U.S. patent application number 14/884883 was filed with the patent office on 2016-04-07 for methods and devices for in situ nucleic acid synthesis.
The applicant listed for this patent is Gen9, Inc.. Invention is credited to Joseph Jacobson, Senthil Ramu, Daniel Schindler.
Application Number | 20160097051 14/884883 |
Document ID | / |
Family ID | 44627167 |
Filed Date | 2016-04-07 |
United States Patent
Application |
20160097051 |
Kind Code |
A1 |
Jacobson; Joseph ; et
al. |
April 7, 2016 |
Methods and Devices for In Situ Nucleic Acid Synthesis
Abstract
Disclosed are compositions, methods and devices for the in situ
synthesis of nucleic acids. In an exemplary embodiment, a
support-bound oligonucleotide is elongated by addition of one or
more nucleotides by hybridization of a partially double-stranded
oligonucleotide, ligation and removal of unwanted nucleotides.
Inventors: |
Jacobson; Joseph; (Newton,
MA) ; Ramu; Senthil; (Cambridge, MA) ;
Schindler; Daniel; (Newton, MA) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Gen9, Inc. |
Cambridge |
MA |
US |
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|
Family ID: |
44627167 |
Appl. No.: |
14/884883 |
Filed: |
October 16, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13700291 |
Jul 12, 2013 |
9187777 |
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PCT/US2011/038079 |
May 26, 2011 |
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14884883 |
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61349585 |
May 28, 2010 |
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Current U.S.
Class: |
506/16 |
Current CPC
Class: |
C12N 15/1068 20130101;
C12N 15/1093 20130101; C12N 15/66 20130101; C12P 19/34
20130101 |
International
Class: |
C12N 15/10 20060101
C12N015/10 |
Claims
1. A composition for synthesizing a plurality of target nucleic
acids on a surface of a solid support, the composition comprising a
plurality of partially double-stranded oligonucleotides, each
having a first strand and a second strand such that each partially
double-stranded oligonucleotide comprises a double-stranded portion
and a 5' overhang on the second strand, wherein the double-stranded
portion comprises at least one predetermined addition nucleotide at
the 3' end of the first strand, and wherein the double-stranded
portion has a predefined sequence that is identical across the
plurality of partially double-stranded oligonucleotides.
2. The composition of claim 1 wherein the double-stranded portion
comprises a restriction enzyme binding site.
3. The composition of claim 1 wherein the shorter strand comprises
at least one ribonucleotide upstream of the at least one
predetermined addition nucleotide.
4. The composition of claim 1 wherein the 5' overhang comprises a
degenerate sequence.
5. The composition of claim 1, further comprising a support having
a plurality of features, each feature comprising a plurality of
single-stranded support-bound oligonucleotides that can be ligated
with the partially double-stranded oligonucleotides at the 3' end
of the predetermined addition nucleotide.
6. The composition of claim 5, wherein the plurality of
single-stranded support-bound oligonucleotides each have sequence
complementarity to the 5' overhang.
7. The composition of claim 5, further comprising an ink jet
programmed to provide the partially double-stranded
oligonucleotides at a select feature.
8. A composition for synthesizing a plurality of target nucleic
acids on a surface of a solid support, the composition comprising:
a support having a plurality of features, each feature comprising a
plurality of single-stranded support-bound oligonucleotides; and a
first partially double-stranded oligonucleotide comprising a first
strand and a second strand forming a double-stranded portion and a
5' overhang on the second strand, wherein the first strand
comprises a first predetermined ligatable addition nucleotide at
the 3' end; wherein the first partially double-stranded
oligonucleotide can be ligated to a first support-bound
oligonucleotide at the first predetermined ligatable addition
nucleotide, thereby generating a first ligation product comprising
the first predetermined addition nucleotide; and wherein the first
ligation product is cleavable thereby generating a first elongated
support-bound oligonucleotide comprising the first predetermined
addition nucleotide.
9. The composition of claim 8 further comprising a second partially
double-stranded oligonucleotide comprising a second predetermined
ligatable addition nucleotide.
10. The composition of claim 9 wherein the first and second
partially double-stranded oligonucleotides comprise in the
double-stranded portion an identical predefined sequence.
11. The composition of claim 8 wherein the double-stranded portion
comprises a restriction enzyme binding site.
12. The composition of claim 8 wherein the first ligation product
is cleavable by a restriction enzyme.
13. The composition of claim 8 wherein the shorter strand comprises
at least one ribonucleotide upstream of the first predetermined
ligatable addition nucleotide.
14. The composition of claim 13 wherein the first ligation product
is cleavable by an RNase.
15. The composition of claim 8 wherein the 5' overhang comprises a
degenerate sequence.
16. The composition of claim 8, wherein the plurality of
single-stranded support-bound oligonucleotides each have sequence
complementarity to the 5' overhang.
17. The composition of claim 8, further comprising an ink jet
programmed to provide the first partially double-stranded
oligonucleotide at a select feature.
18. The composition of claim 8 wherein the first partially
double-stranded oligonucleotide is prepared by hybridizing the
first strand to the second strand, wherein the first strand
comprises at its 5' end a predefined sequence and at its 3' end the
first predetermined ligatable addition nucleotide, and wherein the
second strand comprises at its 3' end a sequence complementary to
the predefined sequence and a nucleotide complementary to the first
predetermined ligatable addition nucleotide.
19. The composition of claim 8 wherein the first partially
double-stranded oligonucleotide comprises a detectable label.
20. The composition of claim 8 further comprising an imaging
system.
Description
RELATED APPLICATIONS
[0001] This application is a divisional patent application of U.S.
application Ser. No. 13/700,291, filed Nov. 27, 2012, which is a 35
U.S.C. .sctn.371 national phase patent application of International
Patent Application No. PCT/US2011/038079, filed May 26, 2011, which
claims the benefit of and priority to U.S. Provisional Application
No. 61/349,585, filed May 28, 2010, the entire disclosures of each
of which applications are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to nucleic acid synthesis and
more particularly to in situ synthesis of nucleic acids such as
oligonucleotides.
BACKGROUND
[0003] Synthetic biopolymers such as oligonucleotides play a
pivotal role in many fields such as molecular biology, forensic
science, and medical diagnostics. Oligonucleotides, in particular,
have become indispensable tools in modern biotechnology.
Oligonucleotides are being used in a wide variety of techniques,
ranging from diagnostic probing methods, PCR, antisense inhibition
of gene expression to nucleic acid assembly. This widespread use of
oligonucleotides has led to an increasing demand for rapid,
inexpensive and efficient methods for synthesizing
oligonucleotides.
[0004] Nucleic acid arrays such as DNA and RNA arrays are useful in
a variety of different fields such as diagnostics (for example
single polymorphism detection), genomics (for example genomic DNA
purification, differential gene expression analysis) and synthetic
biology (for example, gene synthesis).
SUMMARY
[0005] Aspects of the technology provided herein relate to devices,
methods and compositions for synthesizing nucleic acids (e.g.
polynucleotides or oligonucleotides) having a predefined sequence.
Aspects of the invention relate to the devices and methods for the
synthesis of a plurality of nucleic acids and/or libraries of
nucleic acids on a solid support. In one aspect, a device for
synthesizing a nucleic acid having a predetermined sequence is
provided.
