WO2022187334A1

WO2022187334A1 - High-yield probe library construction

Info

Publication number: WO2022187334A1
Application number: PCT/US2022/018480
Authority: WO
Inventors: Siyuan WANG; Miao Liu
Original assignee: Yale University
Priority date: 2021-03-03
Filing date: 2022-03-02
Publication date: 2022-09-09

Abstract

Provided herein are methods and kits for generating one or more single-stranded DNA (ssDNA) oligonucleotides by making single-strand breaks in double-stranded DNA (dsDNA) with a site-specific nicking endonuclease, then extending the nicked dsDNA with a DNA polymerase with strand displacement capability.

Description

HIGH- YIELD PROBE LIBRARY CONSTRUCTION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63/155,984, filed March 3, 2021, the disclosure of which is herein incorporated by reference in its entirety.

FIELD

[0002] Embodiments described herein generally refer to methods for creating a high- yield probe library.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0003] This invention was made with government support under CA260701, CA251037 and HG011245 awarded by National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

[0004] Traditional high-yield probe library construction methods involve the transcription and reverse transcription of RNA to produce single-stranded DNA probes from a common pool. What is needed is a new probe library construction method that is faster and does not require RNase-free materials and handling while maintaining the same high-yield probe output.

SUMMARY

[0005] Provided herein are methods and kits for generating one or more single-stranded DNA (ssDNA) oligonucleotides. Specifically, provided herein are methods and kits for generating ssDNA oligonucleotides by making single-strand breaks in double-stranded DNA (dsDNA) with a site-specific nicking endonuclease. An ssDNA oligonucleotide is created by extending the nicked dsDNA with a DNA polymerase with strand displacement capability, which displaces the ssDNA oligonucleotide while restoring the dsDNA. The process can be repeated to generate additional copies of the ssDNA oligonucleotide. [0006] In some embodiments, provided is a method for generating one or more single- stranded DNA (ssDNA) oligonucleotides, comprising: a) in a reaction mixture comprising one or more double-stranded DNA (dsDNA) oligonucleotides, wherein said dsDNA oligonucleotides comprise at least one recognition sequence for at least one site-specific nicking endonuclease, generating, using said site-specific nicking endonuclease, single-strand breaks in at least some of the dsDNA oligonucleotides to produce nicked dsDNA oligonucleotides; b) extending, by a DNA polymerase with strand displacement capability, the 3’ end of the single-strand break within the nicked dsDNA oligonucleotides to restore said dsDNA oligonucleotides while displacing corresponding ssDNA oligonucleotides from the 5’ end of the single-strand break into the reaction mixture, and c) optionally, repeating steps (a) and (b) one or more times to generate additional copies of the ssDNA oligonucleotides.

[0007] In some embodiments of the method described above, the site-specific nicking endonuclease and the DNA polymerase with strand displacement capability are added to the reaction mixture prior to step (a).

[0008] In some embodiments of the methods described above, steps (a) and (b) are conducted under conditions to prevent annealing of the displaced ssDNA oligonucleotides. In some embodiments, steps (a) and (b) are conducted at the same temperature. In some embodiments, steps (a) and (b) are conducted at different temperatures.

[0009] In some embodiments of the methods described above, step (c) is conducted by repeating steps (a) and (b) cyclically.

[0010] In some embodiments of the methods described above, steps (a), (b), or both are conducted at a high temperature. In some embodiments, step (b) is conducted at a high temperature. In some embodiments, said high temperature is between about 50 °C to about 80 °C.

[0011] In some embodiments of the methods described above, the site-specific nicking endonuclease is a thermostable site-specific nicking endonuclease. In some embodiments, the thermostable site-specific nicking endonuclease is Nt.BstNBI, Nb.BsmI, Nt.BspQI, or Nb.BsrDI.

[0012] In some embodiments of the methods described above, the DNA polymerase with strand displacement capability is a thermostable strand-displacing DNA polymerase. In some embodiment, the thermostable strand-displacing DNA polymerase is Bst 3.0 polymerase, Bsm DNA polymerase (large fragment), or SD DNA polymerase.

[0013] In some embodiments of the methods described above, the volume of the reaction mixture is sufficiently large, e.g ., with an oligonucleotide concentration lower than 0.1 mM, 0.08 mM, 0.05 mM, or 0.02 mM, to prevent annealing of the displaced ssDNA oligonucleotides.

[0014] In some embodiments of the methods described above, the site-specific nicking endonuclease is a non-thermostable site-specific nicking endonuclease. In some embodiments, the non-thermostable site-specific nicking endonuclease is Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nt.BsmAI, Nb.BssSI, Nb.BtsI, orNt.CviPII.

[0015] In some embodiments of the methods described above, the DNA polymerase with strand displacement capability is a non-thermostable strand-displacing DNA polymerase. In some embodiments, the non-thermostable strand-displacing DNA polymerase is phi29 DNA polymerase, Klenow Fragment (3' 5' exo-), or Bsu DNA polymerase (large fragment).

[0016] In some embodiments of the methods described above, the one or more dsDNA oligonucleotides in step (a) is generated by amplifying one or more ssDNA or dsDNA oligonucleotides, wherein (i) the ssDNA or dsDNA oligonucleotides used in amplification comprise at least one recognition sequence for the at least one site-specific nicking endonuclease or a reverse complement thereof, or (ii) the at least one recognition sequence for the at least one site-specific nicking endonuclease is introduced into the dsDNA oligonucleotides during amplification. In some embodiments, the amplification is performed using polymerase chain reaction (PCR). In some embodiments, the at least one recognition sequence for the at least one site-specific nicking endonuclease or a reverse complement thereof is introduced into the dsDNA oligonucleotides through a PCR primer.

