U.S. patent application number 17/019448 was filed with the patent office on 2021-03-04 for microarray synthesis and assembly of gene-length polynucleotides.
This patent application is currently assigned to Gen9, Inc.. The applicant listed for this patent is Gen9, Inc.. Invention is credited to Andrew V. Oleinikov.
Application Number | 20210062185 17/019448 |
Document ID | / |
Family ID | 1000005220480 |
Filed Date | 2021-03-04 |
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United States Patent
Application |
20210062185 |
Kind Code |
A1 |
Oleinikov; Andrew V. |
March 4, 2021 |
MICROARRAY SYNTHESIS AND ASSEMBLY OF GENE-LENGTH
POLYNUCLEOTIDES
Abstract
There is disclosed a process for in vitro synthesis and assembly
of long, gene-length polynucleotides based upon assembly of
multiple shorter oligonucleotides synthesized in situ on a
microarray platform. Specifically, there is disclosed a process for
in situ synthesis of oligonucleotide fragments on a solid phase
microarray platform and subsequent, "on device" assembly of larger
polynucleotides composed of a plurality of shorter oligonucleotide
fragments.
Inventors: |
Oleinikov; Andrew V.;
(Boston, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Gen9, Inc. |
Boston |
MA |
US |
|
|
Assignee: |
Gen9, Inc.
Boston
MA
|
Family ID: |
1000005220480 |
Appl. No.: |
17/019448 |
Filed: |
September 14, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16793632 |
Feb 18, 2020 |
10774325 |
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17019448 |
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15440293 |
Feb 23, 2017 |
10640764 |
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16793632 |
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14701957 |
May 1, 2015 |
10450560 |
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15440293 |
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13617685 |
Sep 14, 2012 |
9051666 |
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14701957 |
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13250207 |
Sep 30, 2011 |
9023601 |
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13617685 |
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12488662 |
Jun 22, 2009 |
8058004 |
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13250207 |
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10243367 |
Sep 12, 2002 |
7563600 |
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12488662 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01J 2219/00378
20130101; B01J 2219/00585 20130101; B01J 2219/00454 20130101; B01J
2219/00626 20130101; B82Y 30/00 20130101; B01J 2219/00689 20130101;
B01J 2219/00677 20130101; B01J 2219/00641 20130101; B01J 2219/00605
20130101; B01J 2219/00722 20130101; C12Q 1/6837 20130101; C40B
40/06 20130101; B01J 2219/005 20130101; B01J 19/0046 20130101; B01J
2219/00675 20130101; B01J 2219/00432 20130101; B01J 2219/00713
20130101; B01J 2219/00659 20130101; B01J 2219/00596 20130101; C40B
50/14 20130101; B01J 2219/00385 20130101; B01J 2219/00639 20130101;
C40B 80/00 20130101; B01J 2219/00608 20130101; C12N 15/1068
20130101; B01J 2219/00527 20130101 |
International
Class: |
C12N 15/10 20060101
C12N015/10; B82Y 30/00 20060101 B82Y030/00; C40B 80/00 20060101
C40B080/00; B01J 19/00 20060101 B01J019/00; C40B 50/14 20060101
C40B050/14; C12Q 1/6837 20060101 C12Q001/6837 |
Claims
1. (canceled)
2. A method for producing a polynucleotide comprising a target
sequence, the method comprising: (a) providing a plurality of
double-stranded oligonucleotides, each double-stranded
oligonucleotide comprising: (i) an internal sequence identical to a
portion of the target sequence, wherein the internal sequence, at
one or both ends, comprises an overlapping sequence region that: is
identical to an overlapping sequence region of another
double-stranded oligonucleotide in the plurality of double-stranded
oligonucleotides; and comprises a Type II restriction endonuclease
cleavage site; and (ii) one or more flanking sequences, wherein
each flanking sequence comprises a Type II restriction endonuclease
recognition site corresponding to the Type II restriction
endonuclease cleavage site of the internal sequence; (b) digesting
the plurality of double-stranded oligonucleotides with a Type II
restriction endonuclease that recognizes the Type II restriction
endonuclease recognition site; and (c) ligating the digested
oligonucleotides to produce the polynucleotide comprising the
target sequence.
3. The method of claim 2, wherein each double-stranded
oligonucleotide in (a) comprises a flanking sequence at each
end.
4. The method of claim 2, wherein the plurality of double-stranded
oligonucleotides are digested in (b) with a Type IIS restriction
endonuclease.
5. The method of claim 2, wherein the Type IIS restriction
endonuclease includes at least one of MylI, BspMI, BsaXI, BsrI,
BmrI, BtsI, and Fok1.
6. The method of claim 2, wherein the method does not comprise PCR
amplification prior to step (c).
7. The method of claim 2, further comprising: purifying the
digested oligonucleotides prior to ligation in (c); amplifying the
polynucleotide comprising the target sequence; or a combination
thereof.
8. A method for producing a polynucleotide comprising a target
sequence, the method comprising: (a) synthesizing on a surface a
plurality of single-stranded oligonucleotides, each single-stranded
oligonucleotide comprising: (i) an internal sequence identical to
or complementary to a portion of the target sequence, wherein the
internal sequence, at one or both ends, comprises an overlapping
sequence region that is identical to or complementary to an
overlapping sequence region of another single-stranded
oligonucleotide in the plurality of single-stranded
oligonucleotides; and (ii) flanking sequences, one at each end of
the internal sequence, each flanking sequence comprising a primer
binding site, and at least one flanking sequence further comprising
a Type II restriction endonuclease recognition site; (b) amplifying
the plurality of single-stranded oligonucleotides; (c) digesting
the amplified oligonucleotides with a Type II restriction
endonuclease that recognizes the Type II restriction endonuclease
recognition site; and (d) ligating the digested oligonucleotides to
produce the polynucleotide comprising the target sequence.
9. The method of claim 8, wherein each single-stranded
oligonucleotide in the plurality of single-stranded
oligonucleotides comprises flanking sequences, one at each end of
the internal sequence, each flanking sequence comprising: (i) a
primer binding site; and (ii) a Type II restriction endonuclease
recognition site.
10. The method of claim 8, wherein the plurality of single-stranded
oligonucleotides has been synthesized on a surface.
11. The method of claim 10, wherein the surface is a microarray or
wherein the surface comprises beads.
12. The method of claim 8, wherein the amplified oligonucleotides
are digested in (c) with a Type IIS restriction endonuclease.
13. The method of claim 12, wherein the Type IIS restriction
endonuclease includes at least one of MylI, BspMI, BsaXI, BsrI,
BmrI, BtsI, and Fok1.
14. The method of claim 8, further comprising: purifying the
digested oligonucleotides prior to ligation in (d); amplifying the
polynucleotide comprising the target sequence; or a combination
thereof.
15. A method for producing a polynucleotide comprising a target
sequence, the method comprising: (a) providing a plurality of
oligonucleotides, each oligonucleotide comprising: (i) an internal
sequence identical to or complementary to a portion of the target
sequence, wherein the internal sequence, at one or both ends,
comprises an overlapping sequence region that is identical to or
complementary to an overlapping sequence region of another
oligonucleotide in the plurality of oligonucleotides; and (ii)
flanking sequences, one at each end of the internal sequence, each
flanking sequence comprising a primer binding site, and at least
one flanking sequence further comprising a Type II restriction
endonuclease recognition site; (b) amplifying the plurality of
oligonucleotides; (c) digesting the amplified oligonucleotides with
a restriction endonuclease that recognizes the Type II restriction
endonuclease recognition site; and (d) ligating the digested
oligonucleotides to produce the polynucleotide comprising the
target sequence.
16. The method of claim 15, wherein each oligonucleotide in the
plurality of oligonucleotides comprises flanking sequences, one at
each end of the internal sequence, each flanking sequence
comprising: (i) a primer binding site; and (ii) a Type IIS
restriction endonuclease recognition site.
17. The method of claim 15, wherein the plurality of
oligonucleotides has been synthesized on a surface.
18. The method of claim 17, wherein the surface is a microarray or
comprises beads.
19. The method of claim 15, further comprising: purifying the
digested oligonucleotides prior to ligation in (d); amplifying the
polynucleotide comprising the target sequence; or a combination
thereof.