[0006] Aspects of the invention relate to a method for synthesizing
a nucleic acid having a predefined sequence, the method comprising:
a) providing a support comprising an anchor oligonucleotide at a
first feature; b) hybridizing a partially double-stranded first
oligonucleotide to the anchor oligonucleotide wherein the first
oligonucleotide comprises a 5' overhang and ligatable predetermined
addition nucleotide at a 3' end of the double-stranded portion; c)
ligating the first oligonucleotide to the anchor oligonucleotide
thereby generating a first ligation product; and d) removing
unwanted nucleotides from the first ligation product thereby
generating a first elongated product comprising the predetermined
terminal nucleotide. Steps b) c) and d) may be repeated to
synthesize the nucleic acids having the predefined sequence. In
some embodiments, the anchor oligonucleotide is support-bound. Yet,
in other embodiments, the anchor oligonucleotide is in solution
within a droplet. In some embodiments, the support comprises a
plurality of features, each feature comprising a plurality of
single-stranded anchor oligonucleotides.
[0007] In some embodiments, the partially double-stranded
oligonucleotides comprises a double-stranded portion and a
single-stranded 5' overhang, wherein the single-stranded overhang
comprises degenerate bases. In some embodiments, the partially
double-stranded oligonucleotides are generated by hybridizing a
first construction oligonucleotide comprising at its 5' end, a
predefined sequence, and at its 3' end, the predetermined ligatable
addition nucleotide, to a longer oligonucleotide comprising from
its 5' end to its 3' end : a single-stranded overhang, a nucleotide
complementary to the predetermined addition nucleotide, and at its
3' end a sequence complementary to the 5' end predefined sequence
of the first construction oligonucleotide. The single-stranded
overhang may be complementary to the 5' end of the anchor
oligonucleotide.
[0008] In some embodiments, the nucleotide upstream of the
predetermined addition nucleotide on the first construction
oligonucleotide is a RNA base. In some embodiments, the step of
removing comprises cleaving the first ligation product using a
RNase.
[0009] In other embodiments, the double-stranded portion of the
partially double-stranded oligonucleotide comprises a restriction
endonuclease binding site. The restriction endonuclease may be a
type II S endonuclease. In some embodiments, removing nucleotides
from the ligation product comprises cleaving the ligation product
with a restriction enzyme. The cleavage provides an elongated
product comprising the predetermined nucleotide addition. In some
embodiments, nucleotides are melted off and washed off from the
elongated product.
[0010] In some embodiments, the partially double-stranded
oligonucleotides comprise a detectable label. The label is
preferably a fluorescent label. In some embodiments, the methods
further comprise analyzing the hybridization step and/or the
ligation step, wherein the presence of the detectable label is
indicative of the completion of the step(s). In some embodiments,
the method comprises analyzing the removal step wherein the absence
of detectable label is indicative of the completion of the step.
The steps of analyzing are preferably performed using an imaging
system such as a CCD.
[0011] In some embodiments, the partially double-stranded first
oligonucleotide is deposited at the first location or feature using
an ink jet device. In some embodiments, a plurality of partially
double-stranded oligonucleotides are provided at a plurality of
different features. The plurality of partially double-stranded
oligonucleotides preferably comprises a 5' single-stranded
overhang, a predetermined addition nucleotide and a double-stranded
portion and wherein the double-stranded portion is identical within
the plurality of oligonucleotides. The plurality of
oligonucleotides may differ only with at least one desired
nucleotide addition such as A, T, G or C. In some embodiments, the
first elongated product comprises one or more predetermined
addition nucleotides.
[0012] Another aspect of the invention relates to a composition for
synthesizing a plurality of nucleic acids on a surface of a solid
support, the composition comprising a plurality of partially
double-stranded oligonucleotide wherein the plurality of partially
double-stranded oligonucleotide comprises a 5' single-stranded
sequence and a double-stranded sequence comprising a predefined
sequence and a predetermined ligatable nucleotide addition at a 3'
end of the shorter strand of the double-stranded sequence, wherein
the double-stranded sequence is identical within the plurality of
oligonucleotides. In some embodiments, the double-stranded portion
comprises a restriction enzyme binding site. In some embodiments,
the shorter strand comprises at least one RNA base upstream of the
predetermined addition nucleotide. In some embodiments, the 5'
single-stranded sequence is a degenerate sequence.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a non-limiting schematic illustration of an
embodiment of a method for synthesizing nucleotide addition
constructs.
[0014] FIG. 2 is a non-limiting schematic illustration of an ink
jet based deposition of nucleotide addition constructs at discrete
features comprising support-bound oligonucleotides.
[0015] FIG. 3 is a non-limiting schematic illustration of the
process of single nucleotide addition to support-bound
oligonucleotides by hybridization of nucleotide addition constructs
to support-bound oligonucleotides and removal of unwanted
sequences.
[0016] FIG. 4 is a non-limiting schematic illustration of the
process of single nucleotide addition to support-bound
oligonucleotides by hybridization of nucleotide addition constructs
to support-bound oligonucleotides followed by a RNAse degradation
step.
[0017] FIG. 5 is a non-limiting schematic illustration of the
process of single nucleotide addition to support-bound
oligonucleotides by hybridization of nucleotide addition constructs
to support-bound oligonucleotides and removal of the unwanted
sequences by cleavage.
[0018] FIG. 6 is a non-limiting schematic illustration of the
process of single nucleotide addition to support-bound
oligonucleotides by hybridization of nucleotide addition constructs
NacT (80) and NacC (90) to support-bound oligonucleotides and
removal of the unwanted sequences by cleavage using a restriction
endonuclease. The partially double-stranded Nucleotide addition
construct NacT has the following sequence:
TABLE-US-00001 (SEQ ID NO: 1) 5' NNNNAGAGGAGC 3' 3' TCTCCTCG 5'
The partially double-stranded Nucleotide addition construct NacC
has the following sequence:
TABLE-US-00002 (SEQ ID NO: 2) 5' NNNNGGAGGAGC 3' 3' CCTCCTCG 5'
[0019] FIG. 7 is a non-limiting schematic illustration of the
process of a single nucleotide addition to support-bound
oligonucleotides by hybridization of nucleotide addition constructs
to support-bound oligonucleotides followed by deoxy-uracil base
excision.
[0020] FIG. 8 is a non-limiting schematic illustration of real time
or single molecule in-situ nucleic acid synthesis.
DETAILED DESCRIPTION OF THE INVENTION
[0021] Aspects of the invention relate to a device and/or methods
for synthesizing a nucleic acids having a desired or predetermined
sequence on a solid support. The device and methods described
herein permits relatively inexpensive, rapid, and high fidelity
construction of essentially any desired nucleic acid. Unless
defined otherwise below, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure belongs. Still,
certain elements are defined herein for the sake of clarity. It
must be noted that, as used in this specification and the appended
claims, the singular forms "a", "an" and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a polynucleotide" can include more than one
polynucleotide.
[0022] Aspects of the invention relate to the synthesis of nucleic
acids and more particularly to the in situ synthesis of nucleic
acids on the surface of a solid support. Oligonucleotides or
polynucleotides of any length can be produced by the devices and
methods described herein. In some embodiments, methods are provided
for generating high numbers of nucleic acids such a DNA, RNA or
oligonucleotides. As used herein the terms "nucleic acid",
"polynucleotide", "oligonucleotide" are used interchangeably and
refer to naturally-occurring or synthetic polymeric forms of
nucleotides. The oligonucleotides and nucleic acid molecules of the
present invention may be formed from naturally occurring
nucleotides, for example forming deoxyribonucleic acid (DNA) or
ribonucleic acid (RNA) molecules. Alternatively, the naturally
occurring oligonucleotides may include structural modifications to
alter their properties, such as in peptide nucleic acids (PNA) or
in locked nucleic acids (LNA). The solid phase synthesis of
oligonucleotides and nucleic acid molecules with naturally
occurring or artificial bases is well known in the art. The terms
should be understood to include equivalents, analogs of either RNA
or DNA made from nucleotide analogs and as applicable to the
embodiment being described, single-stranded or double-stranded
polynucleotides. Nucleotides useful in the invention include, for
example, naturally-occurring nucleotides (for example,
ribonucleotides or deoxyribonucleotides), or natural or synthetic
modifications of nucleotides, or artificial bases. As used herein,
the term monomer refers to a member of a set of small molecules
which are and can be joined together to from an oligomer, a polymer
or a compound composed of two or more members. The particular
ordering of monomers within a polymer is referred to herein as the
"sequence" of the polymer. The set of monomers includes but is not
limited to example, the set of common L-amino acids, the set of
D-amino acids, the set of synthetic and/or natural amino acids, the
set of nucleotides and the set of pentoses and hexoses. Aspects of
the invention described herein primarily with regard to the
preparation of oligonucleotides, but could readily be applied in
the preparation of other polymers such as peptides or polypeptides,
polysaccharides, phospholipids, heteropolymers, polyesters,
polycarbonates, polyureas, polyamides, polyethyleneimines,
polyarylene sulfides, polysiloxanes, polyimides, polyacetates, or
any other polymers.
