Methods and materials to convert oligonucleotide probes to target specific polymerase extension primers

Abstract

The present disclosure provides for methods of generating a single stranded primer from an oligonucleotide probe. For instance, the present disclosure is directed to a method of preparing oligonucleotide primers from oligonucleotide probes, such as oligonucleotide probes which have been amplified, and which include a target specific primer sequence, a forward primer binding site, and a reverse primer binding site. It is believed that the presently disclosed method permits the synthesis of thousands to hundreds of thousands and even a million primers in a cost-effective manner, with good yield, in high quantities and at high purity levels.

Description

Attorney Docket No. : Ventana-0298 WO (PCT Application)METHODS AND MATERIALS TO CONVERT OLIGONUCLEOTIDE PROBES TO TARGET SPECIFIC POLYMERASE EXTENSION PRIMERSCROSS REFERENCE TO RELATED APPLICATIONSThe present disclosure claims the benefit of the filing date of United States Provisional Patent Application No. 63 / 704,598 filed on October 8, 2024, the disclosure of which is hereby incorporated by reference herein in its entirety.FIELD OF THE DISCLOSUREThe present disclosure relates to oligonucleotides and their method of synthesis.BACKGROUND OF THE DISCLOSURE

[0001] Oligonucleotide primers are used in polymerase chain reaction (PCR) and other polymerase extension reactions. These primers specifically target a portion of a nucleic acid molecule, such as a target nucleic acid molecule, and use the nucleic acid molecule as a template to generate a reverse complement of the nucleic acid molecule. Current methods for synthesizing such oligonucleotide primers requires large-scale oligonucleotide chemical synthesis. For instance, these oligonucleotide probes are typically synthesized in physically separated tubes, columns, or wells. Due to this, increasing the number of individual oligonucleotide probes in a massively parallel fashion (e.g., >1000, >10,000, >100,000, >1,000,000 oligonucleotide probes) is challenging and can be quite costly. There remains a need to generate oligonucleotide primers in a cost-effective manner.

[0002] Compared with whole-genome sequencing, targeted sequencing is a cost-effective method for analyzing specific genomic regions. Targeted sequencing has wide application in diagnostics, metagenomic, phylogenetic, ancient and environmental DNA studies, and forensics. In targeted sequencing, regions of interest are enriched by hybridization capture using targetspecific probes or by PCR amplification using target-specific primers, followed by high- throughput next-generation sequencing (NGS).

[0003] In-solution hybridization capture uses oligonucleotide probes, such as biotinylated oligonucleotide probes, to capture target regions in one or more target nucleic acid molecules. Probe synthesizers are able generate tens of thousands, hundreds of thousands, and even millionsAttorney Docket No. : Ventana-0298 WO (PCT Application) of different oligonucleotide probes in a single synthesis run. These oligonucleotide synthesizers, however, generate a small amount of each oligonucleotide probe, which typically requires amplification methods to increase oligonucleotide probe concentration in order to be used in capture reactions. For instance, microarray-based synthesis generates oligonucleotides in femtomole scales; and the templates synthesized at such small scales require amplification before use in hybridization capture.

[0004] The amplification of each oligonucleotide probe requires two common primer binding sites to be incorporated on each of the oligonucleotide probes to enable PCR amplification, namely a forward common primer binding site and a reverse common primer binding site. In order utilize to use oligonucleotide probes amplified in this manner as oligonucleotide primers, such as oligonucleotide primers in PCR or other polymerase extension reactions, a conversion method is needed to generate an extendable 3' end from these oligonucleotide probes.BRIEF SUMMARY OF THE DISCLOSURE

[0005] There remains a need for to cost-effectively synthesize sufficient quantities of a plurality of different oligonucleotide primers for use in PCR and other polymerase extension reactions. The present disclosure provides a simple and cost-effective method of converting oligonucleotide probes into oligonucleotide primers for use in PCR and Primer Extension Target Enrichment (PETE) reactions. In particular, the presently disclosed methods remove at least one of the two common primer binding sites (such as universal primer binding sites) at the 3' end of amplified oligonucleotide probes.

[0006] A first aspect of the present disclosure a method of preparing a single stranded primer from a single stranded template (e.g., an oligonucleotide probe) comprising:

[0007] (a) obtaining a single stranded template including a target specific primer sequence, wherein the target specific primer sequence is flanked by a forward primer binding site and a reverse primer binding site;

[0008] (b) preparing a double stranded template from the obtained single stranded template, where the double stranded template comprises a top strand including the target specific primer sequence flanked by the forward primer binding site and the reverse primer binding site, and a bottom strand including a reverse complement of the target specific primer sequence, wherein the reverse complement of the target specific primer sequence is flanked by a reverseAttorney Docket No. : Ventana-0298 WO (PCT Application) complement of the forward primer binding site and a reverse complement of the reverse primer binding site, wherein the reverse complement of the reverse primer binding site includes one or more uracil nucleotides;

[0009] (c) generating one or more abasic sites at one or more positions in the reverse complement of the reverse primer binding site, wherein each of the generated one or more generated abasic sites correspond to positions of the one or more uracil nucleotides in the reverse complement of the reverse primer binding site;

[0010] (d) introducing nicks at each of the one or more generated abasic sites to remove the reverse complement of the reverse primer binding site from the bottom strand of the double stranded template to form a double stranded molecule including an overhang;

[0011] (e) removing the reverse primer binding site from the double stranded molecule including the overhang to provide a double stranded primer precursor molecule comprising a top strand including the target specific primer sequence flanked by the forward primer binding site, and a bottom strand including the reverse complement of the target specific primer sequence flanked by the reverse complement of the forward primer binding site; and

[0012] (f) optionally generating a single stranded primer from the double stranded primer precursor molecule.

[0013] In some embodiments, the double stranded template is prepared by contacting a sample including the obtained single stranded template with a polymerase, a forward primer, and a reverse primer. In some embodiments, the polymerase is an uracil competent polymerase. In some embodiments, the reverse primer includes one or more uracil nucleotides. In some embodiments, the reverse primer includes a plurality of uracil nucleotides. In some embodiments, the forward primer includes an affinity label. In some embodiments, the affinity label is biotin. In some embodiments, the forward primer includes one or more phosphorothioate bonds.

[0014] In some embodiments, the generating of the one or more abasic sites comprises contacting a sample including the double stranded template with an uracil DNA glycosylase. In some embodiments, the uracil DNA glycosylase is selected from the group consisting of UG1, SUG1, and hSMUGl.

[0015] In some embodiments, the introducing of the nicks at each of the one or more generated abasic sites comprises contacting the sample including the double stranded templateAttorney Docket No. : Ventana-0298 WO (PCT Application) with an endonuclease. In some embodiments, the endonuclease is selected from the group consisting of Endonuclease III, Endonuclease IV, and Endonuclease VIII.

[0016] In some embodiments, the method further comprises digesting single stranded DNA fragments remaining in the sample including the double stranded template following the nicking at each of the one or more generated abasic sites. In some embodiments, the single stranded DNA fragments are digested by contacting the sample including the double stranded template with an exonuclease. In some embodiments, the exonuclease is Recjf.

[0017] In some embodiments, the double stranded primer precursor molecule is prepared by contacting a sample including the double stranded molecule having the overhang with a single stranded DNA exonuclease. In some embodiments, the single stranded DNA exonuclease is a 3' single stranded DNA exonuclease. In some embodiments, the single stranded DNA exonuclease is selected from the group consisting of is exonuclease I, thermolabile exonuclease I, Msz exonuclease, and exonuclease T.

[0018] In some embodiments, the double stranded primer precursor molecule is prepared by contacting a sample including the double stranded molecule including the overhang with a single stranded endonuclease. In some embodiments, the single stranded endonuclease is selected from the group consisting of mung bean nuclease and nuclease Pl.

[0019] In some embodiments, the double stranded primer precursor molecule is prepared by contacting a sample including the double stranded molecule having the overhang with a polymerase that has 3' to 5' exonuclease activity but lacks 5' to 3' exonuclease activity. In some embodiments, the polymerase having 3' to 5' exonuclease activity, but which lacks 5' to 3' exonuclease activity is selected from the group consisting of a Klenow fragment of DNA polymerase I, a Phi 29 DNA polymerase, a DNA polymerase I, a T4 DNA polymerase, and a T7 DNA polymerase.

[0020] In some embodiments, the single stranded primer is generated from the double stranded primer precursor molecule by contacting a sample including the double stranded primer precursor molecule with a double stranded DNA exonuclease. In some embodiments, the double stranded DNA exonuclease is a 5' double stranded DNA exonuclease. In some embodiments, the 5' double stranded DNA exonuclease is selected from the group consisting of a T7 exonuclease, a T5 exonuclease, a lambda exonuclease, and an Exonuclease VIII.Attorney Docket No. : Ventana-0298 WO (PCT Application)

[0021] A second aspect of the present disclosure is a method of preparing a single stranded primer from a single stranded template comprising:

[0022] (a) obtaining a single stranded template having target specific primer sequence, wherein the target specific primer sequence is flanked (a) at a 5' end with a forward primer binding site; and (b) at a 3' end with a reverse primer binding site;

[0023] (b) preparing a double stranded template from the obtained single stranded template, where the double stranded template comprises a first strand including the target specific primer sequence flanked at the 5' end with the forward primer binding site and at the 3' end with the reverse primer binding site, and a second strand including a reverse complement of the target specific primer sequence, wherein the reverse complement of the target specific primer sequence is flanked at a 3' end with a reverse complement of the forward primer binding site and at a 5' end with a reverse complement of the reverse primer binding site, wherein the reverse complement of the reverse primer binding site includes one or more uracil nucleotides;

[0024] (c) degrading the reverse complement of the reverse primer binding site in the second strand of the double stranded template to generate a double stranded template including a 3' overhang;

[0025] (d) removing the reverse primer binding site from the first strand of the double stranded template including the 3' overhang to provide a double stranded primer precursor molecule comprising a first strand including the target specific primer sequence flanked at the 5' end with the forward primer binding site, and a second strand including the reverse complement of the target specific primer sequence flanked by at the 3' end with the reverse complement of the forward primer binding site; and

[0026] (e) optionally generating a single stranded primer from the double stranded primer precursor molecule.