20. The method of claim 15, wherein the amplified oligonucleotides
are digested in (c) with a Type IIS restriction endonuclease.
21. The method of claim 20, wherein the Type IIS restriction
endonuclease includes at least one of MylI, BspMI, BsaXI, BsrI,
BmrI, BtsI, and Fok1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 16/793,632 filed Feb. 18, 2020, which is a continuation of U.S.
patent application Ser. No. 15/440,293 filed Feb. 23, 2017, now
U.S. Pat. No. 10,640,764, which is a continuation of U.S. patent
application Ser. No. 14/701,957 filed May 1, 2015, now U.S. Pat.
No. 10,450,560, which is a continuation of U.S. patent application
Ser. No. 13/617,685 filed Sep. 14, 2012, now U.S. Pat. No.
9,051,666, which is a continuation of U.S. patent application Ser.
No. 13/250,207 filed Sep. 30, 2011, now U.S. Pat. No. 9,023,601,
which is a continuation of U.S. patent application Ser. No.
12/488,662 filed Jun. 22, 2009, now U.S. Pat. No. 8,058,004, which
is a continuation of U.S. patent application Ser. No. 10/243,367
filed Sep. 12, 2002, now U.S. Pat. No. 7,563,600, the content of
all of which applications is incorporated herein by reference in
their entirety.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention provides a process for in vitro
synthesis and assembly of long, gene-length polynucleotides based
upon assembly of multiple shorter oligonucleotides synthesized in
situ on a microarray platform. Specifically, the present invention
provides a process for in situ synthesis of oligonucleotide
sequence fragments on a solid phase microarray platform and
subsequent, "on chip" assembly of larger polynucleotides composed
of a plurality of smaller oligonucleotide sequence fragments.
BACKGROUND OF THE INVENTION
[0003] In the world of microarrays, biological molecules (e.g.,
oligonucleotides, polypeptides and the like) are placed onto
surfaces at defined locations for potential binding with target
samples of nucleotides or receptors. Microarrays are miniaturized
arrays of biomolecules available or being developed on a variety of
platforms. Much of the initial focus for these microarrays have
been in genomics with an emphasis of single nucleotide
polymorphisms (SNPs) and genomic DNA detection/validation,
functional genomics and proteomics (Wilgenbus and Lichter, J Mal.
Med. 77:761, 1999; Ashfari et al., Cancer Res. 59:4759, 1999;
Kurian et al., J Pathol. 187:267, 1999; Hacia, Nature Genetics 21
suppl.:42, 1999; Hacia et al., Mol. Psychiatry 3:483, 1998; and
Johnson, Curr. Biol. 26:R171, 1998).
[0004] There are, in general, three categories of microarrays (also
called "biochips" and "DNA Arrays" and "Gene Chips" but this
descriptive name has been attempted to be a trademark) having
oligonucleotide content. Most often, the oligonucleotide
microarrays have a solid surface, usually silicon-based and most
often a glass microscopic slide. Oligonucleotide microarrays are
often made by different techniques, including (1) "spotting" by
depositing single nucleotides for in situ synthesis or completed
oligonucleotides by physical means (ink jet printing and the like),
(2) photolithographic techniques for in situ oligonucleotide
synthesis (see, for example, Fodor U.S. Patent '934 and the
additional patents that claim priority from this priority document,
(3) electrochemical in situ synthesis based upon pH based removal
of blocking chemical functional groups (see, for example,
Montgomery U.S. Pat. No. 6,092,302 the disclosure of which is
incorporated by reference herein and Southern U.S. Pat. No.
5,667,667), and (4) electric field attraction/repulsion of
fully-formed oligonucleotides (see, for example, Hollis et al.,
U.S. Pat. No. 5,653,939 and its duplicate Heller U.S. Pat. No.
5,929,208). Only the first three basic techniques can form
oligonucleotides in situ e.g., building each oligonucleotide,
nucleotide-by-nucleotide, on the microarray surface without placing
or attracting fully formed oligonucleotides.
[0005] With regard to placing fully formed oligonucleotides at
specific locations, various micro-spotting techniques using
computer-controlled plotters or even ink-jet printers have been
developed to spot oligonucleotides at defined locations. One
technique loads glass fibers having multiple capillaries drilled
through them with different oligonucleotides loaded into each
capillary tube. Microarray chips, often simply glass microscope
slides, are then stamped out much like a rubber stamp on each sheet
of paper of glass slide. It is also possible to use "spotting"
techniques to build oligonucleotides in situ. Essentially, this
involves "spotting" relevant single nucleotides at the exact
location or region on a slide (preferably a glass slide) where a
particular sequence of oligonucleotide is to be built. Therefore,
irrespective of whether or not fully formed oligonucleotides or
single nucleotides are added for in situ synthesis, spotting
techniques involve the precise placement of materials at specific
sites or regions using automated techniques.
[0006] Another technique involves a photolithography process
involving photomasks to build oligonucleotides in situ,
base-by-base, by providing a series of precise photomasks
coordinated with single nucleotide bases having light-cleavable
blocking groups. This technique is described in Fodor et al., U.S.
Pat. No. 5,445,934 and its various progeny patents. Essentially,
this technique provides for "solid-phase chemistry, photolabile
protecting groups, and photolithography . . . to achieve
light-directed spatially-addressable parallel chemical
synthesis."
[0007] The electrochemistry platform (Montgomery U.S. Pat. No.
6,092,302, the disclosure of which is incorporated by reference
herein) provides a microarray based upon a semiconductor chip
platform having a plurality of microelectrodes. This chip design
uses Complimentary Metal Oxide Semiconductor (CMOS) technology to
create high-density arrays of microelectrodes with parallel
addressing for selecting and controlling individual microelectrodes
within the array. The electrodes turned on with current flow
generate electrochemical reagents (particularly acidic protons) to
alter the pH in a small "virtual flask" region or volume adjacent
to the electrode. The microarray is coated with a porous matrix for
a reaction layer material. Thickness and porosity of the material
is carefully controlled and biomolecules are synthesized within
volumes of the porous matrix whose pH has been altered through
controlled diffusion of protons generated electrochemically and
whose diffusion is limited by diffusion coefficients and the
buffering capacities of solutions. However, in order to function
properly, the microarray biochips using electrochemistry means for
in situ synthesis has to alternate anodes and cathodes in the array
in order to generated needed protons (acids) at the anodes so that
the protons and other acidic electrochemically generated acidic
reagents will cause an acid pH shift and remove a blocking group
from a growing oligomer.
Gene Assembly
[0008] The preparation of arbitrary polynucleotide sequences is
useful in a "postgenomic" era because it provides any desirable
gene oligonucleotide or its fragment, or even whole genome material
of plasmids, phages and viruses. Such polynucleotides are long,
such as in excess of 1000 bases in length. In vitro synthesis of
oligonucleotides (given even the best yield conditions of
phosphoramidite chemistry) would not be feasible because each base
addition reaction is less than 100% yield. Therefore, researchers
desiring to obtain long polynucleotides of gene length or longer
had to turn to nature or gene isolation techniques to obtain
polynucleotides of such length. For the purposes of this patent
application, the term "polynucleotide" shall be used to refer to
nucleic acids (either single stranded or double stranded) that are
sufficiently long so as to be practically not feasible to make in
vitro through single base addition. In view of the exponential
drop-off in yields from nucleic acid synthesis chemistries, such as
phosphoramidite chemistry, such polynucleotides generally have
greater than 100 bases and often greater than 200 bases in length.
It should be noted that many commercially useful gene cDNA's often
have lengths in excess of 1000 bases.
[0009] Moreover, the term "oligonucleotides" or shorter term
"oligos" shall be used to refer to shorter length single stranded
or double stranded nucleic acids capable of in vitro synthesis and
generally shorter than 150 bases in length. While it is
theoretically possible to synthesize polynucleotides through single
base addition, the yield losses make it a practical impossibility
beyond 150 bases and certainly longer than 250 bases.
[0010] However, knowledge of the precise structure of the genetic
material is often not sufficient to obtain this material from
natural sources. Mature cDNA, which is a copy of an mRNA molecule,
can be obtained if the starting material contains the desired mRNA.