[0023] In some embodiments, the methods provided herein use
oligonucleotides that are immobilized on a surface or substrate
(e.g., support-bound oligonucleotides). As used herein the term
"support" and "substrate" are used interchangeably and refers to a
porous or non-porous solvent insoluble material on which polymers
such as nucleic acids are synthesized or immobilized. As used
herein "porous" means that the material contains pores having
substantially uniform diameters (for example in the nm range).
Porous materials include paper, synthetic filters etc. In such
porous materials, the reaction may take place within the pores. The
support can have any one of a number of shapes, such as pin, strip,
plate, disk, rod, bends, cylindrical structure, particle, including
bead, nanoparticles and the like. The support can have variable
widths. The support can be hydrophilic or capable of being rendered
hydrophilic and includes inorganic powders such as silica,
magnesium sulfate, and alumina; natural polymeric materials,
particularly cellulosic materials and materials derived from
cellulose, such as fiber containing papers, e.g., filter paper,
chromatographic paper, etc.; synthetic or modified naturally
occurring polymers, such as nitrocellulose, cellulose acetate,
poly(vinyl chloride), polyacrylamide, cross linked dextran,
agarose, polyacrylate, polyethylene, polypropylene,
poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene
terephthalate), nylon, poly(vinyl butyrate), polyvinylidene
difluoride (PVDF) membrane, glass, controlled pore glass, magnetic
controlled pore glass, ceramics, metals, and the like etc.; either
used by themselves or in conjunction with other materials. In some
embodiments, oligonucleotides are synthesized on an array format.
For example, single-stranded oligonucleotides are synthesized in
situ on a common support wherein each oligonucleotide is
synthesized on a separate or discrete feature (or spot) on the
substrate. In preferred embodiments, single-stranded
oligonucleotides are bound to the surface of the support or
feature. As used herein the term "array" refers to an arrangement
of discrete features for storing, routing, amplifying and releasing
oligonucleotides or complementary oligonucleotides for further
reactions. In a preferred embodiment, the support or array is
addressable: the support includes two or more discrete addressable
features at a particular predetermined location (i.e., an
"address") on the support. Therefore, each oligonucleotide molecule
of the array is localized to a known and defined location on the
support. The sequence of each oligonucleotide can be determined
from its position on the support.
[0024] Various types of microarray manufacturing devices and
technologies have been described e.g. combinatorial array, ink
jetting, direct surface printing. There is a variety of methods
known to synthesize such nucleic acid molecules having a predefined
sequence. Oligonucleotide synthesis can be performed through
massively parallel custom syntheses on microchips (Zhou et al.
(2004) Nucleic Acids Res. 32:5409; Fodor et al. (1991) Science
251:767). Nucleic acid arrays are comprised of a surface to which
are attached to a set of oligonucleotides of specific or predefined
sequence, most typically in known location or address.
Pre-synthesized oligonucleotides may be attached to the surface or
a solid support or oligonucleotides may be synthesized in-situ.
Given the difficulty of separately synthesizing and then attaching
large numbers of different oligonucleotides to a surface, in-situ
synthesized arrays are typically of significantly greater density
that pre-synthesized oligonucleotides arrays, with spot densities
currently up to several million. In order to create an in-situ
synthesized array means are needed for directing the order of
nucleotide addition to the various positions on a surface.
Practically, all of these procedures rely on synthesis via chemical
reactions such as phosphoramidite chemistry and/or rely on both
acid labile and photolabile deprotection chemistries. For acid
labile chemistries, the local acid condition may be varied by means
which include the direct ink jet of an acid, the stamping of an
acid or the electrochemical generation of an acid or
photogeneration of an acid. For photolabile chemistries the local
optical condition may be varied by means of a steerable laser,
photomask or DMD. Spurious chemical reactions cause random base
errors in oligonucleotides. The techniques suffer from the
requirement of running organic chemistries which are sensitive to
moisture as well from dupurination errors which come from acid
exposure or from bond cleavage errors which come from repeated UV
exposure. Accordingly, there is a need in the art to provide for a
method and devices for the in situ synthesis of pluralities of
nucleic acids using a small number of biologically-based and
enzymatic reagents.
[0025] In some embodiments, oligonucleotides are attached, spotted,
immobilized, surface-bound, supported or synthesized on the
discrete features of the surface or array. Oligonucleotides may be
covalently attached to the surface or deposited on the surface.
Arrays may be constructed, custom ordered or purchased from a
commercial vendor (e.g., Agilent, Affymetrix, Nimblegen). Various
methods of construction are well known in the art e.g., maskless
array synthesizers, light directed methods utilizing masks, flow
channel methods, spotting methods etc. In some embodiments,
construction and/or selection oligonucleotides may be synthesized
on a solid support using maskless array synthesizer (MAS). Maskless
array synthesizers are described, for example, in PCT application
No. WO 99/42813 and in corresponding U.S. Pat. No. 6,375,903. Other
examples are known of maskless instruments which can fabricate a
custom DNA microarray in which each of the features in the array
has a single-stranded DNA molecule of desired sequence. Other
methods for synthesizing construction and/or selection
oligonucleotides include, for example, light-directed methods
utilizing masks, flow channel methods, spotting methods, pin-based
methods, and methods utilizing multiple supports. Light directed
methods utilizing masks (e.g., VLSIPS.TM. methods) for the
synthesis of oligonucleotides is described, for example, in U.S.
Pat. Nos. 5,143,854, 5,510,270 and 5,527,681. These methods involve
activating predefined regions of a solid support and then
contacting the support with a preselected monomer solution.
Selected regions can be activated by irradiation with a light
source through a mask much in the manner of photolithography
techniques used in integrated circuit fabrication. Other regions of
the support remain inactive because illumination is blocked by the
mask and they remain chemically protected. Thus, a light pattern
defines which regions of the support react with a given monomer. By
repeatedly activating different sets of predefined regions and
contacting different monomer solutions with the support, a diverse
array of polymers is produced on the support. Other steps, such as
washing unreacted monomer solution from the support, can be
optionally used. Other applicable methods include mechanical
techniques such as those described in U.S. Pat. No. 5,384,261.
