Heteroduplex Thermostable Ligation Assembly (HTLA) and / or Circular Heteroduplex Thermostable Ligation Assembly (CHTLA) to generate double-stranded DNA fragments with single-stranded sticky ends

Abstract

The present invention relates primarily to a process for generating single-stranded overhangs (sticky ends) of user-defined length and sequence in double-stranded DNA molecules, which process is independent of restriction endonuclease and DNA exonuclease activity, and the formed heteroduplex DNA is joined by one or more ligations using a DNA ligase, resulting in the generation of double-stranded DNA fragments with single-stranded overhangs for joining and generating larger linear or covalently closed circular DNA molecules, optionally with variable regions.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 323,542, filed March 25, 2022, the entire contents of which are incorporated by reference herein for all purposes. [Background technology]

[0002] 2. Background of the Invention Technical Field The present invention relates primarily to a process for generating single-stranded overhangs (sticky ends) of user-defined length and sequence in double-stranded DNA molecules, which process is independent of restriction endonuclease and DNA exonuclease activity, and the heteroduplex DNA formed is joined by one or more ligations using a DNA ligase, resulting in the generation of double-stranded DNA fragments with single-stranded overhangs for joining and generating larger linear or covalently closed circular DNA molecules, optionally with variable regions.

[0003] Related Technology In the prior art, assembling multiple DNA fragments in the correct order has been achieved using restriction enzyme-based cloning, but the process is often severely limited by the availability of compatible, appropriately positioned restriction enzyme sites. All ordered in vitro assembly methods require complementary single-stranded DNA (sticky) cohesive ends to guide the ordering of the DNA fragments. Methods are primarily differentiated by the approach to generating the sticky ends and their length. For example, most type II restriction endonucleases used in conventional DNA cloning recognize palindromic sequences in double-stranded DNA and cleave phosphodiester bonds within this palindromic sequence, leaving staggered cohesive (sticky) ends with overhangs up to 5 nucleotides long. To achieve assembly in the correct order, each DNA fragment to be joined must have a unique sticky end, generated, for example, by the enzymatic activity of a different restriction endonuclease. In practice, it is generally accepted that the number of fragments that can be joined in this manner to generate a circular DNA in a plasmid vector is less than 10. Type IIS restriction endonucleases recognize specific DNA sequences that can be cleaved at a precise distance from the recognition site in any DNA sequence, whether palindromic or not, but also generate short sticky ends of 5 nucleotides or less, most often 4 nucleotides. For example, type IIS restriction endonucleases form the basis of so-called Golden Gate cloning. Given that the four nucleotide overhangs can be repeated 256 times, theoretically up to 128 DNA fragments can be joined in the correct order in a single-pot Golden Gate reaction. However, the practical limit is 24 fragments, given that DNA ligase accepts and ligates some sticky ends with imperfect Watson-Crick base pairing sequences. Another prominent assembly technique, called Gibson assembly, is based on homology and uses bacteriophage exonucleases, namely T5 and T7 exonucleases, to generate partially single-stranded DNA molecules. However, the use of Gibson assembly is limited, as the number of fragments that can be joined in an ordered sequence is generally considered to be less than 20. Summary of the Invention [Problem to be solved by the invention]

[0004] To overcome the shortcomings of conventional assembly techniques, the present invention provides a more efficient, high-fidelity technique for generating DNA assemblies that are not limited by the number of linked fragments. [Means for solving the problem]

[0005] Summary of the Invention The present invention provides an efficient DNA assembly process that generates ligatable single-stranded overhangs of user-defined sequence and length (from one to several thousand nucleotides long forming heteroduplex DNA with 5' or 3' overhangs), i.e. sticky-ended DNA molecules are generated by a process of the present invention called Heteroduplex Thermostable Ligase Assembly (HTLA) process that forms linear or closed circular DNA molecules from double-stranded or single-stranded DNA precursor molecules, when such a process is performed for more than one cycle by a process called Circular Heteroduplex Thermostable Ligase Assembly (CHTLA) starting from double-stranded DNA precursors, a mixture of linear DNA molecules with sticky ends, circular DNA molecules, and / or blunt-ended linear DNA molecules are formed, and when CHTLA is performed starting from single-stranded precursors, exclusively linear and / or circular DNA molecules with sticky ends are formed. Herein, we define a "heteroduplex" DNA molecule as a substantially double-stranded molecule, in which the "top" strand is derived from one double- or single-stranded precursor DNA molecule and the "bottom" strand is derived from a different double- or single-stranded DNA molecule, and are joined by Watson-Crick base pairing in a process known as annealing or hybridization of complementary DNA strands.

[0006] In one aspect, the present invention provides a method for forming a sticky end block (SEB) having a 5' or 3' overhang, the method comprising: providing at least three precursor single strands, double strands, or mixtures of DNA fragments thereof, which are designed to assemble correctly to produce a defined DNA sequence; introducing at least three precursor DNA fragments into a buffered medium; applying heat at a melting temperature to melt at least three precursor DNA fragments; and This involves lowering the melting temperature to allow annealing in the presence of a thermostable ligase to generate a heteroduplex DNA formed by base pairing of complementary regions juxtaposing the DNA ends, which are then ligated, thereby generating a new larger double-stranded DNA product whose single-stranded ends have 5' or 3' overhangs called sticky end blocks (SEBs), or, if the overhangs at the ends of the SEB are complementary, a covalently closed circular DNA.

[0007] A precursor DNA fragment as used herein is selected from the group of double-stranded DNA molecules, single-stranded DNA molecules, or a mixture of both. The nucleotide bases in said DNA fragment may be native adenine, guanine, cytosine, thymine, or any chemically modified form thereof, which may be incorporated into the DNA molecule by chemical synthesis or by the action of an enzyme, i.e., DNA polymerase, or may appear chemically or enzymatically on one or more bases after synthesis.

[0008] The above-mentioned method of forming sticky end blocks (SEBs) with 5' or 3' overhangs includes a precursor DNA fragment and a thermostable DNA ligase enzyme in a buffer medium containing a buffer, such as Tris-HCL, MgCl2, KCl, NAD, DTT, and Triton X-100, to maintain a pH of about 4.0 to about 10, preferably about 6 to 10, more preferably about 7.5 to about 9. The melting temperature can be in the range of about 37°C to 100°C, more preferably above 60°C, and the melting time is in the range of about 30 seconds to about 10 minutes, more preferably in the range of 1 to 5 minutes. Annealing is performed by lowering the temperature to 5°C to 60°C below the melting temperature, more preferably to about 10°C to 40°C below the melting temperature, and the time of such annealing step is about 30 seconds to 10 minutes, more preferably about 4 minutes to 6 minutes.

[0009] Furthermore, for the generation of a mixture of SEB and blunt-ended DNA, the above method can be repeated multiple times, e.g., at least 2-12 times or more, using double-stranded DNA precursors. Note that in each cycle beyond 2, the formed blunt-ended product may also contribute to the formation of the desired SEB.

[0010] In another aspect, the present invention provides a method for forming a sticky end block (SEB) having a 5' or 3' overhang, the method comprising: providing at least three precursor single strands, double strands, or mixtures of DNA fragments thereof, which are designed to assemble correctly to produce a defined DNA sequence; introducing at least three precursor DNA fragments into a buffered medium containing Tris-HCL, MgCl2, KCl, NAD, DTT, and Triton X-100 at a pH of about 7.5 to about 9; applying heat at a temperature that causes melting of at least three precursor DNA fragments (wherein the temperature for melting is determined by the size of the sequence and can range from about 37° C. to 100° C., and the heating time ranges from 30 seconds to 10 minutes); and and reducing the temperature to anneal in the presence of a thermostable ligase, where the annealing is carried out at a temperature 10°C to 40°C below the melting temperature for a time period ranging from 4 minutes to 10 minutes to generate heteroduplex DNA formed by base pairing of complementary regions with single-stranded ends, thereby forming sticky end blocks with 5' or 3' overhangs.

