CROSS-REFERENCE TO RELATED APFUCATIONS

[01] This application claims tbe benefit of U.S. Provisional Patent Application No.

62/362^75, filed July U, 2016, which la mcorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

[02] The preaent application li being filed along with a Sequence Listing in dectronic fimnat The Sequence Lilting la provided a* a file entitled ADI^PCT_ST25, created June 1, 2017, which ii 8 KB in size. The information in the electronic format of the Sequence

Lilting ia Incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[03] The preaent invention generally ralatee to improved very rapid methods of assembly of two or more DNA molecules in the field of recombinant DNA technology, methods of rapid Ugadon-mdependent cloning, and kits for rapid Hgatioo-tadependent dotting and thdr uses.

BACKGROUND OF THE INVENTION

[04] DNA cloning is the procedure of making copies (or clones) of particular DNA sequences, usually by insertion into a cloning vector and replication into a host organism. Molecular cloning regroups the sd of operations needed to assemble and clone recombinant DNA fragments for the purpose of DNA cloning; it usually involves methods to generate DNA fragments and varied strategics to assemble them with a vector mat can be further amplified Into the host Traditionally DNA doning was made with the use of restriction enzymes (Cohen ft aL 1973). Typically the DNA to done is cut on each end by a restriction enzyme, the vector is cut by the same enzyme, usually in an area named the multi-cloning she (MCS) and the two fragments are joined together by treatment with a DNA ligase. The use of two restriction enzymes with noo-compslible overhangs allows directional doning in which the DNA fragments is inserted into the vector in only one direction.

[OS] Cloning methods were later devdoped with the availability of the polymerase chain reaction (PCR). PCR enables <k novo synthesis of DNA fragments hi vitro by T^nwrt^ ¹ amplification of a DNA sequence. A PCR uses a DNA template, two small oligonudeotides called primera whose sequence matches the DNA template on each end of the sequence to amplify, and a series of multlstep temperature cycles ooniprieing a denaturatlon step to separate the two DNA strands, an annealing step where primen find their homologous sequence on DNA, and finally a step of synthesis or elongation by the means of a DNA polymerase in the presence of dNTPs. If restriction sites are introduced in the primer sequence, PCR fragments can be oloned by traditional restriction enzyme cloning.

Alternatively a blunt-ended PCR product can be cloned directly in an open vector after ligation with the DNA ligase, but not directionally. The use of Taq DNA polymerase often leaves an extra A nucleotide on the 3' end which limits the blunt-end ckmmg efficiency. This problem led to me popular T/A cloning method in which a free T overhang generated by a restriction enzyme digestion on the vector conveniently creates a one-base oomplementary overhang for cloning. All these methods require at some point the use of a DNA Ugase to join DNA fragments, even when multiple PCR fragments are combined by Splicing by Overlap Extension PCR (Horton RM et al 1989).

[06] The need for restriction sites and the inherent length of traditional cloning has led to the development of numerous alternate strategics that did not require a DNA ligation step.

For purpose of clarity and relevance to this invention, we will define ligation-lndopendent cloning or "LIC", as any clonmg method that uses the complementary annealing of sequence-specific single-stranded stretches of DNA located on each end of the DNA fragments for the joining reaction; the stretches must be long enough to hold the DNA assembly together through the transformation process. Note that a few modem cloning methods often use the term ^H seamless doning" to be differentiated from the older ligation- independent cloning term, still the event at the source of the DNA joining reaction is the annealing between two ooinpleroentary stretches of single-stranded DNA (s&DNA) that are physically created during the process. Some authors also use the term "homologous reoomblnatlon" to highlight the sequence homology between the DNA fragments but the joining reaction Is again physically accomplished by the annealing of complementary stretches of ssDNA. The procedure is de facto well-adapted for the cloning of PCR inserts where homologies can be incorporated in the primer sequence (LIC-PCR). In most applications, the stretches of saDNA are generated by the 3* to 5' exonuclease activity found in proof-reading polymerases, such as for example T4 DNA polymerase, but other enzymes have been used with success such as lambda exonuclease, an enzyme with a 5'- >3' exo activity (Tsenl999). The filling of the gaps left by the Miwwltag of partially overlapping ssDNA tails and ligation per set can occur either in vitro before transformation or in vivo after transformation in the bacterial host with the help of the DNA repair system. It has been shown indeed mat the host bacterium fills up the resulting sequence gaps and ligates the construct very efficiently. There are very few by-products, in particular no by- products, resulting from improper ligations. The sequetioe-dependeaoy of the process nukes the cloning very reliable and efficient The procedure mimics to some extent natural reenmbination processes used by live cells to repair, mix, and/or exchange DNA molecules.

[07] The first documented report of LIC was made by Aslanidis (Aalanidis et al 1990).

Specially designed vectors with two 12-nuoleotide (nt) stretches of DNA containing four nucleotides but one, located on each side of the cloning she were used Digestion with a 3'- >5' exonuclease in the presence of the complementary nucleotide triphosphate would continue until the first occurrence of the missing base was encountered (event which kicks off the polymerase activity), thus allowing fine control of the stretches of homologous ssDNA (Aalanidis et aL 1990; Haun et al. 1992; Knijper et al. 1992). In the SLIC method (Li et al. 2007), all dNTPS were initially excluded to allow sequenoe-independent overlaps to be created, and dCTP was men added to bring the T4 DNA polymerase to a stall at the first O occurrence and the LIC proceeded as usual.

[08] The minimal length requirement for LIC was found to be 10-nt long (Aslanidis et al 1994). Improved cloning efficiency was observed between 15-nt and 30-nt (Sharon et al, US Patent 6,372,429), but stretches as long as 100 nt have been reported (Elledge, US Patent Application US 2007/0292954 Al). m a similar situation intermediate between classical cloning and LIC, Stoker et al. wore able to apply the Aslanidls method to generate a 2-ot overhang but a final step of DNA ligation was still required for oloning (Stockcr et al. 1990).

[09] An alternative method to generate ssDNA tails for cloning PCR products is to mix PCR fragments of unequal length. The method was used In rolling circle PCR (RCPCR) where both vector and insert are «npiifl«H and annealed before transfbotion (Jones et al. 1990a and 1990b). In their "enzyme- free dotting" method, Tillct et al. mixed PCR products of different lengths to achieve similar results (Tillet et aL 1999); in a comparable approach, the "mixed-PCR" reaction used multiple nested PCR products (Tachibana et aL 2009). The iPCR method achieved the same goal by using incomplete PCR products generated towards the end of amplification and found In any PCR reaction (lA etaL 2007).