Additional methods applicable to synthesis of construction and/or
selection oligonucleotides on a single support are described, for
example, in U.S. Pat. No. 5,384,261. For example, reagents may be
delivered to the support by either (1) flowing within a channel
defined on predefined regions or (2) "spotting" on predefined
regions. Other approaches, as well as combinations of spotting and
flowing, may be employed as well. In each instance, certain
activated regions of the support are mechanically separated from
other regions when the monomer solutions are delivered to the
various reaction sites. Flow channel methods involve, for example,
microfluidic systems to control synthesis of oligonucleotides on a
solid support. For example, diverse polymer sequences may be
synthesized at selected regions of a solid support by forming flow
channels on a surface of the support through which appropriate
reagents flow or in which appropriate reagents are placed. Spotting
methods for preparation of oligonucleotides on a solid support
involve delivering reactants in relatively small quantities by
directly depositing them in selected regions. In some steps, the
entire support surface can be sprayed or otherwise coated with a
solution, if it is more efficient to do so. Precisely measured
aliquots of monomer solutions may be deposited dropwise by a
dispenser that moves from region to region. Pin-based methods for
synthesis of oligonucleotides on a solid support are described, for
example, in U.S. Pat. No. 5,288,514. Pin-based methods utilize a
support having a plurality of pins or other extensions. The pins
are each inserted simultaneously into individual reagent containers
in a tray. An array of 96 pins is commonly utilized with a
96-container tray, such as a 96-well microtiter dish. Each tray is
filled with a particular reagent for coupling in a particular
chemical reaction on an individual pin. Accordingly, the trays will
often contain different reagents. Since the chemical reactions have
been optimized such that each of the reactions can be performed
under a relatively similar set of reaction conditions, it becomes
possible to conduct multiple chemical coupling steps
simultaneously.
[0026] In another embodiment, a plurality of oligonucleotides may
be synthesized on multiple supports. One example is a bead based
synthesis method which is described, for example, in U.S. Pat. Nos.
5,770,358; 5,639,603; and 5,541,061. For the synthesis of molecules
such as oligonucleotides on beads, a large plurality of beads is
suspended in a suitable carrier (such as water) in a container. The
beads are provided with optional spacer molecules having an active
site to which is complexed, optionally, a protecting group. At each
step of the synthesis, the beads are divided for coupling into a
plurality of containers. After the nascent oligonucleotide chains
are deprotected, a different monomer solution is added to each
container, so that on all beads in a given container, the same
nucleotide addition reaction occurs. The beads are then washed of
excess reagents, pooled in a single container, mixed and
re-distributed into another plurality of containers in preparation
for the next round of synthesis. It should be noted that by virtue
of the large number of beads utilized at the outset, there will
similarly be a large number of beads randomly dispersed in the
container, each having a unique oligonucleotide sequence
synthesized on a surface thereof after numerous rounds of
randomized addition of bases. An individual bead may be tagged with
a sequence which is unique to the double-stranded oligonucleotide
thereon, to allow for identification during use.
[0027] Pre-synthesized oligonucleotide and/or polynucleotide
sequences may be attached to a support or synthesized in situ using
light-directed methods, flow channel and spotting methods, inkjet
methods, pin-based methods and bead-based methods set forth in the
following references: McGall et al. (1996) Proc. Natl. Acad. Sci.
U.S.A. 93:13555; Synthetic DNA Arrays In Genetic Engineering, Vol.
20:111, Plenum Press (1998); Duggan et al. (1999) Nat. Genet.
S21:10; Microarrays: Making Them and Using Them In Microarray
Bioinformatics, Cambridge University Press, 2003; U.S. Patent
Application Publication Nos. 2003/0068633 and 2002/0081582; U.S.
Pat. Nos. 6,833,450, 6,830,890, 6,824,866, 6,800,439, 6,375,903 and
5,700,637; and PCT Publication Nos. WO 04/031399, WO 04/031351, WO
04/029586, WO 03/100012, WO 03/066212, WO 03/065038, WO 03/064699,
WO 03/064027, WO 03/064026, WO 03/046223, WO 03/040410 and WO
02/24597; the disclosures of which are incorporated herein by
reference in their entirety for all purposes. In some embodiments,
pre-synthesized oligonucleotides are attached to a support or are
synthesized using a spotting methodology wherein monomers solutions
are deposited dropwise by a dispenser that moves from region to
region (e.g., ink jet). In some embodiments, oligonucleotides are
spotted on a support using, for example, a mechanical wave actuated
dispenser.
[0028] One aspect of the invention relates to compositions useful
for the in situ synthesis of a plurality of oligonucleotides having
a predefined sequence onto a support. Another aspect of the
invention relates to a device for synthesizing a plurality of
oligonucleotides having a predetermined sequence on a solid
support. In some aspects of the invention, the compositions
described herein are particularly useful for fabricating an
addressable oligonucleotide array by in situ synthesis of
oligonucleotides on a solid support. In one such embodiment, at
each of the multiple different addresses on the support, the in
situ synthesis steps may be repeated so as to form a support
comprising a plurality of oligonucleotides (e.g. same or different
oligonucleotide sequences) at one or more different addresses on
the support. In some embodiments, the compositions of the invention
are deposited as droplets at those addresses using, for example, a
pulse jet printing system. The oligonucleotides can be produced by
disposing solutions on particular addressable positions in a
specific order in an iterative process. As used herein, the term
"predefined sequence" or "predetermined sequence" are used
interchangeably and means that the sequence of the polymer is known
and chosen before synthesis or assembly of the polymer. In
particular, aspects of the invention are described herein primarily
with regard to the preparation of nucleic acids molecules, the
sequence of the oligonucleotide or polynucleotide being known and
chosen before the synthesis or assembly of the nucleic acid
molecules. In one embodiment, "oligonucleotides" are short nucleic
acid molecules. For example, oligonucleotides may be from 10 to
about 300 nucleotides, from 20 to about 400 nucleotides, from 30 to
about 500 nucleotides, from 40 to about 600 nucleotides, or more
than about 600 nucleotides long. However, shorter or longer
oligonucleotides may be used. Each oligonucleotide may be designed
to have a different length.
[0029] In one aspect of the invention, a device for synthesizing a
plurality of nucleic acids having a predetermined sequence is
provided. The device can include a support having a plurality of
features, each feature having a plurality of anchor
oligonucleotides. In some embodiments, the plurality anchor
oligonucleotides having a predefined sequence are immobilized at
different discrete features of a solid support. In some
embodiments, the anchor oligonucleotides are single-stranded. In
some embodiments, the plurality of anchor oligonucleotide sequences
may comprise degenerate sequences. In some embodiments, the anchor
oligonucleotides are support-bound. In some embodiments, the device
comprises a solid support having a plurality of spots or features,
and each of the plurality of spots includes a plurality of
support-bound oligonucleotides. In some embodiments, the anchor
oligonucleotides are covalently linked through their 3' end to the
solid support. Yet, in other embodiments the anchor
oligonucleotides are covalently linked through their 5' end to the
solid support.
[0030] In some embodiments, the anchor or support-bound
oligonucleotides are immobilized through their 3' end. It should be
appreciated that by 3' end, it is meant the sequence downstream to
the 5' end and by 5' end it is meant the sequence upstream to the
3' end. For example, an oligonucleotide may be immobilized on the
support via a nucleotide sequence (e.g., a degenerate binding
sequence), a linker or spacer (e.g., a moiety that is not involved
in hybridization). In some embodiments, the anchor oligonucleotide
comprises a spacer or linker to separate the anchor oligonucleotide
sequence from the support. Useful spacers or linkers include
photocleavable linkers, or other traditional chemical linkers. In
one embodiment, oligonucleotides may be attached to a solid support
through a cleavable linkage moiety. For example, the solid support
may be functionalized to provide cleavable linkers for covalent
attachment to the oligonucleotides. The linker moiety may be of six
or more atoms in length. Alternatively, the cleavable moiety may be
within an oligonucleotide and may be introduced during in situ
synthesis. A broad variety of cleavable moieties are available in
the art of solid phase and microarray oligonucleotide synthesis
(see e.g., Pon, R., Methods Mol. Biol. 20:465-496 (1993); Verma et
al., Annu Rev. Biochem. 67:99-134 (1998); U.S. Pat. Nos. 5,739,386,
5,700,642 and 5,830,655; and U.S. Patent Publication Nos.
