# Patent

## Title
Methods for ligation independent cloning of DNA

## Abstract

The present invention provides methods for assembling DNA molecules in a predetermined order to produce a DNA construct useful in Agrobacterium mediated transformation in plants. The method employs ligation independent cloning of separate DNA elements where one of the DNA elements contains T-DNA borders from Agrobacterium tumefaciens. The invention further provides methods for assembling a construct containing an inverted repeat. Using this approach, DNA constructs are constructed rapidly, efficiently and directionally.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims benefit of priority under 35 U.S.C. 119(e) to provisional application Ser. No. 60/634,779 filed Dec. 9, 2004.


INCORPORATION OF SEQUENCE LISTING
Two copies of the sequence listing (Seq. Listing Copy 1 and Seq. Listing Copy 2) and a computer-readable form of the sequence listing, all on CD-R's, each containing the file named 38-21(53344)B_seqListing.txt, which is 71,680 bytes (measured in MS-WINDOWS) and was created on Mar. 26, 2008, are hereby incorporated by reference.
BACKGROUND OF THE INVENTION
Disclosed herein are methods for assembling DNA molecules in a predetermined order in a DNA construct and methods of using such constructs.
Many methods have been developed and used for assembling DNA molecules. Examples of these are cloning by restriction endonuclease digestion followed by ligation of compatible ends, T-A cloning directly from a polymerase chain reaction product, recombination-based cloning and ligation-independent cloning of polymerase chain reaction products.
Ligation-independent cloning is a highly efficient and cost effective method for assembling DNA molecules using non-covalent, bi-molecular association of terminal, single-stranded DNA segments to assemble DNA molecules. Terminal, single-stranded DNA segments can be incorporated into DNA molecules by a variety of methods. Examples of methods for producing terminal, single-stranded DNA segments include, but are not limited to, (a) ligation of adapter molecules containing a terminal, single-stranded DNA segment, (b) incorporation of nicking endonuclease sites during polymerase chain reaction, (c) exonuclease digestion and (d) incorporation of deoxy-uridine residues during polymerase chain reaction which can be deaminated by the enzyme, uracil-N glycosylase. Such methods are described in Aslanidis and de Jong, Nucleic Acids Research, 18 (20), pages 6069-6074 (1990); U.S. Pat. No. 5,580,759 (Yang, et al.); U.S. Pat. No. 5,137,814 (Rashtchian et al.) and U.S. Pat. No. 6,495,318 (Harney), all of which are incorporated herein by reference. Once assembled, the DNA construct is repaired and replicated in the bacterial host using the host's endogenous systems.
Conventional cloning methods can be time consuming and often rely on a series of sub cloning steps to produce the final DNA construct. DNA constructs used in plant transformation contain a number of necessary components for replication, transformation and selection. For example, the DNA construct must have an origin of replication and selectable marker for the bacterial hosts, both for Escherichia coli as well as Agrobacterium tumefaciens. For plant transformation, the construct must also contain a right and left border sequence to allow for the transfer and stable incorporation of the DNA from Agrobacterium to the plant host cells. The DNA construct will also usually contain a eukaryotic promoter, which drives the expression of protein coding sequences and transcriptional terminators. DNA constructs typically also comprise selectable or screenable markers used in identifying the transformed plant host cells. The final DNA construct can be quite large. Therefore, a need exists for developing simpler and widely applicable methods for producing constructs used in plant transformation.
SUMMARY OF THE INVENTION
This invention provides methods for assembling DNA molecules in a predetermined order to produce a DNA construct useful in Agrobacterium mediated transformation in plants. More particularly, the method employs ligation independent cloning of separate DNA elements where one of the DNA elements contains T-DNA borders from Agrobacterium tumefaciens. One aspect of this invention provides a method for assembling DNA molecules in a predetermined order in a DNA construct, said method comprising:
(a) providing at least two double-stranded DNA molecules each of which has terminal, single-stranded, DNA segments in a length of from 10 to 30 nucleotides extending from the 5′ and 3′ termini, wherein terminal, single-stranded DNA segments on each double-stranded DNA molecule do not hybridize to each other, wherein a terminal, single-stranded DNA segment on a first and second double-stranded DNA molecule hybridize to each other or to a single-stranded DNA oligomer of from about 20 to at least about 40 nucleotides to allow for specific annealing and linkage of the DNA molecules in a predetermined order, and wherein one of said double-stranded DNA molecules contains T-DNA borders from Agrobacterium tumefaciens; and

(b) incubating said DNA molecules under conditions suitable to promote the specific annealing and assembling of the DNA molecules in a predetermined order.