[0027] In some embodiments, the double stranded template is prepared by contacting a sample including the obtained single stranded template with a polymerase, a forward primer, and a reverse primer. In some embodiments, the polymerase is an uracil competent polymerase. In some embodiments, the reverse primer includes one or more uracil nucleotides. In some embodiments, the reverse primer includes a plurality of uracil nucleotides. In some embodiments, the forward primer includes an affinity label. In some embodiments, the affinity label is biotin. In some embodiments, the forward primer includes one or more phosphorothioate bonds. In someAttorney Docket No. : Ventana-0298 WO (PCT Application) embodiments, the reverse complement of the reverse primer binding site in the second strand of the double stranded template is degraded by contacting the sample including the double stranded template with an uracil DNA glycosylase and an endonuclease. In some embodiments, the uracil DNA glycosylase is selected from the group consisting of UG1, SUG1, and hSMUGl. In some embodiments, the endonuclease is selected from the group consisting of Endonuclease III, Endonuclease IV, and Endonuclease VIII.

[0028] In some embodiments, the double stranded template is prepared by contacting a sample including the obtained single stranded template with a polymerase, a forward primer, and a reverse primer; wherein the reverse primer includes one or more uracil nucleotides; and wherein the reverse primer further includes a recognition site for a nicking enzyme; and wherein the reverse compliment of the reverse primer binding site includes the recognition site for the nicking enzyme. In some embodiments, the reverse complement of the reverse primer binding site in the second strand of the double stranded template is degraded by contacting the sample including the double stranded template with a nicking enzyme specific for the recognition site.

[0029] In some embodiments, the double stranded primer precursor molecule is prepared by contacting a sample including the double stranded molecule including the 3' overhang with a single stranded DNA exonuclease. In some embodiments, the single stranded DNA exonuclease is a 3' single stranded DNA exonuclease. In some embodiments, the single stranded DNA exonuclease is selected from the group consisting of exonuclease I, thermolabile exonuclease I, Msz exonuclease, and exonuclease T.

[0030] In some embodiments, the double stranded primer precursor molecule is prepared by contacting a sample including the double stranded molecule including the 3' overhang with a single stranded endonuclease. In some embodiments, the single stranded endonuclease is selected from the group consisting of mung bean nuclease and nuclease Pl.

[0031] In some embodiments, the double stranded primer precursor molecule is prepared by contacting a sample including the double stranded molecule including the 3' overhang with a polymerase that has 3' to 5' exonuclease activity but lacks 5' to 3' exonuclease activity. In some embodiments, the polymerase having 3' to 5' exonuclease activity, but which lacks 5' to 3' exonuclease activity is selected from the group consisting of a Klenow fragment of DNA polymerase I, a Phi 29 DNA polymerase, a DNA polymerase I, a T4 DNA polymerase, and a T7 DNA polymerase.Attorney Docket No. : Ventana-0298 WO (PCT Application)

[0032] In some embodiments, the method further comprises digesting any remaining single stranded DNA fragments. In some embodiments, the remaining single stranded DNA fragments are digested by contacting the sample including the double stranded template with an exonuclease. In some embodiments, the exonuclease is Recjf.

[0033] In some embodiments, the single stranded primer is generated from the double stranded primer precursor molecule by contacting a sample including the double stranded primer precursor molecule with a double stranded DNA exonuclease. In some embodiments, the double stranded DNA exonuclease is a 5' double stranded DNA exonuclease. In some embodiments, the 5' double stranded DNA exonuclease is selected from the group consisting of a T7 exonuclease, a T5 exonuclease, a lambda exonuclease, and an Exonuclease VIII.

[0034] A third aspect of the present disclosure is a single stranded primer, the single stranded primer including a 5' forward primer binding site contiguous with a target specific primer sequence, wherein the single stranded primer is prepared according to a process comprising:

[0035] (a) obtaining a single stranded template having a target specific primer sequence, wherein the target specific primer sequence is flanked (a) at a 5' end with a forward primer binding site; and (b) at a 3' end with a reverse primer binding site;

[0036] (b) preparing a double stranded template from the obtained single stranded template, where the double stranded template comprises a first strand including the target specific primer sequence flanked at the 5' end with the forward primer binding site and at the 3' end with the reverse primer binding site, and a second strand including a reverse complement of the target specific primer sequence, wherein the reverse complement of the target specific primer sequence is flanked at a 3' end with a reverse complement of the forward primer binding site and at a 5' end with a reverse complement of the reverse primer binding site, wherein the reverse complement of the reverse primer binding site includes one or more uracil nucleotides;

[0037] (c) degrading the reverse complement of the reverse primer binding site in the second strand of the double stranded template to generate a double stranded template including a 3' overhang;

[0038] (d) removing the reverse primer binding site from the first strand of the double stranded template including the 3' overhang to provide a double stranded primer precursor molecule comprising a first strand including the target specific primer sequence flanked at the 5' end with the forward primer binding site, and a second strand including the reverse complementAttorney Docket No. : Ventana-0298 WO (PCT Application) of the target specific primer sequence flanked by at the 3' end with the reverse complement of the forward primer binding site; and

[0039] (e) generating a single stranded primer from the double stranded primer precursor molecule.

[0040] In some embodiments, the single stranded primer includes an affinity label. In some embodiments, the affinity label is biotin. In some embodiments, the affinity label is included within the forward primer binding site.

[0041] A fourth aspect of the present disclosure is a pair of single stranded primers, wherein each single stranded primer of the pair of single stranded primers comprises a 5' forward primer binding site contiguous with a target specific primer sequence, and wherein each single stranded primer of the pair of single stranded primers is prepared according to a process comprising:

[0042] (a) obtaining a single stranded template having target specific primer sequence, wherein the target specific primer sequence is flanked (a) at a 5' end with a forward primer binding site; and (b) at a 3' end with a reverse primer binding site;

[0043] (b) preparing a double stranded template from the obtained single stranded template, where the double stranded template comprises a first strand including the target specific primer sequence flanked at the 5' end with the forward primer binding site and at the 3' end with the reverse primer binding site, and a second strand including a reverse complement of the target specific primer sequence, wherein the reverse complement of the target specific primer sequence is flanked at a 3' end with a reverse complement of the forward primer binding site and at a 5' end with a reverse complement of the reverse primer binding site, wherein the reverse complement of the reverse primer binding site includes one or more uracil nucleotides;

[0044] (c) degrading the reverse complement of the reverse primer binding site in the second strand of the double stranded template to generate a double stranded template including a 3' overhang;

[0045] (d) removing the reverse primer binding site from the first strand of the double stranded template including the 3' overhang to provide a double stranded primer precursor molecule comprising a first strand including the target specific primer sequence flanked at the 5' end with the forward primer binding site, and a second strand including the reverse complementAttorney Docket No. : Ventana-0298 WO (PCT Application) of the target specific primer sequence flanked by at the 3' end with the reverse complement of the forward primer binding site; and

[0046] (e) generating a single stranded primer from the double stranded primer precursor molecule; and

[0047] wherein a first single stranded primer of the pair of single stranded primers includes an affinity label; and wherein a second single stranded primer of the pair of single stranded primers does not include an affinity label. In some embodiments, the affinity label is biotin. In some embodiments, the affinity label is included within the forward primer binding site.

[0048] A fifth aspect of the present disclosure is a kit comprising (i) an uracil competent polymerase; (ii) a forward primer; and (iii) a reverse primer, the reverse primer including one or more uracil bases. In some embodiments, the forward primer includes one or more phosphorothioate bonds. In some embodiments, the forward primer includes an affinity label. In some embodiments, the affinity label is biotin.

[0049] In some embodiments, the kit further comprises an uracil DNA glycosylase and an endonuclease. In some embodiments, the uracil DNA glycosylase is selected from the group consisting of UG1, SUG1, and hSMUGl. In some embodiments, the endonuclease is selected from the group consisting of Endonuclease III, Endonuclease IV, and Endonuclease VIII.

[0050] In some embodiments, the kit further comprises a 3' single stranded DNA exonuclease, a single stranded endonuclease, or a polymerase that has 3' to 5' exonuclease activity, and which lacks 5' to 3' exonuclease activity. In some embodiments, the 3' single stranded DNA exonuclease is selected from the group consisting of exonuclease I, thermolabile exonuclease I, Msz exonuclease, and exonuclease T. In some embodiments, the single stranded endonuclease is mung bean nuclease or nuclease Pl. In some embodiments, the polymerase that has 3' to 5' exonuclease activity and which lacks 5' to 3' exonuclease activity is a T4 DNA polymerase.

[0051] In some embodiments, the kit further comprises a 5' double stranded DNA exonuclease. In some embodiments, the 5' double stranded DNA exonuclease is selected from the group consisting of a T7 exonuclease, a T5 exonuclease, a lambda exonuclease, and an Exonuclease VIII.

[0052] In some embodiments, the kit further comprises Recjf.