[0017] In some embodiments of the methods described above, the one or more dsDNA oligonucleotides in step (a) is generated by primer binding and extension using one or more ssDNA oligonucleotides as template, wherein (i) the ssDNA oligonucleotides comprise at least one recognition sequence for the at least one site-specific nicking endonuclease or a reverse complement thereof, or (ii) the at least one recognition sequence for the at least one site-specific nicking endonuclease is introduced into the dsDNA oligonucleotides through primer binding and extension. In some embodiments, the primer binding and extension is performed for one round.

In some embodiments, the extension is performed with a DNA polymerase. [0018] In some embodiments of the methods described above, the one or more dsDNA oligonucleotides in step (a) are generated by annealing two or more ssDNA oligonucleotides with reverse-complementary sequences. In some embodiments, said dsDNA oligonucleotides comprise one recognition sequence for one site-specific nicking endonuclease.

[0019] In some embodiments of the methods described above, the ssDNA oligonucleotides generated by the method have an average length of between 10 and 1000 nucleotides. In some embodiments, the ssDNA oligonucleotides generated by the method have an average length of between 10 and 200 nucleotides.

[0020] In some embodiments of the methods described above, the ssDNA oligonucleotides generated by the method are purified.

[0021] In some embodiments of the methods described above, the method does not use RNase-free materials and/or handling.

[0022] In some embodiments of the methods described above, the method is used for generating an ssDNA oligonucleotide library.

[0023] In some embodiments, provided is a kit for generating one or more ssDNA oligonucleotides, comprising a site-specific nicking endonuclease, a DNA polymerase with strand displacement capability, and optionally one or more reaction buffers and/or instructions for use.

[0024] In some embodiments of the kit described above, the site-specific nicking endonuclease is a thermostable site-specific nicking endonuclease. In some embodiments, the thermostable site-specific nicking endonuclease is Nt.BstNBI, Nb.BsmI, Nt.BspQI, or Nb.BsrDI.

[0025] In some embodiments of the kits described above, the site-specific nicking endonuclease is a non-thermostable site-specific nicking endonuclease. In some embodiments, the non-thermostable site-specific nicking endonuclease is Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nt.BsmAI, Nb.BssSI, Nb.BtsI, orNt.CviPII.

[0026] In some embodiments of the kits described above, the DNA polymerase with strand displacement capability is a thermostable strand-displacing DNA polymerase. In some embodiments, the thermostable strand-displacing DNA polymerase is Bst 3.0 polymerase, Bsm DNA polymerase (large fragment), or SD DNA polymerase.

[0027] In some embodiments of the kits described above, the DNA polymerase with strand displacement capability is a non-thermostable strand-displacing DNA polymerase. In some embodiments, the non-thermostable strand-displacing DNA polymerase is phi29 DNA polymerase, Klenow Fragment (3' 5' exo-), or Bsu DNA polymerase (large fragment).

[0028] In some embodiments of the kits described above, the kit further comprises means for purifying the ssDNA oligonucleotides.

BRIEF DESCRIPTION OF THE FIGURES

[0029] FIG. 1 depicts an overview of a method for producing high-yield probe libraries using PCR, a site-specific nicking endonuclease, and a DNA polymerase.

[0030] FIG. 2 depicts a denaturing gel electrophoresis image of a representative probe library.

[0031] FIGs. 3 A, 3B and 3C depict the application of the new probe construction strategy to multiplexed error-robust FISH (MERFISH). MERFISH probes constructed with the new strategy were used to label and distinguish 129 different RNA species in a mouse fetal liver tissue section. The raw MERFISH images show distinct single RNA molecule fluorescence spots (FIG. 3 A). The analyzed MERFISH images show the RNA species identities of each RNA molecule (FIGs. 3B and 3C).

DETAILED DESCRIPTION

[0032] The present disclosure generally relates to methods and kits for generating one or more single-stranded DNA (ssDNA) oligonucleotides. Advanced molecular techniques, such as multiplexed DNA/RNA fluorescence in situ hybridization (FISH), can require large oligonucleotide probe libraries. The methods and kits described herein are capable of high-yield ssDNA oligonucleotide generation. In some embodiments, the methods and kits generate ssDNA oligonucleotides by making single-strand breaks in double-stranded DNA (dsDNA) with one or more site-specific nicking endonucleases. An ssDNA oligonucleotide is created by extending the nicked dsDNA with a DNA polymerase with strand displacement capability, which displaces the ssDNA oligonucleotide while restoring the dsDNA. The process can be repeated to generate additional copies of the ssDNA oligonucleotides.

Definitions [0033] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In cases where the present specification and a document incorporated by reference include conflicting and/or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and/or inconsistent disclosure with respect to each other, then the document having the later effective date shall control.

[0034] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

[0035] As used in this specification and the appended claims, the singular forms “a,”

“an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

[0036] The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of up to ±10% from the specified value, as such variations are appropriate to perform the disclosed methods. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0037] “Polynucleotide,” synonymously referred to as “nucleic acid molecule,” “nucleotides” or “nucleic acids,” refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotide” also embraces relatively short nucleic acid chains, often referred to as “oligonucleotides.” Polynucleotides and oligonucleotides herein include, without limitation unless otherwise indicated, single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single- stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. The terms “polynucleotide” and “oligonucleotide” also include DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications may be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides and oligonucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.

[0038] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. If aspects or embodiments of the disclosure are described as “comprising”, or versions thereof ( e.g ., comprises), a feature, embodiments also are contemplated “consisting of’ or “consisting essentially of’ the feature.

[0039] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0040] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0041] The terms and expressions which have been employed herein are used as terms of description and not of limitation, and use of such terms and expressions do not exclude any equivalents of the features shown and described or portions thereof, and various modifications are possible within the scope of the technology claimed. [0042] The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of statistical analysis, molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such tools and techniques are described in detail in e.g ., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York; Ausubel et al. eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Bonifacino et al. eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, NJ; Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, NJ; and Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, NJ. Additional techniques are explained, e.g. , in U.S. Patent No. 7,912,698 and U.S. Patent Appl. Pub. Nos. 2011/0202322 and 2011/0307437.