[10] Many other ways to create ssDNA tails have been described. The use of nicking endonuclease (enzymes that cut only one strand) has been reported (Yang 2010). The use of deoxyuracil residues (dU) incorporated in primers and secondary enzymatic removal by the uracil DNA gtyoosylase has been reported several times, although for the generation of smaller sticky tails (see e.g. Bitinaite et aL 2007). The presence of abasic sites (sites with no base) induces a stall of the DNA polymerase and, when properly located in primers, creates stretches of saDNA at the ends of the PCR products; this approach was used in the AS-PCR (autostlcky PCR, Gal et al. 1999). The use of primers contaliilag ribonucleotides has been applied to LIC as well (Donahue et al. 2002).

[11] The vaootnia virus DMA polymerase can catalyze tn vtvo the formation of DNA ooncateners (Wilier et aL 2000). This DNA joining reaction is dependent of the exonucleeae activity of the polymerase and is preceded by the formation of complementary ssDNA tails (Hamilton et al 2007). The reaction can proceed at room temperature without the need for any particular step to inactivate the exonuolease and has been applied to the cloning of DNA fragments (Evans et al. US Patent 7,375,860).

[12] It was shown early after the discovery of LIC that ssDNA stretches can be generated by controlled 3'->5' exonuclease digestion. Typically one used the 3'->5' exonuclease activity of T4 DNA polymerase or of Exonuclease ΓΠ. The method required a delicate control of the reaction because of the high processivity of those enzymes and variations from batch to batch between enzyme preparations. The last point was reported by Kuijper et aL (1992) who, while proceeding with LIC following the original Aslanidis method, noticed mat each lot of T4 DNA polymerase had to be tested, likely for oontatninatlon by endonuolease activity. In Hsiao et al. (1993), linearized vectors and PCR inserts were purified and mixed in equal molar amounts, then treated by ΒχοΙΠ for 30 s to 1 min on ice; after adding TE (Tris/EDTA buffer), the remaining ΒχοΙΠ was immediately removed by phenol/chloroform extraction. Kaluz et aL (1992) reported a similar approach but only the insert was treated by ExoDI and, following phenol/ehlorofocm extraction, ligated to a cut vector o/n at 16°C; in this case a Ugatkm step was necessary because of the very short overhangs on the vector side. Li et aL (1997) also used ΕχοΓϋ followed by phonol/chloro farm extraction before annealing and transformation and demonstrated thai both 5' overhangs and gaps can be left for repair by the host bacteria.

[13] A very similar approach using T4 DNA polymerase was published in 1993 (Yang et al 1993), Following controlled treatment for 2 min at 37°C, the T4 DNA polymerase was inactivated at 70% for 10 min. The authors then ffllod-in the gaps after annealing using again T4 polymerase in the presence of dNTP. It was suggested that the fill-in step may be omitted but the assumption was not verified experimentally. The method was patented under US Patent 5,580,759. The Gibson assembly goes one step further by ligating the DNA assembly before transformation, thus enabling the cloning of very large DNA molecules (Gibson 2011).

[14] The present invention addresses a need in the art for more efficient and faster methods in cloning techniques. Practically, we have practiced the art of DNA cloning using the LIC method described by Evans et al (US Patent 5,580,759) and discovered novel oondltiona enabling very efficient cloning of DNA fragments with LIC reactions completed

In 10 min or less.

SUMMARY OF THE INVENTION

[IS] The inventor has discovered that the exonuclease activity of known exooucleaaes, such as T4 DNA polymerase, can be inactivated by heat at a much more tepid rate than currently reported in the literature. This property can be applied to greatly shorten me time to done DNA by LIC, which allows the processing of many more samples In less time whether manual or automated processes are used.

[16] T4 DNA polymerase is the 3' _^ > 5" exonucleaso most often used to create single- stranded homologous recesses to assemble DNA fragments, It is reported widely in the literature that hoat inactrvatian of T4 DNA polymerase takes between 10 min and 20 min at 70"C or 75°C. T4 DNA polymerase has two distinct enzymatic activities, a 5'- >3' DNA polymerase activity which requires a DNA template, dNTP and a primer to catalyze the synthesis of a template-dependent DNA strand, and a 3'->5' oxonuclcase activity, which is tenmlate-indepciident After careful examination of the literature, the rate of inactivation and temperature dependence of the 3'->5" exonuclease activity of T4 DNA polymerase has not been studied or reported, and most likely all requirements for heat-inactivation of this enzyme are indeed based solely on the results of inactivation studies of the 5'->3' DNA polymerase activity. Comforting this observation is that activity measurements for purified T4 DNA polymerase by suppliers are reported for a 5'->3' polymerization activity.

Logically, people who used T4 DNA polymerase for DNA cloning by LIC followed the supplier-recommended inactivation temperature in their experiments. For example, U.S. Patent 5-580,759 requires a heat Inactivation of 10 min at 70°C for T4 DNA polymerase enzyme.

[17] We discovered two novel conditions to inactivate T4 DNA polymerase exonuclease activity by heat treatment First, incubation of T4 DNA polymerase between 50"C and 65°C leads to inactivation of the exoaoclease activity; this is up to 15°C lower than existing art Second, we discovered that a brief heat pulse at around 50°C and higher leads to Inactivation of ate exonudesse activity within a very brief period of time as short as 10 s. This represents up to a two log difference shorter compered with what is currently accepted

In tbo field for T4 DNA polymerase.

[18] The present invention provides improved methods for LIC and kits for LIC comprising a step of heat inactivation at temperature lower than 65°C. In some embodiments tho heat inactivatioa of T4 DNA polymerase is between about 50°C and 65°C; in some embodiments the heat inactivation of T4 DNA polymerase is between about 50"C and 60°C; in some embodiments the host iiiacttvaHon of T4 DNA polymerase is between about 50°C and 55°C.

[19] The preaent invention provides improved methods for LIC end kits for LIC comprising a step of rapid heat inscdvation of the exonudease. In some embodiments the heat inactivatioQ of T4 DNA polymerase is for a period of less than ton minutes between about 50°C and 9S°C. In some embodiments the heat inactivation is for a period of less than five minutes at a temperature between about SQ°C and 95 ^B C. In some embodiments the heat inactivation is for a period of less than one minute at a temperature between about 50°C and 95°C. In some embodiments the heat inactivation is for a period of inactivation no longer than about 10 a between about 50°C and 95Ό.

[20] The time gained to inactivate the exonuclease activity translates into much shorter time to complete an LIC reaction which allows many more samples to be processed in less time than prior methods currently in use. In some embodiments, the present invention provides methods for rapid LIC that can be completed in less than ten minutes. In some embodiments, the present invention provides methods for rapid LIC that can be completed in less man five minute, In some embodiments, me present invention provides methods for rapid LIC that can be completed in no longer than three wHimt». in some embodiments, the present invention provides methods for rapid LIC that can be completed in about one minute.