In an embodiment of the method, one of the molecules is a plasmid backbone which contains right and left T-DNA borders. Sense and antisense versions of a construct can be generated by repeating steps (a) and (b) with one of the DNA molecules modified with terminal, single-stranded DNA segments interchanged to the opposite sides of the double-stranded DNA molecule. A DNA construct containing an inverted repeat can be produced using DNA molecules that comprise the same or a portion of the same DNA molecule with two sets of terminal, single-stranded DNA segments interchanged to the opposite sides of the double-stranded DNA molecule. The resulting DNA molecules are then capable of being assembled in a manner that produces the inverted repeat. Furthermore, the terminal, single-stranded DNA segments for the inverted repeat construct can be designed in such a manner as to allow the production of restriction endonuclease sites within the region joining the repeats. This enhancement allows one skilled in the art to digest and cleave the DNA construct in between the repeats facilitating the sequence confirmation of the repeat region. Without cleavage of the construct in this manner, DNA sequencing becomes contaminated by conflicting information as the sequence reaction proceeds through the repeat region.
The DNA molecules in this method encodes a biological functionality selected from the group consisting of promoter or portion thereof, origin of replication, selectable marker, transcriptional regulatory element, cellular localization signal, protein processing sequence, plastid targeting sequence, structural gene or molecule thereof, transcription termination signal, translational regulatory signal, recombination elements, mutagenized genes, artificial genes, chimeric genes, protein domain encoded regions or portion there of, tagging epitopes, DNA spacer regions, synthetic multiple cloning sites, unique restriction enzyme or DNA cleavage sites, and sites for covalent or noncovalent attachment of a biological or a chemical molecule.
Another aspect of this invention provides a method for assembling an inverted repeat of DNA molecules in a predetermined order in a DNA construct, said method comprising:
(a) providing at least two double-stranded DNA molecules each of which has terminal, single-stranded, DNA segments in a length of from 10 to 30 nucleotides extending from the 5′ and 3′ termini, wherein terminal, where single-stranded DNA segments on each double-stranded DNA molecule do not hybridize to each other, and where a terminal, single-stranded DNA segments on a first and a second double-stranded DNA molecule hybridize to each other or to a single-stranded DNA oligomer of from about 20 to at least about 40 nucleotides to allow for specific annealing and linkage of the DNA molecules in a predetermined order, and where two of the at least two double-stranded DNA molecules are substantially identical and have a common terminal single-stranded DNA segment that allows assembly of an inverted repeat of DNA; and

(b) incubating said DNA molecules under conditions suitable to promote the specific annealing and assembling of the DNA molecules in an inverted repeat order.

The terminal, single-stranded DNA segments for the inverted repeat construct can be designed in such a manner as to allow the production of restriction endonuclease sites within the region joining the repeats. This enhancement allows one skilled in the art to digest and cleave the DNA construct in between the repeats facilitating the sequencing of the repeat region.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1a and 1b illustrates alternative methods to assemble DNA molecules using terminal, single-stranded DNA segments. Terminal single stranded segments labeled 2, 3, 8 and 9 are represented by SEQ ID NOS: 30, 31, 32 and 33, respectively. A single-stranded oligomer used to bridge single-stranded DNA segments 8 and 10 is represented as SEQ ID NO: 34.
FIG. 2 illustrates a plasmid backbone useful in the methods of this invention. The circularized plasmid backbone is provided as SEQ ID NO: 1.
FIG. 3 illustrates the production of a plasmid vector with terminal, single-stranded segments. The single-stranded segments are comprised of SEQ ID NO:2 which is bisected after restriction endonuclease and nicking endonuclease digestion along with heating to remove the nicked short complementary strands, resulting in the production of the terminal, single-stranded segments.
FIG. 4 illustrates a DNA construct assembled from two DNA molecules and is Presented as SEQ ID NO: 8.
FIG. 5 illustrates PCR amplification to produce DNA with terminal, single-stranded segments using primers provided as SEQ ID NO: 4 and SEQ ID NO: 5, followed by PCR amplification using primers provided as SEQ ID NO: 6 and SEQ ID NO: 7.
FIG. 6 illustrates the assembly of DNA molecules into a plasmid.
FIG. 7 illustrates an amplification and assembly scheme for producing an inverted repeat. The terminal, single-stranded DNA segments A. B. C and D in the two Waxy DNA molecules are produced using the primers presented as SEQ ID NOS: 10 and 14 (for terminal, single-stranded DNA segment A); SEQ ID NOS: 11 and 15 (for terminal, single-stranded DNA segment B); SEQ ID NOS: 13 and 17 (for terminal, single-stranded DNA segment C) and; SEQ ID NOS: 12 and 16 (for terminal, single-stranded DNA segment D).
FIG. 8 illustrates a DNA construct assembled from three DNA molecules and is presented as SEQ ID NO; 18.
FIG. 9 illustrates the spacer region within an inverted repeat. The region between the repeats (referred herein as TAG-ART.Spacer Region 2) is provided as SEQ ID NO: 19.
FIG. 10a illustrates an antisense molecule with terminal, single-stranded DNA segments. The terminal, single-stranded DNA segments E and F are produced using the primers presented as SEQ ID NOS: 21 and 23 (for terminal, single-stranded DNA segment E) and SEQ ID NOS: 20 and 22 (for terminal, single-stranded DNA segment F).
FIG. 10b illustrates an amplification scheme for making a fusion amplicon with terminal, single-stranded DNA segments. The terminal, single-stranded DNA segments G and H are produced using the primers presented as SEQ ID NO: 27 (for terminal, single-stranded DNA segment G) and SEQ ID NOS: 25 and 28 (for terminal, single-stranded DNA segment H).
FIG. 11 illustrates an assembly scheme for producing an inverted repeat. The terminal, single-stranded DNA segments, E, F, G and H are produced using the primers presented as SEQ ID NOS: 21 and 23 (for terminal, single-stranded DNA segment E); SEQ ID NOS: 20 and 22 (for terminal, single-stranded DNA segment F); SEQ ID NO: 27 (for terminal, single-stranded DNA segment G) and; SEQ ID NOS: 25 and 28 (for terminal, single-stranded DNA segment H).
FIG. 12 illustrates a DNA construct assembled from three DNA molecules and is presented as SEQ ID NO: 29.