Methods

[0043] Methods for generating single-stranded DNA (ssDNA) oligonucleotides are provided herein. In some embodiments, any of the methods can be used for generating an ssDNA oligonucleotide library. In some embodiments, the method comprises a reaction mixture comprising one or more dsDNA oligonucleotides. The method uses the one or more dsDNA oligonucleotides as templates from which to generate the ssDNA oligonucleotides. In some embodiments, the dsDNA oligonucleotides comprise at least one recognition sequence. In some embodiments, the dsDNA oligonucleotides comprise one recognition sequence. In some embodiments, the dsDNA oligonucleotides comprise two or more recognition sequences. In some embodiments, the recognition sequence is a nucleotide sequence recognized by at least one site-specific nicking endonuclease.

[0044] Those skilled in the art understand that there are multiple ways to generate the dsDNA for use in the methods described herein. In some embodiments, the one or more dsDNA oligonucleotides are generated by amplifying one or more ssDNA or dsDNA oligonucleotides. In some embodiments, the ssDNA or dsDNA oligonucleotides used in amplification comprise at least one recognition sequence for the at least one site-specific nicking endonuclease or a reverse complement thereof. In some embodiments, the at least one recognition sequence for the at least one site-specific nicking endonuclease is introduced into the dsDNA oligonucleotides during amplification. In some embodiments, the amplification is performed using polymerase chain reaction (PCR). In some embodiments, the at least one recognition sequence for the at least one site-specific nicking endonuclease or a reverse complement thereof is introduced into the dsDNA oligonucleotides through a PCR primer. In some embodiments, the one or more dsDNA oligonucleotides are generated by primer binding and extension using one or more ssDNA oligonucleotides as template. In some embodiments, the ssDNA oligonucleotides comprise at least one recognition sequence for the at least one site-specific nicking endonuclease or a reverse complement thereof. In some embodiments, the at least one recognition sequence for the at least one site-specific nicking endonuclease is introduced into the dsDNA oligonucleotides through primer binding and extension. In some embodiments, the primer binding and extension is performed for one round. In some embodiments, the extension is performed with a DNA polymerase. In some embodiments, the one or more dsDNA oligonucleotides are generated by annealing two or more ssDNA oligonucleotides with reverse-complementary sequences. In some embodiments, said dsDNA oligonucleotides comprise one recognition sequence for one site- specific nicking endonuclease.

[0045] In some embodiments, the method comprises at least one site-specific nicking endonuclease. Nicking endonucleases are known in the art, and generally are enzymes that cut one strand of a dsDNA at a specific recognition sequence. By hydrolyzing only one strand of the dsDNA, nicking endonucleases produce dsDNA that is cut at one or more sites in one strand only, instead of cutting both strands. In some embodiments, the at least one site-specific nicking endonuclease recognizes and binds to the at least one recognition sequence on the dsDNA oligonucleotides in the reaction mixture.

[0046] In some embodiments, the site-specific nicking endonuclease is a thermostable site-specific nicking endonuclease. In some embodiments, the thermostable site-specific nicking endonuclease is Nt.BstNBI. In some embodiments, the thermostable site-specific nicking endonuclease is Nb.BsmI. In some embodiments, the thermostable site-specific nicking endonuclease is Nt.BspQI. In some embodiments, the thermostable site-specific nicking endonuclease is Nb.BsrDI. In some embodiments, the site-specific nicking endonuclease is a non-thermostable site-specific nicking endonuclease. In some embodiments, the non- thennostable site-specific nicking endonuclease is Nt. AlwI. In some embodiments, the non- thermostable site-specific nicking endonuclease is Nb.BbvCI. In some embodiments, the non- thermostable site-specific nicking endonuclease is Nt.BbvCI. In some embodiments, the non- theimostable site-specific nicking endonuclease is Nt.BsmAI. In some embodiments, the non- theimostable site-specific nicking endonuclease is Nb.BssSI. In some embodiments, the non- theimostable site-specific nicking endonuclease is Nb.BtsI. In some embodiments, the non- thermostable site-specific nicking endonuclease is Nt.CviPII.

[0047] In some embodiments, the method comprises at least one DNA polymerase. In some embodiments, the method comprises at least one DNA polymerase with strand displacement capability. DNA polymerases with strand displacement capability are known in the art, and generally are polymerases that have the ability to displace downstream nucleotides encountered during polymerization. In some embodiments, a DNA polymerase with strand displacement capability extends the 3’ end of the single-strand break within the nicked dsDNA oligonucleotide. As the DNA polymerase with strand displacement capability extends the 3’ end of the single-strand break, it restores the dsDNA oligonucleotides to its full, non-nicked form. In doing so, the DNA polymerase with strand displacement capability displaces the corresponding ssDNA from the 5’ end of the single-strand break, which then releases back into the reaction mixture. In this way, the DNA polymerase with strand displacement capability both restores the dsDNA and releases the ssDNA oligonucleotide.