[0011] In addition, the precursor DNA fragments may contain one or more random or variable nucleotides. Furthermore, any precursor double-stranded DNA fragments not consumed in the above reaction may be reformed upon annealing. After two or more cycles of heating, annealing, and ligation, in addition to new SEB products, new blunt-ended products are also formed, as described below.

[0012] Additionally, the buffer medium may contain single-stranded binding proteins, enzymes such as DNA helicases or topoisomerases, one or more crowding agents, metal ions, detergents, or other agents that promote annealing of DNA strands.

[0013] In yet another aspect, the present invention provides a method for forming a circular heteroduplex DNA, the method comprising: providing at least three precursor DNA fragments designed to correctly form one or more SEBs that assemble to generate a defined covalently closed circular DNA sequence; introducing at least three precursor DNA fragments into a buffered medium containing Tris-HCL, MgCl2, KCl, NAD, DTT, and Triton X-100 at a pH of about 7.5 to about 9; applying heat at a temperature that causes melting of at least three precursor DNA fragments (wherein the temperature for melting is determined by the size of the sequences and can range from about 60° C. to 100° C., and the heating time ranges from 30 seconds to 10 minutes); reducing the temperature for annealing in the presence of a thermostable ligase, where the annealing is performed at a temperature between 5° C. and 40° C. below the melting temperature for a time period ranging from 1 minute to 10 minutes to generate heteroduplex DNA formed by base pairing of complementary regions with single-stranded ends, thereby forming sticky end blocks with 5' or 3' overhangs; and It involves repeating the heating and annealing steps multiple times to form a mixture of covalently closed circular DNA and blunt-ended linear DNA sequences.

[0014] Optionally, SEBs can be generated in separate reactions and then combined to generate larger SEBs or covalently closed circular DNAs (cccDNAs). For example, if 20 DNA precursors are to be ligated, 5 can be ligated in reaction 1, 5 in reaction 2, 5 in reaction 3, and 5 in reaction 4. The products of the four reactions can then be ligated in combination by a further HTLA or CHTLA reaction, or by direct enzymatic ligation of the designed complementary sticky ends of the products of each of the four reactions, for example using T4 DNA ligase or any other suitable DNA ligase at an appropriate temperature.

[0015] In another embodiment, the desired SEB products from reactions 1-4 can be purified from the precursor DNA, for example, by agarose gel purification or any other separation means, and then combined for HTLA or CHTLA reactions to generate blunt-ended products consisting of SEB or SEB and all 20 precursor DNAs.

[0016] Precursor DNA according to the present invention can range in length from about 20 nucleotides to several thousand nucleotides, more preferably from about 200 to 10,000 nucleotides for double-stranded precursors and from 30 to 200 nucleotides for single-stranded precursors. The number of DNA precursor fragments determines the size and length of the SEB formed, as well as the length of the sticky ends at the ends of the SEB prepared by the methods of the present invention.

[0017] Thus, the present invention provides nucleic acid ligation schemes that require temperature cycling, such as one, two, three or more cycles to lower temperatures, for example, from about 95°C to about 60°C.

[0018] In yet a further aspect, the present invention provides a method of forming a sticky end block (SEB) having a 5' or 3' overhang, the method comprising: Providing at least three single-stranded precursor DNA molecules (oligonucleotides) that are designed to assemble correctly to produce a defined DNA sequence; introducing at least three paired precursor DNAs into a buffered medium; applying heat at a melting temperature to melt at least three paired precursor DNA fragments; and This involves lowering the melting temperature to anneal in the presence of a thermostable ligase to generate heteroduplex DNA formed by base pairing of complementary regions with single-stranded ends, thereby forming sticky end blocks with 5' or 3' overhangs.

[0019] The precursor DNA fragment according to the present invention includes a measurement unit that specifies the length of the DNA in the range of about 0.1 kb to about 100 kb, more preferably about 0.5 kb to about 5 kb. The number of complementary paired precursor DNA fragments determines the size and length of the SEB prepared by the method of the present invention.

[0020] Other features and advantages of the invention will become apparent from the following detailed description, the drawings, and the claims. [Brief description of the drawings]

