U.S. patent application number 11/148401 was filed with the patent office on 2010-01-21 for methods for rapid production of target double-stranded dna sequences.
Invention is credited to Kathryn E. Richmond, Matt J. Rodesch, Michael R. Sussman.
Application Number | 20100016178 11/148401 |
Document ID | / |
Family ID | 35510339 |
Filed Date | 2010-01-21 |
United States Patent
Application |
20100016178 |
Kind Code |
A1 |
Sussman; Michael R. ; et
al. |
January 21, 2010 |
METHODS FOR RAPID PRODUCTION OF TARGET DOUBLE-STRANDED DNA
SEQUENCES
Abstract
It has been previously disclosed that DNA segments can be made
in massively parallel chemical synthesis operations on a common
substrate followed by release of the segments from the substrate
and assembly of the segments into target DNA molecules. Here it is
taught that if the DNA primary constructs are sufficiently long and
properly designed, that the copy numbers of the primary constructs
can be multiplied as needed by a PCR process using as a template
regions at the ends of the primary constructs. The end regions,
called flanking regions, can also be designed so that they may be
cleaved easily from the amplification products. The target
double-stranded DNA can then be assembled from the cleaved
fragments. Hundreds of thousands of oligonucleotides can be
synthesized and assembled into many different individual genes by
this process in a relatively quick and efficient process.
Inventors: |
Sussman; Michael R.;
(Madison, WI) ; Richmond; Kathryn E.; (Madison,
WI) ; Rodesch; Matt J.; (Madison, WI) |
Correspondence
Address: |
QUARLES & BRADY LLP
33 E. MAIN ST, SUITE 900, P.O. BOX 2113
MADISON
WI
53701-2113
US
|
Family ID: |
35510339 |
Appl. No.: |
11/148401 |
Filed: |
June 8, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60578195 |
Jun 9, 2004 |
|
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|
Current U.S.
Class: |
506/26 ;
435/91.2 |
Current CPC
Class: |
C12Q 1/686 20130101;
C12Q 2533/107 20130101; C12Q 1/686 20130101 |
Class at
Publication: |
506/26 ;
435/91.2 |
International
Class: |
C40B 50/06 20060101
C40B050/06; C12P 19/34 20060101 C12P019/34 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with United States government
support awarded by the following agency: DOD ARPA DAAD
19-02-2-0026. The United States has certain rights in this
invention.
Claims
1. A method for generating a double-stranded DNA sequence, the
method comprising the following steps in the order set forth below:
(a) synthesizing in parallel a plurality of single-stranded
oligonucleotide primary constructs, each primary construct
comprising (i) an internal region, wherein a plurality of internal
regions define the DNA sequence and wherein each internal region
has at least one portion overlapping and complementary to a portion
of an internal region of at least one different primary construct,
and (ii) a 5' and a 3' flanking region flanking the 5' and the 3'
end of the internal region, each of the flanking regions including
both a primer recognition site and a recognition site for a
restriction enzyme to cut the primary construct at each of the
junctions of said internal region and said flanking regions; (b)
selectively amplifying at least a subset of the plurality of the
single-stranded primary constructs of step (a) to generate multiple
copies by conducting a polymerase chain reaction (PCR) process on
said single-stranded primary constructs using a set of PCR primers
that target the primer recognition site(s) in said flanking regions
to create at least one amplified pool of double-stranded primary
constructs; (c) removing said flanking regions, each of which
includes the primer recognition site and the restriction enzyme
recognition site, from the amplified double-stranded primary
constructs of step (b) by digesting said constructs with the
restriction enzyme, thereby generating a plurality of
double-stranded internal regions; and (d) conducting repetitive
denaturing, annealing and extension procedures on the internal
regions of step (c) in the presence of a polymerase and dNTPs, but
without the presence of primers so that no amplification occurs, to
allow the overlapping internal regions to hybridize and assemble,
thereby generating said double-stranded DNA sequence.
2. The method of claim 1 wherein step (a) is performed by using an
automated gene synthesizer instrument to construct the primary
constructs as single stranded DNA probes in a microarray on a
common substrate and by then detaching the primary constructs from
the substrate.
3. The method of claim 1 wherein the flanking regions at the ends
of all of the primary constructs are the same and wherein in step
(b) a single set of PCR primers is used to amplify all of the
primary constructs.