[0048] In some embodiments, the DNA polymerase with strand displacement capability is a thermostable strand-displacing DNA polymerase. In some embodiment, the thermostable strand-displacing DNA polymerase is Bst 3.0 polymerase. In some embodiment, the thermostable strand-displacing DNA polymerase is Bsm DNA polymerase (large fragment). In some embodiment, the thermostable strand-displacing DNA polymerase is SD DNA polymerase. In some embodiments, the DNA polymerase with strand displacement capability is a non thermostable strand-displacing DNA polymerase. In some embodiments, the non-thermostable strand-displacing DNA polymerase is phi29 DNA polymerase. In some embodiments, the non thermostable strand-displacing DNA polymerase is Klenow Fragment (3’ 5’ exo-). In some embodiments, the non-thermostable strand-displacing DNA polymerase is Bsu DNA polymerase (large fragment). [0049] In some embodiments, the method for generating one or more ssDNA oligonucleotides comprises a reaction mixture comprising one or more dsDNA oligonucleotides described herein, wherein the dsDNA oligonucleotides comprise at least one recognition sequence for at least one site-specific nicking endonuclease. When present in the reaction mixture, at least one site-specific nicking endonuclease of any of the types described herein generate single-strand breaks in at least some of the dsDNA oligonucleotides to produce nicked dsDNA oligonucleotides. In some embodiments, at least one DNA polymerase with strand displacement capability of any of the types described here is also present in the reaction mixture. In some embodiments, the at least one DNA polymerase with strand displacement capability extends the 3’ end of the single-strand break within the nicked dsDNA oligonucleotides, restoring the dsDNA oligonucleotides while displacing corresponding ssDNA oligonucleotides from the 5’ end of the single-strand break into the reaction mixture. In some embodiments, the steps of a) generating single-strand breaks in at least some of the dsDNA oligonucleotides to produce nicked dsDNA oligonucleotides; and b) extending 3’ end of the single-strand break within the nicked dsDNA oligonucleotides, restoring the dsDNA oligonucleotides while displacing corresponding ssDNA oligonucleotides from the 5’ end of the single-strand break into the reaction mixture can be repeated one or more times to generate additional copies of the ssDNA oligonucleotides. In some embodiments, said repetition is conducted cyclically.

[0050] In some embodiments, the dsDNA oligonucleotides, the site-specific nicking endonuclease and the DNA polymerase with strand displacement capability are present in the reaction mixture prior to the execution of any of the method steps described herein. In some embodiments, the dsDNA oligonucleotides, the site-specific nicking endonuclease or the DNA polymerase with strand displacement capability are added to the reaction mixture at different times during the execution of any of the method steps described herein. For example, in some embodiments, the dsDNA oligonucleotides, the site-specific nicking endonuclease and the DNA polymerase with strand displacement capability are all added to the reaction mixture prior to the step of generating single-strand breaks in at least some of the dsDNA oligonucleotides to produce nicked dsDNA oligonucleotides.

[0051] In some embodiments, any of the steps of the methods described herein are conducted under conditions to prevent annealing of the displaced ssDNA oligonucleotides. In some embodiments, the steps of a) generating single-strand breaks in at least some of the dsDNA oligonucleotides to produce nicked dsDNA oligonucleotides; and b) extending 3’ end of the single-strand break within the nicked dsDNA oligonucleotides, restoring the dsDNA oligonucleotides while displacing corresponding ssDNA oligonucleotides from the 5’ end of the single-strand break into the reaction mixture are conducted under conditions to prevent annealing of the displaced ssDNA oligonucleotides.

[0052] In some embodiments, the steps of a) generating single-strand breaks in at least some of the dsDNA oligonucleotides to produce nicked dsDNA oligonucleotides; and b) extending 3’ end of the single-strand break within the nicked dsDNA oligonucleotides, restoring the dsDNA oligonucleotides while displacing corresponding ssDNA oligonucleotides from the 5’ end of the single-strand break into the reaction mixture are conducted at the same temperature. In some embodiments, the steps of a) generating single-strand breaks in at least some of the dsDNA oligonucleotides to produce nicked dsDNA oligonucleotides; and b) extending 3’ end of the single-strand break within the nicked dsDNA oligonucleotides, restoring the dsDNA oligonucleotides while displacing corresponding ssDNA oligonucleotides from the 5’ end of the single-strand break into the reaction mixture are conducted at different temperatures. In some embodiments, the step of a) generating single-strand breaks in at least some of the dsDNA oligonucleotides to produce nicked dsDNA oligonucleotides is conducted at high temperature. In some embodiments, the step of b) extending 3’ end of the single-strand break within the nicked dsDNA oligonucleotides, restoring the dsDNA oligonucleotides while displacing corresponding ssDNA oligonucleotides from the 5’ end of the single-strand break into the reaction mixture is conducted at high temperature. In some embodiments, the steps of a) generating single-strand breaks in at least some of the dsDNA oligonucleotides to produce nicked dsDNA oligonucleotides; and b) extending 3’ end of the single-strand break within the nicked dsDNA oligonucleotides, restoring the dsDNA oligonucleotides while displacing corresponding ssDNA oligonucleotides from the 5’ end of the single-strand break into the reaction mixture are conducted at high temperature. In some embodiments, the high temperature is between about 50 °C to about 80 °C. In some embodiments, the volume of the reaction mixture is sufficiently large, e.g., with an oligonucleotide concentration lower than 0.1 mM, to prevent annealing of the displaced ssDNA oligonucleotides.

[0053] In some embodiments, the ssDNA oligonucleotides generated by any of the methods described herein have an average length of between 10 and 1000 nucleotides. In some embodiments, the ssDNA oligonucleotides generated by any of the methods described herein have an average length of between 10 and 900 nucleotides. In some embodiments, the ssDNA oligonucleotides generated by any of the methods described herein have an average length of between 10 and 800 nucleotides. In some embodiments, the ssDNA oligonucleotides generated by any of the methods described herein have an average length of between 10 and 700 nucleotides. In some embodiments, the ssDNA oligonucleotides generated by any of the methods described herein have an average length of between 10 and 600 nucleotides. In some embodiments, the ssDNA oligonucleotides generated by any of the methods described herein have an average length of between 10 and 500 nucleotides. In some embodiments, the ssDNA oligonucleotides generated by any of the methods described herein have an average length of between 10 and 400 nucleotides. In some embodiments, the ssDNA oligonucleotides generated by any of the methods described herein have an average length of between 10 and 300 nucleotides. In some embodiments, the ssDNA oligonucleotides generated by any of the methods described herein have an average length of between 10 and 200 nucleotides. In some embodiments, the ssDNA oligonucleotides generated by any of the methods described herein have an average length of between 10 and 100 nucleotides. In some embodiments, the ssDNA oligonucleotides generated by any of the methods described herein have an average length of between 100 and 1000 nucleotides. In some embodiments, the ssDNA oligonucleotides generated by any of the methods described herein have an average length of between 200 and 1000 nucleotides. In some embodiments, the ssDNA oligonucleotides generated by any of the methods described herein have an average length of between 300 and 1000 nucleotides. In some embodiments, the ssDNA oligonucleotides generated by any of the methods described herein have an average length of between 400 and 1000 nucleotides. In some embodiments, the ssDNA oligonucleotides generated by any of the methods described herein have an average length of between 500 and 1000 nucleotides. In some embodiments, the ssDNA oligonucleotides generated by any of the methods described herein have an average length of between 600 and 1000 nucleotides. In some embodiments, the ssDNA oligonucleotides generated by any of the methods described herein have an average length of between 700 and 1000 nucleotides. In some embodiments, the ssDNA oligonucleotides generated by any of the methods described herein have an average length of between 800 and 1000 nucleotides. In some embodiments, the ssDNA oligonucleotides generated by any of the methods described herein have an average length of between 900 and 1000 nucleotides.

