots are prepared by
means of oligonucleotide or DNA chip array synthesis, as is well
known in the art (e.g. Affymetrix, Combimatrix). In order for the
oligonucleotides deployed on the surface of such a DNA array chip
to serve as the oligonucleotide spots employed in the present
invention, the oligonucleotide array chip should ideally be
interfaced to the microfluidic chamber. This fluidic-microarray
interfacing can be achieved by a variety of methods, including, but
not limited to, pressure-bonding or adhesive-bonding. In the case
of pressure-bonding, force is applied to sandwich the microarray
and fluidic together sufficiently to prevent the leakage of fluid
from the channels or other device features while the fluids are
being manipulated. In the case of adhesive-bonding, a patterned
adhesive of some kind such as, but not limited to, a double-sided
adhesive that has been patterned with a laser, is used to bond the
fluidic surface to the microarray surface. In other preferred
embodiments, materials such as, but not limited to, CYTOP, a
fluoropolymer, parylene, a vapor-deposited polymer, or
poly(dimethylsiloxane) (PDMS), a silicone-based elastomer, can be
utilized as the adhesive material. Again, the bonding must be
sufficient to prevent the leakage of fluid from channels or other
device features while the fluids are being manipulated.
Additionally, bonding between the fluidic and the microarray must
occur with appropriate registration, so that features on the
microarray, specifically the oligonucleotide spots but also
potentially other features, are properly aligned to the desired
features of the fluidic device.
[0042] It is necessary in general to control the temperature of the
fluidic device, and subsequently any fluid volumes within a device,
in order to facilitate the necessary chemical and biological
processes. The method for temperature control in the preferred
embodiment involves placing the hybrid fluidic-microarray device
onto a thermal heating block, for example a standard PCR
thermocycler with in situ adapter, but any of the methods known in
the art would be suitable and may be advantageously employed in the
present invention.
[0043] Protein Synthesis. Recently it has been demonstrated that
proteins can be expressed in cell-free environments, such as a
microfluidic volume, by employing commercially available in vitro
transcription/translation mixtures [Tabuchi et al., Proteomics 2,
430 (2002)]. Such a capability can readily be coupled with the
described gene synthesis protocols. FIG. 3 is a schematic
representation of an embodiment of a fluidic architecture according
to the present invention where, upon completion of parallel gene
synthesis, the synthesized genes are subsequently expressed in
vitro. As shown in FIG. 3, chambers 302, 304, 306 contain sets of
oligonucleotides 308, 310, 312, that represent three distinct genes
when assembled. Reaction mixtures R5, R6, and R7, containing the
necessary reagents to cleave and assemble the three sets of
oligonucleotides, are introduced through inlets 314, 316, 318 by
compacting against valve 320. Once loaded, valve 322 is closed to
isolate the fluid volumes for thermocycling and gene synthesis.
Once assembled, the three reaction mixtures are then introduced
into mixing chambers 324, 326, 328, whereupon they are combined
with in vitro transcription/translation mixtures introduced through
inlet 330. Mixing valves 332 are then employed to mix the gene
segments with the in vitro transcription/translation mix and, after
incubating at the appropriate temperature, transcription and
translation occur. The expressed proteins are then pushed to
chambers 334, 336, 338 for observation. As shown in FIG. 3, the
expressed proteins of the example shown are capable of fluorescing
red, yellow, and green (e.g. DsRed, YFP, and GFP).
[0044] On-chip Electroporation and Shotgun Gene Modification. A
further preferred embodiment of the present invention comprises a
microfluidic system capable of bringing about a large number of
simultaneous point mutations within a target genome by means of
electroporating a pool of N oligonucleotides into a cell, each
oligonucleotide coding for a point mutation, coupled with a
mechanism for recombining the oligonucleotides with corresponding
homologous regions of the cell's genome or other genetic material.
In an example employing this embodiment, there are N regions of a
genome to be homologously recombined with N oligonucleotides, each
coding for a different mutation that it is desired to introduce. If
the efficiency of each individual recombination event is given by
.epsilon., then the probability of swapping out any one region
after R attempts (e.g. after R repeated electroporation cycles) is
given by P.sub.1=1-(1-.epsilon.).sup.R, and the corresponding
probability of the original sequence remaining after R tries is
P.sub.0=(1-C).sup.R. If swapping operations are independent from
each other, then the overall probability of swapping out at least
M.sub.0 regions after R tries is given by: P SUM = M = M 0 N
.times. N ! M ! .times. ( N - M ) ! .times. P 1 M .times. P 0 N - M
##EQU1## Using this relation, it can be shown that, for a fixed R
(i.e. fixed number of attempted insertion cycles (e.g.
electroporation cycles)), there exists a threshold value for
.epsilon., .epsilon..sub.Threshold, above which it becomes
efficient to carry out shotgun modification.