[0053] A sixth aspect of the present disclosure is a kit comprising (i) an uracil competent polymerase; (ii) a forward primer; (iii) a reverse primer, the reverse primer including one or moreAttorney Docket No. : Ventana-0298 WO (PCT Application) uracil bases and a recognition site for a nick enzyme; and (iv) a nicking enzyme specific to the recognition site.

[0054] In some embodiments, the kit further comprises a 3' single stranded DNA exonuclease, a single stranded endonuclease, or a polymerase that has 3' to 5' exonuclease activity, and which lacks 5' to 3' exonuclease activity. In some embodiments, the 3' single stranded DNA exonuclease is selected from the group consisting of exonuclease I, thermolabile exonuclease I, Msz exonuclease, and exonuclease T. In some embodiments, the single stranded endonuclease is mung bean nuclease or nuclease Pl. In some embodiments, the polymerase that has 3' to 5' exonuclease activity and which lacks 5' to 3' exonuclease activity is a T4 DNA polymerase.

[0055] In some embodiments, the kit further comprises a 5' double stranded DNA exonuclease. In some embodiments, the 5' double stranded DNA exonuclease is selected from the group consisting of a T7 exonuclease, a T5 exonuclease, a lambda exonuclease, and an Exonuclease VIII.

[0056] In some embodiments, the kit further comprises RecjfBRIEF DESCRIPTION OF THE FIGURES

[0057] For a general understanding of the features of the disclosure, reference is made to the drawings. In the drawings, like reference numerals have been used throughout to identify identical elements.

[0058] FIG. 1 depicts a single stranded primer having a forward primer binding site flanking a target specific primer sequence.

[0059] FIG. 2 depicts an oligonucleotide probe including a target specific primer sequence flanked by both a forward primer binding site and a reverse primer binding site.

[0060] FIG. 3 provides a flowchart for preparing a single stranded primer from an oligonucleotide probe.

[0061] FIG. 4A depicts a double stranded template wherein the double stranded template comprises a top strand including the target specific primer sequence flanked by the forward primer binding site and the reverse primer binding site, and a bottom strand including a reverse complement of the target specific primer sequence, wherein the reverse complement of the target specific primer sequence is flanked by a reverse complement of the forward primer binding siteAttorney Docket No. : Ventana-0298 WO (PCT Application) and a reverse complement of the reverse primer binding site, wherein the reverse complement of the reverse primer binding site includes one or more uracil nucleotides.

[0062] FIG. 4B depicts the double stranded template of FIG. 4A, where the double stranded template further includes an affinity label (such as biotin) coupled to the forward primer binding site of the top strand of the double stranded template.

[0063] FIG. 5A illustrates a method of generating a double stranded template from a single stranded template and forward and reverse primers, such as reverse primers including one or more uracil nucleotides.

[0064] FIG. 5B illustrates forward and reverse primers annealed to a single stranded template, where the forward primer includes an affinity label (such as biotin).

[0065] FIG. 6 illustrates a double stranded template including an overhang, such as a 3' overhang. In some embodiments, the 3' overhang comprises the reverse primer binding site of the top strand of the double stranded template.

[0066] FIG. 7A depicts a double stranded primer precursor molecule including a top strand including the target specific primer sequence flanked by the forward primer binding site, and a bottom strand including a reverse complement of the target specific primer sequence, wherein the reverse complement of the target specific primer sequence is flanked by a reverse complement of the forward primer binding site.

[0067] FIG. 7B depicts the double stranded primer precursor of FIG. 7A, where the forward primer binding site of the top strand is coupled to an affinity label, such as biotin.

[0068] FIG. 8 provides an electropherogram showing the size of the PCR product (about 60 to about 70bp) prior to the conversion to double stranded template and after the conversion (USERII) as depicted in FIGS. 7A and 7B.

[0069] FIG. 9 provide target enrichment metrics showing performance of the probe to primer converted panel (on the right) and the control standard primer panel (on the left). On-target rate is a measure of % of reads that overlap the target region of interest. Median fragment length is the length of the library fragments that were sequenced, dedup depth is coverage depth of unique sequence molecules based on UMI (unique molecular barcode) information in the library which indicates capture efficiency (higher is better), %bases in 2 fold range is how uniform the coverage is across the targeted regions (higher is better), and %panel exon bases > 300x which is % of target regions covered at least 300x dedup depth (higher is better).Attorney Docket No. : Ventana-0298 WO(PCT Application)DETAILED DESCRIPTION

[0070] 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.

[0071] As used herein, the singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "includes" is defined inclusively, such that "includes A or B" means including A, B, or A and B.

[0072] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0073] The terms "comprising," "including," "having," and the like are used interchangeably and have the same meaning. Similarly, "comprises," "includes," "has," and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of "comprising" and is therefore interpreted to be an open term meaning "at least the following," and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, "a device having components a, b, and c" means that the device includes at least components a, b, and c. Similarly, the phrase: "a method involving steps a, b, and c" means that the method includes at least steps a, b, and c. Moreover, while the steps and processes may be outlined herein in a particular order, the skilled artisan will recognize that the ordering steps and processes may vary.

[0074] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one elementAttorney Docket No. : Ventana-0298 WO (PCT Application) selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "atleast one of A orB," or, equivalently "at least one of A and / or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0075] As used herein, the term "about" refers to a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In some embodiments, the term "about" means within a standard deviation using measurements generally acceptable in the art. In some embodiments, about means a range extending to + / - 10% of the specified value.

[0076] As used herein "amplification" refers to a process in which a copy number increases. Amplification may be a process in which replication occurs repeatedly over time to form multiple copies of a template. Amplification can produce an exponential or linear increase in the number of copies as amplification proceeds. Exemplary amplification strategies include polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), rolling circle replication (RCA), cascade-RCA, nucleic acid-based amplification (NASBA), and the like. Also, amplification can utilize a linear or circular template. Amplification can be performed under any suitable temperature conditions, such as with thermal cycling or isothermally. Furthermore, amplification can be performed in an amplification mixture (or reagent mixture), which is any composition capable of amplifying a nucleic acid target, if any, in the mixture. PCR amplification relies on repeated cycles of heating and cooling (i.e., thermal cycling) to achieve successive rounds of replication. PCR can be performed by thermal cycling between two or more temperature setpoints, such as a higher denaturation temperature and a lower annealing / extension temperature, or among three or more temperature setpoints, such as a higher denaturation temperature, a lowerAttorney Docket No. : Ventana-0298 WO (PCT Application) annealing temperature, and an intermediate extension temperature, among others. PCR can be performed with a thermostable polymerase, such as Taq DNA polymerase. PCR produces an exponential increase in the amount of a product amplicon over successive cycles. PCR is described, for example, in U.S. Pat. No. 4,683,202; U.S. Pat. No. 4,683,195; U.S. Pat. No. 4,000,159; U.S. Pat. No. 4,965,188; U.S. Pat. No. 5,176,995), the disclosures of each are hereby incorporated by reference herein in their entirety.

[0077] As used herein, the term "biological sample," "tissue sample," "specimen" or the like refers to any sample including a biomolecule (such as a protein, a peptide, a nucleic acid, a lipid, a carbohydrate, or a combination thereof) that is obtained from any organism including viruses. Other examples of organisms include mammals (such as humans; veterinary animals like cats, dogs, horses, cattle, and swine; and laboratory animals like mice, rats, and primates), insects, annelids, arachnids, marsupials, reptiles, amphibians, bacteria, and fungi. Biological samples include tissue samples (such as tissue sections and needle biopsies of tissue), cell samples (such as cytological smears such as Pap smears or blood smears or samples of cells obtained by microdissection), or cell fractions, fragments, or organelles (such as obtained by lysing cells and separating their components by centrifugation or otherwise). Other examples of biological samples include blood, serum, urine, semen, fecal matter, cerebrospinal fluid, interstitial fluid, mucous, tears, sweat, pus, biopsied tissue (for example, obtained by a surgical biopsy or a needle biopsy), nipple aspirates, cerumen, milk, vaginal fluid, saliva, swabs (such as buccal swabs), or any material containing biomolecules that is derived from a first biological sample. In certain embodiments, the term "biological sample" as used herein refers to a sample (such as a homogenized or liquefied sample) prepared from a tumor or a portion thereof obtained from a subject.

[0078] As used herein, the term "end" or "ends" refer to the regions of sequence at (or proximal to) either end of a nucleic acid sequence. As used herein, the term "3' region" refers to a region of a nucleotide strand that includes the 3' end of the strand. As used herein, the term "3' end" designates the end of a nucleotide strand that has the hydroxyl group of the third carbon in the sugar-ring of the deoxyribose at its terminus. As used herein, the term "51region" refers to a region of a nucleotide strand that includes the 5' end of the strand. As used herein, the term "5' end" designates the end of a nucleotide strand that has the fifth carbon in the sugar-ring of the deoxyribose at its terminus.Attorney Docket No. : Ventana-0298 WO (PCT Application)

[0079] As used herein, the term "nucleotide" refers to a nucleoside-5'-oligophosphate compound, or structural analog of a nucleoside-5'-oligophosphate, which can act as a substrate or inhibitor of a nucleic acid polymerase. Exemplary nucleotides include, but are not limited to, nucleoside-5'-triphosphates (e.g., dATP, dCTP, dGTP, dTTP, and dUTP); nucleosides (e.g., dA, dC, dG, dT, and dU) with 5'-oligophosphate chains of 4 or more phosphates in length (e.g., 5'- tetraphosphosphate, 5'-pentaphosphosphate, 5'-hexaphosphosphate, 5'-heptaphosphosphate, 5'- octaphosphosphate); and structural analogs of nucleoside-5'-triphosphates that can have a modified base moiety (e.g., a substituted purine or pyrimidine base), a modified sugar moiety (e.g., an O- alkylated sugar), and / or a modified oligophosphate moiety (e.g., an oligophosphate comprising a thio-phosphate, a methylene, and / or other bridges between phosphates).