[21] In a more general manner, our discovery on the heat-dependence of exonuclease activity is likely true for many if not all proofreading polymerases. Noteworthy is the

Inactrvation times reported for enzymes exhibiting only exo activity that have been used for LIC (e.g. exonuclease HI, lambda exonuclease) are similar to the time reported for T4 DNA polymerase and it is not excluded that analysis of their rate of heat-inactivation reveals much shorter inactivation times. Preferably the exonuclease used in the method of the invention is T4 DNA polymerase, however other enzymes with exonuclease activity include, but are not limited to, Ε.00ΙΙ poll, vaccinia virus DNA polymerase, lambda exonuclease, exonudease HI and T7 exonudease. Some embodiments of the present Invention further comprise tho use of costhnulatory factors. In some embodimenta of the present invention, the costimulatory factors are ssDNA binding proteins. Examples of single stranded binding proteins include, but are not limited to, E. coli SSB, RecA and its homolog RAD51 in human, Tth RecA, human replication protein hRPA, herpes simplex virus 1CP8 protein, yRPA, vaccinia virus single strand binding protein, and ET SSB, a thermostable single-stranded DNA binding protein, m some embodiments the ssDNA binding protein is thermostable enough to resist inactivation during the heat pulse given to inactivate the exonuclease. In some embodiments of the present Invention, the costimulatory factor 1B Tth RecA, the ReoA homolog isolated from Thermut thermophthu, a thermostable RecA. In some cmbodlmsnts tho invention fttrthcr comprises All*. In some embodiments the present hrvention provides a kit for oloning comprising T4 polymerase, thermostable RecA, and ATP.

[22] A rapid LIC kit will contain the 2 or more fragments of DNA to assemble, T4 DNA polymerase, Tth ReoA and ATP. Both DNA fragments share homologous sequences, 15 to 20 nucleotides long, on both ends so tint the melting temperature of the homologous sequences will not be higher than 65°C to allow complete denaturation at 7S°C The reagents are combined on ice in a PGR tube and placed on a PGR niachine pre-cooled at 4°C. A short program, overall less than 5 -mm long, consists in a temperature ramp to 75°C, long enough to create recessed ends on all DNA fragments, inactivate the exonuclease activity and denature the homologous tails, followed by a temperature deorease to 37*0 during which Tth RecA associates with ssDNA. Finally the mixture Is incubated for 1 mm at 37°C to allow assembly of homologous ssDNA stretches and cooled at 4"C to stop the reaction. The overall reaction is less than 5 -mm long to as short as between 2 and 3 min on some sntomatnd thermocyelen. The reaction mixture can be used immediately to transform competent bacteria to Isolate recombinant clones containing the 2 or more DNA fragments properly assembled.

BRIEF DESCRIPTION OF THE DRAWINGS

[23] Figure 1A is a schematic representation of the rapid LIC method of the present Invention. Figure IB is an illustration of the temperature ramp and timeline for the rapid LIC reaction.

[24] Figure 2 includes four panels with schematic summaries of distinct applications of the rapid LIC method. Figure 2A illustrates toe cloning of a PCR fragment amplified by primers PI and P2 or a synthetic DNA fragment into an open vector. Figure 2B illustrates the Complementary Homi-PCR (CH-PCR) reaction where the 2 halves of a vector are amplified by PCR and recombined with modifications and/or mutations at the overlapping junctions. Figure 2C illustrates the cloning of 2 short complementary oligonucleotides. Figure 2D Illustrates the cloning of multiple PCR fragments into an open vector. Shared regions of sequence homology are highlighted by small areas with different shades at the ends of DNA fragments; me replication of origin is indicated by a box with the symbol Rep.

[25] Figure 3 is a picture of an electrophoresis gd showing the results from heat inactivatloo of T4 DNA polymerase exonuclease activity described in Example 1 of the present specification. Figure 3 shows the inactivation at different target temperatures of the T4 DNA polymerase exonuclease activity. Bach lane shows DNA digested by T4 DNA polymerase cxonuclease activity after treatment at different temperatures. The far left and right lanes show DNA ladders. Lane 1 Is a control with no T4 DNA polymerase showing the undigested DNA. Lane 2 shows T4 DNA polymerase exonucleaee activity when the target temperature was 30°C. Lanes 3-12 show the exonodease activity after the T4 DNA polymerase reached target teinperaturcs 3S<C 40°C, 45"C, 50°C, 55°C, «FC, 65°C 70°C, 75*0, and no heat respectively.

[26] Figures 4A and 4B are each an electrophoresis gol showing the results of the T4 DNA polymerase cxonuclease inactivatlon using heat pulse which was described in Example 2 of the present specification. Figure 4 shows the rate of T4 DNA polymerase cxonuclease inactivation by a heat pulse. Figure 4A shows the rate of inactivation at 75°C. The far left lane shows a DNA ladder and Lane 1 shows DNA with no T4 DNA polymerase added; Lano 2 shows DNA with T4 polymerase and no heat, Lane 3 to Lane 9 shows DNA with T4 DNA polymerase raised to 75°C for 5 seconds, IS seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, and 10 minutes, respectively. Figure 4B shows the rate of inactivation for a 10 s heat pulse at varied temperatures. The fir left lane shows a DNA ladder and Lane 1 shows DNA with no T4 DNA polymerase added; Lane 2 shows DNA with T4 polymerase and no heat, Lane 3 to Lane IS shows DNA with T4 DNA polymerase raised for 10 a at 35Χ, 40 ^a C, 45°C, 50°C, 55°C, 6QPC, 65°C 70*0, 75°C, 80°C, 85°C, 90°C and 95°C, respectively.

[27] Figure 5 is a graph of the blue cfu and white cfu colony data from the blue-white screening assay in Example 3. These data compare heat Inactivation against no heat inactlvatloa and show the effect of inactivation of T4 DNA polymerase exonuclease on LIC efficiency. The graph shows data from a variable pro-heat inactivation 37°C plateau on cloning efficiency at time points of 0 seconds, 30 seconds, 1 minute, 2 mfrmtw^ and 4 minutes, respectively.

[28] Figure 6 is a graph of the bine cfu and white cfu colony data from the btuo- white screening assay in Example 4 and shows the time-dependence of antiagUng, These data show the effect of a variable post-heat Inactivation 37*0 temperature plateau on cloning efficiency.

[29] Figure 7 Is a graph of the blue era and white cfu colony data from the blue-white screening assay in Example S. It illustrates the *nflimn«* of RecA and ATP on cloning efficiency.

[30] Figure 8 is a graph of the cloning efficiency from the Complementary Hemi-PCR (CH-PCR) assay In Example 6. ] For the purposes of talerpreting of this specification, the following definltloiis will apply and whenever appropriate, terms used In the singular will also Include the plural and vice versa. In the event that any definition set forth below conflicts with the usage of that word In any other document, including any document incorporated herein by reference, the definition set forth below shall always control for purposes of interpreting this specification and its associated claims unless a contrary meaning is dearly intended (for example in the document where the term is originally used). It is noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the," include plural referents unless expressly and unequivocally limited to one re fa out. Hie use of "or" means "and/or" unless stated otherwise. For illustration purposes, but not as a limitation, "X and/or Y" can mean "X" or "Y" or "X and Y". The use of "comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and not intended to be limiting. Furthermore, where the description of one or more embodiments uses the term "comprising," those skilled in the art would understand that, in some specific instances, the embodiment or embodiments can be alternatively described using the i^gmfff "consisting essentially of" and/or "consisting of. The term "and/or" means one or all of the listed elements or a combination of any two or more of the listed element.