[0054] In some embodiments, the ssDNA oligonucleotides generated by any of the methods described herein are purified. Methods of purifying ssDNA oligonucleotides are known in the art, for example, the use of commercially available kits and reagents, high performance liquid chromatography, or polyacrylamide gel electrophoresis. In some embodiments, the step of purification takes place after a set amount of time. In some embodiments, the step of purification takes place after a set number of dsDNA nicking and extension cycles.

[0055] In some embodiments, any of the methods described herein do not use RNase-free materials and/or handling. In some embodiments, any of the methods described herein do not require RNase-free materials and/or handling.

Kits

[0056] Kits for generating one or more ssDNA oligonucleotides are provided herein. In some embodiments, any of the kits described herein can be used for generating an ssDNA oligonucleotide library. In some embodiments, a kit for generating one or more ssDNA oligonucleotides comprises a site-specific nicking endonuclease, and a DNA polymerase with strand displacement capability. In some embodiments, a kit for generating one or more ssDNA oligonucleotides comprises a site-specific nicking endonuclease, a DNA polymerase with strand displacement capability, and a reaction buffer. In some embodiments, any of the kits described herein further comprises instructions for use. In some embodiments, any of the kits described herein can be used to complete or partially complete any of the methods for generating ssDNA oligonucleotides described herein.

[0057] In some embodiments, the site-specific nicking endonuclease is a thermostable site-specific nicking endonuclease. In some embodiments, the thermostable site-specific nicking endonuclease is Nt.BstNBI. In some embodiments, the thermostable site-specific nicking endonuclease is Nb.BsmI. In some embodiments, the thermostable site-specific nicking endonuclease is Nt.BspQI. In some embodiments, the thermostable site-specific nicking endonuclease is Nb.BsrDI. In some embodiments, the site-specific nicking endonuclease is a non-thermostable site-specific nicking endonuclease. In some embodiments, the non thermostable site-specific nicking endonuclease is Nt. AlwI. In some embodiments, the non- thennostable site-specific nicking endonuclease is Nb.BbvCI. In some embodiments, the non- thermostable site-specific nicking endonuclease is Nt.BbvCI. In some embodiments, the non- theimostable site-specific nicking endonuclease is Nt.BsmAI. In some embodiments, the non- theimostable site-specific nicking endonuclease is Nb.BssSI. In some embodiments, the non- theimostable site-specific nicking endonuclease is Nb.BtsI. In some embodiments, the non- theimostable site-specific nicking endonuclease is Nt.CviPII.

[0058] In some embodiments, the DNA polymerase with strand displacement capability is a thermostable strand-displacing DNA polymerase. In some embodiment, the thermostable strand-displacing DNA polymerase is Bst 3.0 polymerase. In some embodiment, the thermostable strand-displacing DNA polymerase is Bsm DNA polymerase (large fragment). In some embodiment, the thermostable strand-displacing DNA polymerase is SD DNA polymerase. In some embodiments, the DNA polymerase with strand displacement capability is a non thermostable strand-displacing DNA polymerase. In some embodiments, the non-thermostable strand-displacing DNA polymerase is phi29 DNA polymerase. In some embodiments, the non thermostable strand-displacing DNA polymerase is Klenow Fragment (3' 5' exo-). In some embodiments, the non-thermostable strand-displacing DNA polymerase is Bsu DNA polymerase (large fragment).

[0059] In some embodiments, the reaction buffer has a pH of about 6.5 to about 9.0. In some embodiments, the reaction buffer has a pH of about 7.0 to about 9.0. In some embodiments, the reaction buffer has a pH of about 7.5 to about 9.0. In some embodiments, the reaction buffer has a pH of about 8.0 to about 9.0. In some embodiments, the reaction buffer has a pH of about 8.5 to about 9.0. In some embodiments, the reaction buffer has a pH of about 6.5 to about 8.5. In some embodiments, the reaction buffer has a pH of about 6.5 to about 8.0. In some embodiments, the reaction buffer has a pH of about 6.5 to about 7.5. In some embodiments, the reaction buffer has a pH of about 6.5 to about 7.0. In some embodiments, the buffer is concentrated. In some embodiments, the buffer is not concentrated. In some embodiments, the buffer is a thermostable buffer. In some embodiments, any of the kits described herein further comprise one or more means for purifying ssDNA oligonucleotides.