[0080] As used herein, the "polymerase" as used herein, refers to an enzyme that catalyzes the process of replication of nucleic acids. More specifically, DNA polymerase catalyzes the polymerization of deoxyribonucleotides alongside a DNA strand, which the DNA polymerase "reads" and uses as a template. The newly polymerized molecule is complementary to the template strand and identical to the template's partner strand.

[0081] As used herein, the term "primer" refers to an oligonucleotide, either natural or synthetic, that is capable, upon forming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3' end along the template so that an extended duplex is formed. Extension of a primer is usually carried out with a nucleic acid polymerase, such as a DNA or RNA polymerase. The sequence of nucleotides added in the extension process is determined by the sequence of the template polynucleotide. Usually, primers are extended by a DNA polymerase. Primers usually have a length in the range of from 14 to 40 nucleotides, or in the range of from 18 to 36 nucleotides. Primers are employed in a variety of nucleic amplification reactions, for example, linear amplification reactions using a single primer, or polymerase chain reactions, employing two or more primers. Guidance for selecting the lengths and sequences of primers for applications is well known to those of ordinary skill in the art, as evidenced by the following reference that is incorporated by reference herein in its entirety: Dieffenbach, editor, PCR Primer: A Laboratory Manual, 2ndEdition (Cold Spring Harbor Press, New York, 2003).Attorney Docket No. : Ventana-0298 WO (PCT Application)

[0082] As used herein, the phrase "primer binding site" refers to a region or site including a sequence that can be used for amplifying and / or sequencing a nucleic acid molecule (or a fragment thereof).

[0083] As used herein, the term "sequence," when used in reference to a nucleic acid molecule, refers to the order of nucleotides (or bases) in the nucleic acid molecules. In cases, where different species of nucleotides are present in the nucleic acid molecule, the sequence includes an identification of the species of nucleotide (or base) at respective positions in the nucleic acid molecule. A sequence is a property of all or part of a nucleic acid molecule. The term can be used similarly to describe the order and positional identity of monomeric units in other polymers such as amino acid monomeric units of protein polymers.

[0084] As used herein, the term "sequence complementarity" refers to a property shared between two nucleic acid sequences, such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary.

[0085] As used herein, the term "universal" refers to a nucleic acid molecule (e.g., primer or other oligonucleotide) that can be added to any nucleic acid molecule and perform its function irrespectively of the sequence of the nucleic acid molecule. The universal molecule may perform its function by hybridizing to the complement, e.g., a universal primer to a universal primer binding site in a universal primer.

[0086] As used herein, the phrase "universal primer binding site" refers to a region or site including universal sequence that can be used for amplifying and / or sequencing a nucleic acid molecule (or a fragment thereof).

[0087] As used herein, the phrase "universal primer binding sequence" refers to a DNA sequence that may be appended to one or more nucleic acid molecules, for example by ligation, PCR, or ligation mediated PCR. Once added to the one or more nucleic acid molecules, primers specific to the universal primer binding sequence can be used to amplify the one or more nucleic acid molecules using a single pair of amplification primers. Universal priming sequences are typically not related to target sequences within one or more nucleic acid molecules.

[0088] OVERVIEWAttorney Docket No. : Ventana-0298 WO (PCT Application)

[0089] The present disclosure is directed to a method of preparing oligonucleotide primers (also referred to herein as "single stranded primers") (see, FIG. 1) from oligonucleotide probes, such as oligonucleotide probes which have been amplified, and which include a target specific primer sequence, a forward primer binding site, and a reverse primer binding site (see, FIG. 2). It is believed that the presently disclosed method permits the synthesis of thousands to hundreds of thousands and even a million primers in a cost-effective manner, with good yield, in high quantities (e.g., quantities sufficient to perform 100s to 1000s of PCR or PETE reactions), and at high purity levels.

[0090] METHODS OF PREPARING SINGLE STRANDED PRIMERS FROM OLIGONUCLEOTIDE PROBES

[0091] The present disclosure provides methods of preparing single stranded primers from oligonucleotide probes. With reference to FIG. 3, the method of the present disclosure comprises: (a) obtaining an oligonucleotide probe (101); (b) preparing a double stranded template from the obtained oligonucleotide probe (102); (c) creating an overhang in the first / top strand of the generated double stranded template (103); (d) removing the overhang in the first / top strand of the generated double stranded template to provide a double stranded primer precursor molecule (104); and (e) converting the double stranded primer precursor molecule to a single stranded primer (105).

[0092] Obtaining / Synthesis of a Single Stranded Template

[0093] In some embodiments, a single stranded template is obtained, wherein the single stranded template includes (a) a target specific primer sequence, (b) a forward primer binding site, and (c) a reverse primer binding site (step 101). In some embodiments, the target specific primer sequence is flanked on a first end with the forward primer binding site; and flanked on a second end with the reverse primer binding site. In some embodiments, the target specific primer sequence is flanked on a 5' end with the forward primer binding site; and flanked on a 3' end with the reverse primer binding site. The configuration of the single stranded template is illustrated in FIG. 2. In some embodiments, the forward and / or or the reverse primer binding site is a universal primer binding site.

[0094] In some embodiments, the single stranded template is derived from a target specific primer sequence which has been amplified, such as amplified with forward and reverse primers (see FIGS. 5 A and 5B); whereby the amplification provides for the single stranded template (see FIG. 2) which is flanked on a first end with a forward primer binding site (derived from the forwardAttorney Docket No. : Ventana-0298 WO (PCT Application) primer used during amplification); and flanked on a second end with a reverse primer binding site (derived from the reverse primer used during amplification

[0095] Preparation of a Double Stranded Template

[0096] After the single stranded template is obtained, a double stranded template is generated from the single stranded template (step 102). In some embodiments, the double stranded template is the reverse complement of the single stranded template, with the proviso that one end of the double stranded template includes uracil nucleotides instead of thymine nucleotides. In some embodiments, the end of the double stranded template corresponding to the end of the single stranded template that includes the reverse primer binding site includes uracil nucleotides instead of thymine nucleotides.

[0097] In some embodiments, the generated double stranded template comprises a first / top strand including the target specific primer sequence flanked by the forward primer binding site and the reverse primer binding site, and a second / bottom strand including a reverse complement of the target specific primer sequence, wherein the reverse complement of the target specific primer sequence is flanked by a reverse complement of the forward primer binding site and a reverse complement of the reverse primer binding site, wherein the reverse complement of the reverse primer binding site includes one or more uracil nucleotides (see, FIG. 4A). In other embodiments, the double stranded template comprises a first / top strand including the target specific primer sequence flanked at the 5' end with the forward primer binding site and at the 3' end with the reverse primer binding site, and a second / bottom strand including a reverse complement of the target specific primer sequence, wherein the reverse complement of the target specific primer sequence is flanked at a 3' end with a reverse complement of the forward primer binding site and at a 5' end with a reverse complement of the reverse primer binding site, wherein the reverse complement of the reverse primer binding site includes one or more uracil nucleotides. In some embodiments, the first / top strand includes a s specific binding entity or an affinity label, such as a specific binding entity or an affinity label at a 5' end of the first / top strand (see, FIG. 4B).

[0098] In some embodiments, the double stranded template is generated by contacting the oligonucleotide probe with a polymerase, a forward primer, a reverse primer, and / or other amplification reagents (see, FIG. 5A). In some embodiments, the polymerase is one that is engineered to tolerate uracil residues, i.e., an uracil competent polymerase. In some embodiments,Attorney Docket No. : Ventana-0298 WO (PCT Application) the uracil competent polymerase is derived from an Archaeal B-family (family B) DNA polymerase. In some embodiments, the double stranded template is prepared using KAPA HiFi HotStart Uracil+ReadyMix Kit (available from Roche Molecular Systems, Pleasanton, CA). . In some embodiments, the uracil competent polymerase is Q5U®Hot Start High-Fidelity DNA Polymerase, Phusion U Hot Start DNA Polymerase, Equinox Uracil Tolerant Library Amplification Kit.

[0099] In some embodiments, the double stranded template is generated by extending the oligonucleotide probe, in the presence of a uracil competent polymerase, with a forward primer and a reverse primer, wherein the forward primer anneals to the forward primer binding site of the single stranded template, wherein the reverse primer anneals to the reverse primer binding site of the single stranded template, and wherein the reverse primer includes one or more uracil nucleotides. In some embodiments, the reverse primer includes two or more uracil nucleotides. In some embodiments, the reverse primer includes three or more uracil nucleotides. In some embodiments, the reverse primer includes four or more uracil nucleotides. In some embodiments, the reverse primer includes a plurality of uracil nucleotides (see, FIG. 5A).

[0100] In some embodiments, the forward primer includes a specific binding entity or an affinity label (see, FIG. 5B). In some embodiments, the specific binding entity or affinity label is selected from biotin, an antibody, an antibody fragment, a receptor, a lectin, etc. In other embodiments, the specific binding entity or affinity label is biotin. In some embodiments, the specific binding entity or affinity label is located at a 5' end of the forward primer.