[32] The section headings used herein are for organizational purposes only and are not to be construed as limiting the described subject matter in any way. All literature cited in this specification, including but not limited to, patents, patent applications, articles, books, and treatises are expressly incorporated by referenco in their entirety for any purpose. In the event mat any of the incorporated literature contradicts any term defined heroin, this specification controls. While the present†f are described in conjunction with various embodiments, it is not intended that the present **»αΜ ^η & be limited to such embodiments. On the contrary, the present twMng. encompass various alternatives, riwdfications, and cqiihraJents, as will be appreciated by those of skill in the art

[33] The practice of the present invention may employ conventional techniques and descriptions of bacteriology, molecular biology (including recombinant techniques), cell biology, and biochemistry, which are within the skill of the art Such conventional techniques include PCR, extension reaction, oligonucleotide synthesis and oligonucleotide ■wMdinE Specific illustrations of suitable techniques can be had by reference to the example herein below. However, other equivalent conventional procedures can, of course, also be used. Such conventional techniques and descriptions can be found m standard laboratory manuals such as Genome Analysis: A Laboratory Manual Series (Vols. I-IV), PCR Primer A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press, 1989), Gait, 'Oligonucleotide Synthesis: A Practical

Approach" 1984, IRL Press, London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3rd Ed, W. H. Freeman Pub., New York, N.Y. and Berg et aL (2002) Biochemistry, 5th Ed., W. H. Freeman Pub., New York, N.Y. all of wfaioh are herein Incorporated in their entirety by reference for all purposes.

[34] As used herein, "amplify" refers to the process of enzymatically increasing the amount of a specific nucleotide sequence. This amplification Is not limited to but is generally accomplished by PCR. As used herein, ^u denaturat[on ^N refers to the separation of two complementary nucleotide strands from an annealed state. DenBturation can be induced by a number of factors, such as, for example, ionic strength of me buffer, temperature, or cbemicals that disrupt base pairing interactions.

[35] As used herein, the term "amplifying" refers to a process whereby a portion of a nucleic add la replicated using, for example, any of a broad range of primer extension reactions. Exemplary primer extension reactions include, but are not limited to, PCR. Unless specifically stated, "amplifying" refers to a single replication or to an arithmetic, logarithmic, or exponential amplification.

[36] As used herein, "annealing" refers to the specific interacdoa between strands of nucleotides wherein the strands bind to one another substantially baaed on complementarity between the strands as determined by Watson-Crick base pairing. It is not necessary that complementarity be 100% for annealing to occur.

[37] The terms "amplification cycle" and "PCR cycle" are used Interchangeably herein and as used herein refers to the denaturing of a double-stranded polynucleotide sequence foil owed by imwwHng of a primer sequence to its complementary sequence and extension of the primer sequence.

[38] The terms "polymerase" and "nucleic acid polymerase" are used interchangeably and as used herein refer to any polypeptide that catalyzes the synthesis or sequencing of a polynucleotide using an existing polynucleotide as a template.

[39] The term "polynucleotide" refers in particular to doublo-standed DNA, double- stranded RNA, hybrid DNA/RNA duplex, single-stranded DNA and single-stranded RNA.

[40] As used herein, "DNA polymerase" refers to a nucleic add polymerase that catalyzes the synthesis or sequencing of DNA using an existing polynucleotide as a template.

[41] As used herein, the term "exonuclease" refers to any polypeptide mat catalyzes the sequential cleavage of nucleotides one at a time from one end of a polynucleotide chain.

[42] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices and materials are now described.

[43] All publications and patents mentioned herein are incorporated herein by reference for all purposes, including me purpose of describing and disclosing, for example, the constructs and methodologies that are described in the publications, which might be used in connection with the presently described invention. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. DETAILED DESCRIPTION OF THE INVENTION

Rapid LIC

[44] The present invention relates to a novel method to clone DNA by LIC using rapid heat inactfvation of the exonuolease enabling the joining of two or more DNA fragments In a very short experimental time. This Invention is a direct Improvement of the LIC method developed by Yang and collaborators (Yang etal. 1993 and US Patent 5,580,759).

[45] hi the most common application of DNA cloning by the Yang method, two DNA molecules share sequence homologies so that the ends of one fragment are complementary to the ends of the other fragment These DNA fragments can be generated either by digestion with restriction enzymes of larger fragments, PCR of a DNA template, or direct synthesis with no other sequence requirements than the terminal sequence homologies. The method consists in a controlled digestion of each fragment by a strand-specific exonudease (og. 3' -5' exonuclease activity of T4 DNA polymerase) creating single stranded DNA overhangs on each end. Following inactivation of the exonuolease activity by prolonged heat treatment, the overhangs are men annealed together respectively of their sequence complementarity to create circular Joined DNA molecules that can be used to transform bacterial host and clone the DNA. The initial assumption that gaps left by imperfect armeelmg could be repaired by the bacterial host, thus omitting in vitro fill-in reaction and ligation, has been verified experimentally since.

[46] The method of the present invention is called rapid LIC. The principle of rapid LIC and its timeline are illustrated on Figure LA and IB. In me most common application, the rapid LIC method will join two DNA molecules for the purpose of DNA cloning. The first step incorporates two DNA molecules sharing sequence homologies so that each end of one fragment is homologous to one end of the other fragment. The second stop utilizes an exonuclease to create strand-specific recessed ends on both ends of each DNA fragment; the exonuclease is then rapidly inactivated by a short heat pulse. The third step consists in an annenHng reaction to create circular close DNA molecules and Is followed by a transformation of a bacterial host to Initiate DNA repair and replication.