However, it is not always known if the particular mRNA is present
in a sample or the amount of the mRNA might be too low to obtain
the corresponding cDNA without significant difficulties. Also,
different levels of homology or splice variants may interfere with
obtaining one particular species of mRNA. On the other hand many
genomic materials might be not appropriate to prepare mature gene
(cDNA) due to exon-intron structure of genes in many different
genomes.
[0011] In addition, there is a need in the art for polynucleotides
not existing in nature to improve genomic research performance. In
general, the ability to obtain a polynucleotide of any desired
sequence just knowing the primary structure, for a reasonable
price, in a short period of time, will significantly move forward
several fields of biomedical research and clinical practice.
[0012] Assembly of long arbitrary polynucleotides from
oligonucleotides synthesized by organic synthesis and individually
purified has other problems. The assembly can be performed using
PCR or ligation methods. The synthesis and purification of many
different oligonucleotides by conventional methods (even using
multi-channel synthesizers) are laborious and expensive procedures.
The current price of assembled polynucleotide on the market is
about $12-25 per base pair, which can be considerable for
assembling larger polvnucleotides. Very often the amount of
conventionally synthesized oligonucleotides would be excessive.
This also contributes to the cost of the final product.
[0013] Therefore, there is a need in the art to provide
cost-effective polynucleotides by procedures that are not as
cumbersome and labor-intensive as present methods to be able to
provide polynucleotides at costs below $1 per base or 1-20 times
less than current methods. The present invention was made to
address this need.
SUMMARY OF THE INVENTION
[0014] The present invention provides a process for the assembly of
oligonucleotides synthesized on microarrays into a polynucleotide
sequence. The desired target polynucleotide sequence is dissected
into pieces of overlapping oligonucleotides. In the first
embodiment these oligonucleotides are synthesized in situ, in
parallel on a microarray chip in a non-cleavable form. A primer
extension process assembles the target polynucleotides. The primer
extension process uses starting primers that are specific for the
appropriate sequences. The last step is PCR amplification of the
final polynucleotide product. Preferably, the polynucleotide
product is a cDNA suitable for transcription purposes and further
comprising a promoter sequence for transcription.
[0015] The present invention provides a process for assembling a
polynucleotide from a plurality of oligonucleotides comprising:
[0016] (a) synthesizing or spotting a plurality of oligonucleotide
sequences on a microarray device or bead device having a solid or
porous surface, wherein a first oligonucleotide is oligo 1 and a
second oligonucleotide is oligo 2 and so on, wherein the plurality
of oligonucleotide sequences are attached to the solid or porous
surface, and wherein the first oligonucleotide sequence has an
overlapping sequence region of from about 10 to about 50 bases that
is the same or substantially the same as a region of a second
oligonucleotide sequence, and wherein the second oligonucleotide
sequence has an overlapping region with a third oligonucleotide
sequence and so on; [0017] (b) forming complementary oligo 1 by
extending primer 1, wherein primer 1 is complementary to oligo 1;
[0018] (c) disassociating complementary oligo 1 from oligo 1 and
annealing complementary oligo 1 to both oligo 1 and to the
overlapping region of oligo 2, wherein the annealing of
complementary oligo 1 to oligo 2 serves as a primer for extension
for forming complementary oligo 1+2; [0019] (d) repeating the
primer extension cycles of step (c) until a full-length
polynucleotide is produced; and [0020] (e) amplifying the assembled
complementary full length polynucleotide to produce a full length
polynucleotide in desired quantities.
[0021] Preferably, the solid or porous surface is in the form of a
microarray device. Most preferably, the microarray device is a
semiconductor device having a plurality of electrodes for
synthesizing oligonucleotides in situ using electrochemical means
to couple and decouple nucleotide bases. Preferably, the primer
extension reaction is conducted through a sequential process of
melting, annealing and then extension. Most preferably, the primer
extension reaction is conducted in a PCR amplification device using
the microarray having the plurality of oligonucleotides bound
thereto.
[0022] The present invention further provides a process for
assembling a polynucleotide from a plurality of oligonucleotides
comprising: [0023] (a) synthesizing in situ or spotting a plurality
of oligonucleotide sequences on a microarray device or bead device
each having a solid or porous surface, wherein the plurality of
oligonucleotide sequences are attached to the solid or porous
surface, and wherein each oligonucleotide sequence has an
overlapping region corresponding to a next oligonucleotide sequence
within the sequence and further comprises two flanking sequences,
one at the 3' end and the other at the 5' end of each
oligonucleotide, wherein each flanking sequence is from about 7 to
about 50 bases and comprising a primer region and a sequence
segment having a restriction enzyme cleavable site; [0024] (b)
amplifying each oligonucleotide using the primer regions of the
flanking sequence to form double stranded (ds) oligonucleotides;
[0025] (c) cleaving the oligonucleotide sequences at the
restriction enzyme cleavable site; and [0026] (d) assembling the
cleaved oligonucleotide sequences through the overlapping regions
to form a full length polynucleotide.
[0027] Preferably, the flanking sequence is from about 10 to about
20 bases in length. Preferably, the restriction enzyme cleavable
site is a class ii endonuclease restriction site sequence capable
of being cleaved by its corresponding class II restriction
endonuclease enzyme. Most preferably, the restriction endonuclease
class II site corresponds to restriction sites for a restriction
endonuclease class II enzyme selected from the group consisting of
Mly I, BspM I, Bae I, BsaX I, Bsr I, Bmr I, Btr I, Bts I, Fok I,
and combinations thereof. Preferably, the flanking sequence further
comprises a binding moiety used to purify cleaved oligonucleotides
from flanking sequences. Preferably, the process further comprises
the step of labeling the flanking sequence during the amplification
step (b) using primer sequences labeled with binding moieties. Most
preferably, a binding moiety is a small molecule able to be
captured, such as biotin captured by avidin or streptavidin, or
fluorescein able to be captured by an anti-fluorescein
antibody.
[0028] The present invention further provides a process for
assembling a polynucleotide from a plurality of oligonucleotides
comprising: [0029] (a) synthesizing in situ or spotting a plurality
of oligonucleotide sequences on a microarray device or bead device
each having a solid or porous surface, wherein the plurality of
oligonucleotide sequences are attached to the solid or porous
surface, and wherein each oligonucleotide sequence has an
overlapping region corresponding to a next oligonucleotide sequence
within the sequence, and further comprises a sequence segment
having a cleavable linker moiety; [0030] (b) cleaving the
oligonucleotide sequences at the cleavable linker site to cleave
each oligonucleotide complex from the microarray or bead solid
surface to form a soluble mixture of oligonucleotides, each having
an overlapping sequence; and [0031] (c) assembling the
oligonucleotide sequences through the overlapping regions to form a
full length polynucleotide.
[0032] Preferably, the cleavable linker is a chemical composition
having a succinate moiety bound to a nucleotide moiety such that
cleavage produces a 3'hydroxy nucleotide. Most preferably, the
cleavable linker is selected from the group consisting of
5'-dimethoxytrityl-thymidine-3'succinate,
4-N-benzoyl-5'-dimethoxytrityl-deoxycytidine-3'-succinate,
1-N-benzoyl-5'-dimethoxytrityl-deoxyadenosine-3'-succinate,
2-N-isobutyryl-5'-dimethoxytrityl-deoxyguanosine-3'-succinate, and
combinations thereof.
[0033] The present invention further provides a process for
assembling a polynucleotide from a plurality of oligonucleotides
comprising: [0034] (a) synthesizing in situ or spotting a plurality
of oligonucleotide sequences on a microarray device or bead device
each having a solid or porous surface, wherein the plurality of
oligonucleotide sequences are attached to the solid or porous
surface, and wherein each oligonucleotide sequence has a flanking
region at an end attached to the solid or porous surface, and a
specific region designed by dissecting the polynucleotide sequence
into a plurality of overlapping oligonucleotides, wherein a first
overlapping sequence on a first oligonucleotide corresponds to a
second overlapping sequence of a second oligonucleotide, and
wherein the flanking sequence comprises a sequence segment having a
restriction endonuclease (RE) recognition sequence capable of being
cleaved by a corresponding RE enzyme; [0035] (b) hybridizing an
oligonucleotide sequence complementary to the flanking region to
form a double stranded sequence capable of interacting with the
corresponding RE enzyme; [0036] (c) digesting the plurality of
oligonucleotides to cleave them from the microarray device or beads
into a solution; and [0037] (d) assembling the oligonucleotide
mixture through the overlapping regions to form a full length
polynucleotide.