[0101] In some embodiments, the forward primer includes one or more phosphorothioate bonds. It is believed that the phosphorothioate bond substitutes a sulfur atom for a non-bridging oxygen in a phosphate backbone of an oligonucleotide. In some embodiments, the forward primer includes one or more phosphorothioate bond. In some embodiments, the forward primer includes between 1 and about 10 phosphorothioate bonds. In other embodiments, the forward primer includes one or more phosphorothioate bonds includes between 1 and about 8 phosphorothioate bonds. In yet other embodiments, the forward primer includes one or more phosphorothioate bonds includes between 1 and about 4 phosphorothioate bonds. In further embodiments, the one or more phosphorothioate bonds are located at one end of the forward primer.

[0102] In some embodiments, the reverse primer includes a recognition site for a nicking enzyme, such as a nicking endonuclease. In some embodiments, a recognition site is located at anAttorney Docket No. : Ventana-0298 WO (PCT Application) end of the reverse primer (see, FIG. 5B). As used herein, the term "nicking endonuclease" refers to an endonuclease having nicking activity that can recognize a specific nucleotide sequence and cleave only one strand of a double-stranded nucleic acid having the abovementioned nucleotide sequence. In some embodiments, the nicking endonuclease can cleave the phosphodiester bond of one strand of a double-stranded DNA molecule.

[0103] In some embodiments, the number of bases in the nicking endonuclease recognition site is at least 3, such as at least 4, such as at least 5, such as at least 6, or such as at least 7. Suitable endonucleases include, but are not limited to, Nb.BbvCI (the number ofbases in the recognition site is 7: 5'-GC / TGAGG-3'), Nb.BsmI (the number of bases in the recognition site is 6: 5'-NG / CATTC-3'), Nb.BtsI (the number of bases in the recognition site is 6: 5'-NN / CACTGC-3'), Nb.BsrDI (the number of bases in the recognition site is 6: 5'- NN / CATTGC-31), Nt.BspQI (the number of bases in the recognition site is 7: 5'-GCTCTTCN / -3'), Nt.BbvCI (the number of bases in the recognition site is 7: 5'-CC / TCAGC-3'), Nt.AIwI (the number of bases in the recognition site is 5: 5'-GGATCNNNN / N-3'), Nt.BsmAI (the number of bases in the recognition site is 5: 5'-GTCTCN / N-3'), Nt.BstNBI (the number of bases in the recognition site is 5: 5'-GAGTCNNNN / N-3'), Nt.CviPII (the number of bases in the recognition site is 3: 5'- / CCD-3'), Nb.Mval269I (the number of bases in the recognition site is 6: 5'-G / CATTC-3'), Nt.BpulOI (the number of bases in the recognition site is 7: 5'-CC / TNAGC- 3') and Nb.BssSI (the number of bases in the recognition site is 6: 5'-C / TCGTG-3') (the number of bases in the recognition site and its sequence is shown in each parenthesis above). Here, depicts a cleavage site; "N" is an A, T, G or C nucleotide; and "D" is an A, T or G nucleotide.

[0104] Additional nicking endonuclease recognition sites and methods of effectuating nicking with an endonuclease are disclosed by Joneja et. al., "Linear nicking endonuclease- mediated strand-displacement DNA amplification." Anal Biochem. 2011 Jul l;414(l):58-69, the disclosure of which is hereby incorporated by reference herein in its entirety. Yet additional nicking enzyme recognition sequences are identified in U.S. Patent No. 10,570,441, the disclosure of which is hereby incorporated by reference herein in its entirety.

[0105] Degradation of the Reverse Complement of the Reverse Primer Binding Site in the Second / Bottom Strand of the Double Stranded TemplateAttorney Docket No. : Ventana-0298 WO (PCT Application)

[0106] Following the preparation of the double stranded template (stepl02), the reverse complement of the reverse primer binding site in the second / bottom strand of the double stranded template is degraded (step 103).

[0107] In some embodiments, the reverse complement of the reverse primer binding site in the second / bottom strand of the double stranded template is degraded using a combination of enzymes that function to (i) introduce one or more apurinic / apyrimidinic (abasic) sites into the reverse complement of the reverse primer binding site in the second / bottom strand of the double stranded template; and (ii) introduce a cut or “nick” at each of the one or more introduced abasic sites in the reverse complement of the reverse primer binding site in the second / bottom strand of the double stranded template. In other embodiments, the reverse complement of the reverse primer binding site in the second / bottom strand of the double stranded template is degraded using a nicking enzyme or nicking endonuclease, provided that the reverse primer used to generate the double stranded template (step 102) includes a recognition site as described herein.

[0108] Generation and Cutting of Abasic Sites

[0109] In some embodiments, the abasic sites are generated using one or more DNA glycosylases. As used herein, the term "glycosylase" refers to an enzyme that hydrolyzes a glycosyl compound. In some examples, glycosylases are "monofunctional," meaning that they lack additional activity beyond glycosylase activity. In other embodiments, the glycosylates are "bifunctional," meaning that the DNA glycosylases also may cut the phosphodiester bond of DNA.

[0110] In some embodiments, the one or more DNA glycosylates are one or more uracil DNA glycosylase (UDGs). Specifically, the UDG catalyzes the excision of an uracil base, forming an abasic (apyrimidinic) site while leaving the phosphodiester backbone intact. In some embodiments, UG1, SUG1, or hSMUGl may be used to generate abasic sites at one or more uracil bases. In some embodiments, the UDG includes the recombinant UDG protein described in U.S. Patent Numbers 5,888,795, and 7,723,093, the disclosures of which are hereby incorporated by reference herein in their entireties.[0U1] In some embodiments, the abasic sites at the uracil bases may then be processed by an endonuclease to generate cuts in the phosphodiester backbone. In some embodiments, the endonuclease is one of Endonuclease III, Endonuclease IV, or Endonuclease VIII.

[0112] In some embodiments, the UDG and the endonuclease are introduced to the sample including the double stranded template separately. In other embodiments, the UDG and theAttorney Docket No. : Ventana-0298 WO (PCT Application) endonuclease are introduced to the sample including the double stranded template simultaneously. In some embodiments, a USER is introduced to the sample including the double stranded template. It is believed that USER (Uracil-Specific Excision Reagent) Enzyme generates a single nucleotide gap at the location of an uracil. It is also believed that USER Enzyme is a mixture of Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease VIII. It is also believed that UDG catalyzes the excision of an uracil base, forming an abasic (apyrimidinic) site while leaving the phosphodiester backbone intact. It is believed that the lyase activity of Endonuclease VIII breaks the phosphodiester backbone at the 3' and 5' sides of the abasic site so that base-free deoxyribose is released.

[0113] In some embodiments, degradation is performed using a USER Enzyme, a Thermolabile USER II Enzyme, or a Thermostable USER III Enzyme, each of which is available from New England Biolabs. In addition to different optimal reaction temperatures (37°C for USER and Thermolabile USER II Enzyme and 65°C for Thermostable USER III Enzyme) and ability to be heat inactivated (Thermolabile USER II Enzyme only), the different USER Enzymes generate different 3' and 5' termini after cleavage. USER Enzyme (NEB #M5505) contains Endonuclease VIII and leaves a 3' and 5' phosphate after cleavage. Thermolabile USER II Enzyme (NEB M5508) contains Endonuclease III and leaves a 3 '-phospho-a, P-unsaturated aldehyde and 5' phosphate after cleavage. Thermostable USER III Enzyme (NEB #M5509) contains Endonuclease IV and leaves a 3'-hydroxyl and 5'-deoxyribose phosphate, (see https: / / www.neb.com / en- us / products / m5505-user-enzyme). In some embodiments, the reasons with USER were incubated at about 37°C for about 15 minutes followed by heat inactivation at about 65°C for about 10 minutes.

[0114] Nicking Enzymes

[0115] In those embodiments where the double stranded template includes a nicking enzyme recognition site, such as a recognition site at an end of the reverse complement of the reverse primer binding site and adjacent to the target specific primer sequence, a nicking enzyme may be introduced to cut the reverse complement of the reverse primer binding site from the second / bottom strand of the double stranded template. In some embodiments, the nicking enzyme specific for the recognition site included within second / bottom strand of the double stranded template. Exemplary nicking enzymes include, but are not limited to, N.Bst9I, N.BstSEI, Nb.BbvCI(NEB), Nb. Bpul0I(F ermantas), Nb.BsmI(NEB), Nb.BsrDI(NEB), Nb.BtsI(NEB),Attorney Docket No. : Ventana-0298 WO(PCT Application)Nt.AlwI(NEB), Nt.BbvCI(NEB), Nt.BpulOI(Fermentas), Nt.BsmAI, Nt.BspD6I, Nt.BspQI(NEB), Nt.BstNBI(NEB), and Nt.CviPII(NEB). Examples of nicking enzyme recognition sequences acted upon by nicking endonucleases are identified in U.S. Patent No. 10,570,441, the disclosure of which is hereby incorporated by reference herein in its entirety.

[0116] Optional Further Digestion

[0117] In some embodiments, an exonuclease capable of digesting single-stranded DNA is introduced to digest any single stranded DNA fragments, i.e., fragments generated from the degradation of the reverse complement of the reverse primer binding site. Any exonuclease may be utilized provided it is single strand specific. In some embodiments, the exonuclease is a Recjf exonuclease. Red enzyme is an Mg2+-dependent single-stranded DNA (ssDNA) exonuclease that degrades its substrates in the 5' to 3' direction. Red has a strong specificity for ssDNA and no 3'- 5' exonuclease activity. Double-stranded DNA (dsDNA) is neither a substrate nor a competitive inhibitor of ssDNA exonuclease activity of Red. RecJf is a recombinant fusion protein of Red and maltose binding protein (MBP). It has the same enzymatic properties as wild-type Red. Fusion to MBP enhances RecJf solubility. RecJf is available from NEB under catalogue number M0264S.