[0021] [Figure 1]FIG. 1 illustrates a heteroduplex thermostable ligation assembly (HTLA) for generating a 100-base long sticky end block (SEB) with 10-base sticky ends from three 5' phosphorylated double-stranded DNA precursors according to the present invention. Precursor A has partial sequence identity with precursor C, as shown in blue (nucleotides 11-50 of the 100-base sequence). Precursor B has complete sequence identity to precursor C, as shown in purple (nucleotides 51-90 of the 100-base sequence). When the double-stranded precursors are melted, the "top strands" of precursors A and B can anneal to the "bottom" strand of precursor C, as shown, and vice versa (not shown). In the resulting heteroduplex, at the junction between the last nucleotide of precursor A (base number 50) and the first nucleotide of precursor B (base number 51), a so-called nick in the phosphodiester backbone is sealed by the enzymatic activity of a DNA ligase. The resulting SEB is a 100-base long molecule in which 80 Watson-Crick base pairs are formed with a 10-base long 5' sticky end. Note that a "reverse" SEB with a 3' overhanging sticky end is also formed (not shown). [Diagram 2]FIG. 2 illustrates a circular heteroduplex thermostable ligation assembly (CHTLA) for generating sticky end blocks (SEBs) according to the present invention. Note that the figure illustrates the generation of SEBs from four phosphorylated double-stranded DNA precursors (i.e., PCR products generated using 5'-phosphorylated oligonucleotide primers), two generated primarily from DNA region 1 and two generated primarily from DNA region 2. The "top" strand of each precursor is shown in solid lines and the "bottom" strand is shown in dashed lines. Note that self-annealing of the phosphorylated DNA precursors (grey dashed arrows) simply regenerates the precursor molecule. Two SEBs are shown, one with a 5' overhanging sticky end and one with a 3' overhanging sticky end, encompassing all of DNA region 1 and DNA region 2. Although four are shown here, SEBs could theoretically be generated by circular ligation from an unlimited number of PCR precursors. Note also that any cycle other than cycle 1 can form blunt-ended blocks encompassing most, but not all, of DNA regions 1 and 2, as shown. These blunt-ended products can also serve as precursors for forming SEBs in subsequent cycles. [Diagram 3] FIG. 3 shows an example of an HTLA reaction with four double-stranded DNA precursors whose nucleotide sequences are shown. Note that the capital letters in the precursors indicate the nucleotides that make up the sticky ends of the product SEB, and the lowercase letters indicate the nucleotides that are Watson-Crick base paired in the product SEB. In the product SEB, SEQ ID NO:9 is generated by ligation of SEQ ID NO:1 to SEQ ID NO:3. SEQ ID NO:10 is generated by ligation of SEQ ID NO:6 to SEQ ID NO:8. Similarly, SEQ ID NO:11 is formed by ligation of SEQ ID NO:5 to SEQ ID NO:7, and SEQ ID NO:12 is generated by ligation of SEQ ID NO:2 to SEQ ID NO:4. [Figure 4]Figure 4 shows CHTLA to assemble a partial open reading frame (ORF) of human gene SAP130. Four ~550 bp double-stranded DNA precursors were ligated using HiFi Taq Ligase (lane 3) or Ampligase® (lane 4) for 10 cycles of CHTLA to generate the partial SAP130 ORF. Note the complete conversion of the ~550 bp precursor (solid arrow) to a 1.1 kb product (solid arrow) using HiFi Taq Ligase. [Diagram 5] Figure 5 shows the product of CHTLA to assemble the complete ORF of human gene SAP130. Ten 450-750 bp double-stranded DNA precursors (lane 2, green arrows) were ligated in a 10-cycle CHTLA reaction using HiFi Taq ligase, yielding a 3.05 kb product (lane 3, green arrow). The CHTLA product was digested with Xba I+Sal I (lane 4) to yield the predicted 2.6 kb product (orange arrow), supporting correct assembly. The 2.6 kb band was excised and cloned into pBluescriptSK-. Correct assembly was further confirmed by restriction digestion of DNA from the resulting 2 / 2 colonies (data not shown). [Figure 6A] FIG. 6A shows a heteroduplex thermostable ligase assembly (HTLA) according to the present invention; * indicates 5'-PO4. [Figure 6B] Figure 4B shows a gel image from 5-precursor HTLA to generate the 3.0 kb ORF of human SAP130. The colored bars above the gel image indicate the arrangement of duplexed and offset double-stranded DNA precursors 1.6, 1.45, 1.25, 1.0, and 0.8 kilobase pairs in length. Lane 1 is the double-stranded DNA precursor only; lane 2 is the HTLA reaction product using Ampligase thermostable ligase; lane 3 is the HTLA reaction product using HiFi Taq thermostable ligase; lane 4 is a DNA marker; white arrows are the SEB products. [Figure 7]FIG. 7 shows the circular heteroduplex thermostable ligase assembly (CHTLA) according to the present invention and illustrates the results of the first and second cycles. [Figure 8] Figure 8 shows a strategy for performing HTLA with a mixture of double-stranded and single-stranded precursors (oligonucleotides) and for incorporating oligonucleotides with randomized regions according to the invention into SEB. Note that four of the DNA precursors are double-stranded and two are single-stranded oligonucleotides. In the illustrated oligonucleotides, N refers to any of the four standard nucleotides (i.e., A, T, G, or C). Identical DNA sequences of the precursors are indicated by identical colors. [Figure 9A] Figures 9A and 9B show a strategy for generating HTLA precursors with perfect blunt ends according to the present invention. Note that "normal" PCR generates "short" incomplete products that can be one, two or more nucleotides, leaving overhangs. These products are not suitable for HTLA, as they will leave gaps in the heteroduplex DNA (SEB) that forms the rectangle in Figure 9A. [Figure 9B] As shown in Figure 9B, it is highly desirable to generate "perfect" blunt-ended DNA precursors by "blunting" the ends of the PCR-generated DNA precursors using a type IIS restriction enzyme (i.e., MlyI) prior to their use in the HTLA or CHTLA reaction. [Figure 10A] Figures 10A and 10B show the construction of an 8.8 kb plasmid from four HTLA / CHTLA-generated SEBs. Figure 10A shows the strategy for generating an 8.8 kilobase pair (kb) circular DNA molecule by ligation of four SEBs generated by HTLA or CHTLA. Note that one of the SEBs contains a plasmid vector for propagating the DNA in bacteria. [Figure 10B]Figure 10B shows the generation of four SEBs by CHTLA: lane 1, 2.9 kb vector SEB was generated in one cycle of HTLA reaction from four PCR precursors (1.2 kb, 1.7 kb, 1.9 kb, and 1.0 kb). Three "insert" SEBs were generated by 10 cycles of CHTLA (10 cycles of 95°C for 1 min and 60°C for 5 min) as follows: SEB 1 (final 1.7 kb) was generated from 0.35 kb, 1.35 kb, 0.95 kb, and 0.75 kb precursors; SEB 2 (final 2.5 kb) was generated from 1.6 kb, 0.9 kb, 1.7 kb, and 0.8 kb precursors; SEB 3 (final 1.7 kb) was generated from 0.6 kb, 0.6 kb, 1.1 kb, and 1.1 kb precursors. Four independently generated SEBs were ligated at room temperature using T4 DNA ligase. SEB sticky ends = 6 nucleotides. The T4 ligation products were transformed into the bacterial strain DH5α. Correct clones identified by colony PCR were confirmed by restriction digestion (Xho I + Sac I) of miniprep DNA. [Figure 11] FIG. 11 shows a strategy to generate sticky end blocks with randomized sequences near their ends (SEBREs) by introducing randomized DNA oligonucleotides at the SEB "ends" (just inside the sticky ends) in accordance with the present invention. Note that four double-stranded precursors (precursor I-IV) and two single-stranded precursors (randomized oligos I and II) are shown. One application of this strategy would be to create DNA libraries in which two regions are randomized or sequence restricted. In the randomized oligos, the L portion constitutes the ligatable portion of the sticky end, the R region has the randomized sequence, and the H region is homologous to either precursor I or precursor IV. Note also that the vectors shown are generated by HTLA to contain sticky ends complementary to the L regions of randomized oligos I and II. Ligation of the SEBREs into the SEB vector results in closed circular DNA molecules with gaps in the randomized regions. [Figure 12]12 shows a strategy for generating a sticky end block (SEBRI) with an internally randomized sequence by introduction of an internal randomized oligo in accordance with the present invention. In this strategy, precursor I is generated by polymerase chain reaction using an oligonucleotide primer with a randomized sequence. Precursor III will have a wild-type sequence in the region corresponding to the randomized region of precursor I. The SEBRI thus generated will have a mismatch in the randomized region, which will be repaired using the endogenous bacterial mismatch repair system, for example, upon transformation into bacteria. [Figure 13A] Figures 13A, 13B, and 13C show that the CHTLA of the present invention efficiently produces circular DNA. Figure 13A shows that the commercially available circular plasmid pBluescript II (SK-) (2.961 kb) can be digested with restriction enzymes (RE) to generate four blunt-ended DNA precursors using Pvu II alone or Eco RV+Xmn I (E+X). [Figure 13B] Lane PvuII in FIG. 13B shows that Pvu II digestion produces two blunt-ended DNA fragments of approximately 2.51 kb and 0.45 kb, and lanes E+X in FIG. 13B show that XmnI+PvuII digestion produces two blunt-ended DNA fragments of approximately 1.95 kb and 1.01 kb. FIG. 13B shows the products of a 10-cycle CHTLA reaction using the four blunt-ended DNA precursors produced by the restriction enzyme digestion shown in FIG. 13A, where the thermostable DNA ligase was 9Degree North ligase (9N). The product of the HTLA reaction is shown in lane 9N in FIG. 13B. A product with an apparent size of approximately 4.5 kb is clearly visible on the gel (white arrow), which is the expected location of a 3 kilobase pair closed circular DNA. The HLTA reaction products were then transformed into E. coli and plasmid DNA from the resulting E. coli colonies that had taken up the HTLA product DNA was digested with Eco RV+Xmn I restriction enzymes. [Figure 13C]FIG. 13C shows that 5 / 5 colonies resulting from transformation of the HTLA products into E. coli produced the DNA banding pattern expected for correct assembly. [Figure 14] Figure 14 shows the strategy and one cycle results for generating SEB with three DNA precursors using the HTLA system of the present invention. Note that capital letters (A, B, C) indicate the "top" strand of the DNA precursors, and lower case letters (a, b, c) indicate the "bottom" strand. Two SEB products are shown, one with a 5' overhanging sticky end and one with a 3' overhanging sticky end. [Figure 15] Figure 15A shows a strategy to generate SEB with three DNA precursors using the HTLA system of the present invention, and Figure 15B shows the heteroduplex that can be formed in a cycle regime. Note that ligation occurs only on the trimolecular heteroduplex to generate the SEB product shown in Figure 15C. Note that capital letters (A, B, C) indicate the "top" strand of the DNA precursors, and lower case letters (a, b, c) indicate the "bottom" strand. [Figure 16] Figure 16 shows a strategy to generate a mixture of SEB and blunt-ended products using three DNA precursors and multiple cycles using the CHTLA system of the present invention. Note that capital letters (A, B, C) indicate the "top" strand of the DNA precursors, and lower case letters (a, b, c) indicate the "bottom" strand. [Figure 17] Figure 17 shows the strategy and one cycle results for generating SEB with four DNA precursors using the HTLA system of the present invention. Note that capital letters (A, B, C) indicate the "top" strand of the DNA precursors, and lower case letters (a, b, c) indicate the "bottom" strand. [Figure 18] Figure 18 shows a strategy to generate a mixture of SEB and blunt-ended products using four DNA precursors and multiple cycles using the CHTLA system of the present invention. Note that capital letters (A, B, C) indicate the "top" strand of the DNA precursors, and lower case letters (a, b, c) indicate the "bottom" strand. [Figure 19]Figure 19 shows CHTLA performed with four single-stranded 5'-phosphorylated DNA precursor oligonucleotides. Note that the heating / melting step denatures base pairs due to self-complementarity within the individual oligonucleotides. Note that the product formed is exclusively a single SEB; no blunt-ended products are formed, for example, when the DNA precursors are all oligonucleotides. [Figure 20] FIG. 20 shows the strategy for joining seven single-stranded DNA oligonucleotides (I-VII) to form a 248-base long SEB (240 base pairs formed and 4-nucleotide sticky ends present (colored lines)), and the products of 5-cycle and 10-cycle CHTLA reactions with the seven DNA oligonucleotides (gel images on the left, 5X and 10X), and the results of colony PCR (16 colonies) when the 5X HTLA products were ligated into a plasmid vector with compatible 4-base sticky ends and transformed into Escherichia coli (E. coli). Note that the colony PCR results demonstrated that 15 / 16 of the resulting clones carried the expected size of a 240 base pair insert, with an exception shown in lane 12. Sanger DNA sequencing of DNA from the colonies represented in lane 11 and lane 12 showed that the colony represented in lane 11 had the expected 240 bp DNA sequence and the colony represented in lane 12 had tandem repeats of the 240 bp DNA sequence. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Detailed Description of the Invention The present invention provides a heteroduplex thermostable ligation assembly (HTLA) method for generating sticky ends of user-defined length and sequence that is independent of restriction endonuclease and DNA exonuclease activity. The formed heteroduplex DNA is joined by one or more ligation cycles with a thermostable DNA ligase to generate sticky end blocks, a mixture of sticky end blocks and blunt-ended products, or covalently closed circular DNA (cccDNA), any of the foregoing may or may not contain variable regions, entirely in vitro using the HTLA or circular heteroduplex thermostable ligation assembly (CHTLA) methods of the present invention.