[0038] Preferably, the flanking sequence is from about 10 to about
20 bases in length. Preferably, the restriction enzyme cleavable
site is a class II endonuclease restriction site sequence capable
of being cleaved by its corresponding class II restriction
endonuclease enzyme. Most preferably, the restriction endonuclease
class II site corresponds to restriction sites for a restriction
endonuclease class II enzyme selected from the group consisting of
Mly I, BspM I, Bae I, BsaX I, Bsr I, Bmr I, Btr I, Bts I, Fok I,
and combinations thereof. Preferably, the process further comprises
a final step of amplifying the polynucleotide sequence using
primers located at both ends of the polynucleotide.
[0039] The present invention further provides a process for
creating a mixture of oligonucleotide sequences in solution
comprising: [0040] (a) synthesizing in situ or spotting a plurality
of oligonucleotide sequences on a microarray device or bead device
each having a solid or porous surface, wherein the plurality of
oligonucleotide sequences are attached to the solid or porous
surface, and wherein each oligonucleotide sequence further
comprises two flanking sequences, one at the 3' end and the other
at the 5' end of each oligonucleotide, wherein each flanking
sequence is from about 7 to about 50 bases and comprising a primer
region and a sequence segment having a restriction enzyme cleavable
site; [0041] (b) amplifying each oligonucleotide using the primer
regions of the flanking sequence to form a double stranded (ds)
oligonucleotides; and [0042] (c) cleaving the double stranded
oligonucleotide sequences at the restriction enzyme cleavable
site.
[0043] Preferably, the flanking sequence is from about 10 to about
20 bases in length. Preferably, the restriction enzyme cleavable
site is a class II endonuclease restriction site sequence capable
of being cleaved by its corresponding class II restriction
endonuclease enzyme. Most preferably, the restriction endonuclease
class II site corresponds to restriction sites for a restriction
endonuclease class II enzyme selected from the group consisting of
Mly I, BspM I, Bae I, BsaX I, Bsr I, Bmr I, Btr I, Bts I, Fok I,
and combinations thereof. Preferably, the flanking sequence further
comprises a binding moiety used to purify cleaved oligonucleotides
from flanking sequences. Preferably, the process further comprises
the step of labeling the flanking sequence during the amplification
step (h) using primer sequences labeled with binding moieties. Most
preferably, a binding moiety is a small molecule able to be
captured, such as biotin captured by avidin or streptavidin, or
fluorescein able to be captured by an anti-fluorescein
antibody.
[0044] The present invention further provides a process for
creating a mixture of oligonucleotide sequences in solution
comprising: [0045] (a) synthesizing in situ or spotting a plurality
of oligonucleotide sequences on a microarray device or bead device
each having a solid or porous surface, wherein the plurality of
oligonucleotide sequences are attached to the solid or porous
surface, and wherein each oligonucleotide sequence has a sequence
segment having a cleavable linker moiety; [0046] (b) cleaving the
oligonucleotide sequences at the cleavable linker site to cleave
each oligonucleotide sequence from the microarray or bead solid
surface to form a soluble mixture of oligonucleotides.
[0047] Preferably, the cleavable linker is a chemical composition
having a succinate moiety bound to a nucleotide moiety such that
cleavage produces a 3'hydroxy nucleotide. Most preferably, the
cleavable linker is selected from the group consisting of
5'-dimethoxytrityl-thymidine-3'succinate,
4-N-benzoyl-5'-dimethoxytrityl-deoxycytidine-3'-succinate,
1-N-benzoyl-5'-dimethoxytrityl-deoxyadenosine-3'-succinate,
2-N-isobutyryl-5'-dimethoxytrityl-deoxyguanosine-3'-succinate, and
combinations thereof.
[0048] The present invention further provides a process for
creating a mixture of oligonucleotide sequences in solution
comprising: [0049] (a) synthesizing in situ or spotting a plurality
of oligonucleotide sequences on a microarray device or bead device
each having a solid or porous surface, wherein the plurality of
oligonucleotide sequences are attached to the solid or porous
surface, and wherein each oligonucleotide sequence has a flanking
region at an end attached to the solid or porous surface, and a
specific region, wherein the flanking sequence comprises a sequence
segment having a restriction endonuclease (RE) recognition sequence
capable of being cleaved by a corresponding RE enzyme; [0050] (b)
hybridizing an oligonucleotide sequence complementary to the
flanking region to form a double stranded sequence capable of
interacting with the corresponding RE enzyme; [0051] (c) digesting
the plurality of oligonucleotides to cleave them from the
microarray device or beads into a solution.
[0052] Preferably, the flanking sequence is from about 10 to about
20 bases in length. Preferably, the restriction enzyme cleavable
site is a class II endonuclease restriction site sequence capable
of being cleaved by its corresponding class II restriction
endonuclease enzyme. Most preferably, the restriction endonuclease
class II site corresponds to restriction sites for a restriction
endonuclease class II enzyme selected from the group consisting of
Mly I, BspM I, Bae I, BsaX I, Bsr I, Bmr I, Btr I, Bts I, Fok I,
and combinations thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0053] FIGS. 1A-1C show schematics of gene assembly on a microarray
device surface or porous matrix. In FIG. 1A, the target gene
sequence is dissected into number of overlapping oligonucleotides.
The 3' and 5' are the ends of the shown strand. FIG. 1A also shows,
relative to the target sequence, primer Pr1; extension product of
primer Pr1, which is complementary to oligonucleotide 1; and
extension product of complementary oligonucleotide 1, which is
complementary to oligonucleotides 1+2. FIG. 1B illustrates one
embodiment of the initial steps of an assembly process. In step 1
of assembly, Primer Pr1 is annealed to oligonucleotide 1 and
extended by appropriate polymerase enzyme into product
complementary to oligonucleotide 1. The second step is melting,
re-annealing and extension (i.e., amplification) to lead to
production of larger amount of Pr1 extension product (complementary
oligonucleotide 1), re-association of the complementary
oligonucleotide 1 with oligonucleotide 1, and to annealing of the
complementary oligonucleotide 1 with oligonucleotide 2 followed by
its extension into product complementary to oligonucleotides 1+2.
FIG. 1C shows a continuation of the assembly process from FIG. 1B.
Specifically, step 3 of the process (i.e., melting, re-annealing
and extension) leads to the same products as step 2 plus a product
complementary to oligonucleotides 1+2+3. Cycles (steps) are
repeated until a full-length complementary polynucleotide is
formed. The final step is preparation of the final target
polynucleotide molecule in desirable amounts by amplification
(i.e., PCR) using two primers complementary to the ends of this
molecule (PrX and PrY).
[0054] FIG. 2 shows a second embodiment of the inventive gene
assembly process using oligonucleotides synthesized in situ onto a
microarray device, each having a flanking sequence region
containing a restriction enzyme cleavage site, followed by a PCR
amplification step and followed by a REIT restriction enzyme
cleavage step.
[0055] FIGS. 3A-3C show schematics for gene assembly using oligos
synthesized and then cleaved from a microarray device.
Specifically, in FIG. 3A, oligonucleotide sequences are connected
to the microarray device through a cleavable linker (CL) moiety. An
example of a cleavable linker moiety is provided in FIG. 3A. The
cleavable linkers are molecules that can withstand the
oligonucleotide synthesis process (i.e., phosphoramidite chemistry)
and then can be cleaved to release oligonucleotide fragments.
Chemical cleavage at cleavable linker CL recreates usual 3' end of
specific oligos 1 through N. These oligonucleotides are released
into a mixture. The mixture of oligonucleotides is subsequently
assembled into full-length polynucleotide molecules. FIG. 3B,
oligonucleotide sequences are connected to the microarray device
through additional flanking sequence containing a restriction
enzyme (RE) sequence site. Another oligonucleotide sequence,
complementary to the flanking sequence region, is hybridized to the
oligonucleotides on the microarray device. This recreates a "ds" or
double-stranded oligonucleotide structure, each having a RE
sequence recognition region in the flanking sequence region.