[0045] FIG. 4 is a graphical representation of the probability of
fabricating clones containing 95% of N inserts as a function of
insertion probability for a system undergoing 20 insertion cycles
according to this aspect of the present invention. If M.sub.0 is
set to 95% of N (e.g., if N=326, then M.sub.0=310) then we have for
small R approximately: .epsilon..sub.Threshold.about.10e.sup.-5
{square root over (80R)}
[0046] for a method of shotgun modification. Table 1 shows (using
the exact expression) the number of insertion cycles R as a
function of insertion efficiency, .epsilon., required to guarantee
that 50% of clones will contain 95% or more of the desired N
regions. TABLE-US-00001 TABLE 1 .epsilon. R .92 1 .71 2 .39 5 .22
10 .025 100 .01 290 .001 2900
[0047] A preferred embodiment for carrying out the shotgun
modification procedure according to this aspect of the present
invention comprises a microfluidic system that includes
electroporation means, as known in the art, and that takes as input
a pool of N oligonucleotides with homology to the regions of the
existing genetic material of a cell, further encoding N mutations
that it is desired to introduce, and cells containing the
recombination protein beta or a functional homolog thereof The
oligonucleotides permeate the cell by means of electroporation and
are incorporated into the native cell's genetic material by the
beta protein mechanism, as described by Ellis et al., Proc Nal.
Acad. Sci. USA 98:6742-6, 2001. As shown previously, if .epsilon.
exceeds .epsilon..sub.Threshold, then repeated electroporations of
the oligonucleotide will converge such that the majority of the
cells will contain a preponderance of the desired point
mutations.
[0048] A simple example of the results of shotgun modification is
shown in Table 2. A modified strain of E. coli MG1655 bearing the
beta gene under control of a heat-sensitive repressor was induced
to produce beta protein, and treated to make electrocompetent as
common to the art. A mixture of three distinct oligonucleotides was
transported into the cell culture by electroporation, and the
chromosomes of the cells were modified by these oligonucleotides.
In this example, each of the three oligonucleotides encoded a point
mutation that would restore function to a previously inactivated
gene (for the antibiotic resistance marker genes kan, cat, and
bla). Growth of the resulting cell culture and plating onto
appropriate growth media with combinations of the appropriate
antibiotics revealed the efficiencies with which cells underwent
one, two, or three modifications. TABLE-US-00002 TABLE 2 Cell
sample 1 Cell sample 2 Bla.sup.R (single modification) 10.2% 9.3%
Cat.sup.R (single modification) 19.9% 12.6% Kan.sup.R (single
modification) 15.1% 18.6% Bla.sup.R Cat.sup.R (double modification)
1.9% 1.7% Bla.sup.R Kan.sup.R (double modification) 3.1% 4.3%
Cat.sup.R Kan.sup.R (double modification) 3.6% 3.9% Bla.sup.R
Cat.sup.R Kan.sup.R (triple modification) 0.55% 0.54%
[0049] Another embodiment of the invention is designed to produce
the desired oligonucleotides in vivo using a specially designed DNA
construct, for example, but not limited to, a bacterial artificial
chromosome (BAC). This construct contains the desired sequences
encoding one or more such oligonucleotides, as desired for shotgun
modification described above. One way to produce such
single-stranded oligonucleotides is to employ promoter and
transcription terminator elements to first generate a RNA version
of the desired oligonucleotide, followed by reverse transcription
to generate complementary a DNA-RNA hybrid, followed by digestion
of the RNA strand. A plurality of such elements would be encoded in
BAC form. The advantage of such an approach is that the DNA
construct generated in vitro only needs to be electroporated once.
Following electroporation, production of the desired
oligonucleotide (or oligonucleotides) would proceed in vivo,
followed by action of beta protein or a functional homolog to
incorporate the encoded change (or changes) into the host
chromosome. This process may proceed continually simply by allowing
the cells to grow in continuous culture, until reaching the point
where the desired changes are achieved.
[0050] On-chip Error Correction by Hybridization by Selection.
Another preferred embodiment of the present invention is a
microfluidic gene synthesis system that includes on-board error
correction. It has been shown that errors in gene synthesis can be
substantially reduced by purifying oligonucleotide building blocks
prior to synthesis. In particular, a process known as hybridization
by selection [Tian et al., Nature 432: 1050-4, 2004] can be
employed within a microfluidic chip, wherein oligonucleotides
released from the microarray are hybridized to oligonucleotides
containing complementary sequences, followed by washing and elution
of non-hybridized oligonucleotides [Tian et. al., Nature, 432, 1050
(2004)]. By repeating this process for multiple cycles, error-free
oligonucleotides can be preferentially retained, thus enabling the
synthesis of DNA constructs with fewer errors.
[0051] This process is shown in FIG. 5, which is a schematic
representation of an embodiment of on-chip hybridization by
selection, according to one aspect of the present invention,
wherein, upon release from the chip, oligonucleotides in solution
are hybridized to spots of oligonucleotides containing
complementary sequences, followed by washing and elution of
non-hybridized oligonucleotides. This is repeated for multiple
cycles, facilitating the preferential selection of error-free
oligonucleotides for DNA synthesis. In FIG. 5, reaction mixture R8,
containing only reagents necessary for oligonucleotide cleavage, is
introduced into chamber 502, whereupon oligonucleotides 504, 506,
508, 510, 512, 514 are released from the surface of the microarray.