4. The method of claim 1 wherein the restriction enzyme used in
step (c) is MlyI.
5. The method of claim 1 wherein in step (b) at least two different
sets of primers are used to amplify at least two subsets of primary
constructs.
6. The method of claim 1 wherein the step (c) is performed multiple
times using multiple different restriction enzymes.
7. (canceled)
8. The method of claim 1 further comprising the step of isolating
from the products of step (d) the longest DNA molecule
produced.
9. A method for making a target doubled-stranded DNA sequence, the
method comprising the following steps in the order set forth below:
(a) synthesizing on a microarray a plurality of single stranded
oligonucleotide primary constructs, each primary construct being at
least 70 nucleotides in length and including an internal region of
at least 40 nucleotides, wherein--a plurality of internal regions
define the DNA sequence and wherein each internal region has at
least one portion overlapping and complementary to a portion of an
internal region of at least one different primary construct, and a
5' and 3' flanking region flanking the 5' and 3' end of said
internal region, each of the flanking regions being a region of at
least 15 nucleotides and including a primer recognition site and
including a recognition site for a restriction enzyme at each of
the junctions of said internal region and said flanking regions;
(b) selectively amplifying at least a subset of the single-stranded
primary constructs by conducting a polymerase chain reaction (PCR)
process on said single-stranded primary constructs of step (a)
using a set of PCR primers that target the primer recognition
site(s) in said flanking regions to create at least one amplified
pool of double-stranded primary constructs; (c) removing said
flanking regions, each of which includes the primer recognition
site and the restriction enzyme recognition site . . . from the
amplified double-stranded primary constructs of step (b) by
digesting said constructs with the restriction enzyme, thereby
generating a plurality of double-stranded internal regions; and (d)
conducting repetitive denaturing, annealing and extension
procedures on the internal regions of step (c) in the presence of a
polymerase and dNTPs, but without the presence of primers so that
no amplification occurs to allow overlapping internal regions to
hybridize and assemble thereby generating said double-stranded DNA
sequence.
10. The method of claim 9 wherein step (a) is performed by using an
automated gene synthesizer instrument to construct the primary
constructs as single stranded DNA probes in a microarray on a
common substrate and by then detaching the primary constructs from
the substrate.
11. The method of claim 9 wherein the flanking regions at the ends
of all of the primary constructs are the same and wherein in step
(b) a single set of PCR primers is used to amplify all of the
primary constructs.
12. The method of claim 9 wherein the restriction enzyme used in
step (c) is MlyI.
13. The method of claim 9 wherein in step (b) at least two
different sets of primers are used to amplify at least two subsets
of primary constructs.
14. The method of claim 9 wherein the step (c) is performed
multiple times using multiple different restriction enzymes.
15. (canceled)
16. The method of claim 9 further comprising the step of isolating
from the products of step (d) the longest DNA molecule
produced.
17. The method according to claim 1 wherein the plurality of
primary constructs comprise a first set of primary constructs
comprising internal regions having two distinct portions, each one
overlapping and complementary to portions of two different internal
regions of other primary constructs and a second set of primary
constructs with internal regions comprising one portion overlapping
and complementary to a portion of the internal region of the first
set of primary constructs.
18. The method according to claim 1 wherein each of the internal
regions of the primary constructs comprise two distinct portions,
each portion overlapping and complementary to portions of two
different internal regions of two other primary constructs.
19. The method according to claim 9 wherein the plurality of
primary constructs comprise a first set of primary constructs
internal regions having two distinct portions, each one overlapping
and complementary to portions of two different internal regions of
other primary constructs and a second set of primary constructs
with internal regions comprising one portion overlapping and
complementary to a portion of the internal region of the first set
of primary constructs.
20. The method according to claim 9 wherein each of the internal
regions comprise two portions, each portion overlapping and
complementary to portions of two different internal regions of two
other primary constructs.