[47] Figure 2 Illustrates the main applications of rapid LIC. In one embodiment (Panel A) one DNA fragment Is a vector opened by digestion with restriction enzymes and the other DNA fiagment Is a PCR product The primers PI and P2 are made of two separate areas; the 5' tenninal regions are homologous to me DNA sequence to amplify and the 3' proximal region share sequence homologies with me vector sequence on either side of the restriction outs. Instead of a PCR product, the fiagment to Insert in the vector could be also a synthetic DNA fiagment or the product of the assembly by SOE-PCR of multiple PCR fragments. In another embodiment (Panel B) the DNA fragments are amplified from a single vector and a new vector is created by me rapid LIC method; by carefully choosing the proximal and distal regions of the primers, it is possible to create a deletion, introduce a mutation or insert new sequences in the original vector. We call this method CH-PCR for Complementary Hemi-PCR. In another embodiment (Panel C) one DNA fragment is a vector open after digestion with restriction enzymes and the other results from the annealing of two partially overlapping oligonucleotides. Most if not all DNA cloning methods using exonuclease recession cannot done small oligonucleotides because of their rapid disappearance from the reaction mixture; because the rapid LIC method is very fast, h is still efficient to run it twice back to back; during the first run the out vector is added and recessed ends are generated; after adding the two oligonucleotides, the reaction is run again but the oligonucleotides are not destroyed because the exonuclease activity has been inactivated. In another embodiment (Panel D) multiple DNA fragments are cloned at once in an open vector. Each DNA fragment, including the vector itself; is termmated by a small sequence that is homologous to the beginning of the next fragment; indeed all homology regions taken two by two among all DNA fragments create a unique close circular DNA molecule after conjoint annealing.

Rapid Exonuclease mactivation

[48] It is widely reported in the literature that heat inactivation of T4 DNA polymerase requires a exposure of between 10 and 20 min at temperatures between 70°C and 7S°C. For example 10 min at 70°C Is reported in U.S. Patent 5,580,759; New England Biolabs (Ipswich, MA), a reference company in the field of molecular biology, indicates 75°C for 20 min for inactivation, while Thermo Fisher Scientific Inc. and Promega Corporation (Fltchburg, WI) recommend 10 min at 75°C; the lowest temperature reported is 65°C for 10 min by EURx Ltd. (Poland), a molecular biology supplier. We discovered that T4 DNA polymerase exonuclease activity was Instead inactivated at temperatures as low as 50°C; that la 15°C to 20°C below most recommend temperatures for inact-Yation (Example 1). It is not excluded that in these conditions, the 5'->3' polymerase activity still remains potent Therefore the exonuclease activity of T4 DNA polymerase appears extremely sensitive to elevated temperature. This novel characteristic led to the analysis of the Inactlvation of T4 DNA polymerase exonuclease activity by brief heat pulse. Thne-oourae experiments revealod mat complete inactlvation was occurring between 5 s and 15 s exposure of the exonuclease at 75°C (Example 2, Panel A); for a constant heat pulse of 10 s, Inactivation appears at 50°C and above (Example 2, Panel B). This represents up to a two-log difference with what is currently accepted in the field. Because a major trend in modem molecular biology is the shortening of experimental time, mis discovery opens the possibility to shorten dramatically the time required for cloning DNA.

[49] The DNA polymerases that may be used in the method of the invention include all DNA polymerases having Intrinsic exonuclease activity, preferably 3'->5' exonuclease activity, that can be inactivated rapidly by heat in the conditions indicated in the above embodiments. Such polymerases may be easily identified by assaying the heat-induced inactlvation of the exonuclease activity as described in Example 1 and Example 2A and 2B. This group Includes but is not limited to T4 DNA polymerase, E.coli poll, Klenow fragment, vaccinia virus DNA polymerase, lambda exonuclease, exonuclease HI, and T7 exonuclease.

Length of homology

[SO] The length of the complementary nucleotide sequences located on the ends of each DNA molecule may be between 5 and about 100 nucleotides, preferably between about 10 and about 35 nucleotides, and most preferably between IS and 20 nucleotides.

[SI] In some embodiments, the length of the complementary nucleotide sequences is between 15 and 20 nucleotides with a melting temperature (Tm) equal or lower man 65°C. The Tm Is defined as the temperature at which the folded fraction is 0.5 (Mergoy and Lacroix 2003). The conditions to determine Tm are those classically used for preparing a PCR with a sodium concentration of 50 mM and a primer concentration between 02 uM and 0.25 μΜ; Tm of short oligonucleotides between 10 and 100 nucleotides can be estimated with good accuracy using nearest neighbor calculations (Breslauer et aL 1986; SantaLucia J Jr. 1998). Tools to do these calculations are widely available; for example OllgoAnafyzer is made available by Integrated Technologies, Inc. and OligoCalc Is a free and widely available oligonucleotide properties calculator software (Klbbo 2007)· In this range of Tm, the oonesponding folded heterodimers are melted at 75°C. Taking the examples of mis invention and estimating Tm using OllgoAnalyzer and enthalpies using OligoCalc, we determined the unfolded fraction at 75°C using the equations reported by BOttcher et el. (2015) (Table \\ All sequences In this range of lengths and temperatures havo an unfolded fraction near 100%; oligonucleotides longer than 20 with Tm lower than 65"C are still almost completely unfolded while those with a Tm above 65°C, in this a case the model oligonucleotides polyG(15) and poIyG(20) , havo a significant folded fraction. It should also be noted that because of the experimental difficulties of analyzing melting curves, it is virtually impossible to measure folded fractions experimentally above 97% (Mergny and Lacroix 2003). Because of the much lower concentration of DNA fragments in the rapid LIC reaction mixture than in a PCR reaction, the actual Tm is in fact lower than the values used In these calculations, which are in consequence underestimating the exact unfolded fraction. Therefore, successful cloning events using the rapid LIC invention in this embodiment will result from annealing events mat occurred after the heat pulse and full denaturation of all DNA fragments.

Costimulatory factors

[52] Some embodiments of the present invention further comprise coetimulatory factors.

In some embodiments of the present invention, the costimulatory factors are single strand DNA binding proteins (SSB). Examples of SSB include, but are not limitod to, RecA in K colt and its homolog RAD51 in human. RecA is an ssDNA-dependent ATPase that catalyses the pairing and exchange of DNA strands bearing sequence homologies; its association with DNA is tighter in the presence of ATP (reviewed in Kowalozykowski 1992). We also discovered that the rate of rapid LIC by homologous recombination is increased by RecA alone and further enhanced after adding ATP. In some embodiments of the present invention, the costimulatory factor is Tth RecA, the RecA homolog isolated from Thermua thermopktttis, a thermostable RecA whose activity can survive a heat pulse at 75°C. In some embodiments the invention further comprises ATP. In some embodiments the present invention provides a kit for cloning comprising T4 DNA polymerase, thermostable RecA, and ATP.