DETAILED DESCRIPTION OF THE INVENTION
The sequence listing comprises:
SEQ ID NO: 1—nucleotides in the plasmid backbone shown in FIG. 2;
SEQ ID NO: 2—nucleotides in an adapter molecule;
SEQ ID NO: 3—nucleotides coding for A. thaliana CGPG3721;
SEQ ID NO: 4-7—of nucleotides for primers;
SEQ ID NO: 8—nucleotides in the assembled plasmid shown in FIG. 6;
SEQ ID NO: 9—nucleotides coding for a segment of maize Waxy;
SEQ ID NO: 10-17—nucleotides for primers;
SEQ ID NO: 18—nucleotides in the assembled plasmid shown in FIG. 8;
SEQ ID NO: 19—nucleotides in a spacer region between inverted repeats as illustrated in FIG. 9;
SEQ ID NO: 20-25—nucleotides for primers;
SEQ ID NO: 26—nucleotides in a spacer region between inverted repeats as illustrated in FIG. 11;
SEQ ID NO: 27-28—nucleotides for primers; and
SEQ ID NO: 29—nucleotides in the assembled plasmid shown in FIG. 12.
SEQ ID NO: 30—nucleotide sequence of terminal, single-stranded DNA segment, indicated by “2” in FIG. 1a. 
SEQ ID NO: 31—nucleotide sequence of terminal, single-stranded DNA segment, indicated by “3” in FIG. 1a. 
SEQ ID NO: 32—nucleotide sequence of terminal, single-stranded DNA segment, indicated by “8” in FIG. 1b. 
SEQ ID NO: 33—nucleotide sequence of terminal, single-stranded DNA segment, indicated by “9” in FIG. 1b. 
SEQ ID NO: 34—nucleotide sequence of a single-stranded oligomer 6 used to bridge terminal, single-stranded DNA segment 8 and 9 in FIG. 1b. 
SEQ ID NO: 35—nucleotide sequence of an inserted DNA molecule, indicated as “15” in FIG. 6 represented by nucleotide 10,258 through nucleotide 1 of the plasmid presented as SEQ ID NO: 8.
As used herein “T-DNA” means transfer-DNA which integrates into a genome by Agrobacterium-mediated transformation. For Agrobacterium-mediated transformation T-DNA is typically flanked by T-DNA borders within a plasmid, for example, a binary vector plasmid which is transferred into an Agrobacterium tumefaciens strain carrying a disarmed tumor inducing (Ti) plasmid.
As used herein, the term “assembling” means a process in which DNA molecules are joined through hybridization of terminal, single-stranded DNA segments. The terminal, single-stranded DNA segments are preferably non-palindromic sequences, which can be produced either through polymerase chain reaction or ligation. The terminal, single-stranded DNA segments enable users to specify the precise organization and orientation of DNA molecules in a construct. In one embodiment of the method, the terminal, single-stranded DNA segments are added during polymerase chain reaction. Oligonucleotides are synthesized to contain a sequence of nucleotides, which will be complementary to another terminal, single-stranded DNA segment. Within the oligonucleotide sequence, uridine residues are substituted for thiamine residues in specific positions. Amplification is performed using a thermal stable polymerase capable of reading through uridine residues in the template. After polymerase chain reaction, the resulting product is treated with Uracil-DNA glycosylase (UDG), which specifically deaminates the uridine residues. The DNA strand containing the uridine residues becomes unstable after UDG treatment in the positions containing uridine. Following heat treatment, the double-stranded DNA molecule becomes single-stranded in the region containing the uridine residues. In another embodiment of the method, a terminal single-stranded DNA segment can be introduced using nicking endonucleases. Nicking endonucleases hydrolyze only one strand of the double-stranded DNA molecule. A nicking endonuclease site can be incorporated into the DNA molecule either through conventional cloning methods available to those skilled in the art or through polymerase chain reaction. Oligonucleotides for polymerase chain reaction can be designed to contain the recognition sequence for any of several commercially available nicking endonucleases. After polymerase chain reaction amplification, the polymerase chain reaction product is treated with the appropriate nicking enzyme. After enzyme treatment, the product is incubated at a temperature sufficient to cause loss of the hydrolyzed strand, resulting in a terminal, single-stranded DNA segment. In another embodiment of the method, terminal, single-stranded DNA segments can be introduced by ligation of adapter molecules to the DNA molecule. Assembling of the DNA molecules can occur directly through the hybridization of the terminal, single-stranded DNA segments.
For example, as depicted in FIG. 1a, DNA molecule 1 with a terminal, single-stranded DNA segment 2 (presented as SEQ ID NO: 30) anneals to the terminal, single-stranded DNA segment 3 (presented as SEQ ID NO: 31) of DNA molecule 4. Alternatively, an oligomer can be used to bridge two terminal, single-stranded DNA segments, as depicted in FIG. 1b where single-stranded oligomer 6 (presented as SEQ ID NO: 34) is used to bridge DNA molecules 7 and 10 by annealing to their respective terminal, single-stranded DNA segments 8 (presented as SEQ ID NO: 32) and 9 (presented as SEQ ID NO: 33).
As used herein, the term “DNA construct” refers to an assembled DNA molecule, for example, an assembled plasmid which is capable of autonomous replication within the bacterial hosts, Escherichia coli and Agrobacterium tumefaciens and contains elements necessary for stable integration of DNA contained within the plasmid into the plant host cells.
As used herein, the term “plasmid backbone” describes a DNA molecule, which contains at least the elements necessary for autonomous replication within the bacterial hosts, Escherichia coli and Agrobacterium tumefaciens and contains right and left borders necessary for stable integration of DNA into the plant host cell. The plasmid backbone will also contain a selectable marker for bacterial selection and a different selectable marker used in identifying transformed plant cells. FIG. 2 and Table 1 illustrate DNA elements that are typically contained in a plasmid backbone. For replication in the bacterial host cells, two origins of replication are present. One origin of replication is used for plasmid replication in an Escherichia coli host cell (OR-Ec.ori-ColE1) A repressor of the ColE1 origin of replication (rop) is also present to keep the copy number of the plasmid low to minimize any potential toxic effects from the assembled DNA construct. Another origin of replication is provided for replication within the Agrobacterium host cell, the RK2 vegetative origin of replication. (OR-Ec.oriV-RK2) A gene conferring resistance to the antibiotic, spectinomycin and its associated promoter and terminator is used for selection of bacterial transformants and to maintain the plasmid within the bacterial hosts (antibiotic marker). Right and left borders (RB) and (LB), essential for the transfer and integration of DNA from the Agrobacterium host to the plant cell are contained within the backbone.
During Agrobacterium-mediated transformation DNA between the right and left borders will be transferred and integrated into the DNA of the host plant cell. Between the right and left borders in FIG. 2, there is an nptII (neomycin phosphotransferase II) selectable marker transcription unit and an incomplete transcription unit. The nptII selectable marker transcription unit confers resistance to the antibiotic kanamycin and is used for selecting transformed plant host cells. The nptII unit comprises the cauliflower mosaic virus 35S RNA gene promoter, the nptII coding region and nopaline synthase transcriptional terminator. The incomplete transcription unit comprises a rice actin promoter unit which includes a 5′UTR exon and intron (P-Os.Act). The promoter is used to control the transcription of an operably linked DNA molecule (for example, a peptide coding region, an antisense DNA molecule or an inverted repeat DNA molecule) that can be inserted between the promoter and the transcriptional terminator of the potato proteinase inhibitor II gene (T-St.Pis4). The elements of the plasmid are summarized in Table 1 where the element position identifies a position in SEQ ID NO: 1.