2003/0186226 and 2004/0106728). A suitable cleavable moiety may be
selected to be compatible with the nature of the protecting group
of the nucleoside bases, the choice of solid support, and/or the
mode of reagent delivery, among others. In an exemplary embodiment,
the oligonucleotides cleaved from the solid support contain a free
3'-OH end. Alternatively, the free 3'-OH end may also be obtained
by chemical or enzymatic treatment, following the cleavage of
oligonucleotides. The cleavable moiety may be removed under
conditions which do not degrade the oligonucleotides. Preferably
the linker may be cleaved using two approaches, either (a)
simultaneously under the same conditions as the deprotection step
or (b) subsequently utilizing a different condition or reagent for
linker cleavage after the completion of the deprotection step.
[0031] In other embodiments, the anchor oligonucleotides are in
solution. For example, the anchor oligonucleotides may be provided
within a discrete volume such as a droplet or microdroplet at
different discrete features. In some embodiments, discrete
microvolumes of between about 0.5 pL and about 100 nL may be used.
However, smaller or larger volumes may be used. In some
embodiments, a mechanical wave actuated dispenser may be used for
transferring volumes of less than 100 nL, less than 10 nL, less
than 5 nL, less than 100 pL, less than 10 pL, or less than 0.5
pL.
[0032] The device can further include a member for providing a
droplet to a first spot (or feature) having a plurality of
support-bound oligonucleotides. In some embodiments, the droplet
can include one or more compositions comprising oligonucleotides
(referred herein as nucleotide addition constructs) having a
specific or predetermined nucleotide to be added and/or reagents
that allow one or more of hybridizing, denaturing, chain extension
reaction, ligation, and digestion, so as to produce a first nucleic
acid product which includes the first nucleotide addition. In some
embodiments, different compositions or different nucleotide
addition constructs may be deposited at different addresses on the
support during any one iteration so as to generate an array of
predetermined oligonucleotide sequences (the different features of
the support having different predetermined oligonucleotide
sequences). One particularly useful way of depositing the
compositions is by depositing one or more droplet, each droplet
containing the desired reagent (e.g. nucleotide addition construct
or partially double-stranded oligonucleotide comprising the desired
nucleotide addition) from a pulse jet device spaced apart from the
support surface, onto the support surface. Prior art pulse jet
devices are available commercially for use in ink printing. The
device may also include a member for advancing microfluidic
communication from a first spot to a second spot on the
support.
[0033] One skilled in the art will appreciate that DNA microarrays
can have very high density of oligonucleotides on the surface
(approximately 10.sup.8 molecules per feature), which can generate
steric hindrance to polymerases needed for PCR or polymerase
extension or to the ligase for ligation reactions. Theoretically,
the oligonucleotides are generally spaced apart by about 2 nm to
about 6 nm. For polymerases, a typical 6-subunit enzyme can have a
diameter of about 12 nm. Therefore the support may need to be
custom treated to address the surface density issue such that the
spacing of surface-attached oligonucleotides can accommodate the
physical dimension of the enzyme. For example, a subset of the
oligonucleotides can be chemically or enzymatically cleaved, or
physically removed from the microarray. Other methods can also be
used to modify the oligonucleotides such that when primers are
applied and annealed to the oligonucleotides, at least some 3'
hydroxyl groups of the primers (start of DNA synthesis) are
accessible by polymerase. The number of accessible 3' hydroxyl
groups per spot can be stochastic or fixed. For example, the
primers, once annealed, can be treated to remove some active 3'
hydroxyl groups, leaving a stochastic number of 3' hydroxyl groups
that can be subject to chain extension reactions. In another
example, a large linker molecule (e.g., a concatamer) can be used
such that one and only one start of synthesis is available per
spot, or in a subset of the oligonucleotides per spot.
[0034] In some embodiments, a plurality of nucleotide acid
constructs are provided at different features of the support. In
some embodiments, the nucleic acid constructs (Nac) are partially
double-stranded or duplex oligonucleotides. As used herein, the
term "duplex" refers to a nucleic acid molecule that is at least
partially double-stranded. Each of the plurality of partially
double-stranded oligonucleotides comprises an oligonucleotide
strand comprising an identical or a different predetermined
sequence of X nucleotides, at least one predetermined nucleotide or
nucleotide complementary to the predetermined nucleotide and a
degenerate sequence of Y nucleotides. The terms "nucleoside" or
"nucleotide" are intended to include those moieties which contain
not only the known purine and pyrimidine bases, but also other
heterocyclic bases that have been modified. Such modifications
include methylated purines or pyrimidines, acylated purines or
pyrimidines, alkylated riboses or other heterocycles. In addition,
the terms "nucleoside" and "nucleotide" include those moieties that
contain not only conventional ribose and deoxyribose sugars, but
other sugars as well. Modified nucleosides or nucleotides also
include modifications on the sugar moiety, e.g., wherein one or
more of the hydroxyl groups are replaced with halogen atoms or
aliphatic groups, or are functionalized as ethers, amines, or the
like. In various embodiments, within each feature on the solid
support, each of the plurality of partially double-stranded
oligonucleotides includes an identical predetermined subunit
sequence of X nucleotides, at least one desired or predetermined
nucleotide or a nucleotide complementary to the desired addition
nucleotide and a degenerate sequence of Y nucleotides. In some
embodiments, X is between 2 and 50. More particularly, X is between
3 and 20. In some examples, X is 3, 4, 5, 6, 7, 8, 9, or 10. In
certain embodiments, Y is between 5 and 100. More particularly, Y
is between 5 and 20, or Y is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, or 20. In general, a sequence is called degenerate
if some of its positions have several possible bases. Assuming
.SIGMA.={T, C, A, G} is the DNA alphabet, a sequence (e.g. a
oligonucleotide) can be shown as S=x.sub.1x.sub.2 . . . x.sub.l,
where x.sub.i.OR right..SIGMA., x.sub.i.noteq.O and l is the length
of S. For example, in the oligonucleotide
P*={G}{G}{C,G}{A}{T,C,G}{A} the third position is C or G and the
fifth is C, T or G. The degeneracy of a sequence is the number of
unique sequence combinations it contains, which can be calculated
as d(S)=.PI..sup.l.sub.i=1|x.sub.i|. For example,
d(P*)=1.times.1.times.2.times.1.times.3.times.1=6. In various
embodiments, degenerate sequences can be used to improve the
tolerance of the annealing reaction such that any given
single-stranded oligonucleotide with a free 3'-OH group can bind to
the degenerate binding sequence.
[0035] In some examples, the device has at least 100, 1,000, 4,000,
10,000, 100,000, 1,000,000 or more different features (or "regions"
or "spots") at a particular location (i.e., an "address"). It
should be appreciated that a device may comprise one or more solid
supports. Depending upon the use, any or all of the arrays may be
the same or different from one another and each may contain
multiple spots or features.
[0036] In some embodiments, the duplex oligonucleotides or
partially double-stranded oligonucleotides comprises a nucleotide
addition construct sequence. In some embodiments, the nucleotide
addition sequence comprises at its 5' end a degenerate
single-stranded sequence. The nucleotide addition construct can be
introduced at a first feature so as to hybridize to the anchor
oligonucleotides through its 5' end binding sequence (e.g.
degenerate sequence or specific binding sequence) thereby forming a
first nucleic acid product: a anchor oligonucleotide-nucleotide
addition construct hybrid. In some embodiments, the duplex
oligonucleotide comprising a nucleotide addition construct is
deposited at a feature or location using an ink jet device or a
drop deposition from pulse jets device. As used herein the term
"depositing" means to position, place a composition at a specific
location on the surface to the support. Depositing includes
contacting one composition with another. Depositing may be manual
or automatic, e.g., "depositing" may be accomplished by automated
robotic devices. A "pulse jet" refers to any device which can
dispense drops of a fluid composition onto a support. Pulse jets
operate by delivering a pulse of pressure (such as by a
piezoelectric or thermoelectric element) to liquid adjacent an
outlet or orifice such that a drop can be dispensed therefrom.