[0023] definition As used herein, the term "heteroduplex" DNA molecule refers to a predominantly double-stranded molecule in which the "top" strand is derived from one double- or single-stranded precursor DNA molecule and the "bottom" strand is derived from a different double- or single-stranded DNA molecule and is joined by Watson-Crick base pairing in a process known as annealing or hybridization of complementary DNA strands.

[0024] As used herein, the terms "ligase" and "ligation agent" are used interchangeably and refer to any number of enzymes or non-enzymatic reagents that can join linker probes to target polynucleotides. For example, a ligase is an enzymatic ligation reagent that, under appropriate conditions, forms a phosphodiester bond between the 3'-OH and 5'-phosphate of adjacent nucleotides in a DNA molecule, RNA molecule, or hybrid. Temperature-sensitive ligases include, but are not limited to, bacteriophage T4 ligase and E. coli ligase. Thermostable ligases include, but are not limited to, Afu ligase, Taq ligase, Tfl ligase, Tth ligase, Tth HB8 ligase, Thermus species AK16D ligase, and Pfu ligase. One of skill in the art will appreciate that a number of thermostable ligases, including DNA ligases and RNA ligases, can be obtained from thermophilic or hyperthermophilic organisms, such as certain species of eubacteria and archaea, and that such ligases can be used in the disclosed methods and kits.

[0025] As used herein, the term "duplex sequence" refers to complementary sequences in two polynucleotides, and if the duplex sequence is single stranded (ss), then the duplex sequence on one polynucleotide can hybridize to a complementary ssDNA region of another duplex on another polynucleotide.

[0026] As used herein, the term "overhang" refers to a single-stranded region of double-stranded (ds) DNA at its end, either in the 5' or 3' form depending on the inherent orientation of the DNA. Overhangs are generally generated to various lengths by treating dsDNA with restriction enzymes or exonucleases and / or by the addition of the appropriate dNTPs (dATP, dTTP, dCTP, dGTP) by the action of the enzyme terminal deoxynucleotidyl transferase.

[0027] As used herein, the term double-stranded DNA (dsDNA) refers to an oligonucleotide or polynucleotide consisting of two single strands with 3' overhangs, 5' overhangs, or blunt ends, and all or part of which are complementary to each other; thus, dsDNA may contain single-stranded regions at the ends and may be synthetic or naturally derived from cells or tissues. In one embodiment, dsDNA is a PCR (polymerase chain reaction) product, or a fragment generated from genomic DNA or a plasmid or vector by physical or enzymatic processing thereof.

[0028] As used herein, the term "buffer" refers to an agent that allows a solution to resist changes in pH when an acid or alkali is added to the solution. Examples of suitable non-naturally occurring buffers that can be used in the compositions, kits, and methods of the present invention include, for example, Tris-HCL, MgCl2, KCl, NAD, DTT, Triton X-100 Tris, HEPES, TAPS, and Tricine. Other buffers include, but are not limited to, phosphate, citrate, ammonium, acetate, carbonate, tris(hydroxymethyl)aminomethane (TRIS), 3-(N-morpholino)propanesulfonic acid (MOPS), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), 2-(N-morpholino)ethanesulfonic acid (MES), N-(2-acetamido)-iminodiacetic acid (ADA), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), salts. Examples of buffers that may be used include acetylcholinesterase, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES), 4-(2-hydroxycthyl)-1-piperazineethanesulfonic acid (HEPES), acetamidoglycine, tricine (N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine), glycinamide, and bicine (2-(bis(2-hydroxyethyl)amino)acetic acid) buffers.

[0029] As used herein, the term DNA or RNA is defined as a "polynucleotide" and may include primers, oligonucleotides, nucleic acid strands, and the like. DNA or RNA may be single-stranded or double-stranded, or a mixture thereof. Such DNA or RNA polynucleotides may be synthetic, e.g., synthesized on a DNA synthesizer, or may be naturally occurring, e.g., extracted from a natural source, or derived from cloned or amplified material. Polynucleotides referred to herein may include modified bases. In addition, DNA or RNA sequences may include one or more random or variable nucleotides. The use of randomized (ATCGNNNNATGC) may also include sequence restriction regions, meaning that the sequence restriction limits variation at a position to two or three nucleotide choices (i.e., A or C; A, G, C, etc.) rather than all four (ATGC). Typically, polynucleotides include a 5' phosphate at one end of the strand (the "5' end") and a 3' hydroxyl group at the other end (the "3' end").