Digestion of this ds oligonucleotides with the corresponding RE
enzymes at the RE recognition sites in the flanking sequence
regions releases the specific oligonucleotides 1 through N. When
assembled, oligonucleotide sequences 1 through N form a full-length
polynucleotide molecule. FIG. 3C: Cleavable linker for
oligonucleotide synthesis.
[0056] FIG. 4 shows the assembly of a polynucleotide from three
oligonucleotide fragments wherein each oligonucleotide fragment was
synthesized in situ on a microarray device. The fully assembled
polynucleotide was 172 mers in length, a length not practically
achievable by in situ synthesis. The first embodiment inventive
process was used in this example.
[0057] FIG. 5 shows the oligonucleotide sequences (SEQ ID NO: 1,
SEQ ID NO: 2, SEQ ID NO: 3) used to assemble the 172-mer
polynucleotide (SEQ ID NO: 4) of FIG. 4. The sequences of primers X
and Z are underlined. The Hpa II restriction site is indicated by
italic underlined letters.
[0058] FIG. 6 shows a scheme for preparing the sequences of
flanking regions (SEQ ID NO: 52 and SEQ ID NO: 53) and primers used
for preparation of specific oligonucleotide for assembly using the
REII enzyme MlyI. Primer 1 is complementary to the oligonucleotide
strand on a microarray device and contains a Biotin-TEG
(triethylene glycol) moiety. Primer 2 is the same strand as the
oligonucleotide strand on microarray device and contains Biotin-TEG
moiety. Any sequence between the primers can be used and is just
designated by a string of N's.
[0059] FIG. 7 shows the results of PCR and Mly I digestion of an
oligonucleotide sequence as described in FIG. 6. The clean bands
show the ability to obtain pure oligonucleotides using the second
embodiment of the inventive process to cleave off oligonucleotide
sequences using appropriate restriction enzymes.
[0060] FIG. 8 shows the sequences from nine oligonucleotides
fragments (consecutively numbered 1-9 and having SEQ ID NO: 5, SEQ
ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,
SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively) used
to assemble a 290 bp polynucleotide. The flanking regions are shown
in bold and underlined. The process used for polynucleotide
assembly was the second embodiment. The overlapping regions further
contained a cleavable site as the Mly I recognition site for the
Mly I class II restriction endonuclease.
[0061] FIG. 9 shows a schematic in the top panel for assembling a
polynucleotide from nine oligonucleotides. Nine oligonucleotide
sequences, shown in FIG. 8, were amplified by PCR using primers 1
and 2 (as described in FIG. 6) into ds DNA fragments containing the
same flanking regions and specific overlapping sequences, digested
with Mly I enzyme to remove flanking sequences, and used for
assembly of 290 by DNA fragment. The columns in the gel shown are
M--markers, 1--negative control, assembly without primers FP1 and
FP2, 2--negative control, assembly without specific oligos,
3--assembly of 290 bp fragment from specific oligos plus
amplification with FP1 and FP2 primers. The band in column 3 shows
a high efficiency of the inventive polynucleotide assembly
process.
[0062] FIG. 10 shows a sequence of an assembled polynucleotide in
Example 4, broken down into its component oligonucleotides
(Fragment 1, SEQ ID NO: 14, Fragment 2, SEQ ID NO: 15, Fragment 3,
SEQ ID NO: 16, Fragment 4, SEQ ID NO: 17, Fragment 5, SEQ ID NO:
18, Fragment 6, SEQ ID NO: 19, Fragment 7, SEQ ID NO: 20, Fragment
8, SEQ ID NO: 21, Fragment 9, SEQ ID NO: 22, Fragment 1F, SEQ ID
NO: 23, Fragment 2F, SEQ ID NO: 24, Fragment 3F, SEQ NO: 25,
Fragment 4F, SEQ ID NO: 26, Fragment 5F, SEQ ID NO: 27, Fragment
6F, SEQ ID NO: 28, Fragment 7F, SEQ ID NO: 29, Fragment 8F, SEQ ID
NO: 30, Fragment 9F, SEQ ID NO: 31, Fragment 10F, SEQ ID NO: 32,
Fragment 11F, SEQ ID NO: 33, Fragment 12F, SEQ ID NO: 34, Fragment
13F, SEQ ID NO: 35, Fragment 14F, SEQ ID NO: 36, Fragment 15F, SEQ
ID NO: 37, Fragment 16F, SEQ ID NO: 38, Fragment 17F, SEQ ID NO:
39, Fragment 18F, SEQ ID NO: 40, Fragment 19F, SEQ ID NO: 41,
Fragment 20F, SEQ ID NO: 42, Fragment 21F, SEQ ID NO: 43, Fragment
22F, SEQ ID NO: 44, Fragment 23F, SEQ ID NO: 45, Fragment 24F, SEQ
ID NO: 46, Fragment 25F, SEQ ID NO: 47, Fragment 26F, SEQ ID NO:
48, Fragment 27F, SEQ ID NO: 49, Fragment 28F, SEQ ID NO: 50,
Fragment 29F, SEQ ID NO: 51).
DETAILED DESCRIPTION OF THE INVENTION
[0063] The present invention describes the preparation of a
polynucleotide sequence (also called "gene") using assembly of
overlapping shorter oligonucleotides synthesized or spotted on
microarray devices or on solid surface bead devices. The shorter
oligonucleotides include sequence regions having overlapping
regions to assist in assembly into the sequence of the desired
polynucleotide. Overlapping regions refer to sequence regions at
either a 3' end or a 5' end of a first oligonucleotide sequence
that is the same as part of the second oligonucleotide and has the
same direction (relative to 3' to 5' or 5' to 3' direction), and
will hybridize to the 5' end or 3' end of a second oligonucleotide
sequence or its complementary sequence (second embodiment), and a
second oligonucleotide sequence to a third oligonucleotide
sequence, and so on. In order to design or develop a microarray
device or bead device to be used for polynucleotide assembly, the
polynucleotide sequence is divided (or dissected) into a number of
overlapping oligonucleotides segments, each with lengths preferably
from 20 to 1000 bases, and most preferably from 20 to 200 bases
(FIG. 1A). The overlap between oligonucleotide segments is 5 or
more bases, preferably 15-25 bases to that proper hybridization of
first to second, second to third, third to fourth and so on occurs.
These oligonucleotides (or oligos) are preferably synthesized on a
microarray device using any available method (i.e., electrochemical
in situ synthesis, photolithography in situ synthesis, ink-jet
printing, spotting, etc.). The direction of synthesis relative to
the microarray device surface or porous matrix covering a
microarray device can be from 3' to 5' or from 5' to 3'.
Preferably, in situ synthesis is done in the 3' to 5'
direction.
[0064] In the first embodiment the inventive gene/polynucleotide
assembly process uses oligonucleotides immobilized on a microarray
device. The microarray device itself or a porous reaction layer
with immobilized oligonucleotides can be used for the inventive
gene/polynucleotide assembly process.
[0065] With regard to FIG. 1B, the process comprises several
repeated steps of melting, annealing and extension (FIG. 1B), which
can be performed in any thermal cycler instrument. The cycling
program is similar to the programs used for PCR. At the first step
of gene/polynucleotide assembly, primer Pr1 is added and anneals to
oligonucleotide 1 on the microarray device and then extends by
appropriate polymerase enzyme into product complementary to
oligonucleotide 1 (called complementary oligonucleotide 1). At the
second step of the process the product complementary to
oligonucleotide 1 is melted from oligonucleotide 1, primer Pr1 is
annealed again to the oligonucleotide 1 as well as product
complementary to oligonucleotide 1 is partially re-anneals to
oligonucleotide 1 and partially anneals to oligonucleotide 2 due to
an overlapping sequence region between oligonucleotide 1 and
oligonucleotide 2. Extension of Pr1 leads to production of an
additional amount of Pr1 extension product (complementary
oligonucleotide 1). The annealing of the complementary
oligonucleotide 1 to oligonucleotide 2 followed by its extension
leads to product complementary to oligonucleotides 1+2 (called
complementary oligonucleotides 1+2). Similarly, at step 3 of the
process melting, re-annealing and extension lead to the same
products as at step 2 plus a product complementary to
oligonucleotides 1+2+3. These cycles of melting, annealing and
extension are repeated until full-length polynucleotide is formed.