TABLE 1





Element Name
Position
Element Function







RB
5235-5591
Right border sequence for T-DNA




transfer


P-Os.Act
5609-7009
Promoter from the rice actin gene,




act1 including first exon and first




intron and flanking UTR exon from




the rice actin gene, act1.


T-St.Pis4
7084-8026
Transcription termination sequence




of the potato proteinase inhibitor




II gene.


NptII marker
8075-9507
transcription unit for nptII




marker including promoter and




5′UTR for the CaMV 35S RNA,




coding region for nptII from




E. coli and transcription termi-




nation sequence of from nopaline




synthase gene from Agrobacterium.


IG-St.Pis4
 9519-10265
Intergenic region of the potato




proteinase inhibitor II gene.


LB
 39-480
Left border sequence for T-DNA




transfer.


OR-Ec.oriV-RK2
567-963
Vegetative origin of replication




used by Agrobacterium tumefaciens.


CR-Ec.rop
2472-2663
Coding region for the repressor




of the primer for the ColE1 origin




of replication.


OR-Ec.ori-ColE1
3091-3679
Minimal origin of replication from




the Escherichia coli plasmid,




ColE1.


Antibiotic marker
4210-5098
Transcription unit for antibiotic




marker including promoter for Tn7




adenyltransferase, coding region




for Ec.aadA-SPC/STR, transcription




terminator region from Tn7 adenyl-




transferase conferring spectino-




mycin and streptomycin resistance.









As used herein, the term “substantially identical” describes two DNA molecules which contain DNA sequence that has been derived from the same DNA molecule and contain a region of identity that is at least 19 nucleotides in length.
In the methods of this invention, any DNA molecule can be inserted into a plasmid, i.e., a plasmid backbone. Different promoters, transcriptional terminators and introns can be assembled with a plurality of coding and non-coding DNAs to address a number of user-defined criteria (for example, the expression of a protein encoding DNA or the expression of an inverted repeat). Multiple transcription units (often called gene cassettes) can also be assembled into the plasmid backbone. The methods of this invention can be used to assemble DNA constructs in a high throughput production manner. Any DNA molecule can be designed to contain unique terminal, single-stranded DNA segments, allowing assembly with other DNA molecules in a specific order and orientation. A single plasmid backbone can be used as a template to assemble many different DNA constructs in a production facility by incorporating compatible terminal, single-stranded DNA segments in a collection of DNA molecules chosen for assembly.
The DNA molecules for insertion into a plasmid backbone can be any DNA molecule derived from any source or produced by any means including, but not limited to, amplification such as by polymerase chain reaction or through the use of other polymerases, isolation from natural sources, chemical synthesis, shearing or restriction digest of larger DNA molecules (such as genomic or cDNA), transcription, reverse transcription, and the like. Such DNA molecules may be derived from or comprise DNA from any natural sources such as cells (for example, prokaryotic cells or eukaryotic cells), viruses, tissues, organs (such as organs from any animal, plant, or other source), and organisms or may be derived from or comprise DNA from non-natural or synthetic sources (for example, derivative DNA molecules).
Alternative terminal, single-stranded DNA segments can be created by annealing DNA oligos or DNA adapters that contain a region of homology to a terminal, single-stranded DNA segment. The single DNA strand of the duplex DNA molecule containing the terminal, single-stranded DNA segment can be altered by annealing an oligo to the existing terminal, single-stranded DNA segment.
The following examples are provided to assist those skilled in the art to practice methods of this invention. The examples should not be construed to unduly limit the invention because modifications and variations of the embodiments discussed herein will be apparent to those of ordinary skill in the art without departing from the spirit or scope of the disclosed invention.
Example 1
This example illustrates the introduction of terminal, single-stranded DNA segments into a plasmid backbone. With reference to FIG. 3, a DNA adapter molecule, having the nucleotide sequence of SEQ ID NO:2, was inserted between an AgeI (ACCGGT) and a KpnI (GGTACC) restriction site in the plasmid of FIG. 2 using T4 DNA ligase (Invitrogen, Carlsbad, Calif.), inserting the adapter between promoter P-Os.Act) and the transcription termination sequence (T-St.Pis4). The resulting plasmid was linearized using the restriction endonuclease, HpaI (GTTAAC), then treated with the nicking endonuclease, N.BbvC IA (GCTGAGG). After digestion, the reaction was heated to 65 degree Celsius, causing the nicked strands of DNA to disassociate from their complementary DNA strands providing the resulting linearized, plasmid with two terminal, single-stranded DNA segments. Each terminal, single-stranded segment contained a unique sequence of nucleotides to allow assembly with another DNA molecule in a specific directionality.
Example 2
This example illustrates the addition of two, terminal, single-stranded DNA segments to a protein coding sequence for insertion into the plasmid that has been linearized with terminal single-stranded DNA segments.
DNA encoding the Arabidopsis thaliana protein for a conserved oligomeric Golgi complex component-related protein (CGPG3721) has the nucleotide sequence of SEQ ID NO:3. Polymerase chain reaction was employed to produce the terminal, single-stranded DNA segments in the DNA molecule encoding CGPG3721. As shown in FIG. 5 the sequence of the CGPG3721 coding sequence (SEQ ID NO:3) was used to design the primers with nucleotide sequence of SEQ ID NO: 4 (sense) and 5 (antisense). Each primer comprises one of two terminal, single-stranded DNA segments to be added to the CGPG3721 coding sequence (SEQ ID NO:3). The primers were designed as follows:
Sense Primer:










5′-GCAGTCGCTGTCGTTACCATGGCAATGGAGGTTGGGG-3′







Antisense Primer:
5′-GCGAGTACCGCTGGGTTCTAAGACTCGGGTGTTATCTCAGGAGTC-3′ where, in each primer, the underlined portion of the sequence represents the region that will be made into a terminal, single-stranded DNA segment.
Polymerase chain reaction amplification was performed using a high fidelity thermal polymerase, KOD hot start DNA polymerase (Novagen, Madison Wis.). The polymerase chain reaction was performed in a 25 microliters (μl) volume containing, 1×KOD hot start DNA polymerase buffer, 1M Betaine (Sigma, St. Louis Mo.), 1 mM MgSO4, 250 μM dNTPs, 5 pmols of each primer (primer molecules 11 and 12 in FIG. 5) and 1 unit of KOD hot start DNA polymerase. The polymerase chain reaction was performed in a PTC-225 DNA Engine TetradTM thermal cycler (MJ Research Inc., Waltham Mass.) using the following cycler parameters (“cycle parameter 1”):
1. 94 degree Celsius for 2 minutes
2. 94 degree Celsius for 15 seconds
3. 70 degree Celsius for 30 seconds (−1 degree Celsius per cycle)
4. 72 degree Celsius for 5 minutes
5. Go to step 2, 9 times
6. 94 degree Celsius for 15 seconds
7. 60 degree Celsius for 30 seconds
8. 72 degree Celsius for 5 minutes
9. Go to step 6, 24 times
10. 72 degree Celsius for 10 minutes
11. 10 degree Celsius forever
12. end
A second round of polymerase chain reaction was performed to introduce uridine residues in the region in which the terminal, single-stranded DNA segments were produced (primer molecules 13 and 14 in FIG. 5). Many DNA polymerases are unable to read uridine residues in the template strand of DNA or are unable to polymerize strands using uridine residues. Polymerase chain reaction was therefore performed using an enzyme capable of incorporating and reading uridines, Expand High Fidelityplus PCR System (Roche, Indianapolis Ind.). The primers designed as the second pair of oligonucleotide primers comprising the two terminal, single-stranded DNA segments are provided as SEQ ID NOS: 6 (sense) and 7 (antisense). The primers were designed as follows:
Sense:
5′-GCAGUCGCTGUCGTUACCATG-3′
Antisense:
5′-GCGAGUACCGCUGGGTUCTA-3′
The polymerase chain reaction was performed in a 25 μl volume containing, 1× Expand High Fidelityplus polymerase chain reaction buffer, 1.5 mM MgCl2, 200 μM dNTPs, 5 pmols of each primer, 5 units of Expand High Fidelityplus Enzyme Blend and 1 μl of the first polymerase chain reaction product. The polymerase chain reaction was performed in a PTC-225 DNA Engine TetradTM thermal cycler using the following cycler parameters (“cycle parameter 2”):
1. 94 degree Celsius for 2 minutes
2. 94 degree Celsius for 30 seconds
3. 55 degree Celsius for 30 seconds
4. 72 degree Celsius for 4 minutes
5. Go to step 2, 14 times
6. 72 degree Celsius for 10 minutes
7. 10 degree Celsius forever
8. End
Terminal, single-stranded DNA segments on DNA coding for CGPG3721 were produced by treating the product of the second polymerase chain reaction with Uracil-DNA Glycosylase (New England Biolabs Inc., Beverly, Mass.). The Uracil-DNA Glycosylase reaction was performed in a 10 μl volume containing, 5 μl of the second polymerase chain reaction product, 3.5 μl of deionized water, 0.5 μl 5× Expand High Fidelityplus polymerase chain reaction buffer (containing 7.5 mM MgCl2), and 1 μl of Uracil-DNA Glycosylase (2 units of enzyme). The Uracil-DNA Glycosylase reaction was performed in a PTC-225 DNA Engine TetradTM thermal cycler at 37 degree Celsius for 1 hour.
Example 3
This example illustrates the assembly of a DNA construct. With reference to FIG. 6 the plasmid illustrated in FIG. 4 was prepared by assembling the linearized plasmid 16 which was prepared in Example 1 and the DNA molecule 15, presented as SEQ ID NO: 36 (represented by nucleotide 10,258 through nucleotide 1 of the plasmid presented as SEQ ID NO: 8) which was modified with terminal single-stranded DNA segments in Example 2. One microliter of the linearized plasmid (25 nanograms (ng)) was added to the Uracil-DNA Glycosylase reaction product prepared in Example 2 and incubated in a PTC-225 DNA Engine TetradTM thermal cycler using the following cycler parameters (“cycle parameter 3”):
1. 65° for 5 minutes
2. −0.1°/sec to 37° C.
3. 37° C. forever
4. End
The assembled DNA construct was transformed into ElectroMAXTM DH10B competent cells (Invitrogen, Carlsbad Calif.). A 0.5 μl aliquot from the assembly reaction was mixed with 20 μl of ElectroMAXTM DH 10B competent cells on ice and loaded into a MicroPulser 0.2 mm electroporation cuvette (Bio-Rad Laboratories Inc., Hercules Calif.) for electroporation. The cells were transformed by electroporation using 1.8 kV with a 165-2100 MicroPulser Electroporator (Bio-Rad Laboratories Inc., Hercules Calif.). The transformed cells were incubated in 180 μl of SOC medium (Invitrogen Inc., Carlsbad Calif.) at 37 degree Celsius for 1 hour. The cells were then plated onto LB agar plates containing spectinomycin (75 mg/1) and grown overnight at 37 degree Celsius. Colonies of transformed cells were selected and grown in LB media overnight at 37 degree Celsius. The plasmid DNA construct was isolated using the QIAprep® Spin Miniprep Kit (QIAgen Sciences, Valencia Calif.). DNA sequencing was performed on an Applied Biosystems 3730×1 DNA Analyzer, using Big Dye® Terminator v3.0 for sequence validation. The nucleotide sequence of the assembled plasmid show in FIG. 4 is SEQ ID NO: 8.
Example 4
This example illustrates the assembly of a DNA construct with an inverted repeat, using three DNA molecules each having terminal single-stranded DNA segments. One molecule is a linearized plasmid prepared essentially by the method illustrated in Example 1. The other two molecules were amplified from the same coding sequence but assembled in a manner to allow the production of an inverted repeat. The region between the repeats was produced from terminal, single-stranded DNA segments, resulting in a region between the repeats with restriction endonuclease sites.
With reference to FIG. 7 an inverted repeat DNA molecule with terminal, single-stranded DNA segments was prepared from DNA coding for maize granule-bound glycogen synthase, which is referred to as “Waxy”. PCR was employed to produce unique terminal, single-stranded DNA segments on separate DNA molecules which were designed to be assembled in an inverted repeat of Waxy DNA. The Waxy DNA sequence which was used for primer design is provided as SEQ ID NO:9. The primers designed as the first two pairs of oligonucleotide primers comprising the two terminal, single-stranded DNA segments of each DNA molecule were designed as follows:
SEQ ID NO: 10—Sense primer, Waxy 3