[0037] FIG. 1 shows an exemplary method for producing nucleotide
addition construct (also referred herein as construction duplexes
or partially double-stranded oligonucleotides) which can hybridized
and/or be ligated to an anchor oligonucleotide immobilized onto a
solid support. In a preferred embodiment, the nucleotide addition
constructs (Nac), comprise a first oligonucleotide hybridized to a
second shorter construction oligonucleotide. In preferred
embodiments, the nucleotide addition constructs can be ligated to
the anchor oligonucleotides so as to confer a single or multiple
nucleotide addition in subsequent process steps. In some
embodiments, the first and second oligonucleotide sequences used to
prepare the nucleic acid constructs may be synthetized by standard
phosphoramidite synthesis. The degenerate nucleotide sequence can
be are generated by synthesizing the degenerate base positions with
a mixture of the corresponding nucleotide precursors. FIG. 1a shows
an exemplary composition comprising a first oligonucleotide (10)
comprising from the 5' end to the 3' end: a set of degenerate bases
N followed by a single base which is complimentary to the desired
addition nucleotide, in this case A, followed by a set of specific
nucleotides (labeled p.sub.1, p.sub.2, p.sub.3, p.sub.4 etc . . .
). A second, shorter construction oligonucleotide (12) is provided
comprising starting from the 3' end: the desired addition
nucleotide and a sequence (p'.sub.1, p'.sub.2, p'.sub.3, p'.sub.4
etc) which is complementary to the first nucleotide addition
construct bases p.sub.1, p.sub.2, p.sub.3, p.sub.4. In preferred
embodiments, the first oligonucleotide (10) and the second
oligonucleotide (12) are selectively hybridized under appropriate
hybridization conditions to form duplex (14) having a
double-stranded portion comprising the complementary bases and a 5'
(or 3') single-stranded portion. In some embodiments, the
single-stranded portion or overhang comprises a plurality of
degenerate bases. Yet, in other embodiments the single-stranded
portion or overhang comprises a sequence that is complementary to
the anchor or support-bound oligonucleotide. The terms "hybridizing
specifically to" and "specific hybridization" and "selectively
hybridize to," as used herein refer to the binding, duplexing, or
hybridizing of a nucleic acid molecule preferentially to a
particular nucleotide sequence under stringent conditions. In some
embodiments, the number of degenerate bases may be about 2, about
4, about 5 about 6 about 7 about 8 about 9 about 10, about 20,
about 25, about 30, about 50. Referring to FIG. 1, construction
duplex oligonucleotides are formed using a first oligonucleotide
having a sequence comprising (from 5' to 3') about five degenerate
bases at its 5' end, followed by a desired nucleotide addition
(e.g. A, T, G, C, U), followed by a predefined sequence p1, p2, p3,
p4. For example, the first oligonucleotide sequence (Nac) may be
NNNAp.sub.4p.sub.3p.sub.2p.sub.1, NNNTp.sub.4p.sub.3p.sub.2p.sub.1,
NNNCp.sub.4p.sub.3p.sub.2p.sub.1, or
NNNGp.sub.4p.sub.3p.sub.2p.sub.1 as illustrated in FIG. 1a-d. In
FIG. 1b-d, similar preparations or compositions are provided for
each of the four other possible nucleotide additions such that
construct (14) is the nucleotide addition construct for T (NacT),
construct (24) is the nucleotide addition construct for A (NacA),
construct (34) is the nucleotide addition construct for C (NacC),
and construct (44) is the nucleotide addition construct for G
(NacG).
[0038] In some embodiments, a first oligonucleotide is hybridized
through its degenerate bases to the support-bound oligonucleotide
at a first feature on the solid support. In other preferred
embodiment, a first oligonucleotide is hybridized to a second
oligonucleotide having a sequence complementary to part of the
first oligonucleotide sequences (e.g.
Tp'.sub.4p'.sub.3p'.sub.2p'.sub.1,
Ap'.sub.4p'.sub.3p'.sub.2p'.sub.1,
Cp'.sub.4p'.sub.3p'.sub.2p'.sub.1,
Gp'.sub.4p'.sub.3p'.sub.2p'.sub.1). In a preferred embodiment, the
second oligonucleotide is shorter than the first oligonucleotide
and hybridized to the 3'end of the first oligonucleotide.
[0039] In some embodiments, the bases p.sub.4p.sub.3p.sub.2p.sub.1
hybridized to their complementary bases
p'.sub.4p'.sub.3p'.sub.2p'.sub.1 and form a restriction enzyme
binding site. In another embodiment, the sequence
p'.sub.4p'.sub.3p'.sub.2p'.sub.1 etc. comprises one or more RNA
bases. In a preferred embodiment, at least the nucleotide base
closest to the specific addition base (p'.sub.4) is an RNA base. In
other embodiments, the sequence p'.sub.4p'.sub.3p'.sub.2p'.sub.1
etc. contains one or more uracil bases. In an exemplary embodiment,
at least the nucleotide base closest to the specific addition base
in the sequence of the second oligonucleotide (p'.sub.4) is an
uracil base.
[0040] In some embodiments, nucleotide addition constructs
comprising a desired or predetermined dinucleotide sequence (16
different nucleic acid constructs), or a desired trinucleotide
sequence (64 different nucleic acid constructs), or a desired
tetranucleotide sequence (256 different nucleic acid constructs)
etc. can be generated, added and hybridized to the plurality of
support-bound oligonucleotides.
[0041] FIG. 2 shows an exemplary process for the addition
nucleotide addition constructs at different features on a support.
In an exemplary embodiment, an ink jet based device is used for
depositing specific nucleotide addition constructs (Nac) at
specific locations (or features) on the surface of a support.
Referring to FIG. 2(a), an ink jet device (50) may be loaded with a
specific nucleotide addition construct (e.g. NacA, NacC, NacG,
NacT). FIG. 2a illustrates an exemplary embodiment wherein NacT
(14) is deposited within a droplet (55) onto a first feature on a
support (60) using an ink jet device (50). The ink jet (50) may be
programmed to fire one or more droplets (55) at specific locations
or features of a surface (60) comprising support-bound
oligonucleotides (70). According to preferred embodiments, the
single-stranded degenerate regions of the nucleic acid constructs
(also referred herein as construction duplex) are allowed to anneal
to the single-stranded support-bound oligonucleotides (70) to form
a nucleic acid construct-support-bound oligonucleotide hybrid (FIG.
2b, 80). Referring to FIG. 2c, a second ink jet head (52) is loaded
with a second specific nucleotide addition construct, for example
NacC (44). The ink jet (52) may be programmed to fire one or more
droplets (57) at a second set of specific locations or features on
the surface (60) to bind to the support-bound oligonucleotides
immobilized at that location thereby to form a nucleic acid
construct-support-bound oligonucleotide hybrid (90). One would
appreciate that a limitation on the number of bases that can be
added at one time may depend on the number of inkjet heads that can
be used. In some embodiments, sixty-four ink jets heads may be used
to allow for the addition of 3 bases at a time.