EMBODIMENTS

[0060] Also provided herein are the following non-limited embodiments. [0061] 1. A method for generating one or more single-stranded DNA (ssDNA) oligonucleotides, comprising: a) in a reaction mixture comprising one or more double-stranded DNA (dsDNA) oligonucleotides, wherein said dsDNA oligonucleotides comprise at least one recognition sequence for at least one site-specific nicking endonuclease, generating, using said site-specific nicking endonuclease, single-strand breaks in at least some of the dsDNA oligonucleotides to produce nicked dsDNA oligonucleotides; b) extending, by a DNA polymerase with strand displacement capability, the 3’ end of the single-strand break within the nicked dsDNA oligonucleotides to restore said dsDNA oligonucleotides while displacing corresponding ssDNA oligonucleotides from the 5’ end of the single-strand break into the reaction mixture, and c) optionally, repeating steps (a) and (b) one or more times to generate additional copies of the ssDNA oligonucleotides.

[0062] 2. The method of embodiment 1, wherein the site-specific nicking endonuclease and the DNA polymerase with strand displacement capability are added to the reaction mixture prior to step (a).

[0063] 3. The method of embodiment 1 or embodiment 2, wherein steps (a) and (b) are conducted under conditions to prevent annealing of the displaced ssDNA oligonucleotides.

[0064] 4. The method of any one of embodiments 1-3, wherein steps (a) and (b) are conducted at the same temperature.

[0065] 5. The method of any one of embodiments 1-3, wherein steps (a) and (b) are conducted at different temperatures.

[0066] 6. The method of embodiment 5, wherein step (c) is conducted by repeating steps (a) and (b) cyclically.

[0067] 7. The method of any one of embodiments 1-6, wherein steps (a) and/or (b) are conducted at a high temperature.

[0068] 8. The method of any one of embodiments 1-7, wherein step (b) is conducted at a high temperature.

[0069] 9. The method of embodiment 8, wherein said high temperature is between about

50 °C to about 80 °C. [0070] 10. The method of any one of embodiments 1-9, wherein the site-specific nicking endonuclease is a thermostable site-specific nicking endonuclease.

[0071] 11. The method of embodiment 10, wherein the thermostable site-specific nicking endonuclease is Nt.BsfNBI, Nb.BsmI, Nt.BspQI, or Nb.BsrDI.

[0072] 12. The method of any one of embodiments 1-11, wherein the DNA polymerase with strand displacement capability is a thermostable strand-displacing DNA polymerase.

[0073] 13. The method of embodiment 12, wherein the thermostable strand-displacing DNA polymerase is Bst 3.0 polymerase, Bsm DNA polymerase (large fragment), or SD DNA polymerase.

[0074] 14. The method of any one of embodiments 1-13, wherein the volume of the reaction mixture is sufficiently large, with an oligonucleotide concentration lower than 0.1 mM, to prevent annealing of the displaced ssDNA oligonucleotides.

[0075] 15. The method of any one of embodiments 1-3, 8, and 14, wherein the site- specific nicking endonuclease is a non-thermostable site-specific nicking endonuclease.

[0076] 16. The method of embodiment 15, wherein the non-thermostable site-specific nicking endonuclease is Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nt.BsmAI, Nb.BssSI, Nb.BtsI, or Nt.CviPII.

[0077] 17. The method of any one of embodiments 1-3 and 14-16, wherein the DNA polymerase with strand displacement capability is a non-thermostable strand-displacing DNA polymerase.

[0078] 18. The method of embodiment 17, wherein the non-thermostable strand- displacing DNA polymerase is phi29 DNA polymerase, Klenow Fragment (3' 5' exo-), or Bsu DNA polymerase (large fragment).

[0079] 19. The method of any one of embodiments 1-18, wherein the one or more dsDNA oligonucleotides in step (a) is generated by amplifying one or more ssDNA or dsDNA oligonucleotides, wherein (i) the ssDNA or dsDNA oligonucleotides used in amplification comprise at least one recognition sequence for the at least one site-specific nicking endonuclease or a reverse complement thereof, or (ii) the at least one recognition sequence for the at least one site-specific nicking endonuclease is introduced into the dsDNA oligonucleotides during amplification. [0080] 20. The method of embodiment 19, wherein the amplification is performed using polymerase chain reaction (PCR).

[0081] 21. The method of embodiment 20, wherein the at least one recognition sequence for the at least one site-specific nicking endonuclease or a reverse complement thereof is introduced into the dsDNA oligonucleotides through a PCR primer.

[0082] 22. The method of any one of embodiments 1-18, wherein the one or more dsDNA oligonucleotides in step (a) is generated by primer binding and extension using one or more ssDNA oligonucleotides as template, wherein (i) the ssDNA oligonucleotides comprise at least one recognition sequence for the at least one site-specific nicking endonuclease or a reverse complement thereof, or (ii) the at least one recognition sequence for the at least one site-specific nicking endonuclease is introduced into the dsDNA oligonucleotides through primer binding and extension.

[0083] 23. The method of embodiment 22, wherein the primer binding and extension is performed for one round.

[0084] 24. The method of embodiment 22 or 23, wherein the extension is performed with a DNA polymerase.

[0085] 25. The method of any one of embodiments 1-18, wherein the one or more dsDNA oligonucleotides in step (a) is generated by annealing two or more ssDNA oligonucleotides with reverse-complementary sequences.

[0086] 26. The method of any one of embodiments 1-25, wherein said dsDNA oligonucleotides comprise one recognition sequence for one site-specific nicking endonuclease.

[0087] 27. The method of any one of embodiments 1-26, wherein the ssDNA oligonucleotides generated by the method have an average length of between 10 and 1000 nucleotides.

[0088] 28. The method of any one of embodiments 1-27, wherein the ssDNA oligonucleotides generated by the method have an average length of between 10 and 200 nucleotides.

[0089] 29. The method of any one of embodiments 1-28, wherein the ssDNA oligonucleotides generated by the method are purified.

[0090] 30. The method of any one of embodiments 1-29, wherein the method does not use RNase-free materials and/or handling. [0091] 31. The method of any one of embodiments 1-30, wherein the method is used for generating an ssDNA oligonucleotide library.

[0092] 32. A kit for generating one or more ssDNA oligonucleotides, comprising a site- specific nicking endonuclease, a DNA polymerase with strand displacement capability, and optionally one or more reaction buffers and/or instructions for use.