Overall method

[S3] A rapid LIC cloning reaction will contain two fragments or more of DNA to assemble, T4 DNA polymerase, Tth RecA and ATP. Both DNA fragments are sharing homologous sequences, between S and about 100 nucleotides on both ends, preferably between about 10 and about 35 nucleotides, and most preferably between IS and 20 nucleotides, with aTm each equal or lower than 65°C. The Tm are estimated as described previously using nearest-neighbor calculations m PCR conditions. The reagents are combined on ice in a PCR tube and placed on a thermal cycler (or mermooycler or PCR machine) pre-cooled at 4°C A short program, overall leu than 3-mln long, consists In a 1 s plateau at 75°C followed by a 1 min plateau at 37°C before the temperature is cooled back at 4°C (Fig. IB). The ramp from 4°C to 7S°C takes about 30 seconds (s) and is long enough to create recessed ends of all DNA fragments. The time to go back and forth between 70°C and 75°C is about 10 s, long enough to inactivate of the exonuclease and separate all ssDNA homologous tails. The ramp between 75"C and 2TC takes about 30 s during which Tth RecA associates with ssDNA. Finally the mixture is incubated for 1 min at 37°C to allow assembly of homologous asDNA tails and cooled at back at 4°C to stop me reactioa The overall reaction from the start of the temperature ramp to the return at 4°C is no longer than 3 min (see timing chart below). The reaction mixture can be used immediately to transform competent bacteria to isolate recombinant clones containing the DNA fragments properly assembled. It is possible to replace the thermal cycler by short heat pulses using pro-heated water baths and complete the reaction in shorter times. Inactivation of T4 DNA polymerase will occur after 10-15 s treatment at 50°C and above; melting of ssDNA tafls will occur above 65°C, at best around 75*C in a few seoonds. Association with Tth RecA will be optimal between 65°C and 75°C while armealing between complementary ssDNA tails will mostly occur below 65°C.

Timing Chart.

Time was counted from the start of the temperature raise and measured at passage at 7S°C (time 1), start of 1 minute plateau at 37°C (time 2), end of 37°C plateau (time 3) and program arrest (back to 4*C, time 4) on varied instruments:

Kits

[54] Also provided are Idts. Such Idts can include the compositions of the present invention and, in certain embodiments, instructions for administration. Such kits can facilitate performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and mixed immediately before use. Components include, but are not limited to DNA fragments, a vector, an exonuclease, an SSB, ATP, and a concentrated reaction buffer, each as described herein. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. Hie pack may, for example, oomprlse metal or plastic foil such as a blister pack. Such packaging of the components separately can also, In oertaln Instances, permit long-term storage without losing activity of the components.

[55] Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophQized active component packaged separately. For example, sealed glass ampoules may contain a lyophilizod component and in a separate ampoule, sterile water, sterile saline or sterile each of which has been packaged under a neutral non-reacting gas, such as nitrogen. Ampoules may consist of any suitable material, such as glass, organio polymers, such as polycarbonate, polystyrene, ceramic, metal or any other material typically employed to hold reagents. Other examples of suitable containers include bottles mat may be fabricated from similar substances as ampoules, and envelopes that may consist of foil-lined Interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the Kke. Qmtainers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.

[56] In certain embodiments, kits can be supplied with instructional materials.

Instructions may be printed on paper or other substrate, and/or may be supplied as an dectronlo-readabte medium, such as a floppy disc, mini-CD-ROM, CD-ROM, DVD-ROM, Zip disc videotape, audio tape, and the like. Detailed instructions may not be physically associated whh the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit

EXPERIMENTAL EXAMPLES

[57] The following examples are offered to illustrate, but not to limit the claimed Invention.

[58] It is understood mat the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of mis application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference herein in their entirety for all purposes. Example 1 - Heat-Mediated T4 DNA Polymerase Exonuclease Inactivation [59] This example denunstrates the rapid inactivaHon of T4 DNA polymerase oxonuclease activity by exposure to heat above 50°C and below 70°C. It Is widely acoepted and taught in the literature that T4 DNA polymerase requires at niiiiirnum 10 minutes of incubation at 70°C to beoomo inactive. These previously reported values are believed to be relevant for both polymerase and oxonuclease activities. This experiment tested variable target temrjerature values programmed into a PCR machine initially set at 4°C. After loading the samples containing only T4 DNA polymerase, the thermal cycler rose to a target temperature value and after a one (1) second plateau, samples were rapidly cooled back to 4°C. A test DNA fragment was then added to each sample and after IS mm incubation at 37"C, the DNA was analyzed by electrophoreais on a 1-5% agarose gel. Any remaining exonuclease activity would digest the DNA from both ends, thus creating a smear on the gel, while no exonuclease activity would leave the test DNA Intact as a discrete band on the geL

[60] The test DNA fragment added was Z900. Z900 was generated by amplifying a 966 bp fragment from pUC119 vector with the primers laczbwjr and laczbwj. The PCR product was re-suspended in water at -50 ng/μΐ after purification over a NucleoSpin column from Macherey Nagei (Germany).

[62] The mixture was dietributed Into PCR tubes by 15 ul aliquot; the tubes were men successively placed In a PCR machine pre-cooled at 4°C and the temperature was rapidly increased to one of the variable target temperatures (30°C, 35°C 40 ^B C 45°C, 50°C, 55°C, 6QPC, 65"C, 70°C, 75°C, no heat). Once me temperature had reached the expected value, the samples were brought back to 4°C after a one (1) second-long plateau and then placed on ice.

[63] Then 0J μΐ of lOx reaction bufEor and 4.5 ul of purified Z900 DNA (-200 ng) were mixed with 5 μΐ of reaction buffer for each of the variable target temperature points and incubated for 15 minutes at 37°C. The digested DNA was then analyzed by efectrophoresla on a 1.5% agarose geL

[64] The results from this experiment are shown in the gel electrophoresis In Figure 3.

The lanes are numbered from left (lane 1) to right (lane 12) and flanked by DNA ladders. The DNA fragment was clearly visible in the absence of T4 DNA polymerase (lane 1) and exhibited significant digestion m absence of beat treatment (lane 12). Bxonuolease activity was easily visible at 30°C to 45°C and showed a dear transition around 50°C (lane 6). At

55% and above no exonudease activity was detectable.

[65] This experiment shows that the exonuolease activity of T4 DNA polymerase was rapidly and irreversibly inactivated above 50°C.

[66] This experiment was designed to analyze the rate of heat inactivation of T4 DNA polymerase exonudease activity.

[67] First a mixture containing T4 DNA polymerase 5 ul, lOx buffer 5 ul and water 40 μΐ was kept on ioe. For each time measurement, 5 μΐ were aliquoted into a tube and incubated for a given period of time in a thermostated block and put back on ice. Then 5 ul of a mixture containing purified Z900 DNA, lOx buffer 6 ul and water 30 μΐ was added to each tube and assayed for exonudease activity after 15 minutes at 37°C before analysis by gd electrophoresis (100 ng DNA per tube, 1 tube per lane). Results are shown in Figure 4, Panel A. The time points measured were no heat, 5 seconds, 15 seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, and 10 minutes with no T4 DNA polymerase and no heat as controls. The electrophoresis gd in Figure 4, Panel A shows in its lanes respectively 1) no T4 DNA polymerase, 2) T4 DNA porymerase with no temperature increase, 3) T4 DNA polymerase 5 seconds at 75"C, 4) T4 DNA polymerase 15 seconds at 75"C 5) T4 DNA polymerase 30 seconds at 75°C, 6) T4 DNA polymerase 1 minute at 7S°C 7) T4 DNA polymerase 2 minutes at 75°C, 8) T4 DNA pdymerase 5 minutes at 7S°C 9) T4 DNA polymerase 10 minutes at 75°C. mectivation of the exonudease activity occurred rapidly between 5 s and IS s exposure to 75°C.