[0042] FIG. 3 shows an exemplary process to generate the addition
of a single nucleotide to an anchor or support-bound
oligonucleotide. In some embodiment, after formation of a nucleic
acid construct-support-bound oligonucleotide hybrid, at least a
portion of the nucleic acid construct-support-bound oligonucleotide
hybrid is degraded to expose the nucleotide addition at the 5' end
of the anchor oligonucleotide. As illustrated in FIG. 3 and FIG. 4,
the surface of the solid support comprises a plurality of different
nucleotide addition constructs hybridized to a plurality of
support-bound oligonucleotides at a plurality of different
locations on surface 60. In an exemplary embodiment, bases
p'.sub.1,p'.sub.2,p'.sub.3 etc. contain at least one RNA base. In a
preferred embodiment, at least the nucleotide closest to the
specific addition nucleotide (in this example downstream of
p'.sub.4) is an RNA base. FIG. 3a illustrates a NacT-support-bound
oligonucleotide hybrid (80) and a NacC-support-bound
oligonucleotide hybrid (90) bound at specific locations or features
on surface (60). In a subsequent step, and referring to FIG. 3b and
FIG. 4b, a ligase (for example a high temperature ligase) is
provided under conditions promoting the ligation of the 3' end of
the nucleic acid construct to the support-bound oligonucleotide. By
ligation is meant any method of joining two or more nucleotides to
each other. Ligation can include chemical or enzymatic ligation,
including DNA ligase I, DNA ligase II, DNA ligase III, DNA ligase
IV, E. Coli DNA ligase, T4 DNA ligase, T4 RNA ligase 1, T4 RNA
ligase 2, T7 ligase, T3 DNA ligase, thermostable ligase (taq
ligase) and the like. In some embodiments, a ribonuclease (for
example an RNase) or alkaline phosphatase is added to degrade said
bases p'.sub.1,p'.sub.2 etc. Any small number of bases (84, 94)
which remain hybridized to the elongated product (86, 96) may be
melted off and washed away. FIG. 4d illustrates the resulting
elongated product comprising the anchor oligonucleotides, including
the desired (predetermined) single nucleotide additions (for
example T, (86) and C, (96)). One would appreciate that the step of
hybridizing a duplex construction oligonucleotide to the
support-bound oligonucleotide immobilized to the support, ligating
the duplex construction oligonucleotide to the anchor
oligonucleotide, degrading at least a portion of the duplex
construction oligonucleotide (for example, using a RNase) can be
repeated so as to yield nucleic acids of a desired length and
predetermined sequence.
[0043] In other aspect of the invention, the double-stranded
portion of the nucleotide addition constructs (bases p1,p2,p3 etc.
hybridized to their complementary bases p'.sub.1,p'.sub.2,p'.sub.3
etc) are designed to provide the sequence corresponding to the
binding site for a restriction enzyme. FIG. 5 and FIG. 6 illustrate
an exemplary embodiment wherein NacT-support-bound oligonucleotide
hybrid (80) and a NacC-support-bound oligonucleotide hybrid (90)
are immobilized at specific locations or features on surface (60).
As described above, and referring to FIG. 5b and FIG. 6b, a ligase
(for example, a high temperature ligase) is provided at the
specific features under conditions promoting the ligation of the 3'
end of the Nac to the support-bound oligonucleotide, thereby
forming a duplex oligonucleotide (82, 92). In some embodiments, the
duplex oligonucleotide comprises a binding site for a restriction
enzyme. In a subsequent step, an appropriate restriction enzyme, is
provided under the appropriate conditions to promote a
double-strand cut. In a preferred embodiment, the restriction
enzyme is a type II endonuclease capable of cleaving the
double-stranded sequence just above the desired nucleotide addition
thereby forming a product comprising the support-bound
oligonucleotide hybrid with a single base addition (FIG. 6, 85 and
95). Accordingly, the elongated support-bound oligonucleotide
comprises a single nucleotide addition. One would appreciate that
depending on the cleavage site, the elongated support-bound
oligonucleotide may comprise 2, 3, 4 or more desired nucleotides
addition. In some embodiments, the endonuclease is BspQI or BsaI.
The term "type-IIs restriction endonuclease" refers to a
restriction endonuclease having a non-palindromic recognition
sequence and a cleavage site that occurs outside of the recognition
site (e.g., from 0 to about 20 nucleotides distal to the
recognition site). Type IIs restriction endonucleases may create a
nick in a double-stranded nucleic acid molecule or may create a
double-stranded break that produces either blunt or sticky ends
(e.g., either 5' or 3' overhangs). Examples of Type IIs
endonucleases include, for example, enzymes that produce a 3'
overhang, such as, for example, Bsr I, Bsm I, BstF5 I, BsrD I, Bts
I, Mnl I, BciV I, Hph I, Mbo II, Eci I, Acu I, Bpm I, Mme I, BsaX
I, Bcg I, Bae I, Bfi I, TspDT I, TspGW I, Taq II, Eco57 I, Eco57M
I, Gsu I, Ppi I, and Psr I; enzymes that produce a 5' overhang such
as, for example, BsmA I, Ple I, Fau I, Sap I, BspM I, SfaN I, Hga
I, Bvb I, Fok I, BceA I, BsmF I, Ksp632 I, Eco31 I, Esp3 I, Aar I;
and enzymes that produce a blunt end, such as, for example, Mly I
and Btr I. Type-IIs endonucleases are commercially available and
are well known in the art (New England Biolabs, Beverly,
Mass.).
[0044] In some embodiments, and referring to FIG. 5c-d and FIG.
6c-d, the sequence that remain hybridized to the product (85 and
86) may be melted off and washed off the feature thereby producing
the support-bound oligonucleotides having desired or predetermined
single base additions (86 and 96). These steps may be repeated to
add a plurality of nucleotides and until the target oligonucleotide
having the predefined sequence is produced.
[0045] In some embodiments, and referring to FIG. 7, the sequence
p'.sub.1,p'.sub.2,p'.sub.3 of the partially double-stranded
oligonucleotide may contain at least one uracil base. In a
preferred embodiment, the nucleotide base the closest to the
desired nucleotide addition (for example p'.sub.4 in FIG. 7) is a
uracil base. Referring to FIG. 7a, a NacT-anchor oligonucleotide
hybrid (80) and a NacC-anchor oligonucleotide hybrid (90) are
resident (e.g. immobilized) at specific locations on surface 60.
Referring to FIG. 7b , a ligase (e.g. high temperature ligase) is
provided under conditions promoting the ligation of the 3' end of
the nucleotide addition construct to the support-bound
oligonucleotide. In FIG. 7c, a mixture of uracil DNA glycosylase
(UDG) and endonuclease VIII (from Enzymatics.RTM.) is added to the
composition containing a uracil-containing oligonucleotide
sequence. UDG can catalyze the excision of the uracil base,
creating an abasic site with an intact phosphodiester backbone
while the lyase activity of endonuclease VIII breaks the
phosphodiester backbone at both the 3' and the 5' sides of the
abasic site separating the synthesis nucleotide(s) from the carrier
oligonucleotide. In a subsequent step, unwanted nucleotides may be
melted off and washed off thereby producing an elongated anchor or
support-bound oligonucleotide. In some embodiments, the elongated
support-bound oligonucleotide comprises a single base addition
(FIG. 7, 86 and 96). Yet, in other embodiments, the elongated
support-bound oligonucleotide comprises a dinucleotide, a
trinucleotide etc . . . addition. These steps may be repeated to
allow the synthesis of the nucleic acids (e.g. oligonucleotides)
having the predefined sequence and length.
[0046] Aspect of the invention relates to high fidelity in situ
oligonucleotide synthesis. Devices and methods to selectively
isolate the correct nucleic acid sequence from the incorrect
nucleic acid sequences are provided herein. One should appreciate
that the combined repetitive yield of the ligation and
digestion/degradation steps may limit the ultimate length of the
oligonucleotide to be synthesized. For instance, it is possible
that the nucleotide addition construct fails to hybridize to the
support-bound oligonucleotide, or that the nucleotide addition
construct fails to ligate to the support-bound oligonucleotide or
that the resulting nucleotide addition construct-support-bound
oligonucleotide hybrid is not properly digested or cleaved, leading
to an error-containing oligonucleotide such as an oligonucleotide
including a deletion or an addition in the intended synthesized
strand. Accordingly, some embodiments relate to the removal of
oligonucleotides from the synthesis process if the nucleotide
addition constructs (Nac) fails to hybridize and/or to be ligated
to the support-bound oligonucleotide. In some embodiments, after
every ligation step, the surface may be treated with alkaline
phosphatase. One would appreciate that oligonucleotides that were
successfully ligated will have a phosphate at their 5' end. The 5'
end phosphate can be removed with impunity. However, one skilled in
the art would appreciate that the support-bound oligonucleotides
which failed to hybridize and/or ligate with nucleotide addition
constructs will lose its 5' phosphate and will no longer be
available to undergo ligation. This can prevent these support-bound
oligonucleotides from further participating in the synthesis
process thereby preventing the synthesis of error-containing
nucleic acid sequences.