[0093] 33. The kit of embodiment 32, wherein the site-specific nicking endonuclease is a thermostable site-specific nicking endonuclease.

[0094] 34. The kit of embodiment 33, wherein the thermostable site-specific nicking endonuclease is Nt.BsfNBI, Nb.BsmI, Nt.BspQI, or Nb.BsrDI.

[0095] 35. The kit of embodiment 32, wherein the site-specific nicking endonuclease is a non-thermostable site-specific nicking endonuclease.

[0096] 36. The kit of embodiment 35, wherein the non-thermostable site-specific nicking endonuclease is Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nt.BsmAI, Nb.BssSI, Nb.BtsI, or Nt.CviPII.

[0097] 37. The kit of any one of embodiments 32-36, wherein the DNA polymerase with strand displacement capability is a thermostable strand-displacing DNA polymerase.

[0098] 38. The kit of embodiment 37, wherein the thermostable strand-displacing DNA polymerase is Bst 3.0 polymerase, Bsm DNA polymerase (large fragment), or SD DNA polymerase.

[0099] 39. The kit of any one of embodiments 32-36, wherein the DNA polymerase with strand displacement capability is a non-thermostable strand-displacing DNA polymerase.

[0100] 40. The kit of embodiment 39, wherein the non-thermostable strand-displacing DNA polymerase is phi29 DNA polymerase, Klenow Fragment (3' 5' exo-), or Bsu DNA polymerase (large fragment).

[0101] 41. The kit of any one of embodiments 32-40, further comprising means for purifying the ssDNA oligonucleotides.

EXAMPLES

[0102] The following examples are illustrative, but not limiting, of the methods described herein.

Example 1: High-Yield Probe Library Construction [0103] Multiplexed DNA/RNA fluorescence in situ hybridization (FISH) or targeted in situ sequencing techniques often require the construction of single-stranded DNA oligonucleotide probe library with high yield. Here, a new high-yield probe library construction method is described.

[0104] An overview of the construction method is shown in FIG. 1. First, the method begins with a single-stranded DNA oligonucleotide pool. Such oligonucleotide pool can be commercially synthesized from multiple sources, but cannot be directly used as probe libraries in multiplexed FISH or in situ sequencing experiments due to the low yield of the oligonucleotides. To construct the high-yield probe libraries, the oligonucleotide pool is first amplified through polymerase chain reaction (PCR), which yields whole double-stranded DNA products (labeled double-stranded DNA template in FIG. 1). Alternatively, the double-stranded DNA template may be generated by annealing single-stranded DNA oligonucleotides with reverse complementary sequences, or by one round of primer binding and extension with DNA polymerase instead of PCR.

[0105] Next, a site-specific thermostable nicking endonuclease (Nt.BstNBI) and a thermostable DNA polymerase (Bst 3.0 polymerase) with strand displacement capability were applied to the double stranded DNA pool. The nicking endonuclease generates a single strand break on the double stranded DNA template, producing nicked double-stranded oligonucleotides. The specific nicking site can be included in the original single-stranded DNA oligonucleotide sequences, or can be added to the double-stranded DNA template through the PCR primer. The DNA polymerase extends the 3’ end of the nick, displacing a single-stranded DNA oligonucleotide from the 5’ end of the nick. The nicking and strand displacement polymerization happen repeatedly in this pooled reaction, so that many copies of the single-stranded DNA oligonucleotides are generated, which can be purified and used as probes.

[0106] The method is conducted with a high reaction temperature to reduce the chance of the product single-stranded probes to anneal with each other at high concentration due to partial homology, resulting in unwanted extension. Alternatively, increasing the reaction volume to alleviate unwanted probe annealing is also possible. In this case, non-thermostable nicking endonucleases and strand-displacing DNA polymerases can be used, for example, phi29 DNA polymerase. [0107] FIG. 2 shows a denaturing gel electrophoresis image of a representative probe library constructed by following the procedure shown in FIG. 1. The brightest band in the product lane corresponds to the expected probe length. FIGs. 3A, 3B and 3C show the application of the new probe construction strategy to multiplexed error-robust FISH (MERFISH). MERFISH probes constructed with the new strategy were used to label and distinguish 129 different RNA species in a mouse fetal liver tissue section. The raw MERFISH images show distinct single RNA molecule fluorescence spots (FIG. 3 A). The analyzed MERFISH images show the RNA species identities of each RNA molecule (FIGs. 3B and 3C).

[0108] From the foregoing, it will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made. Accordingly, the various embodiments disclosed herein are not intended to be limiting.

Claims

WHAT IS CLAIMED IS:

1. A method for generating one or more single-stranded DNA (ssDNA) oligonucleotides, comprising: a) in a reaction mixture comprising one or more double-stranded DNA (dsDNA) oligonucleotides, wherein said dsDNA oligonucleotides comprise at least one recognition sequence for at least one site-specific nicking endonuclease, generating, using said site-specific nicking endonuclease, single-strand breaks in at least some of the dsDNA oligonucleotides to produce nicked dsDNA oligonucleotides; b) extending, by a DNA polymerase with strand displacement capability, the 3’ end of the single-strand break within the nicked dsDNA oligonucleotides to restore said dsDNA oligonucleotides while displacing corresponding ssDNA oligonucleotides from the 5’ end of the single-strand break into the reaction mixture, and c) optionally, repeating steps (a) and (b) one or more times to generate additional copies of the ssDNA oligonucleotides.

2. The method of claim 1, wherein the site-specific nicking endonuclease and the DNA polymerase with strand displacement capability are added to the reaction mixture prior to step (a).

3. The method of claim 1 or claim 2, wherein steps (a) and (b) are conducted under conditions to prevent annealing of the displaced ssDNA oligonucleotides.