The number of cycles should be equal or more than the number of
oligos on microarray device. After formation, the final target
polynucleotide molecule is amplified by a PCR process with two
primers complementary to the ends of this molecule to the desirable
amounts.
[0066] In a second embodiment, a plurality of oligonucleotides that
together comprise (with overlapping regions) the target
polynucleotide sequence are synthesized on a microarray device (or
can be synthesized on beads as a solid substrate), wherein each
oligonucleotide sequence further comprises flanking short sequence
regions, wherein each flanking sequence region comprises one or a
plurality of sequence sites for restriction endonuclease,
preferably endonuclease class II (ERII) enzymes. Each
oligonucleotide is amplified by PCR using appropriate
oligonucleotide primers to the flanking sequence regions to form a
preparation of a plurality of oligonucleotides. The preparation of
oligonucleotides is treated then with appropriate REII enzyme(s)
(specific to the restriction sequences in the flanking sequence
regions) to produce flanking fragments and overlapping
oligonucleotides that, together comprise the desired polynucleotide
sequence. Flanking fragments and PCR primers are removed from the
mixture, if desired, by different methods based on size or specific
labeling of the PCR primers. The oligonucleotides resembling the
desired target polynucleotide then assembled into the final target
polynucleotide molecule using repetition of the primer extension
method and PCR amplification of the final molecule.
[0067] Specifically, in the second embodiment, the assembly process
initially uses oligonucleotides immobilized on a microarray device
or beads, via immobilization techniques, such as spotting or
ink-jet printing or by direct in situ synthesis of the microarray
device using various techniques, such as photolithography or
electrochemical synthesis. The overlapping oligonucleotide
sequences are designed having an overlapping region and one or two
flanking sequence regions comprising a restriction class II
recognition site (FIG. 2A). The assembled oligonucleotides together
comprise the target polynucleotide sequence.
[0068] The length of flanking sequences is at least the length of
REII recognition site. The flanking sequences are designed to have
minimal homology to the specific oligonucleotide sequences regions
on the microarray device. The flanking sequences can be the same
for each oligonucleotide fragment, or be two or more different
sequences. For example, a pair of appropriate primers, called Pr1
and Pr2, was designed to amplify each oligonucleotide on a
microarray device (FIG. 2) by PCR. Each primer may contain a
binding moiety, such as biotin, that does not affect their ability
to serve as primers. After PCR amplification the amplified ds copy
of each oligonucleotide was present in the reaction mixture. This
reaction mixture was treated with the appropriate REII enzyme or
enzymes specific for the restriction sites in the flanking sequence
regions. The digestion sites for REII were designed, after
cleavage, to produce the desired specific oligonucleotide sequence
fragments that, when assembled will form the target polynucleotide
sequence. As a result of digestion a mixture of specific double
stranded (ds) overlapping oligonucleotide sequence fragments
resembling the structure of desired target polynucleotide, and ds
flanking sequences were formed. If desired, these flanking
sequences and residual primers are removed from the mixture using
specific absorption through specific moieties introduced in the
primers (such as, for example, by absorption on avidin beads for
biotin-labeled, primers), or based on the size difference of the
specific oligos and flanking sequences and primers. The mixture of
specific oligonucleotide sequences resembling target gene sequence
is used to assemble the final target polynucleotide molecule using
repeated cycles of melting, self-annealing and polymerase extension
followed by PCR amplification of the final target polynucleotide
molecule with appropriate PCR primers designed to amplify. This
final PCR amplification step is routinely done in the art and
described in, for example, Mullis et al., Cold Spring Harb. Symp.
Quant. Biol. 51 Pt 1:263-73, 1986; and Saiki et al,, Science
239:487-91, 1988. PCR amplification steps generally follow
manufacturer's instructions. Briefly, A process for amplifying any
target nucleic acid sequence contained in a nucleic acid or mixture
thereof comprises treating separate complementary strands of the
nucleic acid with a molar excess of two oligonucleotide primers and
extending the primers with a thermostable enzyme to form
complementary primer extension products which act as templates for
synthesizing the desired nucleic acid sequence. The amplified
sequence can be readily detected. The steps of the reaction can be
repeated as often as desired and involve temperature cycling to
effect hybridization, promotion of activity of the enzyme, and
denaturation of the hybrids formed.
[0069] In another embodiment for the assembly step, oligonucleotide
sequences that together comprise the target polynucleotide molecule
are assembled using a ligase chain reaction as described in Au et
al., Biochem. Biophys. Res. Commun. 248:200-3, 1998. Briefly, short
oligonucleotides are joined through ligase chain reaction (LCR) in
high stringency conditions to make "unit fragments" (Fifty
microliters of reaction mixture contained 2.2 mM of each oligo, 8
units Pfu DNA ligase (Stratagene La Jolla, Calif.) and reaction
buffer provided with the enzyme. LCR was conducted as follows:
95.degree. C. 1 min; 55.degree. C. 1.5 min, 70.degree. C. 1.5 min,
95.degree. C. 30 sec for 15 cycles; 55.degree. C. 2 min; 70.degree.
C. 2 min, which are then fused to form a full-length gene sequence
by polymerase chain reaction.
[0070] In another embodiment the ds oligonucleotide sequences are
assembled after preparation by chain ligation cloning as described
in Pachuk et al., Gene 243:19-25, 2000; and U.S. Pat. No. 6,143,527
(the disclosure of which is incorporated by reference herein).
Briefly, chain reaction cloning allows ligation of double-stranded
DNA molecules by DNA ligases and bridging oligonucleotides.
Double-stranded nucleic acid molecules are denatured into
single-stranded molecules. The ends of the molecules are brought
together by hybridization to a template. The template ensures that
the two single-stranded nucleic acid molecules are aligned
correctly. DNA ligase joins the two nucleic acid molecules into a
single, larger, composite nucleic acid molecule. The nucleic acid
molecules are subsequently denatured so that the composite molecule
formed by the ligated nucleic acid molecules and the template cease
to hybridize to each. Each composite molecule then serves as a
template for orienting unligated, single-stranded nucleic acid
molecules. After several cycles, composite nucleic acid molecules
are generated from smaller nucleic acid molecules. A number of
applications are disclosed for chain reaction cloning including
site-specific ligation of DNA fragments generated by restriction
enzyme digestion, DNAse digestion, chemical cleavage, enzymatic or
chemical synthesis, and PCR amplification.
[0071] With regard to the second embodiment of the inventive
process (illustrated in FIG. 2), a target polynucleotide gene
sequence (either strand) is divided into number of overlapping
oligonucleotide sequences by hand or with a software program, as
shown in FIGS. 1A-1C. These oligonucleotide sequences, plus
flanking sequences A and B (having one or a plurality of
restriction enzyme sites in the flanking region sequence), are
synthesized (in situ) on microarray device, or on a bead solid
surface using standard in situ synthesis techniques, or spotted
(pre-synthesized) onto a microarray device using standard
oligonucleotide synthesis procedures with standard spotting (e.g.,
computer-aided or ink jet printing) techniques. The oligonucleotide
sequences are amplified, preferably using a PCR process with a pair
of primers (Pr1 and Pr2). The primers are optionally labeled with
specific binding moieties, such as biotin. The resulting amplified
mixture of different amplified oligonucleotide sequences are double
stranded (ds). The mixture of ds oligonucleotide sequences are
treated with an appropriate restriction enzyme, such as an REII
restriction enzyme (e.g.,Mly I enzyme), to produce mixture of
different double stranded (ds) overlapping oligonucleotide
sequences that can be assembled into the structure of the desired
polynucleotide (gene) and ds flanking sequences. Optionally, the
flanking sequences and residual primers are removed from the ds
oligonucleotide sequence mixture, preferably by a process of
specific absorption using specific binding moieties introduced in
the primers (e.g., biotin), or by a process of size fractionation
based on the size differences of the specific oligonucleotide
sequences and flanking sequences. The mixture of specific
oligonucleotide sequences is assembled, for example, by a process
of repeated cycles of melting, self-annealing and polymerase
extension followed by PCR amplification of the final molecule with
appropriate PCR primers designed to amplify this complete molecule
(e.g., as described in Mullis et al., Cold Spring Harb. Symp.