[0030] The nucleic acid used herein can be any nucleic acid, for example, human nucleic acid, bacterial nucleic acid, or viral nucleic acid. The nucleic acid sample can be, for example, a nucleic acid sample derived from one or more cells, tissues, or bodily fluids, for example, blood, urine, semen, lymph, cerebrospinal fluid, or amniotic fluid, or other biological samples, for example, tissue culture cells, buccal swabs, mouthwash, stool, tissue sections, biopsy aspirates, and archaeological samples, for example, bone or mummified tissue. The nucleic acid can be, for example, DNA, RNA, or the DNA product of reverse transcribed RNA. Nucleic acids may be derived from any source, including, but not limited to, eukaryotes, plants, animals, vertebrates, fish, mammals, humans, non-humans, bacteria, microorganisms, viruses, biological sources, serum, plasma, blood, urine, semen, lymph, cerebrospinal fluid, amniotic fluid, biopsy, needle aspiration biopsy, cancer, tumor, tissue, cell, cell lysate, crude cell lysate, tissue lysate, tissue culture cells, buccal swab, mouthwash, stool, mummified tissue, forensic sources, autopsy, archaeological sources, infectious diseases, hospital-acquired infections, production sources, drug formulations, biomolecule products, protein formulations, lipid formulations, carbohydrate formulations, inanimate objects, air, soil, sap, metals, fossils, excavated materials, and / or other terrestrial or extraterrestrial materials and sources.

[0031] The invention disclosed herein uses precursor DNA fragments that are subjected to one or more cycles of thermal denaturation, hybridization, and ligation using a thermostable DNA ligase in a thermal cycler in a specific buffer. Recognizing the urgent need for more efficient and high fidelity techniques for large-scale DNA assembly, Heteroduplex Thermostable Ligase Assembly (HTLA) was developed by the inventors. HTLA is a simple assembly platform that generates ligatable single-stranded overhangs of user-defined length to generate sticky end blocks (SEBs) for assembly into higher order linear or circular structures. The HTLA / CHLTA process generates products called sticky end blocks (SEBs) consisting of double-stranded DNA molecules with single-stranded ends, as shown in Figures 1 and 2.

[0032] In particular, the precursors in FIG. 1 and all precursors described herein include the generation of SEB by HTLA or CHTLA, where the precursor DNA is specifically designed. For example, consider the simple case of designing three double-stranded precursors (A, B, and C) required to generate a 100 base pair SEB that is 80 base pairs double stranded, has a 10 base sticky end at the 5' end, and a 10 base sticky end at the 3' end. Precursor A can be a double-stranded molecule consisting of nucleotides 1-50. Precursor B can be a double-stranded molecule consisting of nucleotides 51-90. Precursor C can be a double-stranded molecule consisting of nucleotides 11-100. Thus, precursors A and C are identical in the DNA sequence of nucleotides 11-50, and precursors B and C are identical in the DNA sequence of nucleotides 11-90. Upon melting and reannealing, the so-called top strands of precursors A and B can anneal to the so-called bottom strand of precursor C due to the complementarity of the nucleotide sequences of nucleotides 11-50 of precursor A and 51-90 of precursor B, resulting in a double-stranded region of 80 base pairs. Nucleotides 1-10 of precursor A remain single-stranded due to the lack of complementary bases in precursor C. Similarly, nucleotides 91-100 of precursor C remain single-stranded due to the lack of complementary bases in precursor B. The so-called nick in the phosphodiester backbone between nucleotides 50 and 51 on the top strand of the heteroduplex can be sealed by DNA ligase. Thus, a 100 base pair SEB is formed, an 80 nucleotide long region is double-stranded (from nucleotides 11-90) and the 5' and 3' ends have sticky ends 10 nucleotides long. Note that reverse heteroduplex formation also occurs, i.e., the bottom strands of precursors A and B can form complementary base pairs with the top strand of precursor C and be ligated to form SEB. It is evident that in addition to the formation of the SEB product, the three precursors A, B, and C can also be reformed by reannealing their complementary strands.

[0033] A schematic of SEB generation is shown in Figure 2, where 5'-phosphorylated double (and offset) synthetic DNA (i.e., PCR products, de novo synthetic DNA (single-stranded or double-stranded)), standard restriction enzyme-generated fragments, or combinations thereof, are denatured in a thermocycler and annealed in the presence of a thermostable ligase. Upon annealing, a heteroduplex DNA with 5' and 3' overhangs is formed in addition to the reannealed input DNA (precursor). For example, four precursor PCR fragments are generated to join the SEB product from left to right, from region 1 to region 2. The left end of precursor A1 coincides with the left end of region 1, and its right end falls short of the right end of region 1. The left end of precursor B1 is offset inward from the left end of region 1. The range of this offset determines the length of the left sticky end (i.e., 4 to over 100 bp). The right end of precursor B1 extends beyond the right end of region 1 into region 2. The extent of invasion into region 2 determines the length of the duplex between precursor B1 and precursor A2, one of the second pair of precursor PCR products (A2 and B2) representing region 2. The left end of precursor A2 is in region 1 and is duplexed with precursor B1. The right end of precursor A2 is short of the right end of region 2. The left end of precursor B2 is recessed inward from the left end of precursor A2, and its right end coincides with the right end of region 2. The extent of offset of the right ends of precursors A2 and B2 determines the length of the right sticky end (i.e., 4 to over 100 bp).

[0034] When the four precursor PCR fragments are melted and reannealed, there are eight possible outcomes. The single strands of the four precursor PCR fragments can come together again, regenerating the input precursor (dashed arrows, Figure 2). In addition, four heteroduplexes can be formed: two A1 / B1 heteroduplexes (i.e., the top strand of A1 anneals to the bottom strand of B1 and vice versa) and two A2 / B2 heteroduplexes (i.e., the top strand of A2 anneals to the bottom strand of B2 and vice versa). Note that the offset between both ends of A1 and B1 results in one heteroduplex with a 5' single-stranded overhang and one heteroduplex with a 3' single-stranded overhang. The same applies to the A2 / B2 heteroduplex. The precursors are designed such that the overhang at the right end of the A1 / B1 heteroduplex is perfectly complementary to the overhang at the left end of the A2 / B2 heteroduplex. This complementarity allows the A1 / B1 heteroduplex to be ligated to the A2 / B2 heteroduplex to generate an SEB product composed of region 1 and region 2, which are double-stranded DNA molecules with non-complementary sticky ends. Note that ligation of the heteroduplex reduces the pool of precursor PCR fragments. If a thermostable ligase is used, then after the ligation reaction, the temperature can be raised to 98 degrees or higher to melt both the precursor PCR fragments and the product SEB. Upon cooling, ligation of the A1 / B1 and A2 / B2 heteroduplexes occurs again, generating more product SEB and further depleting the pool of precursor PCR fragments. After several cycles, a significant portion of the precursor PCR fragments are converted to a single SEB product consisting of DNA region 1 bound to DNA region 2, which is mostly double-stranded but has non-complementary single-stranded ends. Note that this illustration illustrates the formation of a relatively simple SEB from four PCR precursors that bind DNA region 1 to DNA region 2. More complex SEBs can also be formed by HTLA from 5 to over 100 precursors.