[0118] Removal of the Reverse Primer Binding Site from the First / Top Strand of the Double Stranded Template to Form a Double Stranded Primer Precursor Molecule

[0119] After the reverse complement of the reverse primer binding site in the second / bottom strand of the double stranded template is degraded (steps-with), the reverse primer binding site of the first / top strand of the double stranded template is removed to form a double stranded primer precursor molecule (step 104).

[0120] In some embodiments, the reverse primer binding site of the first / top strand of the double stranded template is removed using a 3' single stranded DNA exonuclease or a single stranded endonuclease, which results in a double stranded primer precursor molecule comprising a first / top strand including the target specific primer sequence flanked by the forward primer binding site, and a second / bottom strand including the reverse complement of the target specific primer sequence flanked by the reverse complement of the forward primer binding site (see, FIG. 6A and 6B). In some embodiments, the 3' single stranded DNA exonuclease is exonuclease I, thermolabile exonuclease I, Msz exonuclease, or exonuclease T. In some embodiments, the endonuclease is mung bean nuclease or nuclease Pl.Attorney Docket No. : Ventana-0298 WO(PCT Application)

[0121] In other embodiments, the reverse primer binding site of the first / top strand of the double stranded template is removed using a polymerase that has 3' to 5' exonuclease activity, and which lacks 5' to 3' exonuclease activity. Suitable DNA polymerases include, but are not limited to, the Klenow fragment of DNA polymerase I, Phi 29 DNA polymerase, DNA polymerase I, T4 DNA polymerase, Amplitaq FS (Applied Biosystems), reverse transcriptase, and T7 DNA polymerase. In other embodiments, the polymerase is Pfu, Vent, Deep Vent and UITma (see Cline J, Braman JC, Hogrefe HH. PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Res. 1996 Sep 15;24(18):3546-51. doi: 10.1093 / nar / 24.18.3546. PMID: 8836181; PMCID: PMC146123, the disclosure of which is hereby incorporated by reference herein in its entirety).

[0122] In other embodiments, the polymerase that has 3' to 5' exonuclease activity, and which lacks 5' to 3' exonuclease activity is KAPA HiFi HotStart DNA Polymerase (available from Roche Molecular Systems, Pleasanton, CA).

[0123] Preparation of a Single Stranded Primer from the Double Stranded Primer Precursor Molecule

[0124] After the double stranded primer precursor molecule is formed (step 104), the double stranded primer precursor molecule is converted into a single stranded primer (step 105). In some embodiments, the conversion of the double stranded primer precursor molecule into the single stranded primer is performed using an 5' double stranded DNA exonuclease, i.e., one having 5' to 3' activity and specificity to double stranded nucleic acid molecules. In some embodiments, the 5' double stranded DNA exonuclease is a T7 exonuclease. In other embodiments, the 5' double stranded DNA exonuclease is a T5 exonuclease. In other embodiments, the 5' double stranded DNA exonuclease is a lambda exonuclease. In some embodiments, the 5' double stranded DNA exonuclease is an Exonuclease VIII (truncated).

[0125] KITS

[0126] The present disclosure also provides for kits including one or more components for preparing single stranded primers, such as single stranded primers which may be used in PCR or other primer extension reactions. In some embodiments, the kit includes (i) an uracil competent polymerase; (ii) a forward primer; and (iii) a reverse primer, the reverse primer including one or more uracil bases. In other embodiments, the kits include (i) KAPA HiFi HotStart Uracil+ReadyMix Kit, (ii) a forward primer; and (iii) a reverse primer, the reverse primer includingAttorney Docket No. : Ventana-0298 WO (PCT Application) one or more uracil bases. In some embodiments, the forward primer includes one or more phosphorothioate bonds, such as 1 or more, such as 2 or more, such as 3 or more, such as 4 or more phosphorothioate bonds. In some embodiments, the forward primer includes an affinity label or specific binding entity. In some embodiments, the affinity label is biotin. In some embodiments, the reverse primer further includes a recognition site for a nicking endonuclease.

[0127] In some embodiments, the kit further comprises an uracil DNA glycosylase and an endonuclease. In some embodiments, the uracil DNA glycosylase is UG1, SUG1, or hSMUGl. In some embodiments, the endonuclease is selected from Endonuclease III, Endonuclease IV or Endonuclease VIII. In some embodiments, the uracil DNA glycosylase is UG1, SUG1, or hSMUGl; and the endonuclease is selected from Endonuclease III, Endonuclease IV or Endonuclease VIII.

[0128] In some embodiments, the kit further includes a 3' single stranded DNA exonuclease, a single stranded endonuclease, or a polymerase that has 3' to 5' exonuclease activity, and which lacks 5' to 3' exonuclease activity. In some embodiments, the 3' single stranded DNA exonuclease is exonuclease I, thermolabile exonuclease I, Msz exonuclease, or exonuclease T. In some embodiments, the single stranded endonuclease is mung bean nuclease or nuclease Pl. In some embodiments, the polymerase that has 3' to 5' exonuclease activity and which lacks 5' to 3' exonuclease activity is a T4 DNA polymerase.

[0129] In some embodiments, the kit further includes a 5' double stranded DNA exonuclease. In some embodiments, the 5' double stranded DNA exonuclease is selected from a T7 exonuclease, a T5 exonuclease, a lambda exonuclease, or an Exonuclease VIII.

[0130] In some embodiments, the kit includes (i) an uracil competent polymerase; (ii) a forward primer; (iii) a reverse primer, the reverse primer including one or more uracil bases; and (iv) a USER enzyme. In some embodiments, the USER enzyme is selected from a USER Enzyme, Thermolabile USER II Enzyme, or a Thermostable USER III Enzyme (each available from New England Bioscience). In some embodiments, the kit further includes a 3' single stranded DNA exonuclease, a single stranded endonuclease, or a polymerase that has 3' to 5' exonuclease activity, and which lacks 5' to 3' exonuclease activity. In some embodiments, the 3' single stranded DNA exonuclease is exonuclease I, thermolabile exonuclease I, Msz exonuclease, or exonuclease T. In some embodiments, the single stranded endonuclease is mung bean nuclease or nuclease Pl. In some embodiments, the polymerase that has 3' to 5' exonuclease activity and which lacks 5' to 3'Attorney Docket No. : Ventana-0298 WO(PCT Application) exonuclease activity is a T4 DNA polymerase. In some embodiments, the kit further includes a 5' double stranded DNA exonuclease. In some embodiments, the 5' double stranded DNA exonuclease is selected from a T7 exonuclease, a T5 exonuclease, a lambda exonuclease, or an Exonuclease VIII.

[0131] In some embodiments, the kit includes (i) an uracil competent polymerase; (ii) a forward primer; (iii) a reverse primer, the reverse primer including one or more uracil bases and a recognition site for a nick enzyme; and (iv) a nicking enzyme specific to the recognition site. In some embodiments, the kit further includes a 3' single stranded DNA exonuclease, a single stranded endonuclease, or a polymerase that has 3' to 5' exonuclease activity, and which lacks 5' to 3' exonuclease activity. In some embodiments, the 3' single stranded DNA exonuclease is exonuclease I, thermolabile exonuclease I, Msz exonuclease, or exonuclease T. In some embodiments, the single stranded endonuclease is mung bean nuclease or nuclease Pl. In some embodiments, the polymerase that has 3' to 5' exonuclease activity and which lacks 5' to 3' exonuclease activity is a T4 DNA polymerase. In some embodiments, the kit further includes a 5' double stranded DNA exonuclease. In some embodiments, the 5' double stranded DNA exonuclease is selected from a T7 exonuclease, a T5 exonuclease, a lambda exonuclease, or an Exonuclease VIII.

[0132] METHODS OF ENRICHING A SAMPLE FOR ONE OR MORE TARGET NUCLEIC ACID MOLECULES

[0133] The present disclosure also provides for methods of performing primer extension target enrichment (PETE) using the single stranded primers prepared in accordance with the methods described herein.

[0134] A primer extension target enrichment (PETE) workflow may be utilized to enrich a sample for one or more target nucleic acid molecules. PETE workflows are described in United Patent Application Publication Nos. 2021 / 0207211 and 2020 / 0392483; in United States Patent Nos. 10,907,204 and 11,499,180; and in International Publication Nos. WO / 2018 / 013710 and WO / 2022 / 008578, the disclosures of which are each incorporated by reference herein in their entireties. Commercial PETE workflows are available from Roche (e.g., HAPA HyperPETE Workflow).

[0135] By way of example only, a PETE workflow may be utilized to enrich a sample for one or more target nucleic acid molecules by: a) providing a reaction mixture comprising theAttorney Docket No. : Ventana-0298 WO (PCT Application) sample and a first single stranded primer (including any of those prepared according to the methods described herein), wherein the sample comprises single-stranded target nucleic acid molecule having a 3' and a 5' end and non-target nucleic acid molecules; b) hybridizing a first single stranded primer to the single-stranded target nucleic acid molecules in the reaction mixture, wherein the first single stranded primer hybridizes at least 6 nucleotides from the 3' end of the single-stranded target nucleic acid molecule and comprises an affinity ligand; c) extending the hybridized first target-specific primer with a DNA polymerase to form a first double-stranded product comprising the target nucleic acid molecule hybridized to the extended first single stranded primer, wherein the hybridized target nucleic acid molecule comprises a single-stranded overhang region of at least 6 consecutive nucleotides at the 3' end; d) removing single-stranded target and non-target nucleic acid molecules from the reaction mixture by capturing the affinity ligand of the first doublestranded product; e) hybridizing a second single stranded primer to the single-stranded overhang region at the 3' end of the hybridized target polynucleotide of the captured first double stranded product, wherein the second single stranded primer comprises a 3' hybridizing region and a barcode region; and f) extending the hybridized second target-specific primer with a DNA polymerase, wherein the DNA polymerase comprises strand displacement activity, 5'-3' double stranded DNA exonuclease activity, or a combination thereof, thereby displacing or degrading the extended first single stranded primer and forming a second double-stranded product comprising a barcode, wherein the second double-stranded product comprises the target nucleic acid molecule hybridized to an extended second single stranded primer, wherein the extended second single stranded primer comprises: i) a complement of at least a portion of the target nucleic acid molecule; and, ii) a single-stranded 5' overhang region comprising the barcode.