The mixture containing these oligonucleotides is then pushed into
mixing chamber 516, which encloses oligonucleotide spots 518, 520,
522, 524, 526, 528, which are complementary to oligonucleotides
504, 506, 508, 510, 512, 514. The reaction mixture temperature is
then set to ideal conditions for annealing to occur, at which point
mixing valves 530 are employed to circulate the released
oligonucleotides over their anchored complementary counterparts,
thus facilitating rapid hybridization.
[0052] Once hybridized, oligonucleotides that are not hybridized,
and thus are likely to contain mutations, are then eluted out of
outlet 532. This process is repeated for multiple cycles, at which
point the temperature of the reaction mixture is raised so that the
preferentially retained oligonucleotides de-hybridize. They are
then pushed into reaction chamber 534, whereupon reaction mixture
R9, containing the necessary reagents for gene synthesis, is
introduced. Thermocycling is then conducted to synthesize the
desired DNA construct from relatively error-free oligonucleotide
building blocks. Again, it should be noted that, in other preferred
embodiments, reaction mixture R8 may alternatively already contain
oligonucleotides 518, 520, 522, 524, 526, and 528, thus eliminating
the need for cleavage of oligonucleotides or a microarray
substrate.
[0053] In a preferred embodiment, the microfluidic devices shown in
FIGS. 1A-B, 2A-C, 3, and 5, which contain within them various
chambers, channels, and valves, are composed of a polymeric
material such as, but not limited to, PDMS, a silicone-based
elastomer, and are constructed by methods of fabrication well known
in the art of multi-layer soft lithography. The microfluidic device
could also be composed of other polymeric materials, including, but
not limited to, polyethylene, polypropylene, and
polymethylmethacrylate. Hard materials including, but not limited
to, glass and silicon can also be employed, and these can be
machined via methods well-known to those skilled in the art of
microfabrication. Furthermore, the microfluidic chamber and all
connected channels that will contact reaction mixtures may
optionally be coated with additional materials including, but not
limited to, CYTOP, a fluoropolymer, or parylene, a vapor deposited
polymer. Such coatings can be achieved via methods well-known to
those skilled in the art of microfabrication. In a preferred
embodiment, the valves shown in FIGS. 1A-B, 2A-C, 3, and 5 are
fabricated and operated via methods known to those skilled in the
art of multilayer soft lithography [e.g. Unger, et al. Science,
288, 113 (2000)] or similar valving technologies, utilizing
polymeric materials such as, but not limited to, PDMS in
combination with hard materials such as, but not limited to, glass
[e.g. Grover, Sensors and Actuators B. 89,315 (2003)].
[0054] In other preferred embodiments, instead of cleaving
oligonucleotides from the surface of the microarray,
oligonucleotides may alternatively be introduced in the reaction
mixtures. For example, in FIG. 1A, oligonucleotides 104, 106, 108,
110, 112, 114, 116, 118, 120 can already be present in reaction
mixture R1 at the appropriate concentration (e.g. 25 nM each), thus
enabling synthesis of the desired gene without a microarray. In
such an embodiment, the "floor" of the microfluidic chamber may be
composed of a variety of materials including, but not limited to,
glass, silicon, PDMS, CYTOP, or parylene, and does not require the
presence of oligonucleotides on the surface of the reaction chamber
floor. Similarly, for the embodiments described in FIGS. 1B, 2A-C,
and 3, the oligonucleotides may also be introduced in the reaction
mixtures, thus not requiring the presence of the oligonucleotide
spots shown.
[0055] FIG. 6 is a polyacrylamide gel electrophoresis (PAGE) image
showing successful parallel synthesis of genes along with negative
controls in multiple chambers. In FIG. 6, gel image 610 verifies
the synthesis of GFP 620 and DsRed 630 DNA constructs by the method
of the present invention, wherein 42 and 26 oligonucleotides,
respectively, were included in the reaction mixture and assembled,
in parallel, in two chambers of a microfluidic device. Gel 610 also
depicts successful negative controls 640, 650, wherein reaction
mixtures that did not contain a complete set of oligonucleotides
were thermocycled in parallel in separate chambers, thus
experiencing the same temperature gradients, did not yield the
desired DNA constructs. In the presence of construction
oligonucleotides, DNA constructs GFP and dsRed (993 and 733 bp
respectively) and OR128-1 and ble (942 and 461 bp respectively) are
synthesized and amplified. Without construction oligonucleotides,
no product bands are generated. Molecular weight markers 660 (M)
are also shown, with 500, 750, and
[0056] bp positions indicated.
[0057] While a preferred embodiment of the present invention is
disclosed, many other implementations will occur to one of ordinary
skill in the art and are all within the scope of the invention.
Each of the various embodiments described above may be combined
with other described embodiments in order to provide multiple
features. Furthermore, while the foregoing describes a number of
separate embodiments of the apparatus and method of the present
invention, what has been described herein is merely illustrative of
the application of the principles of the present invention. Other
arrangements, methods, modifications, and substitutions by one of
ordinary skill in the art are therefore also considered to be
within the scope of the present invention, which is not to be
limited except by the claims that follow.
* * * * *