[0047] One would appreciate that if nucleotide addition
construct-anchor oligonucleotide hybrid is not properly digested or
cleaved, a sequence error such as a deletion may be introduced. For
example, incomplete cleavage in one round of synthesis can prevent
nucleotide addition in a next round and successful addition in a
next round would yield to a product sequence comprising a base pair
deletion. In some embodiments, alternating the use of restriction
enzyme specificity during the synthesis process may serve as a
blocking agent. In some embodiments, a first restriction enzyme can
be used after a first addition (e.g. after ligation of a first
nucleotide addition construct) and a second restriction enzyme can
be used after a second addition (e.g. after ligation of a second
nucleotide addition construct), each restriction enzyme having a
sequence specificity. The ligation products will be cleaved by the
restriction enzyme only if the contain the appropriate
sequence.
[0048] In some embodiments, a set of labeled nucleotide addition
constructs (Nac) consisting for example of NacT (140), NacA (150),
NacC (160) and NacG (170) are produced (FIG. 8). In certain
exemplary embodiments, a detectable label can be used to detect one
or more nucleotides and/or oligonucleotides described herein.
Examples of detectable markers include various radioactive
moieties, enzymes, prosthetic groups, fluorescent markers,
luminescent markers, bioluminescent markers, metal particles,
protein-protein binding pairs, protein-antibody binding pairs and
the like. In some embodiments, a fluorescent label such as a
flourophore or a chemiluminescent label is associated or attached
to the nucleotide addition construct. Examples of fluorescent
proteins include, but are not limited to, yellow fluorescent
protein (YFP), green fluorescence protein (GFP), cyan fluorescence
protein (CFP), umbelliferone, fluorescein, fluorescein
isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein,
dansyl chloride, phycoerythrin and the like. Examples of
bioluminescent markers include, but are not limited to, luciferase
(e.g., bacterial, firefly, click beetle and the like), luciferin,
aequorin and the like. Examples of enzyme systems having visually
detectable signals include, but are not limited to, galactosidases,
glucorimidases, phosphatases, peroxidases, cholinesterases and the
like. Identifiable markers also include radioactive compounds such
as .sup.125I, .sup.35S, .sup.14C, or .sup.3H. Identifiable markers
are commercially available from a variety of sources. Preferably
the oligonucleotide probes or nucleotides are fluorescently labeled
with four different fluorophores, each fluorophore being associated
to a particular base or nucleotide.
[0049] Some aspects of the invention relate to the analysis,
detection and characterization of the newly synthesized
oligonucleotides using an analysis device. In some embodiments, the
analysis device uses a CCD (or CMOS) optical sensor to allow rapid
imaging of the array. Surface-bound fluorescent labels from the
array fluoresce in response to the light. In some embodiments, the
fluorescent constructs are employed in combination with CCD imaging
systems. In some embodiments, the imaging system allows the
detection and/or measurement of labeled molecules, and/or
localization of the fluorescent signal. A computer can transform
the data into another format for presentation. The resulting data
can be displayed as an image with color in each region varying
according to the light emission.
[0050] In an exemplary embodiment, an ink jet (50) is loaded with a
specific nucleotide addition constructs, for example fluorophore
labeled NacT 140 (FIG. 8a). Ink jet (50) may be programmed to fire
one or more droplets (55) at specific locations of a surface (65)
which comprises anchor oligonucleotides (70). In some preferred
embodiments, the density of anchor oligonucleotides at each feature
is about one anchor oligonucleotide per optically resolvable
surface areal patch.
[0051] FIG. 8c illustrates the use of an ink jet to deposit labeled
nucleotide addition constructs to features comprising support-bound
oligonucleotides to form a Nac-support-bound oligonucleotide hybrid
(180). To confirm that the formation of the labeled
Nac-support-bound oligonucleotide hybrid, the fluorophore labeled
Nac may be excited such that it emits a photon (200) which may be
detected by a detector (210). In some embodiments, the detector
(210) is a fluorescent array detector such as a CCD (or CMOS)
imaging system which enables the imaging of a specific a region of
the support's surface or the entire surface in parallel. If no
fluorescence signal is detected, it is an indication that
nucleotide addition construct coupling to the anchor
oligonucleotide did not take place and the ink-jet can then be
directed to deposit a nucleotide addition construct an additional
time. Conversely, the same system may be used to confirm proper
nucleotide addition construct digestion or cleavage by observing
the disappearance of fluorescence signal after the digestion or
cleavage step. Additional steps may be taken to confirm proper
ligation by constructing a nucleotide addition construct that has
two separate fluorophores, one on the 3' up strand (e.g. upper
strand) and one on the 5' up strand (e.g. lower strand), for
example, each of different colors. In some embodiments, the
hybridization of the initial nucleotide addition construct to
support-bound oligonucleotide can be confirmed by fluorescently
observing the presence of both fluorophores. In some embodiments,
the ligase can be added and the 3' up strand of the nucleotide
addition construct can be melted off. If proper ligation has taken
place, one will observe the disappearance of the 3' up fluorophore
but not of the 5' up fluorophore. The 3' up oligonucleotide may
then be re-hybridized and followed by either restriction enzyme
cleavage or endonuclease digestion as discussed above (FIGS. 5 and
6). After restriction enzyme cleavage or endonuclease digestion
followed by melting, the fluorescent signal from both fluorophores
should disappear. One would appreciate that aspects of the
invention provide specific feedback or readouts for the different
step in the oligonucleotide synthesis such as i) hybridization of
the nucleotide addition construct to the anchor oligonucleotide,
ii) proper ligation to the anchor oligonucleotide iii) cleavage of
the unwanted portion of the nucleotide addition construct. In some
embodiments, in order to confer a high effective repetitive yield
for such in-situ nucleic acid synthesis, hybridization, ligation or
cleavage/digestion may be repeated upon failure of any of the
feedback readouts detailed above.
[0052] In some embodiments, the oligonucleotides are used for
assembling a target polynucleotide having a predefined sequence. In
some embodiments, the target polynucleotide may be assembled using
an assembly procedure that may include several parallel and/or
sequential reaction steps in which a plurality of different nucleic
acids or oligonucleotides are synthesized or immobilized,
amplified, and are combined in order to be assembled (e.g., by
extension or ligation) to generate a longer nucleic acid product to
be used for further assembly, cloning, or other applications (see
U.S. provisional application 61/235,677 and PCT application
PCT/US09/55267 which are incorporate herein by reference in their
entirety).
[0053] The present invention provides among other things novel
methods and devices for synthesis of nucleic acids. While specific
embodiments of the subject invention have been discussed, the above
specification is illustrative and not restrictive. Many variations
of the invention will become apparent to those skilled in the art
upon review of this specification. The full scope of the invention
should be determined by reference to the claims, along with their
full scope of equivalents, and the specification, along with such
variations.
Sequence CWU 1
1
2112DNAArtificial SequenceSynthetic construct 1nnnnagagga gc
12212DNAArtificial SequenceSynthetic construct 2nnnnggagga gc
12
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