[0136] EXAMPLES

[0137] Purpose

[0138] The initial proof of concept experiment involved producing single stranded primers from probe templates through PCR amplification and an enzymatic conversion process; and then evaluating their performance in targeted capture compared to primers manufactured through the standard phosphorami dite method of oligonucleotide synthesis.

[0139] Materials

[0140] NA12878 Cell Line DNA

[0141] KAPA HyperPETE HotSpot Capture Panel Probe TemplateAttorney Docket No. : Ventana-0298 WO (PCT Application)

[0142] Universal Forward Primer

[0143] Universal Reverse Primer

[0144] KAPA HiFi Hotstart Uracil + Readymix

[0145] lOmM dNTP Mix

[0146] 500mM MgC12

[0147] DMSO

[0148] Betaine

[0149] Nuclease-free H20

[0150] Thermolabile USER® II Enzyme

[0151] Monarch® PCR & DNA Cleanup Kit (5 pg)

[0152] KAPA HyperPETE Hotspot Panel

[0153] KAPA HyperPETE Reagent Kit

[0154] KAPA HyperCapture Bead Kit

[0155] KAPA HyperPlus Library Preparation Kit

[0156] KAPA UDI Primer Mixes

[0157] KAPA Universal UMI Adapters

[0158] Tapestation High Sensitivity D1000

[0159] NextSeq 500 / 550 Mid Output Kit v2.5 (300 Cycles)

[0160] Method

[0161] The KAPA HyperPETE HotSpot Capture Panel Probe Template was ordered and consists of synthesized ssDNA probes with a forward primer binding site, HyperPETE targetspecific primer sequences, and a reverse primer binding site.

[0162] The template was used at lOpg input into PCR Amplification with the universal forward and reverse primers (see Tables 1 and 2).Attorney Docket No. : Ventana-0298 WO (PCT Application)Table 1: Probe to Primer PCR Master Mix.Table 2: Probe to Primer PCT Thermocycling Profile

[0163] Following amplification, an aliquot of the product was quality checked using the Tapestation High Sensitivity DI 000 kit.

[0164] Ten reactions with the USERII enzyme were set up and included 10 pmol of the amplified product, 1 pL of Therm olabile USER® II Enzyme, 5uL of rCutSmart™ Buffer, and nuclease-free water for a total volume of 50 pL / reaction. The reactions were incubated at 37°C for 15 minutes followed by heat inactivation at 65°C for 10 minutes.Attorney Docket No. : Ventana-0298 WO (PCT Application)

[0165] The USERII treated products were then purified and concentrated using the Monarch® PCR & DNA Cleanup Kit (5 pg).

[0166] An aliquot of the final product was quality checked using the Tapestation High Sensitivity DI 000 kit.

[0167] The KAPA HyperPETE Somatic Tissue DNA Workflow vl.O Instructions for Use were followed for lOng of non-formalin compromised DNA using cell line DNA NA12878. The input volume of this product to use in the capture extension step of HyperPETE was calculated based on the nmol of total primer used in each 50uL reaction for the KAPA HyperPETE HotSpot Panel, which is 0.0102364 nmol. Based on the concentration of the final Probe to Primer HotSpot Capture Panel product, 4.74 pL was the input volume. The standard synthesized KAPA HyperPETE Hot Spot Release Panel was used in the release primer steps. Three replicates of the Probe to Primer HotSpot Capture Panel product and three replicates of the standard synthesized KAPA HyperPETE Hot Spot Capture and Release Panels were included in the test.

[0168] The HyperPETE libraries were pooled and sequenced on a NextSeq500 with the NextSeq 500 / 550 Mid Output Kit v2.5 (300 Cycles). The data was processed using an internal analysis pipeline.

[0169] Overall, the results demonstrate that the primers were successfully synthesized in accordance with the methods described herein; and that the resulting primers synthesized according to the disclosed methods worked similarly to the standard primer panel (see target enrichment FIGS. 8 and 9).

[0170] Although the present disclosure has been described with reference to several illustrative embodiments, it should be understood that numerous other modifications and embodiments can be devised by those skilled in the art that will fall within the spirit and scope of the principles of this disclosure. More particularly, reasonable variations and modifications are possible in the component parts and / or arrangements of the subject combination arrangement within the scope of the foregoing disclosure, the drawings, and the appended claims without departing from the spirit of the disclosure. In addition to variations and modifications in the component parts and / or arrangements, alternative uses will also be apparent to those skilled in the art.

Claims

Attorney Docket No. : Ventana-0298 WO (PCT Application)CLAIMS1. A method of preparing a single stranded primer from a single stranded template comprising: a. obtaining a single stranded template including a target specific primer sequence, wherein the target specific primer sequence is flanked by a forward primer binding site and a reverse primer binding site; b. preparing a double stranded template from the obtained single stranded template, where the double stranded template comprises a top strand including the target specific primer sequence flanked by the forward primer binding site and the reverse primer binding site, and a bottom strand including a reverse complement of the target specific primer sequence, wherein the reverse complement of the target specific primer sequence is flanked by a reverse complement of the forward primer binding site and a reverse complement of the reverse primer binding site, wherein the reverse complement of the reverse primer binding site includes one or more uracil nucleotides; c. generating one or more abasic sites at one or more positions in the reverse complement of the reverse primer binding site, wherein each of the generated one or more generated abasic sites correspond to positions of the one or more uracil nucleotides in the reverse complement of the reverse primer binding site; d. nicking each of the one or more generated abasic sites to remove the reverse complement of the reverse primer binding site from the bottom strand of the double stranded template to form a double stranded molecule including an overhang, wherein the double stranded molecule including the overhang comprises the top strand including the target specific primer sequence flanked by the forward primer binding site and by the reverse primer binding site, and the bottom strand including the reverse complement of the target specific primer sequence, wherein the reverse complement of the target specific primer sequence is flanked by the reverse complement of the forward primer binding site e. removing the reverse primer binding site from the double stranded molecule including the overhang to provide a double stranded primer precursor molecule comprising a top strand including the target specific primer sequence flanked by the forward primer binding site, and a bottom strand including the reverse complementAttorney Docket No. : Ventana-0298 WO (PCT Application) of the target specific primer sequence flanked by the reverse complement of the forward primer binding site; and f. optionally generating a single stranded primer from the double stranded primer precursor molecule.

2. The method of claim 1, wherein the preparing of the double stranded template comprises contacting a sample including the obtained single stranded template with a polymerase, a forward primer, and a reverse primer.

3. The method of any one of the preceding claims, wherein the polymerase is an uracil competent polymerase.

4. The method of any one of the preceding claims, wherein the reverse primer includes one or more uracil nucleotides.

5. The method of any one of the preceding claims, wherein the reverse primer includes a plurality of uracil nucleotides.

6. The method of any one of the preceding claims, wherein the forward primer includes an affinity label.

7. The method of claim 6, wherein the affinity label is biotin.

8. The method of any one of the preceding claims, wherein the forward primer includes one or more phosphorothioate bonds.

9. The method of any one of the preceding claims, wherein the generating of the one or more abasic sites comprises contacting a sample including the double stranded template with an uracil DNA glycosylase.

10. The method of claim 9, wherein the uracil DNA glycosylase is selected from the group consisting of UG1, SUG1, and hSMUGl.

11. The method of any one of the preceding claims, wherein the nicking of the one or more generated abasic sites comprises contacting the sample including the double stranded template with an endonuclease.

12. The method of claim 11, wherein the endonuclease is selected from the group consisting of Endonuclease III, Endonuclease IV, and Endonuclease VIII.

13. The method of any one of the preceding claims, further comprising digesting single stranded DNA fragments remaining in the sample including the double stranded template following the nicking at each of the one or more generated abasic sites.Attorney Docket No. : Ventana-0298 WO (PCT Application)14. The method of claim 13, wherein the single stranded DNA fragments are digested by contacting the sample with an exonuclease.

15. The method of claim 14, wherein the exonuclease is Recij.

16. The method of any one of the preceding claims, wherein the preparing of the double stranded primer precursor molecule comprises contacting a sample including the double stranded molecule including the overhang with a single stranded DNA exonuclease.

17. The method of claim 16, wherein the single stranded DNA exonuclease is a 3' single stranded DNA exonuclease.

18. The method of claim 16, wherein the single stranded DNA exonuclease is selected from the group consisting of is exonuclease I, thermolabile exonuclease I, Msz exonuclease, or exonuclease T.

19. The method of any one of claims 1 - 15, wherein the preparing of the double stranded primer precursor molecule comprises contacting a sample including the double stranded molecule including the overhang with a single stranded endonuclease.

20. The method of claim 19, wherein the single stranded endonuclease is selected from the group consisting of mung bean nuclease and nuclease Pl.

21. The method of any one of claims 1 - 15, wherein the preparing of the double stranded primer precursor molecule comprises contacting a sample including the double stranded molecule including the overhang with a polymerase that has 3' to 5' exonuclease activity but lacks 5' to 3' exonuclease activity.