21. A method of generating a double-stranded DNA sequence, the
method comprising the following steps in the order set forth below:
(a) synthesizing in parallel a plurality of single stranded
oligonucleotide primary constructs, each primary construct
comprising a 5'-flanking region, an internal region, and a 3'
flanking region, wherein (i) a plurality of the internal regions
define the DNA sequence and wherein each single stranded internal
region has a sequence complementary to the next internal region,
(ii) each of the two flanking regions includes a primer recognition
site and a recognition site for a restriction enzyme at each of the
junctions of the internal region and flanking region; (b)
selectively amplifying at least a subset of the plurality of the
single-stranded primary constructs of step (a) to generate multiple
copies by conducting a polymerase chain reaction (PCR) process on
said single-stranded primary constructs using a set of PCR primers
that target the primer recognition site(s) in said flanking regions
to create at least one amplified pool of double-stranded primary
constructs; (c) removing said flanking regions, each of which
includes the primer recognition site and the restriction enzyme
recognition site, from the amplified double-stranded primary
constructs of step (b) by digesting said constructs with the
restriction enzyme thereby generating a plurality of
double-stranded internal regions, wherein the enzyme is a type II
restriction enzyme that makes a blunt end after cleavage of the DNA
sequence; and (d) conducting repetitive denaturing, annealing and
extension procedures on the internal regions of step (c) in the
presence of a polymerase and dNTPs, but without the presence of
primers so that no amplification occurs, se-as to allow the
overlapping internal regions to hybridize and assemble by
extension, thereby generating said double-stranded DNA
sequence.
22. The method of claim 1 or 9 or 21 further comprising assembling
a longer double-stranded DNA by repeating the denaturing, annealing
and extension procedures in the presence of polymerase and dNTPs.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. provisional
patent application Ser. No. 60/578,195 filed Jun. 9, 2004.
BACKGROUND OF THE INVENTION
[0003] The invention pertains to the field of molecular biology and
techniques and apparatus for the manufacture of DNA molecules of
defined or desired sequences. The ex vivo manufacture of DNA
molecules makes possible the use of those DNA molecules in vivo to
synthesize any desired peptides, proteins or assemblies of proteins
or combinations of nucleic acids, as may be desired, and to perform
a large variety of genetic experiments in living organisms.
[0004] In modern biotechnology it is common to create DNA sequences
chemically, that is to say apart from any living organism. The DNA
sequences are assembled and replicated in vitro using cell free
techniques and ultimately are recombined or reassembled into DNA
sequences which can be inserted into organisms for biological
purposes. It has become commonplace to synthesize short DNA
sequences, referred to as oligonucleotides, directly from
individual nucleosides and to construct larger DNA sequences from
smaller oligonucleotides.
[0005] It has also been proposed that one may create larger DNA
molecules by the making many smaller, but properly designed, DNA
molecules in parallel, and then permitting those smaller molecules,
or fragments, to self-assemble and thereby make a longer
oligonucleotide. This can be done most conveniently by using a
maskless array synthesizer (MAS) instrument, of the type disclosed
in published PCT application WO 99/42813 to create a number of
single stranded DNA sequences in a massively parallel synthesis
operation. The single stranded DNA sequences thus created can then
be cleaved from the substrate upon which they are constructed and
permitted to anneal and form much larger DNA segments. This process
and the general principles behind its operation are described in
published PCT application PCT/US02/15951, the disclosure of which
is incorporated herein by reference. A unique attribute of the MAS
instrument of particular interest for this process is that fact
that the instrument can create microarrays in which the
oligonucleotides attach in the array are quite long, as long as 60
to 100 nucleotides. It is believed that only this style of
microarray instrument permits the synthesis of oligonucleotides of
this length in a microarray.
[0006] While these methods permit the synthesis of long DNA
segments, using the massively parallel synthesis capabilities of
the MAS style of instrument, there is one drawback to the
methodology described in the aforementioned published PCT
applications. The amount of DNA assembled by this synthesis,
without other procedures, is relatively small. While the amount of
DNA is so small as to be difficult to measure physically, it is
believed that approximately 20 picomoles of DNA are synthesized on
a typical DNA microarray made by an MAS instrument. The fact that
the amount of DNA is this small makes the physical handling and
amplification of the DNA a relatively sophisticated procedure. The
DNA is so small an amount that if the DNA is handled at a
concentration consistent with most DNA reaction conditions, the
volume becomes much smaller than the volumes suitable for handling
in most fluid handling operations. Conversely, if the solution is
diluted to a reasonable volume, then the DNA molecules are so
dilute in the solution that the normal enzymes and other agents
used for altering and manipulating DNA are difficult to use because
of the dilution of the DNA. Accordingly, the process for the
massive parallel synthesis of DNA fragments would be improved if
methodologies existed to dramatically increase the amount of DNA
which is the product of such a process.