[0035] In one embodiment, to link DNA fragments in an ordered sequence, multiple SEBs are generated in separate reactions, purified (i.e., by gel purification), and designed such that their final single-stranded overhangs are complementary, i.e., the right sticky end of SEB1 is perfectly homologous to the left sticky end of SEB2, etc. Such designed multiple SEBs can be acyclically ligated by conventional DNA ligase (i.e., T4 DNA ligase) to generate a larger DNA molecule consisting of multiple joined SEBs, as shown in Figure 10. When a vector (plasmid, cosmid, BAC, YAC, etc.) is included as an SEB and its sticky ends are complementary to the first and last SEBs of the ordered sequence, a complete closed circular vector that can be propagated in bacteria or yeast is obtained, as shown in Figure 10.

[0036] In another embodiment, multiple SEBs are generated multiplexed in a single reaction such that their overhangs are homologous, and because the SEBs are generated from precursor DNA fragments (i.e., they are not purified individually but ligated), they are designed to be joined in the correct order by HTLA / CHTLA alone. SEBs that join DNA region 1 and DNA region 2, DNA region 2 and DNA region 3, DNA region 3 and DNA region 4, etc., and even tens to thousands of DNA regions in one reaction vessel can be generated by HTLA / CHTLA using DNA precursors. In this case, if a vector (plasmid, cosmid, BAC, YAC, etc.) is included as an SEB and its sticky ends are complementary to the first and last SEBs in the ordered sequence, a complete closed circular vector that can be propagated in bacteria or yeast is obtained. An example demonstrating CHTLA to join five DNA regions in one reaction vessel using 10 DNA precursors is shown in FIG. 5.

[0037] In another embodiment, the vector sequence is not included in the precursor sequence and the first and last DNA elements of the ordered linear assembly have complementary sticky ends, in this case cccDNA, also known as DNA minicircles, are formed by the HTLA / CHTLA process.

[0038] Figures 6A and 6B show the HTLA of the present invention. Figure 6A shows that four (or more) offset partially duplexed phosphorylated precursors from two (or more) DNA regions to be assembled are melted and the heteroduplexes are ligated during temperature gradient annealing. The shaded boxes indicate the desired sticky end blocks (SEBs). Figure 6B shows a gel image of 5-precursor HTLA to generate the 3.0 kb ORF of human gene SAP130. Lane 1, 1.6, 1.45, 1.25, 1.0, and 0.8 kb precursors; lane 2, HTLA reaction products using Ampligase thermostable ligase; lane 3, HTLA reaction products using HiFi Taq thermostable ligase; lane 4 DNA marker. Arrows, SEB products. * indicates 5'-PO4.

[0039] 7 shows the steps of the circular heteroduplex thermostable ligase assembly (CHTLA) of the present invention. The SEB product at the end of cycle 1 is melted in cycle 2. A mixture of SEB and blunt end block (BEB) is formed upon annealing in cycles 2 and beyond. * indicates 5'-PO4.

[0040] In another embodiment, oligonucleotides can be incorporated into the SEB in addition to HTLA / CHTLA. The ability to include oligos with randomized regions is crucial. This allows for the generation of large pools of DNA with identical sequences except for precise positions that are randomly mutated or have limited sequence variation (i.e., only purines at a given position, or only pyrimidines at a given position). One application of this is to create bacteriophage libraries of phages with large genomes that have mutated receptor binding motifs, as described below. In particular, such libraries can screen for commercially valuable phages in many applications. Using this strategy, we have generated, cloned, and sequenced a 1.6 kb SEB, as shown in Figure 8. Five out of five had mutations at both ends.

[0041] In another embodiment, the PCR precursors are "blunted" to generate DNA ends of well-defined nature, which can be completely blunt ends or sticky ends of defined sequence as shown in FIG. 9. The PCR reaction generates a significant portion of products with undefined and incomplete 3' ends. Such "short" or incomplete PCR products are ineffective to function as precursors in the HLTA or CHTLA reaction. For example, to generate blunt-ended products, a type IIS restriction enzyme (i.e., MlyI) recognition sequence is included in the PCR primers used to generate the PCR-generated DNA precursors for the HLTA or CHTLA reaction. After PCR, the products are cleaved with a type IIS enzyme to generate a pool of completely blunt-ended precursors for the HLTA or CHTLA assembly reaction. In another example, to generate DNA precursors with defined sticky ends, the type IIS enzymes BsaI or PaqCI or BsmBI or BspQI would be used to digest the PCR-generated DNA precursors before use in the HTLA or CHTLA reaction.

[0042] To demonstrate that multiple SEBs can be generated, purified, and ligated to generate ordered DNA assemblies in a plasmid vector, four SEBs were generated that, when correctly assembled, would result in a circular plasmid of 8.8 kb (Figure 10). The HTLA products are shown in Figure 10B. The strategy used for 4-SEB ligation involves a 2.9 kb vector SEB generated in a one-cycle HTLA reaction from four PCR precursors (1.2 kb, 1.7 kb, 1.9 kb, and 1.0 kb). The three SEB inserts were assembled in 10 cycles of CHTLA (95°C for 1 min, 60°C for 5 min × 10) as follows: SEB 1 (final 1.7 kb), from 0.35 kb, 1.35 kb, 0.95 kb, and 0.75 kb precursors; SEB 2 (final 2.5 kb), from 1.6 kb, 0.9 kb, 1.7 kb, and 0.8 kb precursors; SEB 3 (final 1.7 kb), from 0.6 kb, 0.6 kb, 1.1 kb, and 1.1 kb precursors. Reaction mixture DNA was purified and ligated at room temperature with T4 DNA ligase. SEB sticky ends = 6 nucleotides. T4 ligation products were converted to DH5α. The precursor offset ensured perfect complementarity between the heteroduplexes, and their juxtaposed ends were readily ligated to generate SEB. Although the CHTLA reaction generates multiple dead-end intermediate products, the yield of the correct size SEB is high enough to generate a clear band that can be gel-purified (white arrow, Figure 10B). This simple strategy of generating dsDNA with complementary overhangs for use as building blocks for larger assemblies has never been considered before.

[0043] Single-cycle and multiple-cycle HTLA products are shown in Figure 10B. Correctly ordered assemblies were identified by colony PCR and confirmed by restriction enzyme digestion (Figure 10B). Correct clones identified by colony PCR are confirmed in Figure 10B, and these data demonstrate that CHTLA-generated SEBs can be efficiently joined into higher-order assemblies. Note that the yield of correctly sized SEBs, consisting of a mixture of sticky- and blunt-ended products, is high enough to produce a sharp band that can be purified by conventional agarose gel purification or other means (Figure 10B). These data demonstrate that HTLA- and CHTLA-generated SEBs can be efficiently joined into higher-order assemblies.

[0044] In another embodiment, consecutive strands of SEB are generated multiplexed in a single reaction such that their overhangs are complementary and are designed to be joined in the correct order by circular ligation alone because SEB is generated from precursor fragments (i.e., they are not purified individually but are ligated, e.g., by T4 ligase).Despite the fact that many methods are available for generating randomized DNA libraries, no robust method is currently available for generating random mutations in multiple regions of large circular DNA (>50 kb) in a seamless manner.

[0045] The method of the present invention will not only be useful for generating phage libraries, but also be a powerful method for generating other viral libraries in which mutations at several distributed positions may be beneficial, for example, adeno-associated viral libraries that are screened to find tissue-tropic isolates for gene therapy applications. These objectives were achieved in the present invention by incorporating single-stranded oligos with degenerate regions into SEB in the CHTLA reaction.