Quant. Biol. 51 Pt 1:263-73, 1986; and Saiki et al., Science
239:487-91, 1988).
[0072] In yet another embodiment of the inventive process
(illustrated in FIGS. 3A-3B), the oligonucleotide sequences
comprising the target polynucleotide sequence are synthesized on a
microarray device or bead solid support, each oligonucleotide
having a cleavable linker moiety synthesized within the sequence,
such that after synthesis, oligonucleotides can be cleaved from the
microarray device into a solution. Examples of appropriate
cleavable linker moieties are shown in FIG. 3A. In addition to this
method of cleavage, a sequence containing RE enzyme site can be
synthesized at the ends of oligonucleotides attached to the
microarray device. These oligonucleotides on the microarray device
then hybridize with an oligonucleotide complementary to this
additional flanking sequence and treated with an RE enzyme specific
for the RE enzyme site. This process releases oligonucleotide
fragments resembling the structure of the target polynucleotide.
This set of oligonucleotides then can be assembled into the final
polynucleotide molecule using any one of the methods or combination
of the methods of ligation, primer extension and PCR.
[0073] In a third embodiment of the inventive process, a plurality
of oligonucleotides that can be assembled into a full length
polynucleotide are synthesized on a microarray device (or beads
having a solid surface) having specific cleavable linker moieties
(FIG. 3A) or capable of being cleaved from the solid support of the
microarray device or beads by a chemical treatment. The net effect
is to recreate the functional 3' ends and 5' ends of each specific
oligonucleotide sequence. After treatment to cleave them, the
oligonucleotides (each having overlapping regions) are released
into a mixture and used for full-length polynucleotide gene
assembly using any of the gene assembly processes described
herein.
[0074] Specifically, in the third embodiment and as illustrated in
FIGS. 3A-3B, a target polynucleotide sequence is dissected into
number of overlapping oligonucleotide sequences by a software
program or on paper, but not necessarily physically in a
laboratory. These oligonucleotide sequences are physically
synthesized on a microarray device. In alternative A, the
oligonucleotide sequences are connected to the microarray device
through cleavable linker moiety. Chemical cleavage under basic
conditions (e.g., through addition of ammonia), at cleavable linker
CL recreates the usual 3' end of the specific oligonucleotide
sequences 1 through N. Oligonucleotide sequences 1 through N are
released into a mixture. The mixture of oligonucleotide sequences
is used for polynucleotide assembly.
[0075] In alternative B, oligonucleotide sequences are connected to
a microarray device through additional flanking sequence regions
containing a restriction enzyme (RE) sequence site. A second
oligonucleotide fragment, complementary to the flanking sequence,
is hybridized to the oligonucleotides on the microarray device.
This recreates a ds structure at the flanking sequence region,
including the RE recognition site. Digestion of this ds DNA
structure with RE enzyme specific to the RE recognition site in the
flanking sequence region will release specific oligonucleotides 1
through N into a mixture solution. The oligonucleotides l through N
are able to assemble into a polynucleotide molecule in
solution.
[0076] In another example of alternative B, oligonucleotides that
together assemble into the polynucleotide are synthesized on a
microarray device, each having a flanking sequence on the
microarray side. The flanking sequence further comprises a
restriction endonuclease (RE) recognition site (see FIG. 3B).
Oligonucleotides complementary to the flanking sequence region are
added and hybridized to the oligonucleotides on microarray device.
After hybridization a RE (restriction enzyme specific to the RE
sequence in the flanking region) is added to the microarray device.
Specific oligonucleotide sequences are released from the microarray
device as a result of RE digestion into a mixture. The mixture of
specific oligonucleotide sequences ARE assembled into the
full-length polynucleotide sequence.
Example 1
[0077] This example illustrates assembly of 172-mer polynucleotide
sequence from non-cleavable oligonucleotide sequences synthesized
on a microarray device according to the first embodiment inventive
process (FIGS. 4 and 5). Three oligonucleotides (sequences shown in
FIG. 5) were synthesized in situ on a microarray device according
to an electrochemical process (see U.S. Pat. No. 6,093,302, the
disclosure of which is incorporated by reference herein). The
oligonucleotide sequences synthesized were amplified by a PCR
reaction with primers X (complementary to the strand of oligo #1)
and Z (same strand as Oligo #3) (FIG. 5). After 45 cycles of PCR
using a PCR kit with AmplyGold.RTM. enzyme (Applied Biosystems) a
correct DNA fragment of 172 by was synthesized (FIG. 4). Its
subsequent digestion confirmed the specificity of this enzyme with
Hpa II producing two fragments of 106 bp and 68 bp.
Example 2
[0078] This example illustrates the second embodiment of the
inventive process for preparing oligonucleotides for assembly into
full-length polynucleotides by PCR and REII (restriction enzyme)
digestion. A single oligonucleotide sequence was synthesized on a
microarray device according to the procedure in Example 1 (see
FIGS. 2 and 6). The oligonucleotide sequence further comprised 2
flanking sequences, each having a recognition site for a Mly I
restriction enzyme. This microarray device was subject to a PCR (25
cycles) reaction with two primers (shown in FIG. 7) to produce an
amplified PCR fragment mixture. The amplified PM fragment obtained
was digested by Mly I restriction enzyme and purified by a PCR
purification kit (Qiagen) to produce specific oligonucleotides
ready for assembly (FIG. 7). Similarly, this specific
oligonucleotide was purified from the flanking sequences by
absorption of the digestion mixture by Streptavidin-agarose
(Sigma).
Example 3
[0079] This example illustrates the assembly of a 290 bp
polynucleotide sequence from 9 oligonucleotide sequences, each
having flanking sequences containing a MlyI restriction site. Each
of the nine different oligonucleotide sequences was synthesized on
a microarray device through an in situ electrochemistry process as
described in example 1 herein.
[0080] The microarray device containing the nine specific
oligonucleotide sequences (with flanking sequences as shown in FIG.
8) was used for PCR amplification of each oligonucleotide sequence
using two primers, Primer 1 and 2, described in FIG. 6 to form a
mixture of ds oligonucleotide sequences. The primers were
complementary to the flanking sequences. The mixture of the
amplified ds oligonucleotide sequences was digested by Mly I
enzyme. Specific ds oligonucleotide sequences were purified and
then assembled into the final 290 bp polynucleotide sequence in two
steps as described in FIG. 2 and shown schematically in FIG. 9. At
the first step of assembly 20 cycles of melting-annealing-extension
were used. The final product was amplified using two primers FP1
and FP2 (FIG. 9) in 25 cycles of PCR into a 290 bp polynucleotide
DNA.
Example 4
[0081] This example illustrates the creation of a cDNA
polynucleotide sequence capable of coding on expression for fusion
protein MIP-GFP-FLAG (Macrophage Inflammation Protein-Green
Fluorescence Protein-FLAG peptide) using thirty-eight overlapping
oligonucleotide sequences (FIG. 10). The 38 oligonucleotides were
synthesized on a microarray device using an electrochemical in situ
synthesis approach, as described in example 1. Each oligonucleotide
sequence contained a cleavable linker moiety (see FIG. 3A) at their
3' end. After simultaneous deprotection and cleavage of these
oligonucleotide sequences by concentrated ammonia, the mixture of
oligonucleotide sequences was purified by gel-filtration through
the spin column. The purified oligonucleotide sequences were
assembled into a polynucleotide by a process shown schematically in
FIGS. 3A-3B. The resulting DNA polynucleotide was 965 bp and
contained both a T7 RNA-polymerase promoter and a coding sequence
for MIP-GFP-FLAG fusion protein. The polynucleotide assembled in
this example was used in a standard transcription/translation
reaction and produced the appropriate MIP-GFP-FLAG fusion protein.
The translated protein was purified from the reaction mixture using
anti-FLAG resin (Sigma). The functional protein possessed green
fluorescence signal in appropriate blue light. Accordingly, this
experiment demonstrated that the inventive gene assembly process
provided the correct DNA sequence coding for the functional
protein.