BRIEF SUMMARY OF THE INVENTION
[0007] The present invention is summarized in a method for making a
target DNA sequence in which the method starts with the step of
synthesizing in parallel a large number of single stranded DNA
primary constructs, each primary construct including an internal
region and flanking regions on each side of the internal region,
each of the flanking regions including a recognition site for a
restriction enzyme which would cut the primary construct at each of
the junctions of the internal region and the flanking regions. The
next step is amplifying the primary constructs by conducting a PCR
reaction on the primary constructs using primers located in the
flanking regions of the primary constructs to create an amplified
pool of primary constructs. The next step is to digest the
amplified pool of primary constructs with a restriction enzyme to
cleave the internal regions in the pool of amplified constructs
from the flanking regions. Finally, assembly of the target sequence
is performed by adding a polymerase and dNTPs to the product of the
last step and conducting repetitive denaturing, annealing and
extension procedures to assemble the target sequence from the
products of the extension.
[0008] It is an object of the present invention to improve the
process of the synthesis of DNA sequences specifically by massively
parallel chemical synthesis of small segments followed by assembly
of the small segments into larger DNA sequences.
[0009] It is an advantage of the method of the present invention in
that it permits amplification of the number of copies of DNA
synthesized to increase the amount of DNA available for DNA
assemble procedures.
[0010] Other objects advantages and features of the present
invention will become apparent from the following
specification.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0011] FIG. 1 illustrates a DNA sequence of a potential primary
construct for use in the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0012] The present invention is directed toward a method for
increasing the yield of oligonucleotides constructed in a massively
parallel DNA synthesis operation. The method is best used with a
single stranded DNA parallel synthesis technology which permits the
synthesis of relatively long oligonucleotides, here called primary
constructs. The primary constructs are single stranded DNA
molecules, longer than the typical oligonucleotides made on a
microarray, being typically on the order of 70mers or longer. To
understand the concept of the present invention, the generalized
arrangement of the primary construct is illustrated in FIG. 1, with
an exemplary but arbitrary DNA sequence. Each primary construct is
composed of an internal region, shown to be a 40mer in FIG. 1, and
two flanking regions, each illustrated as a 15mer in FIG. 1. The
internal region of the primary construct is a sequence of DNA
intended to be incorporated in the target DNA sequence being
constructed. The flanking regions are regions intended to be
functional in the primary sequence and include both PCR primer
sites and restriction enzyme recognition sites, so that the primary
constructs can be amplified followed by cleavage of the flanking
regions, as will be described in more detail below. A core idea of
this process is that following detachment of the primary constructs
from the substrate, creating a mix pool of single stranded primary
constructs, a PCR reaction is run using primers which recognize
primer regions in the flanking regions on all of the primary
constructs. This amplifies the copy number of each of the numerous
primary constructs in parallel. Then the amplified primary
constructs are all digested with a restriction enzyme, to cleave
off the flanking regions. At this point the internal regions, the
40mers, are released, independent and can be used in the assembly
of the target DNA.
[0013] To facilitate comprehension of the process described here,
the consistent use of common terminology here is appropriate. In
this document, the "target sequence" refers to the ultimate DNA
construct, the synthesis of which is the object of the entire
process here. The "automated gene synthesizer" or "AGS" instrument
refers to an instrument capable of making many different
oligonucleotides in parallel, typically, but not necessarily, on a
common substrate. The "primary construct" refers to the single
stranded oligonucleotides created by the automated gene synthesizer
instrument and then released into solution. The "internal region"
and the "flanking region" are regions of the primary construct, as
illustrated here. The assembly process refers to the methodology
used to assemble the internal regions into the target sequence.
[0014] The process of constructing the target sequence begins with
an automated gene synthesis instrument. This instrument is a device
intended to chemically synthesize a large number of single stranded
DNA molecules in a massively parallel DNA synthesis process. Any of
the commercially used DNA microarray synthesis instruments in use
today that can create long pieces of single stranded DNA (greater
than 60mers) may be adapted for this purpose. DNA microarray are
available commercially and can be custom made by companies in that
business. However, the preferred approach is to use an instrument
of the style described in, among other places, U.S. Pat. No.