[0046] In yet another embodiment, the single stranded oligonucleotide can be one of the components in a HTLA or CHTLA reaction. Additionally, the oligonucleotide can contain a variable region such that the reaction produces SEBs containing variable DNA sequences of known length and potential sequences, for example to generate a library of so-called SEBs that can be incorporated into larger DNA molecules such that the final large DNA contains one or more regions of variable DNA sequence. In one example, oligonucleotides were used to generate variable or randomized regions near the ends of the SEBs. We call this sticky end blocks with randomized ends, or SEBs. RE It is called SEB. RE To demonstrate the feasibility of the generation, two 40-mer oligos were incorporated into a 1.7 kb SEB (one at each end), as shown in Figure 11. Region H=29 bases, R=5 bases, and L=6 bases by 10 cycles of CHTLA. In particular, the length of region L can be about 6-50, and the length of region R can vary from 6-100. SEB RI was ligated into the 2.9 kb vector SEB containing T4 DNA ligase. Positive clones were identified by colony PCR (20 / 20) and successful oligo incorporation (both) was confirmed by DNA sequencing (5 / 5).

[0047] Specifically, a CHTLA reaction was performed to generate a 1.7 kb SEB and two "model" oligos were added. The oligos featured ends homologous to the PCR precursor (Figure 11, region H), ends complementary to the ligation partner SEB (Figure 11, region L), and five central non-complementary bases (Figure 11, region R). RELigation of an oligo-modified SEB bearing the randomized region (note that the randomized region is just internal to the sticky end sequences required to induce ordered ligation) into the SEB-generating vector yielded approximately 1,000 colonies. Colony PCR revealed that 20 / 20 were correct, and sequencing revealed that both oligos were incorporated into 5 / 5 clones. These data demonstrated the feasibility of this approach. Again, because the SEB is generated by ligation of several SEBs, if a vector (plasmid, cosmid, BAC, YAC, etc.) is included whose sticky ends are complementary to the first and last SEBs of the ordered sequence, a complete closed circular vector that can be propagated in bacteria or yeast is obtained.

[0048] During the development of CHTLA, a PCR precursor was generated using degenerate oligos to generate the SEB with an internally located randomized region (SEB RI It was determined that SEB(SEB RI ) was well tolerated and SEB RI is ligated as efficiently as "normal" SEB (Figure 12). This observation has led to a clear technical strategy to introduce random mutations into SEB for library diversification. Specifically, precursors with randomized or variable regions are generated, for example by PCR, and the precursors are used to ligate internal randomized or variable regions of SEB. RI To test the feasibility, two internal mismatched regions were introduced into the 1.7 kb SEB, as shown in Figure 12: region R = 5 bases, region H = 29 bases, and region L = 6 bases. Mismatched region 1 was located at positions 227-231 of the SEB leading strand; mismatched region 2 was located at positions 790-794 of the lagging strand. RIwas ligated into the 2.9 kb SEB vector (6-base sticky ends) using T4 DNA ligase. Correct clones were identified by colony PCR, and 20 colonies were selected for DNA miniprep and DNA sequencing. Interestingly, mismatches at positions 227-231 (leading strand) were present in 17 / 20. In contrast, mismatches at positions 790-794 (lagging strand) were present in 3 / 20, which may have been due to strand bias during DNA repair in vivo. In other experiments, attempts to bias were made by using SEB ligase with deoxyadenosine methylase. RI Repair towards randomized oligos by methylation of adenosines in the . Despite confirmation of successful complete methylation using methylation-sensitive restriction enzyme digestion, no bias was observed, contrary to existing dogma in the E. coli DNA repair field (data not shown).

[0049] Another embodiment addresses the relentless rise of multiple antibiotic-resistant bacteria, renewing intense interest in bacteriophage (phage) therapy for patient treatment. However, phages are a largely untapped resource with the potential to transform not only infectious disease control, but also food preservation, plant pathogen control, biosensor development, biofilm control, and surface disinfection. Phages infect and lyse only bacteria with extraordinary species and strain specificity. Ensuring environmental or patient safety of new isolates is a tedious but essential task, as many phage genomes are large in size (up to 500 kb) and the function of many open reading frames is unknown. Phage genome engineering is also severely limited by the lack of packaging systems in all but a few "benchmark" phages (i.e., lambda, M13). Recent breakthroughs in phage engineering have the potential to revolutionize this paradigm: it is now possible to "reboot" a large proportion of the phages of the common, highly transformable E. coli strain DH10B, opening the door to creating libraries of "wild" phages. To exploit this breakthrough, there is an urgent need to develop efficient means of creating highly complex libraries using fully characterized "chassis" phages that differ in receptor-binding motifs that determine superior bacterial strain specificity. The present invention addresses the need for highly complex library creation by synthesis of large DNAs with variable regions to enable the construction of complex chassis phage libraries. With current large-scale synthetic DNA assembly techniques, it is technically impossible to achieve this goal. Complementary single-stranded cohesive (sticky) ends are required for all ordered DNA assemblies. The present invention provides a method to generate sticky ends of user-defined length and sequence that is independent of restriction endonuclease and DNA exonuclease activity.

[0050] The heteroduplex DNA is joined by multiple ligation cycles using a thermostable DNA ligase, and the present invention provides a novel approach, termed circular heteroduplex thermostable ligation assembly (CHTLA), to generate large covalently closed circular DNAs (cccDNAs) with variable regions, entirely in vitro. In particular, the CHTLA of the present invention can be used to rapidly create large phage genomic libraries of great complexity and transform them into third-party hosts. Such libraries are a valuable resource from which phages can be screened, with commercial potential for any application in which phages are needed to control bacterial growth.

[0051] The present invention provides a "pre-circularised" lambda genome to enhance transformation efficiency. As a proof of principle to demonstrate that CHTLA can generate circular DNA, four precursors were designed to construct a 3 kb circular plasmid (Figure 13B) using 9°N™ (9N) ligase in CHTLA. 7 ) transformation, 10 8 More than 100% (5 / 5) of the colonies were found to have the correct assembly (Figure 13C). This efficiency is several orders of magnitude higher than can be achieved using Gibson assembly or conventional cloning with T4 DNA ligase. To demonstrate this, conventional ligation of the same fragments using T4 ligase resulted in approximately 10 5 10 colonies were obtained, with a series of non-productive ligation events occurring, of which 0 / 5 had correct assemblies (Figure 13B). These data demonstrate that CHTLA dramatically outperforms conventional cloning in generating correctly assembled closed circular DNA, and is supercompetent (i.e., 10 10 / μg) cells using 10 10 These results demonstrate the potential to produce colonies in excess of 100 μg / ml.

[0052] Figure 13 shows that CHTLA of the present invention efficiently generates circular DNA. Specifically, pBluescript II (SK-) (2.961 kb) was digested with Pvu II alone or Eco RV+Xmn I (E+X) to generate all blunt-ended fragments. Pvu II digestion generates two blunt DNAs of approximately 2.51 kb and approximately 0.45 kb, while E+X digestion generates two blunt DNAs of approximately 1.95 kb and 1.01 kb (Figure 13, top panel). The DNA was mixed, purified, and ligated with 9°N™ (9N) thermostable ligase in a 10-cycle CHTLA reaction (Figure 13, left center panel). A product with an apparent size of approximately 4.5 kb was clearly visible on the gel (Figure 13B, lane 9N), which is the expected location of the 3 kb closed circular DNA. The purified ligation mixture (1 μl) was transferred to electrocompetent cells (competency = approximately 5 × 10 7 Transformation of 1 μl of the T4 ligation reaction into 9°N™ (9N) ligase plate (1000 colonies / μg pUC19 DNA) yielded approximately 5,000 colonies under antibiotic selection. In parallel, the same DNA was ligated with T4 DNA ligase at room temperature (Figure 13, middle right panel). A series of DNA fragments was formed, including a product of over 10 kb (Figure 13B, middle right panel, lane T4). Transformation of 1 μl of the T4 ligation reaction yielded approximately 80 colonies. Five colonies were picked from the 9°N™ (9N) ligase plate and five from the T4 ligase plate, and the DNA was restriction digested with Eco RV+Xmn I (lower panel). All five colonies from the 9N plate yielded the correct fragment. 0 / 5 colonies from the T4 ligase plate yielded the correct fragment.