[68] Second, the same experiment was repeated using a constant heat exposure of 10 soconds and a variable target tonperaturc between 35°C and 95°C by 5°C incremont Figure 4, Panel B shows the result of the analysis by gel electrophoresis. Inactivation of the exonucletse activity was observed at 50"C and was complete at SS*C and above.

[69] In this experiment; we analyzed the influence of the length of inoubation at 37% before heat inactivatloa of the T4 DNA exonuclease activity on the efficiency of cloning. A blue-white colony assay wis used following the approach developed by Tbieroe (Thieme tt al. 2001) and used a PCR machine to control temperature.

[70] Blue-white cloning assay

[71] Hie PCR product Z900 was treated by Dpnl restriction enzyme to remove the DNA template and re-suspended in water at 10 ng/μΐ after purification over a Macherey Nsgel NucleoSpin column. The primer iaczbw_s contains a IS nucleotides overlap with the sequence of the pADL-IOb phagemld vector (Antibody Design Labs, San Diego, CA) on the peptide leader pelB side of the first Bgll site with a melting temperature of 58,4*0 (OligoAnatyzer 3.1, Integrated DNA Technologies, Inc., Coral villc, IA) and the primer laczbw_r overlaps on the other side of the second Bgll site with a melting temperature of 59*C. pADL-IOb was cut by Bgll and re-suspended at 20 ng/μΙ in pure water after purification over a Macherey Nagd NucleoSpin column.

[72] The following reaction was prepared on ice ma PGR tube:

[73] The temperature was first raised to a plateau at 37"C of variable length, followed by a one second plateau at 75*C _t and a 10 minute plateau at 37"C before the reaction was cooled down to 4°C. 50 ul of XLIO-Gold bacteria (Agilent Technologies, San Diego, CA) made chemically-competent were transformed with 2 ul of the reaction mixture. After 30 rain incubation on ice, me cells were heet-sliocked for 30 seconds at 42"C, 150 μΙ of SOC medium were added and after 1 hour incubation at 37*C, 100 μΙ of each frsnsfonnatlon were plated in duplicate on agar plates supplemented with ampicillin 100 μκ/ηιΐ, IPTG and

X-gaL Blue and white oolonice were counted the day after.

[74] The results of this experiment are depicted on Figure 5. The shorter the incubation at

37°C, the higher is the number of blue colonies. Longer incubations resulted in lower cloning efficiency. This result ckariy indicated that very short incubation times prior to heat inactivation of T4 DNA polymerase exonuclease activity gave the best conditions for an efficient rapid LIC method. Exemple 4 Time Depedence of Annealing

[75] The same reaction was performed from Example 3 with a 30 second plateau at 37°C before the heat-medlsted inactivation followed by an annealing plateau at 37°C of variable length. The influence of RecA, a tnediator of ssDNA annealing was also studied. Tth RecA, a thermostable form of ReoA that survives the heat pulse at 75°C was used (0 J μΐ Tth RecA from New England Biolabs per reaction).

. [76] The experimental results are shown In Figure 6. There was no apparent difference in cloning efficiency between no plateau at all and up to 10 min incubation at 37°C. These data clearly indicated that a short *tm-¾-ding plateau at 37*0 is sufficient to achieve significant doning efficiency. As expected, in the presence of Tth RecA, the cloning efficiency was higher by about 70% in this experiment

[77] The influence of Tth RecA was further studied with the same assay using a short temperature cycle consisting of a plateau of one second at 75"C starting from a PCR machine pro-cooled and equilibrated at 4"C and a one minute plateau at 37°C before cooling the reaction back at 4°C. The overall cycle was completed between 2 and 3 min fiom the start of the temperature cycle to its return at 4°C. Also analyzed was the influence of ATP 1 mM which regulates the interaction of RecA with single- stranded DNA,

[78] The experiment was done in tetraplicatc (2 duplicates) and the data are shown as a graph on Figure 7. A significant increase In the number of blue transfbrmant was observed;

66% increase on average in the presence of RecA, further Increased by another 47% in the presence of ATP. The overall Increase in the presence of RecA and ATP was 145% higher than the control without RecA and ATP.

[79] In this example, we performed the CH-PCR assay by amplifying by PGR two halves of a phage DNA. The two fragments were joined in the conditions of Example 5 and the suocess of the reaction was quantified by a plaque assay after transform stion in a bacterial

host

[80] VCSM13 phage DNA was amplified with primer ml3g5_j and ml3g2_r (fragment

V, 7270 bp) and phage CM13.9 was amplified with primers ml3g2_s and ml3g5_r

(fragment C, 1448 bp). CM13.9 is a single mutant of M13K07 oontaJning the lrlA

mutation (G->A mutation at position 8247).

[81] Primer sequence and Tm estimation (OligoAnalyzer 3.1)

1 |

[83] The final volume of the PCR reaction was 50 ul. After ampllflcatiQn for 25 cycles

with nnnoaHng temperature at 5TC and 3 min elongation, the template DNA was digested

overnight at 37°C after addition of Dpnl restriction enzyme 1 ul directly in the PCR tube.

Minigel analysis confirmed the amplification the two DNA fragments at the expected size.

After purification over a Machercy Nagel Nuol∞Spin oohimn and elution in water, the final

DMA. concentration was measured by UV srjectrophotometry.

[84] Rapid LIC reaction:

[85] The final volume of the UC reaction was IS μΐ. After mixing all reagents on ice, the tubes were placed in PCR tnachine pre-oooled at 4°C The temperature cycle was first a plateau at 37°C of variable length, a plateau at 75"C for 1 second, a plateau for 10 mm at 37"C, men the temperature was brought back 4°C. XLIO-Oold chemically competent bacterial cells SO μΐ were transformed with 2 ul of the reaction by heat shock; 150 μΐ of SOB medium were added and the mixture was further Incubated for 1 h at 37*0. 100 μΐ of the cells were mixed with 5 ul of TGI Phage Competent™ cells (Antibody Design Labs, San Diego, CA) and 3 ml melted top agar at 50°C and poured on a pro- warmed bottom agar plates. Plates were done in duplicate and plaques were counted the morning after.

[86] The results are shown in Figure 8. The cloning by LIC using our method resulted in a very large number of plaques only when the two complementary pieces of DNA were present in the reaction with very few plaques in both negative controls. There was no detectable influence of the length of the plateau in the timeframe that was tested; therefore the shortest time is tho preferred for the fastest reaction, similarly to the results in Example 3.