6,375,903, the disclosure of which is incorporated herein by
reference. This style of instrument, originally designed to make
DNA microarrays, uses a micromirror device under computer control
to make individually customizable and unique microarrays. The
instrument and the chemistry used with it can be optimized such
that the manufacture of single stranded oligonucleotides is so
efficient, that this style of instrument can make 786,000 different
oligonucleotides, each up to or over 100 nucleotides in length, in
parallel and all in a matter of a few hours. Each manufacture of a
microarray is unique and customized, since it is under computer
control. The instrument permits the manufacture of multiple
microarrays per day, each individually designed and customized.
This is the technology that has been adopted to be used as the
automated gene synthesizer instrument for the process described
here. The microarray synthesized by such an instrument results in
many individual groups of similar single stranded oligonucleotides
secured on a common substrate. To use the oligonucleotides as the
primary constructs in the process here, the oligonucleotides are
separated from the substrate, thus making the primary constructs
used in this process free in solution. This separation from the
substrate can be done by simply using a base or acid labile linkage
between the oligonucleotide and the substrate, and then using a
base or an acid to release the primary constructs.
[0015] Returning to a description of the overall process, the
primary constructs are synthesized in parallel using an automated
gene synthesis instrument, and the primary constructs are then
cleaved from the substrate. As briefly described above, the primary
constructs have particular properties. Each of the primary
constructs created includes at its opposing ends two flanking
regions and has, in its interior, an internal sequence. The
sequence of the internal sequence is determined by the sequence of
the target DNA, the DNA construct to be made by this overall
process. The internal sequences serve the function of the sequences
referred to as DNA molecule segments in the disclosure of
PCT/US02/15951, incorporated by reference above. Thus each internal
region makes up a portion of the total target sequence, and each
internal region is complementary to portions of two other internal
regions on other primary constructs. The flanking regions have
their sequences designed with two important properties in mind. One
is that the flanking regions incorporate primer recognition sites
for PCR primers. The other is that the flanking regions incorporate
a cleavage recognition site for a type II restriction enzyme, that
is to say a restriction enzyme that will cut a DNA sequence at a
cleavage site that is not its recognition site. This is illustrated
again in FIG. 1. Note that the exemplary primary construct of FIG.
1 includes in each flanking region the sequence GAGTC. This is the
recognition site for the restriction enzyme Mly I. The site of
cleavage of the restriction enzyme Mly I is also indicated in FIG.
1, and note that this site is several bases distant from the
recognition site. This same concept may be used with any
restriction enzyme that cleaves DNA at a site adjacent to, but not
within, the recognition site of the enzyme. Mly I is preferred
because its cleavage of double stranded DNA results in a blunt
end.
[0016] So, again, the process begins with the synthesis of the
primary construct on the automated gene synthesizer instrument and
the release of the primary constructs from the substrate. At this
point, there is a solution of single stranded DNA molecules, but a
small number of molecules are made. The next step in the process is
to perform a polymerase chain reaction (PCR) DNA amplification
reaction. The procedures for conducting PCR reactions are well
known in the art. The primers used for the PCR amplification are
selected to bind to the primer recognition sites in the flanking
regions of the primary constructs. The PCR reaction is conducted
for several rounds to thus amplify the number of copies of the
primary constructs to whatever magnitude of copies is convenient
for the remainder of the process. Since the flanking regions on all
of the primary constructs are the same, all of the primary
constructs will be amplified in about the same proportion. This
idea is subject to a variation, It is envisioned that one
alternative is for all the PCR primers and recognition sites to be
the same for all the primary constructs so that there is a single
amplification of all of the pooled primary constructs recovered
from the substrate. Another alternative is to have sets or groups
of PCR primers and recognition sites to permit selective
amplification of subsets or groups of primary constructs by simply
using the PCR primers which target that particular subset or group.
The amplified constructs can then be assembled, as described below,
followed by amplification of other subsets or groups using
different primers.
[0017] As might be surmised at this point, after the amplification
step, the next step in the process is to cleave the flanking
regions from the primary constructs using the restriction enzyme.