5′-AAGTACTGCGATCGCGTTAACGCTAGATTCAAGTCGTCCTTCGATTT



CATCG-3′






SEQ ID NO: 11—Antisense primer, Waxy 1









5′-GCAGTCGCTGTGCGATACCACGTCGGGGCCCTTCTGCTCTTCCA-3′







SEQ ID NO: 12—Sense primer, Waxy 2









5′-AGCGTTAACGCGATCGCAGTACTTGAAGCTCCAAGGATCCTGAGCCT



CAAC-3′






SEQ ID NO: 13—Antisense primer, Waxy 4

5′-GCGAGTACCGCTGGCGATCTAACGTCGGGGCCCTTCTGCTCTTCC-3′ where, in each primer, the underlined portion of the sequence represents the region that will be made into a terminal, single-stranded DNA segment. Polymerase chain reaction amplification was performed essentially as described in Example 2 using cycle parameter 1. A second round of polymerase chain reaction was performed to introduce uridine residues in the region in which the terminal, single-stranded DNA segments were produced. Polymerase chain reaction was performed as previously described in Example 2 using cycle parameter 2. The primers designed as the second pair of oligonucleotide primers comprising the two terminal, single-stranded DNA segments were designed as follows:
SEQ ID NO: 14—Sense primer, Waxy 3U










5′-AAGUACTGCGAUCGCGTUAACGCU-3′







SEQ ID NO: 15—Antisense primer, Waxy 1U











5′-GCAGUCGCUGUGCGAUACC-3′







SEQ ID NO: 16—Sense primer, Waxy 2U











5′-AGCGTUAACGCGAUCGCAGUACUU-3′







SEQ ID NO: 17—Antisense primer, Waxy 4U











5′-GCGAGUACCGCUGGCGAUCTA-3′







The terminal, single-stranded DNA segments A, B, C and D in the two Waxy DNA molecules were produced by treating equal molar amounts of the products of the second polymerase chain reaction with Uracil-DNA glycosylase and assembled essentially as described in Example 3 using cycle parameter 3, where single-stranded DNA segment A is complementary to single-stranded DNA segment D allowing assembly of the antisense and sense oriented Waxy segments into an inverted repeat construct.

Following assembly of the Waxy inverted repeat, the inverted repeat and the linearized plasmid, each having terminal, single-stranded DNA segments, were assembled essentially as described in Example 3 using cycle parameter 3. The final assembled DNA construct as shown in FIG. 8 comprised the elements described in Table 2 where the position is indicated by reference to SEQ ID NO: 18.