[87]

[88] In this example wo cloned a synthetic DNA fragment (scbhieOl, 885 bp) in the vector TGEX-FC (Antibody Design Labs, San Diego, CA). A 3482 bp -long fragment containing the bacterial origin of replication was amplified by PCR using TGEX-Fc plasmld DNA as a template and the primers bsaran s and tgex_S3rev. The areas of homology in the ecbmoOl fiagment are underlined in the hereafter table; the homologies are bom 22-bp long with a Tm of 60.4°C and 64.4°C respectively (OligoAnaryzer 3.1, Integrated DNA Technologies, Inc.). Bxninitiaticm by mlnigol analysis of the PCR reaction showed a unique bond at the expected size; the PCR reaction was purified over a Macherey Nagd NuolooSpin column and ehited in water at the concentration of20ng/uJ.

[89] Primers and DNA fragment

All components were mixed on loe In a single PCR tube and the tube transferred to a PCR machine pro-cooled at 4°C. A temperature cycle made of a 1 second plateau at 75°C followed by a 1 mm plateau at 37°C before cooling back at 4°C was then Initiated; the oyclo was completed in 3 min. Chemically competent XLlO-gold bacteria were transformed and plated on an agar plate supplemented with ampicfllin. The day after, three colonies were picked and grown overnight at 37°C with shaking. Sequence analysis revealed the presence of the synthetic DNA sequence properly inserted in all 3 colonies.

[91] Example 8: Cloniny nf p PCR ftupmmt In » vector,

[92] In this example, we cloned the anti-HEL antibody fragment HyHBLlO soFv (Biophysical Journal Vol. 83, 2946-2968 (2002)) into the TGEX-SCblue vector built in Example 7. The HyHEL-10 scFv fragment was amplified from a phagemid done derived from the pADL-lOb vector (Antibody Design Lobs, San Diego, CA) with tho HyHEL-10

scFv sequenoe inserted in tho double Sfll cloning she using the primers scFvblue_s and

scFvblue r. A single band at the expected size was found by minlgel analysis. After

treatment by Dpnl to cut the methylated template DNA, the PCR product was purified over

a Macherey Nagel NucleoSpin column and elated in water at the concentration of 10 ng/ul.

TQEX-SCblue vector was out by Sfll; the reaction mixture was purified over a Macherey

Nagel NucleoSpin column and eluted in water at the concentration of 75 ng/ul.

[93] Primers and PCR fragment; Tm estimation (OllgoAnalyzer 3.1) are given for the

sequence overlaps with the cut vector (underlined).

1

[94]

[95] The rapid LIC method was Identical to the procedure In Example 7. Fifty microliter (SO μΐ) of chemically competent XLlO-gold cells were transformed by heat shock with 2 ul of the reaction, resuspended In 200 ul SOC medium and, after 1 hour incubation at 37"C with shaking, plated on agar plates supplemented with ampUoillin, IPTO and X-gaL The day after, around half of me colonies were white. Four white colonies were picked, grown overnight In 3 -ml 2xYT medium supplemented with ampldllm, and sequence analysis of two colonies showed proper insertion of the scFv PCS. fragment

[96] Bvamplw ^Q - tqgartkm of tWO ρ ^η ι ^η ρΐ^ιρήργ nllynjiiiclBotfalag In a Vector.

[97] In mis example a large loop in a DNA sequence was inserted. This type of cloning project is known to be difficult because of the presence of secondary structures. The recipient vector contained a lambda tl terminator (HI) where the terminator loop had been replaced by an Xbal site (underlined):

5'-

[98] Two oligonucleotides oontainlng homology areas with the truncated ttl sequence on each side of the Xbal site and complementary on their 3* ends were designed to oomplete the entire ttl terminator sequence after insertion in the Xbal site. Below, the sequence homologies with the vector are underlined while the complementary sequence between the two oligonucleotides have been boxed (length 18, Tm 45.1°C).

[99] Primers

[100] Three hundred nanogram (300 ng) of the recipient vector 23C1HH10S.2 were

digested in a 10 ul reaction volume with Xbal for 3 hours. The cut vector was purified over a Machcrcy Nagel NucleoSpln column and eluted in IS μΐ of water.

[101] Rapid LIC reaction

[102] The rapid LIC method was identical to the procedure in Example 7. After oompletiofl of the temperature cycle, 1 μΐ of an equfanolar mixture of the two oligonucleotides at 1 μΜ each in water was added to the reaction and the temperature cycle used in Example 7 was run a second time. Clone analysis after bacterial transformation revealed a high proportion of parental clones; colony PCR followed by restriction analysis with Xbal of 16 colonies found 2 colonies missing the Xbal site. Sequence analysis of these two clones found the proper insertion of the two oligonucleotides, thus creating a complete ltl terminator.

[103] twylii 1 n- Insertion of two PNA *»Γ""1¾ m a vector.

[104] In mis example, we cloned the variable human heavy chain domain of a human

antibody together with a modified CHI domain of human IgGl into the backbone of the TOEX-HC vector (Antibody Design Labs, San Diego, CA). The resulting plasmid in association whh a light chain expressing vector can be used to express a recombinant Fab fragment.

[105] Primers and DNA fragments

[107] The rapid LIC method was identical to the procedure m Example 7. Fifty microliter (50 μΐ) of chemically oompetent XLlO-gold oaUs were transformed by heat shook with 2 μΐ of the reaction, resospended in 200 ul SOC medium and, after 1 hour incubation at 3TC with shaking, plated on agar plates supplemented with amplicillin, IPTG and X-gal. The day after, around half of the colonies were white. Four colonies were picked, grew overnight in 3 -ml 2xYT medium supplemented with ampicillin, and sequence analysis of two colonies showed proper assembly of the 2 fragments in the DNA vector.

All components were mixed on ioe in a single PCR tube and hmw-wMnl for a short period of time in a water bath pre-heated at 75°C before being brought back on ioe. Each assay was done in duplicate as well as a control with the help of a PCR machine as described in Example 7. Chemically competent XLlO-gold bacteria wen transformed and plated on agar plates supplemented with ampicillin, IPTG and X-gal and incubated overnight at 37°C. The morning after, bhie and white colonies were counted.

[111] Results. Average bhie and white colony counts are given for incubation in a 75°C water bath for 2 s, 5 s, 10 s and for a rapid LIC reaction made using a PCR machine. The percentage of blue colonies, resulting from the proper Insertion of the alpha fragment of the beta-galaotosidase in the vector, were close to 80% on average, below the percentage observed for a reaction done with a PCR machine (95%), but still In the range of a very satisfying cloning efficiency.

[112] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to those of ordinary skill In the art and are to be included within the spirit and purview of this application and soope of the appended claims. All publications, patents, patent applications, and/or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and/or other document were Individually indicated to be inoorporated by reference for all purposes.

The following articles and all articles, patents, and patent applications referenced within this specification are Incorporated by reference as part of this application In their entirety.

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