The entire pool of amplified primary constructs can be digested
together to completion. Again it is preferred that a type II
restriction enzyme which cuts DNA at a site different from the
recognition site is preferred. Referring to the example in FIG. 1,
the Mly I enzyme recognizes the motif GAGTC in the flanking region
and cuts the molecule five bases displaced from the C base of the
recognition site. The use of Mly I is preferred since it leaves a
blunt end after cleavage. This cleanly cuts the entire flanking
region off of the primary construct leaving only the internal
region. Many other suitable restriction enzymes can be used,
including BciVI, BmrI, FauI, BsrDI, AlwI and PleI, although these
molecule may leave overhanging bases that must either be cleaved
off or matched with a complement, depending on whether the overhang
is intended to be in the internal region or the flanking region.
The result of this step is a pool of DNA segments, each the size of
the internal regions. The pool contains multiple copies of each
internal region which was represented in the initial pool or
primary constructs.
[0018] Note that since use of the MAS or AGS instrument permits the
sequence of the oligonucleotides it constructs to be completely
variable. Hence, the sequence of each of the internal regions in
completely selectable and can be precisely defined by the user
before the process starts. The pool of constructs now created thus
consists of an amplified pool of internal regions composed of
sequences selected by the user for assembly into the target
sequence.
[0019] Once the internal regions are cut from the flanking region,
the cut pieces can separated, which can be done by any of several
DNA separation techniques. However, as will become apparent from
the examples below, it is not always necessary to perform any
separation at this stage. Instead, the now independent internal
regions can begin the assembly process. As in the process described
in PCT/US02/15951, the DNA sequences of the internal regions are
each complementary to sequences in the internal regions of other
species in the pool. However, in contrast to the situation in the
published PCT application, the assembly process starts from here
with a pool of double stranded, not single stranded, DNA molecules.
So in the method described here, it is preferred that the assembly
process be aided by a series of denaturing, annealing and extension
steps, with a DNA polymerase in the reaction. In other words, the
process is like a PCR reaction, but without any new primers added,
so that no amplification occurs. Consider what happens in each
annealing and extension step. As the internal regions find their
complements, some 40mer internal regions will match to their exact
40mer complements, but then the double stranded DNA molecule
created by that annealing will not be affected by the DNA
polymerase. Other internal regions will hybridize to a different
internal region that overlaps the first internal region only in
part, i.e. the first step in assembly of the target sequence. When
that occurs, the complex thus created is partially double stranded
and partially single stranded, and the DNA polymerase will add
complements to both single strands to their end. In the example of
an internal region with is a 40mer, and which overlaps another
40mer by 20 bases, the two hybridized strands would each have their
strands extended to create double stranded 60mers. This process can
then be repeated over and over, and in the process longer and
longer assembled molecules result, eventually resulting in the full
length target molecule. The largest molecule created by this
process will be the desired proper target sequence.
[0020] One might wonder why the flanking regions include PCR primer
sites. One can purchase kits for generalized PCR primers which are
intended to amplify all DNA. However, this method is subject to PCR
bias, in which some sequences may amplify better than others. By
using a common PCR primer in all the flanking regions which are
amplified in a common reaction, the possibility of bias in the
amplification process is minimized. Bias is also reduced by having
all the internal region sequences being of the same length (e.g. 40
base pairs) even though they differ in sequence.
EXAMPLES
[0021] Synthesis of Primary Constructs. The Automated Gene
Synthesizer (AGS-1) was used to make a chip containing two 60mer
oligonucleotides on a base-labile linker. The primary construct
oligonucleotides were designed for amplification and subsequent
gene assembly and consisted of (2) flanking 15mer primer sites
containing restriction sites (Mly I; GAGTC(N)5) and internal 30mer
fragments to be used for subsequent gene assembly.
[0022] After production, the primary construct oligonucleotides
were cleaved off the microarray by treatment of the entire
microarray with NH.sub.4OH for 30 minutes. The resulting solution
was then removed from the substrate of the microarray, transferred
to a tube and left for sixteen hours to allow for removal of base
protecting groups. The solution was then dried down in a speed
vacuum centrifuge and the precipitate was subsequently resuspended
in 5 .mu.l sterile Milli-Q water.
[0023] The microarray eluate aliquot (0.2 .mu.l) was used for PCR
amplification using two 15mer PCR primers containing the
restriction enzyme site (Mly I) and in the presence of Pfu
polymerase. The product of amplification was labeled with 32P using
T4 polynucleotide kinase and analyzed by gel electrophoresis on a
1XTBE 20% PAGE Urea gel. After electrophoresis at 1500V for one
hour, the gel was placed in a phosphoimager cassette and scanned
using the STORM Molecular Dynamics system. The results demonstrated
recovery of the intended DNA.