TABLE 2





Element Name
Position
Element Function







RB
11493-11823
Right border sequence for T-DNA




transfer.


P-Zm.SzeinC1
  1-1909
Promoter elements from the 27 KDa




storage protein zein gene from




Maize including 5′UTR and an




enhancer intron from the Zea mays




HSP70 intron with flanking exon




enhancer sequence.


CR-Zm.Waxy
1943-2577
Antisense coding region of the


AS

Zea mays Waxy gene.


Spacer
2578-2601
Spacer region, SEQ ID NO: 19.


CR-Zm.S
2602-3002
Sense coding region of the


Waxy

Zea mays Waxy gene.


T-St.Pis4
3044-3896
Transcription termination sequence




of the potato proteinase inhibitor




II gene.


nptII
4031-5476
Transcription unit for nptII


marker

marker including CaMV 35S promoter,




coding region for nptII and nos




transcription termination sequence.


IG-St.Pis4
5488-6234
Intergenic region of the potato




proteinase inhibitor II gene used




for marker excision.


LB
6297-6738
Left border sequence for T-DNA




transfer.


OR-Ec.oriV-
6825-7221
Vegetative origin of replication


RK2

used by Agrobacterium tumefaciens.


CR-Ec.rop
8730-8921
Coding region for the repressor




of the primer for the ColE1 origin




of replication.


OR-Ec.ori-
9439-9937
Minimal origin of replication


ColE1

from the Escherichia coli plasmid




ColE1 used for replication in




E. coli.


Antibiotic
10468-11356
Transcription unit for antibiotic


marker

resistance.









The assembled DNA construct was transformed into ElectroMAXTM DH10B competent cells as previously described in Example 3.
Sequencing through an inverted repeat results in contamination of the sequence chromatogram due to false priming within the inverted repeat. Therefore, it becomes necessary to linearize the inverted repeat between the repeats prior to sequencing. In the present example, the terminal, single-stranded DNA segments that were used to assemble the Waxy inverted repeat were designed to contain three restriction endonuclease sites which were not present in the plasmid backbone, containing terminal, single-stranded DNA segments or the regions of DNA encoding the Waxy protein. The region between the repeats (referred herein as TAG-ART.Spacer Region 2) is illustrated in FIG. 9 and provided as SEQ ID NO: 19. Two digestion reactions were performed on the assembled construct, pMON94750 using HpaI (GTTAAC) and AsiSI (GCGATCGC) and 400 ng of pMON94750 per reaction. After digestion, the linearized construct was precipitated. DNA sequencing confirmation was performed on an Applied Biosystems 3730×1 DNA Analyzer, using Big Dye® Terminator v3.0 using sequencing primers designed to read from the HSP70 intron and the Pis4 terminator through the Waxy inverted repeat. Using sequence data from both digestions, the full sequence of the Waxy inverted repeat was determined.
Example 5
This example illustrates an alternative assembly of a DNA construct useful for gene suppression of the Waxy gene with a spacer inserted between the anti-sense and sense segment of the Waxy gene.
With reference to FIG. 10a an anti-sense segment of Waxy coding region of about 635 nt (indicated as “CR-Zm.Waxy AS” in Table 3 and in the construct illustrated in FIG. 12) is amplified from the Waxy gene by PCR followed by uracil-DNA glycosylase treatment to produce unique terminal, single-stranded DNA segments E and F. More specifically, the segment for the anti-sense oriented Waxy is cloned using a sense primer (SEQ ID NO:20) and an anti-sense primer (SEQ ID NO:21), where the first 16 and 24 nucleotides of each primer, respectively are designed for single-stranded DNA segments. Polymerase chain reaction amplification is performed essentially as described in Example 2 using cycle parameter 1. A second round of polymerase chain reaction is performed to introduce uridine residues in the region in which the terminal, single-stranded DNA segments are produced. Polymerase chain reaction is performed as previously described in Example 2 using cycle parameter 2. The primers designed as the second pair of oligonucleotide primers comprising the two terminal, single-stranded DNA segments of the antisense DNA molecule are designed as follows:
SEQ ID NO: 22—Sense primer










5′-GCAGUCGCUGUGCGAUACC-3′







SEQ ID NO: 23—Anti-sense primer










5′-AGCGTUAACGCGAUCGCAGUACUU-3′







The terminal, single-stranded DNA segments in the anti-sense segment of Waxy are produced by treating equal molar amounts of the product of the second polymerase chain reaction with Uracil-DNA glycosylase essentially as described in Example 3 using cycle parameter 3.

With reference to FIG. 10b a sense segment of Waxy coding region of about 400 nt (indicated as “CR-Zm.Waxy S” in Table 3 and in the construct illustrated in FIG. 12) is amplified from the same part of the Waxy gene and is fused to a 150 nt “Spacer” having sense sequence of SEQ ID NO: 26 to form a fusion amplicon with unique terminal single-stranded DNA segments G and H. More specifically, the sense segment of Waxy is cloned using a sense primer (SEQ ID NO: 24) and an anti-sense primer (SEQ ID NO: 25), where the first 18 nucleotides of the sense primer are homologous to the 3′ end of the Spacer and where the 18 nucleotides of the anti-sense primer is designed for a single-stranded segment. Polymerase chain reaction amplification is performed essentially as described in Example 2 using cycle parameter 1. The Spacer is synthesized from a synthetic primer of SEQ ID NO:26 and a complementary primer and used in the second round of polymerase chain reaction which produces a fusion amplicon of the Spacer and sense segment of Waxy, followed by introducing uridine residues in the region in which the terminal, single-stranded DNA segments are produced. The primers designed as the second pair of oligonucleotide primers comprising the two terminal, single-stranded DNA segments G (complementary to F) and H of the fusion amplicon are designed as follows:
SEQ ID NO: 27 Sense Primer