[0053] 14 shows the strategy to generate SEB with three DNA precursors using the HTLA system of the present invention, and the results of one cycle. The three precursors Aa, Bb, and Cc are melted, annealed, and ligated to form the sticky end-blocked products Bac and bAC.

[0054] Figure 15 shows the strategy to generate SEB with three DNA precursors using the HTLA system of the present invention, and the HTLA DNA that can be formed in a one-cycle manner. The three precursors Aa, Bb, and Cc are melted, annealed, and ligated to form several non-ligatable heteroduplex DNAs. The sticky end block products are AbC and aBc.

[0055] Figure 16 shows a strategy to generate a mixture of SEB and blunt-ended products using three DNA precursors and multiple cycles using the CHTLA system of the present invention. The three precursors Aa, Bb, and Cc are melted, annealed, and ligated to generate sticky-ended Bac and bAC, and additional cycles of melting, annealing, and ligation form the desired mixture of SEB and blunt-ended products.

[0056] Figure 17 shows the strategy to generate SEB with four DNA precursors using the HTLA system of the present invention, and the results of one cycle. The four precursors Aa, Bb, Cc, and Dd are melted, annealed, and ligated to form several possible heteroduplex DNAs. The sticky end block products are AbCd and aBcD.

[0057] 18 shows a strategy for mixing SEB and blunt-ended products using four DNA precursors and multiple cycles using the CHTLA system of the present invention. The four precursors Aa, Bb, Cc, and Dd are melted, annealed, and ligated to generate sticky ends AbCd and aBcD, and additional cycles of melting, annealing, and ligation form the blunt-ended products.

[0058] As shown in FIG. 19, the DNA precursor in the HLTA or CHTLA reaction is composed entirely of 5' phosphorylated single-stranded DNA oligonucleotides. In this embodiment, the melting step serves to destroy secondary structures within the individual oligonucleotides formed by self-complementarity. Note that in this embodiment, even if HTLA is performed over multiple cycles, the product can be composed entirely of SEBs, i.e., there is no blunt-end product form. The SEBs thus generated in separate reactions can then be joined by typical ligation to form larger assemblies. Alternatively, the final product can be designed as cccDNA. Specifically, seven single-stranded DNA oligonucleotides were designed to generate a 248-base-long SEB using CHTLA, where 240 Watson-Crick base pairs were formed and 4-base sticky ends were present.

[0059] The resulting CHTLA product was conventionally ligated into a restriction enzyme digested vector plasmid and subsequently transformed into E. coli, as shown in Figure 20. Colony PCR of the resulting clones demonstrated that 15 / 16 carried the expected 248 base long plasmid insert, and Sanger DNA sequencing of one clone demonstrated that the insert was the correct DNA sequence.

Claims

1. 1. A method for forming a sticky end block (SEB) having a 5′ or 3′ overhang, comprising: providing at least three precursor single-stranded, double-stranded DNA fragments, or mixtures thereof, designed to assemble correctly to generate a defined DNA sequence; introducing said at least three precursor DNA fragments into a buffered medium; applying heat at a melting temperature to melt the at least three precursor DNA fragments; and The method comprises lowering the melting temperature for annealing in the presence of a thermostable ligase to generate heteroduplex DNA formed by base pairing of complementary regions juxtaposed to the DNA termini for further ligation, thereby generating a new larger double-stranded DNA product with single-stranded ends having 5' or 3' overhangs to form a sticky end block (SEB), or forming a covalently closed circular DNA in which the 5' or 3' overhangs are complementary.

2. 2. The method of claim 1, wherein the pH of the buffered medium is from about 6.0 to about 9.

3. The buffer medium may be Tris-HCL, MgCl ２ 13. The method of claim 1, comprising: KCl, NAD, DTT, and Triton X-100.

4. 2. The method of claim 1, wherein the temperature for melting is determined by the size of the sequence and ranges from about 37°C to 100°C.

5. The method according to claim 1, wherein the heating time for melting is in the range of 30 seconds to 10 minutes.

6. The method of claim 1, wherein annealing is performed at a temperature 5° C. to 40° C. below the melting temperature.

7. The method of claim 1, wherein the annealing time is about 4 minutes to 10 minutes.

8. 2. The method of claim 1, wherein the precursor DNA fragments range in size from about 20 nucleotides to several thousand nucleotides in length and can be single-stranded or double-stranded.

9. The buffer medium may include Tris-HCL, MgCl with a pH of about 7.5 to about 8. ２ , KCl, NAD, DTT, and Triton X-100, the melting temperature is greater than about 80° C. for about 3 minutes, the annealing temperature is about 10° C.-20° C. below the melting temperature for about 4 minutes-6 minutes, and the melting, annealing, and ligation steps are performed multiple times ranging from 2 to 12 times to generate blunt-ended products.

10. 10. The method of claim 1, wherein the melting, annealing, and ligation steps are performed multiple times in the range of 2 to 30 times to generate a cohesive product.

11. 10. The method of claim 9, wherein the sticky end sequence can be ligated with T4 ligase to provide a longer extended sequence.

12. 10. The method of claim 9, wherein the precursor DNA fragments range in size from about 20 nucleotides to several thousand nucleotides in length and can be single-stranded or double-stranded.

13. 10. The method of claim 9, wherein the temperatures associated with melting, annealing, and ligation range from about 95°C to a lower temperature of about 60°C for one, two, or more cycles.

14. 2. The method of claim 1, wherein the defined sequence of DNA comprises one or more random or variable nucleotides.

15. 1. A method for forming a mixture of sticky-ended heteroduplex DNA and blunt-ended DNA products comprising: providing at least three precursor single-stranded, double-stranded DNA fragments, or mixtures thereof, that are designed to assemble correctly to generate a defined DNA sequence; The at least three precursor DNA fragments are diluted with Tris-HCL, MgCl at a pH of about 7.5 to about 9. ２ , introducing into a buffer medium containing KCl, NAD, DTT, and Triton X-100; applying heat at a temperature to cause melting of the at least three precursor DNA fragments, the temperature for melting being determined by the size of the sequence and can range from about 60° C. to 100° C., and the heating time being in the range of 30 seconds to 10 minutes; Reducing the temperature for annealing in the presence of a thermostable ligase, the annealing being carried out at a temperature between 5° C. and 40° C. lower than the temperature for said melting for a time period ranging from 4 minutes to 10 minutes, thereby generating heteroduplex DNA formed by base pairing of complementary regions with single-stranded ends, thereby forming sticky end blocks with 5′ or 3′ overhangs; and repeating said heating and annealing steps multiple times to form a mixture of SEB and blunt-ended DNA sequences.

16. 16. The method of claim 15, wherein the sticky-end complementary DNA sequences are further ligated using a DNA ligase capable of ligating the sticky ends to form an extended nucleotide sequence and / or a circular DNA.

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