[0024] This same process was successfully repeated as noted above
except using a monohydroxysilane slide and its treatment with
NH.sub.4OH for 60 minutes to cleave oligonucleotides off its
surface. This was done to demonstrate that this process is
successful on multiple types of surfaces.
[0025] Synthesis and Assembly of 100 bp sequence. The Automated
Gene Synthesizer (AGS-1) was used to make a chip containing four
70mer oligonucleotides on a base-labile linker. Again, the primary
construct oligonucleotides were designed for amplification and
subsequent gene assembly and consisted of (2) flanking 15mer primer
sites containing restriction sites (Mly I; GAGTC(N)5) and internal
40mer fragments to be used for subsequent gene assembly. The
sequences of the four primary constructs were as follows:
TABLE-US-00001 5'TGCCGGAGTCAGCGTagaggatccccgggtaccggtagaaaaaatgag
taaagg GTGGCGACTCTGACT 3' 70mer-40F1a
5'TGCCGGAGTCAGCGTgaagaacttttcactggagttgtcccaattctt gttga
tGTGGCGACTCTGACT 3' 70mer-40F2a
5'TGCCGGAGTCAGCGTcccGttaacatcaccatctaattcaacaagaat tgggac
GTGGCGACTCTGACT 3' 70mer-40Rev9a
5'TGCCGGAGTCAGCGTactccagtgaaaagttcttctcctttactcatt ttttct
GTGGCGACTCTGACT 3' 70mer-40Rev9b
[0026] In the sequences presented above, the internal regions are
in lower case while the flanking regions are in upper case. The
recognition sites for Mly I are underlined. The cleavage site for
Mly I is between the upper and lower case letters. Note that the
flanking regions are identical in each of the primary constructs.
Note also that the 3' half of the internal region in 40F1a is
complementary to the 3' half of the internal region of 40Rev9b. The
5' half of the internal region 40Rev9b is complementary to the 5'
half of the internal region 40Rev9a, and the 3' half of the
internal region of 49Rev9a is complementary to the 3' half of the
internal region of 40F2a. The 5' end of the internal regions of
40F1a and 40Rev9a are unmatched.
[0027] After production on a microarray, the primary construct
oligonucleotides were cleaved off the substrate of the microarray
by treatment with NH.sub.4OH for 30 minutes. The solution was then
removed from the microarray, transferred to a tube and left for
sixteen hours to allow for removal of base protecting groups. The
eluate was then dried down in a speed vacuum centrifuge and the
precipitate was subsequently resuspended in 5 .mu.l sterile Milli-Q
water and used for gene assembly.
[0028] A chip eluate aliquot (0.2 .mu.l) was used for PCR
amplification, with two 15mer PCR primers containing the
restriction enzyme site (Mly I). After PCR amplification, the
product was digested overnight with the restriction endonuclease
Mly I to remove all of the flanking regions on all of the DNA
strands, leaving multiple copies of the four 40mer internal
regions. The unpurified restriction enzyme digest fragments were
then used for subsequent gene assembly and amplification
reactions.
[0029] Initial target sequence assembly was performed by combining
a fraction of the digested PCR product and Pfu polymerase, buffers,
and dNTPs in a reaction and cycling to denature, anneal and extend
the gene fragments. The assembled sequence was then amplified by
PCR with Pfu polymerase, labeled with .sup.32P using T4
polynucleotide kinase and analyzed by gel electrophoresis on a
1xTBE 20% PAGE Urea gel. After electrophoresis at 1500V for one
hour, the gel was placed in a phosphoimager cassette and scanned
using the STORM Molecular Dynamics system. Noted on the gel was the
assembly of a 100 bp DNA fragment. This fragment could only be the
target sequence resulting from assembly of the internal regions and
extension with the polymerase.