5′-AAGUACTGCGAUCGCGTUAACGCU-3′







SEQ ID NO: 28—Antisense primer










5′-GCGAGUACCGCUGGCGAUCTA-3′







The polymerase chain reaction is performed in a 30 μl volume containing, 1× Expand High Fidelityplus polymerase chain reaction buffer, 1.5 mM MgCl2, 200 μM dNTPs, 5 pmols of each primer, 5 units of Expand High Fidelityplus Enzyme Blend, 1 μl of the first polymerase chain reaction sense DNA molecule product and 1 μl of Spacer molecule (10 ng). The polymerase chain reaction is performed in a PTC-225 DNA Engine TetradTM thermal cycler using the following cycler parameters (“cycle parameter 4”):

1. 94 degree Celsius for 2 minutes
2. 94 degree Celsius for 15 seconds
3. 62 degree Celsius for 30 seconds (−1 degree Celsius/cycle)
4. 72 degree Celsius for 3 minutes
5. Go to step 2, 12 times
6. 94 degree Celsius for 15 seconds
7. 50 degree Celsius for 30 seconds
8. 72 degree Celsius for 3 minutes
9. Go to step 6, 15 times
10. 72 degree Celsius for 5 minutes
11. 10 degree Celsius forever
12. End
The terminal, single-stranded DNA segments in the fusion amplicon are produced by treating equal molar amounts of the product of the second polymerase chain reaction with Uracil-DNA glycosylase. The anti-sense segment with terminal single-stranded DNA segments E and F and the fusion amplicon with terminal single-stranded DNA segments G and H are assembled essentially as described in Example 3 using cycle parameter 3. The assembled inverted repeat molecule is shown in FIG. 11 with remaining terminal single-stranded DNA segments E and H.
The assembled inverted repeat molecule and a linearized plasmid, each having terminal, single-stranded DNA segments, are assembled essentially as described in Example 3 using cycle parameter 3. The final assembled DNA construct as shown in FIG. 12 comprise the elements described in Table 3 where the position is indicated by reference to SEQ ID NO: 29.









TABLE 3





Element Name
Position
Element Function







RB
11493-11823
Right border sequence for T-DNA




transfer.


P-Zm.SzeinC1
  1-1909
Promoter elements from the 27 KDa




storage protein zein gene from




Maize including 5′UTR and an




enhancer intron from the Zea mays




HSP70 intron with flanking exon




enhancer sequence.


CR-Zm.Waxy
1943-2578
Antisense coding region of the


AS

Zea mays Waxy gene.


Spacer
2579-2728
Spacer region, SEQ ID NO: 26.


CR-Zm. Waxy
2729-3129
Sense coding region of the


S

Zea mays Waxy gene.


T-St.Pis4
3171-4024
Transcription termination sequence




of the potato proteinase inhibitor




II gene.


nptII marker
4158-5603
Transcription unit for nptII marker




including CaMV 35S promoter, coding




region for nptII and nos trans-




cription termination sequence.


IG-St.Pis4
5615-6361
Intergenic region of the potato




proteinase inhibitor II gene used




for marker excision


LB
6406-6865
Left border sequence for T-DNA




transfer.


OR-Ec.oriV-
6952-7348
Vegetative origin of replication


RK2

used by Agrobacterium tumefaciens.


CR-Ec.rop
8897-9048
Coding region for the repressor




of the primer for the ColE1 origin




of replication.


OR-Ec.ori-
 9566-10064
Minimal origin of replication


ColE1

from the Escherichia coli plasmid




ColE1 used for replication in




E. coli.


Antibiotic
10595-11483
Transcription unit for antibiotic


marker

resistance.









The assembled DNA construct is transformed into ElectroMAXTM DH10B competent cells as previously described in Example 3.
One skilled in the art will recognize that other cycle parameters can be employed in the polymerase chain reaction amplifications and the Uracil-DNA Glycosylase reaction without departing from the scope and spirit of the present invention. The present invention can be performed within a wide range of equivalent parameters. The present invention is intended to cover any uses, variations, or adaptations of the invention following the principles of the invention in general.

## Claims

1. A method for assembling a DNA construct of DNA molecules with a substantially identical DNA segment in an inverted repeat of said substantially identical DNA segment, said method comprising:
a) providing at least two double-stranded DNA molecules, wherein each molecule has terminal, single-stranded, DNA segments in a length of from 10 to 30 nucleotides extending from the 3′ termini; wherein terminal, single-stranded DNA segments on each double-stranded DNA molecule do not hybridize to each other; wherein a terminal, single-stranded DNA segments on a first double-stranded DNA molecule and a terminal, single-stranded DNA segments extending from a second double-stranded DNA molecule hybridize to each other to allow for specific annealing and linkage of the first and second DNA molecules in a predetermined order; and wherein said first and second double-stranded DNA molecules comprise a substantially identical DNA segment; in reverse complement order and wherein one of said double-stranded DNA molecules comprises a spacer region of DNA that is not substantially identical with DNA in the other molecule;
b) incubating said DNA molecules under conditions suitable to promote the specific annealing and assembling of the DNA molecules to provide a construct of said molecules wherein said substantially identical DNA elements are arranged as inverted repeat segments; and
c) wherein the spacer region is between the first and second double-stranded DNA molecules and comprises restriction endonuclease sites permitting the digestion of inverted repeat segments to facilitate DNA sequencing of said inverted repeat segments.