Sequence CWU 1
1
53182DNAArtificial SequenceSynthetic construct 1taattatgct
gagtgatatc cctttctacc tgtgcggctg gcggacgacg aagtcgaatg 60tggagggccg
tctaaggtgt ct 82284DNAArtificial SequenceSynthetic construct
2ggacgacgaa gtcgaatgtg gagggccgtc taaggtgtct taaagtatcg actgatgaaa
60ctctgctcgt cggtcacgag gttc 84386DNAArtificial SequenceSynthetic
construct 3gtatcgactg atgaaactct gctcgtcggt cacgaggttc cctcgaccac
cgcatgatgt 60ttctgctact gctgttcacg attatc 864142DNAArtificial
SequenceSynthetic construct 4taattatgct gagtgatatc cctttctacc
tgtgcggctg aagtcgaatg tggagggccg 60tctaaggtgt cttaaagtat aactctgctc
gtcggtcacg aggttccctc gaccaccgca 120gctactgctg ttcacgatta tc
142589DNAArtificial SequenceSynthetic construct 5ccatcacgct
gagtcttacg tacgtaatac gactcactat agggaaagtc gccaccatgg 60acacgccgac
gagacgactc ctaatcgaa 89685DNAArtificial SequenceSynthetic construct
6ccatcacgct gagtcttacg cgcctgctgc ttcagctaca cctcccggca gattccacag
60aatttcgaga cgactcctaa tcgaa 85787DNAArtificial SequenceSynthetic
construct 7ccatcacgct gagtcttacg atagctgact actttgagac gagcagccag
tgctccaagc 60ccggtgtcga gacgactcct aatcgaa 87881DNAArtificial
SequenceSynthetic construct 8ccatcacgct gagtcttacg atcttcctaa
ccaagcgaag ccggcaggtc tgtgctgacc 60ccgagacgac tcctaatcga a
819130DNAArtificial SequenceSynthetic construct 9ccatcacgct
gagtcttacg caggcactca gctctacggg gccgtcgccg atgggggtgt 60tctgctggta
gtggtcggcg agctgcatat ttctggaccc actcctcact gagacgactc
120ctaatcgaac 1301083DNAArtificial SequenceSynthetic construct
10ccatcacgct gagtcttacg atatttctgg acccactcct cactggggtc agcacagacc
60tgccgagacg actcctaatc gaa 831184DNAArtificial SequenceSynthetic
construct 11ccatcacgct gagtcttacg ggcttcgctt ggttaggaag atgacaccgg
gcttggagca 60ctggcgagac gactcctaat cgaa 841284DNAArtificial
SequenceSynthetic construct 12ccatcacgct gagtcttacg tgctcgtctc
aaagtagtca gctatgaaat tctgtggaat 60ctgccgagac gactcctaat cgaa
841388DNAArtificial SequenceSynthetic construct 13ccatcacgct
gagtcttacg gggaggtgta gctgaagcag caggcggtcg gcgtgtccat 60ggtggcgacg
agacgactcc taatcgaa 881450DNAArtificial SequenceSynthetic construct
14tacgtaatac gactcactat agggaaagtc gccaccatgg acacgccgac
501546DNAArtificial SequenceSynthetic construct 15cgcctgctgc
ttcagctaca cctcccggca gattccacag aatttc 461648DNAArtificial
SequenceSynthetic construct 16atagctgact actttgagac gagcagccag
tgctccaagc ccggtgtc 481742DNAArtificial SequenceSynthetic construct
17atcttcctaa ccaagcgaag ccggcaggtc tgtgctgacc cc
421847DNAArtificial SequenceSynthetic construct 18agtgaggagt
gggtccagaa atatgtcagc gacctagagc tgagtgc 471944DNAArtificial
SequenceSynthetic construct 19atatttctgg acccactcct cactggggtc
agcacagacc tgcc 442045DNAArtificial SequenceSynthetic construct
20ggcttcgctt ggttaggaag atgacaccgg gcttggagca ctggc
452145DNAArtificial SequenceSynthetic construct 21tgctcgtctc
aaagtagtca gctatgaaat tctgtggaat ctgcc 452249DNAArtificial
SequenceSynthetic construct 22gggaggtgta gctgaagcag caggcggtcg
gcgtgtccat ggtggcgac 492354DNAArtificial SequenceSynthetic
construct 23ggtgaacagc tcctcgccct tgctcaccat ggcactcagc tctaggtcgc
tgac 542452DNAArtificial SequenceSynthetic construct 24catggtgagc
aagggcgagg agctgttcac cggggtggtg cccatcctgg tc 522550DNAArtificial
SequenceSynthetic construct 25ttgtggccgt ttacgtcgcc gtccagctcg
accaggatgg gcaccacccc 502651DNAArtificial SequenceSynthetic
construct 26gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
c 512745DNAArtificial SequenceSynthetic construct 27ttgccgtagg
tggcatcgcc ctcgccctcg ccggacacgc tgaac 452848DNAArtificial
SequenceSynthetic construct 28gagggcgatg ccacctacgg caagctgacc
ctgaagttca tctgcacc 482948DNAArtificial SequenceSynthetic construct
29cagggcacgg gcagcttgcc ggtggtgcag atgaacttca gggtcagc
483054DNAArtificial SequenceSynthetic construct 30accggcaagc
tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggc
543155DNAArtificial SequenceSynthetic construct 31ggggtagcgg
ctgaagcact gcacgccgta ggtcagggtg gtcacgaggg tgggc
553248DNAArtificial SequenceSynthetic construct 32gtgcagtgct
tcagccgcta ccccgaccac atgaagcagc acgacttc 483351DNAArtificial
SequenceSynthetic construct 33gtagccttcg ggcatggcgg acttgaagaa
gtcgtgctgc ttcatgtggt c 513451DNAArtificial SequenceSynthetic
construct 34ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
c 513549DNAArtificial SequenceSynthetic construct 35gggtcttgta
gttgccgtcg tccttgaaga agatggtgcg ctcctggac 493648DNAArtificial
SequenceSynthetic construct 36aaggacgacg gcaactacaa gacccgcgcc
gaggtgaagt tcgagggc 483749DNAArtificial SequenceSynthetic construct
37agctcgatgc ggttcaccag ggtgtcgccc tcgaacttca cctcggcgc
493851DNAArtificial SequenceSynthetic construct 38gacaccctgg
tgaaccgcat cgagctgaag ggcatcgact tcaaggagga c 513951DNAArtificial
SequenceSynthetic construct 39tccagcttgt gccccaggat gttgccgtcc
tccttgaagt cgatgccctt c 514051DNAArtificial SequenceSynthetic
construct 40ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
c 514152DNAArtificial SequenceSynthetic construct 41gttcttctgc
ttgtcggcca tgatatagac gttgtggctg ttgtagttgt ac 524251DNAArtificial
SequenceSynthetic construct 42tatatcatgg ccgacaagca gaagaacggc
atcaaggtga acttcaagat c 514350DNAArtificial SequenceSynthetic
construct 43acgctgccgt cctcgatgtt gtggcggatc ttgaagttca ccttgatgcc
504449DNAArtificial SequenceSynthetic construct 44cgccacaaca
tcgaggacgg cagcgtgcag ctcgccgacc actaccagc 494551DNAArtificial
SequenceSynthetic construct 45acggggccgt cgccgatggg ggtgttctgc
tggtagtggt cggcgagctg c 514651DNAArtificial SequenceSynthetic
construct 46agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
c 514749DNAArtificial SequenceSynthetic construct 47tttgctcagg
gcggactggg tgctcaggta gtggttgtcg ggcagcagc 494850DNAArtificial
SequenceSynthetic construct 48tgagcaccca gtccgccctg agcaaagacc
ccaacgagaa gcgcgatcac 504951DNAArtificial SequenceSynthetic
construct 49ggcggtcacg aactccagca ggaccatgtg atcgcgcttc tcgttggggt
c 515051DNAArtificial SequenceSynthetic construct 50atggtcctgc
tggagttcgt gaccgccgcc gggatcactc tcggcatgga c 515149DNAArtificial
SequenceSynthetic construct 51ggcggccgct ttacttgtac agctcgtcca
tgccgagagt gatcccggc 495220DNAArtificial SequenceSynthetic
construct 52gagacgactc ctaatcgaac 205320DNAArtificial
SequenceSynthetic construct 53ccatcacgct gagtcttacg 20
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