[0030] Synthesis and assembly of 180 bp sequence. The Automated
Gene Synthesizer (AGS-1) was used to make a chip containing eight
different 70mer oligonucleotide primary constructs on a
monohydroxysilane slide. The oligonucleotide primary constructs
were designed for amplification and subsequent gene assembly and
each consisted of (2) flanking regions, all of which were
identical, containing 15mer primer sites and containing restriction
sites (Mly I; GAGTC(N)5). Each primary construct also included the
40mer internal regions to be used for subsequent gene assembly. For
six of the primary constructs, each half of its internal region was
complementary to one half of the internal region of the internal
region of another primary construct. Two primary constructs had
internal regions that were complementary only for one half of their
region to another primary construct internal region.
[0031] We included quality control targets on the periphery of this
chip to be used to evaluate oligonucleotide synthesis quality. So
for this chip, the post-synthesis processing consisted of a 2 hour
soak in EDA/EtOH to remove the side-protecting groups from the
oligos on the chip, a 2 hour hybridization at 45.degree. C. with
the cy3 labeled complement of the QC target, scanning to verify a
successful synthesis, removal of the QC labeled probe by denaturing
at 75.degree. C. for 30 minutes, and then standard cleavage in
NH.sub.4OH for one hour, followed immediately by speedvac dry down
(16 our deprotection not necessary as side protecting groups were
previously removed in the 2 hour EDA/EtOH soak). The precipitate
was subsequently re-suspended in 5 .mu.l sterile Milli-Q water and
used for gene assembly.
[0032] A chip eluate aliquot (0.3 .mu.l) was used for PCR
amplification with two 15mer PCR primers containing the restriction
enzyme site (Mly I). After PCR amplification, the product was
digested overnight with Mly I to remove of the flanking regions,
leaving the four 40mer assembly internal regions. The unpurified
restriction enzyme digest fragments were then used for subsequent
gene assembly and amplification reactions. To perform successful
assemblies with increased numbers of oligos, it was noted that
increased amounts of the unpurified restriction enzyme digested DNA
was needed in the assembly reactions.
[0033] Initial gene assembly was performed by combining a fraction
of the digested PCR product with Pfu polymerase, buffers, and dNTPs
in a reaction vessel, and cycling to denature, anneal and extend
the gene fragments. No other primers were added. The assembled
sequence was then amplified by PCR with Pfu polymerase, and
analyzed by gel electrophoresis on a 3.5% agarose 1XTBE gel. After
electrophoresis at 110V for forty-five minutes, the gel was stained
with ethidium bromide and analyzed. The gel revealed the expected
180 base pair DNA product.
[0034] Synthesis and assembly of 340 bp sequence. Using the methods
described above, the assembly of a 340 base pair DNA sequence was
performed with 16 primary oligonucleotide constructs as primary
constructs and using four different primer sets for the PCR
reactions.
[0035] Synthesis and amplification of 400 sequences using 100
different primers sets. The automated gene synthesis instrument
(AGS) described above was used to make a microarray containing 700
different single stranded DNA sequences, each 70 nucleotides in
length (70mers). Each 70mer included a 40mer internal region for
the ultimate construct and two flanking 15mers containing primer
sites. The 400 sequences consisted of 100 sets of 4 oligos, with
each set using a unique primer site pair. These subsets were then
amplified each with their respective primer sets and the results
run on an gel. The gel showed the expected 70mer band for each of
the 100 oligonucleotides. A subset of the oligos (eight sets of 4
oligos) were then assembled using the methods previously described
and the results were analyzed by gel. The gel showed the expended
bands of 100mer for each of the eight sets. This demonstrates that
the method can be used in parallel with a large number of primer
sets.
Sequence CWU 1
1
5110DNAArtificialSynthetic Oligonucleotides 1gagtcnnnnn
10270DNAArtificialSynthetic Oligonucleotides 2tgccggagtc agcgtagagg
atccccgggt accggtagaa aaaatgagta aaggagtggc 60gactctgact
70370DNAArtificialSynthetic Oligonucleotides 3tgccggagtc agcgtgaaga
acttttcact ggagttgtcc caattcttgt tgaatgtggc 60gactctgact
70470DNAArtificialSynthetic Oligonucleotides 4tgccggagtc agcgtcccgt
taacatcacc atctaattca acaagaattg ggacagtggc 60gactctgact
70570DNAArtificialSynthetic Oligonucleotides 5tgccggagtc agcgtactcc
agtgaaaagt tcttctcctt tactcatttt ttctagtggc 60gactctgact 70
* * * * *