4. The method of any one of claims 1-3, wherein steps (a) and (b) are conducted at the same temperature.

5. The method of any one of claims 1-3, wherein steps (a) and (b) are conducted at different temperatures.

6. The method of claim 5, wherein step (c) is conducted by repeating steps (a) and (b) cyclically.

7. The method of any one of claims 1-6, wherein steps (a) and/or (b) are conducted at a high temperature.

8. The method of any one of claims 1-7, wherein step (b) is conducted at a high temperature.

9. The method of claim 8, wherein said high temperature is between about 50 °C to about 80 °C.

10. The method of any one of claims 1-9, wherein the site-specific nicking endonuclease is a thermostable site-specific nicking endonuclease.

11. The method of claim 10, wherein the thermostable site-specific nicking endonuclease is Nt.BstNBI, Nb.BsmI, Nt.BspQI, or Nb.BsrDI.

12. The method of any one of claims 1-11, wherein the DNA polymerase with strand displacement capability is a thermostable strand-displacing DNA polymerase.

13. The method of claim 12, wherein the thermostable strand-displacing DNA polymerase is Bst 3.0 polymerase, Bsm DNA polymerase (large fragment), or SD DNA polymerase.

14. The method of any one of claims 1-13, wherein the volume of the reaction mixture is sufficiently large, with an oligonucleotide concentration lower than 0.1 mM, to prevent annealing of the displaced ssDNA oligonucleotides.

15. The method of any one of claims 1-3, 8, and 14, wherein the site-specific nicking endonuclease is a non-thermostable site-specific nicking endonuclease.

16. The method of claim 15, wherein the non-thermostable site-specific nicking endonuclease is Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nt.BsmAI, Nb.BssSI, Nb.BtsI, orNt.CviPII.

17. The method of any one of claims 1-3 and 14-16, wherein the DNA polymerase with strand displacement capability is a non-thermostable strand-displacing DNA polymerase.

18. The method of claim 17, wherein the non-thermostable strand-displacing DNA polymerase is phi29 DNA polymerase, Klenow Fragment (3' 5' exo-), or Bsu DNA polymerase (large fragment).

19. The method of any one of claims 1-18, wherein the one or more dsDNA oligonucleotides in step (a) is generated by amplifying one or more ssDNA or dsDNA oligonucleotides, wherein (i) the ssDNA or dsDNA oligonucleotides used in amplification comprise at least one recognition sequence for the at least one site-specific nicking endonuclease or a reverse complement thereof, or (ii) the at least one recognition sequence for the at least one site-specific nicking endonuclease is introduced into the dsDNA oligonucleotides during amplification.

20. The method of claim 19, wherein the amplification is performed using polymerase chain reaction (PCR).

21. The method of claim 20, wherein the at least one recognition sequence for the at least one site-specific nicking endonuclease or a reverse complement thereof is introduced into the dsDNA oligonucleotides through a PCR primer.

22. The method of any one of claims 1-18, wherein the one or more dsDNA oligonucleotides in step (a) is generated by primer binding and extension using one or more ssDNA oligonucleotides as template, wherein (i) the ssDNA oligonucleotides comprise at least one recognition sequence for the at least one site-specific nicking endonuclease or a reverse complement thereof, or (ii) the at least one recognition sequence for the at least one site-specific nicking endonuclease is introduced into the dsDNA oligonucleotides through primer binding and extension.

23. The method of claim 22, wherein the primer binding and extension is performed for one round.

24. The method of claim 22 or 23, wherein the extension is performed with a DNA polymerase.

25. The method of any one of claims 1-18, wherein the one or more dsDNA oligonucleotides in step (a) is generated by annealing two or more ssDNA oligonucleotides with reverse complementary sequences.

26. The method of any one of claims 1-25, wherein said dsDNA oligonucleotides comprise one recognition sequence for one site-specific nicking endonuclease.

27. The method of any one of claims 1-26, wherein the ssDNA oligonucleotides generated by the method have an average length of between 10 and 1000 nucleotides.

28. The method of any one of claims 1-27, wherein the ssDNA oligonucleotides generated by the method have an average length of between 10 and 200 nucleotides.

29. The method of any one of claims 1-28, wherein the ssDNA oligonucleotides generated by the method are purified.

30. The method of any one of claims 1-29, wherein the method does not use RNase-free materials and/or handling.

31. The method of any one of claims 1-30, wherein the method is used for generating an ssDNA oligonucleotide library.

32. A kit for generating one or more ssDNA oligonucleotides, comprising a site-specific nicking endonuclease, a DNA polymerase with strand displacement capability, and optionally one or more reaction buffers and/or instructions for use.

33. The kit of claim 32, wherein the site-specific nicking endonuclease is a thermostable site- specific nicking endonuclease.

34. The kit of claim 33, wherein the thermostable site-specific nicking endonuclease is Nt.BstNBI, Nb.BsmI, Nt.BspQI, or Nb.BsrDI.

35. The kit of claim 32, wherein the site-specific nicking endonuclease is a non-thermostable site-specific nicking endonuclease.

36. The kit of claim 35, wherein the non-thermostable site-specific nicking endonuclease is Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nt.BsmAI, Nb.BssSI, Nb.BtsI, orNt.CviPII.

37. The kit of any one of claims 32-36, wherein the DNA polymerase with strand displacement capability is a thermostable strand-displacing DNA polymerase.

38. The kit of claim 37, wherein the thermostable strand-displacing DNA polymerase is Bst 3.0 polymerase, Bsm DNA polymerase (large fragment), or SD DNA polymerase.

39. The kit of any one of claims 32-36, wherein the DNA polymerase with strand displacement capability is a non-thermostable strand-displacing DNA polymerase.

40. The kit of claim 39, wherein the non-thermostable strand-displacing DNA polymerase is phi29 DNA polymerase, Klenow Fragment (3' 5' exo-), or Bsu DNA polymerase (large fragment).

41. The kit of any one of claims 32-40, further comprising means for purifying the ssDNA oligonucleotides.