22. The method of claim 21, wherein the polymerase having 3' to 5' exonuclease activity but lacks 5' to 3' exonuclease activity is selected from the group consisting of a Klenow fragment of DNA polymerase I, a Phi 29 DNA polymerase, a DNA polymerase I, a T4 DNA polymerase, and a T7 DNA polymerase.

23. The method of any one of the preceding claims, wherein the single stranded primer is generated from the double stranded primer precursor molecule by contacting a sample including the double stranded primer precursor molecule with a double stranded DNA exonuclease.

24. The method of claim 23, wherein the double stranded DNA exonuclease is a 5' double stranded DNA exonuclease.Attorney Docket No. : Ventana-0298 WO (PCT Application)25. The method of 23, wherein the 5' double stranded DNA exonuclease is selected from the group consisting of a T7 exonuclease, a T5 exonuclease, a lambda exonuclease, and an Exonuclease VIII.

26. A method of preparing a single stranded primer from a single stranded template comprising: a. obtaining a single stranded template having target specific primer sequence, wherein the target specific primer sequence is flanked (a) at a 5' end with a forward primer binding site; and (b) at a 3' end with a reverse primer binding site; b. preparing a double stranded template from the obtained single stranded template, where the double stranded template comprises a first strand including the target specific primer sequence flanked at the 5' end with the forward primer binding site and at the 3' end with the reverse primer binding site, and a second strand including a reverse complement of the target specific primer sequence, wherein the reverse complement of the target specific primer sequence is flanked at a 3' end with a reverse complement of the forward primer binding site and at a 5' end with a reverse complement of the reverse primer binding site, wherein the reverse complement of the reverse primer binding site includes one or more uracil nucleotides; c. degrading the reverse complement of the reverse primer binding site in the second strand of the double stranded template to generate a double stranded template including a 3' overhang; d. removing the reverse primer binding site from the first strand of the double stranded template including the 3' overhang to provide a double stranded primer precursor molecule comprising a first strand including the target specific primer sequence flanked at the 5' end with the forward primer binding site, and a second strand including the reverse complement of the target specific primer sequence flanked by at the 3' end with the reverse complement of the forward primer binding site; and e. optionally generating a single stranded primer from the double stranded primer precursor molecule.

27. The method of claim 26, wherein the preparing of the double stranded template comprises contacting a sample including the obtained single stranded template with a polymerase, a forward primer, and a reverse primer.Attorney Docket No. : Ventana-0298 WO (PCT Application)28. The method of any one of claims 26 - 27, wherein the polymerase is an uracil competent polymerase.

29. The method of any one of claims 26 - 28, wherein the reverse primer includes one or more uracil nucleotides.

30. The method of any one of claims 26 - 29, wherein the reverse primer includes a plurality of uracil nucleotides.

31. The method of any one of claims 26 - 30, wherein the forward primer includes an affinity label.

32. The method of claim 31, wherein the affinity label is biotin.

33. The method of any one of claims 26 - 32, wherein the forward primer includes one or more phosphorothioate bonds.

34. The method of any one of claims 26 - 33, wherein the degrading of the reverse complement of the reverse primer binding site in the second strand of the double stranded template comprises contacting the sample including the double stranded template with an uracil DNA glycosylase and an endonuclease.

35. The method of claim 34, wherein the uracil DNA glycosylase is selected from the group consisting of UG1, SUG1, and hSMUGl.

36. The method of claim 34, wherein the endonuclease is selected from the group consisting of Endonuclease III, Endonuclease IV, and Endonuclease VIII.

37. The method of claim 26, wherein the preparing of the double stranded template comprises contacting a sample including the obtained single stranded template with a polymerase, a forward primer, and a reverse primer; wherein the reverse primer includes one or more uracil nucleotides; and wherein the reverse primer further includes a recognition site for a nicking enzyme; and wherein the reverse compliment of the reverse primer binding site includes the recognition site for the nicking enzyme.

38. The method of claim 37, wherein the reverse complement of the reverse primer binding site in the second strand of the double stranded template is degraded by contacting the sample including the double stranded template with a nicking enzyme specific for the recognition site.

39. The method of any one of claims 26 - 38, further comprising digesting remaining single stranded DNA fragments after the degradation of the reverse complement of the reverse primer binding site in the second strand of the double stranded template.Attorney Docket No. : Ventana-0298 WO (PCT Application)40. The method of claim 39, wherein the single stranded DNA fragments are digested by contacting the sample including the double stranded template with an exonuclease.

41. The method of claim 40, wherein the exonuclease is Recij.

42. The method of any one of claims 26 - 41, wherein the preparing of the double stranded primer precursor molecule comprises contacting a sample including the double stranded molecule including the 3' overhang with a single stranded DNA exonuclease.

43. The method of claim 42, wherein the single stranded DNA exonuclease is a 3' single stranded DNA exonuclease.

44. The method of claim 42, wherein the single stranded DNA exonuclease is selected from the group consisting of is exonuclease I, thermolabile exonuclease I, Msz exonuclease, or exonuclease T.

45. The method of any one of claims 26 - 41, wherein the preparing of the double stranded primer precursor molecule comprises contacting a sample including the double stranded molecule including the 3' overhang with a single stranded endonuclease.

46. The method of claim 45, wherein the single stranded endonuclease is selected from the group consisting of mung bean nuclease and nuclease Pl.

47. The method of any one of claims 26 - 41, wherein the preparing of the double stranded primer precursor molecule comprises a sample including the double stranded molecule including the 3' overhang with a polymerase that has 3' to 5' exonuclease activity but lacks 5' to 3' exonuclease activity.

48. The method of claim 47, wherein the polymerase having 3' to 5' exonuclease activity, but which lacks 5' to 3' exonuclease activity is selected from the group consisting of a Klenow fragment of DNA polymerase I, a Phi 29 DNA polymerase, a DNA polymerase I, a T4 DNA polymerase, and a T7 DNA polymerase.

49. The method of any one of claims 26 - 48, wherein the single stranded primer is generated from the double stranded primer precursor molecule by contacting a sample including the double stranded primer precursor molecule with a double stranded DNA exonuclease.

50. The method of claim 49, wherein the double stranded DNA exonuclease is a 5' double stranded DNA exonuclease.Attorney Docket No. : Ventana-0298 WO (PCT Application)51. The method of 50, wherein the 5' double stranded DNA exonuclease is selected from the group consisting of a T7 exonuclease, a T5 exonuclease, a lambda exonuclease, and an Exonuclease VIII.

52. A single stranded primer, the single stranded primer including a 5' forward primer binding site contiguous with a single stranded template, wherein the single stranded primer is prepared according to a process comprising: a. obtaining a single stranded template having target specific primer sequence, wherein the target specific primer sequence is flanked (a) at a 5' end with a forward primer binding site; and (b) at a 3' end with a reverse primer binding site; b. preparing a double stranded template from the obtained single stranded template, where the double stranded template comprises a first strand including the target specific primer sequence flanked at the 5' end with the forward primer binding site and at the 3' end with the reverse primer binding site, and a second strand including a reverse complement of the target specific primer sequence, wherein the reverse complement of the target specific primer sequence is flanked at a 3' end with a reverse complement of the forward primer binding site and at a 5' end with a reverse complement of the reverse primer binding site, wherein the reverse complement of the reverse primer binding site includes one or more uracil nucleotides; c. degrading the reverse complement of the reverse primer binding site in the second strand of the double stranded template to generate a double stranded template including a 3' overhang; d. removing the reverse primer binding site from the first strand of the double stranded template including the 3' overhang to provide a double stranded primer precursor molecule comprising a first strand including the target specific primer sequence flanked at the 5' end with the forward primer binding site, and a second strand including the reverse complement of the target specific primer sequence flanked by at the 3' end with the reverse complement of the forward primer binding site; and e. generating a single stranded primer from the double stranded primer precursor molecule.

53. The single stranded primer of claim 52, wherein the single stranded primer includes an affinity label.Attorney Docket No. : Ventana-0298 WO (PCT Application)54. The single stranded primer of claim 53, wherein the affinity label is biotin.

55. The single stranded primer of any one of claims 53 - 54, wherein the affinity label is included within the forward primer binding site.

56. A pair of single stranded primers, wherein each single stranded primer of the pair of single stranded primers comprises a 5' forward primer binding site contiguous with a target specific primer sequence, and wherein each single stranded primer of the pair of single stranded primers is prepared according to a process comprising: a. obtaining a single stranded template having target specific primer sequence, wherein the target specific primer sequence is flanked (a) at a 5' end with a forward primer binding site; and (b) at a 3' end with a reverse primer binding site; b. preparing a double stranded template from the obtained single stranded template, where the double stranded template comprises a first strand including the target specific primer sequence flanked at the 5' end with the forward primer binding site and at the 3' end with the reverse primer binding site, and a second strand including a reverse complement of the target specific primer sequence, wherein the reverse complement of the target specific primer sequence is flanked at a 3' end with a reverse complement of the forward primer binding site and at a 5' end with a reverse complement of the reverse primer binding site, wherein the reverse complement of the reverse primer binding site includes one or more uracil nucleotides; c. degrading the reverse complement of the reverse primer binding site in the second strand of the double stranded template to generate a double stranded template including a 3' overhang; d. removing the reverse primer binding site from the first strand of the double stranded template including the 3' overhang to provide a double stranded primer precursor molecule comprising a first strand including the target specific primer sequence flanked at the 5' end with the forward primer binding site, and a second strand including the reverse complement of the target specific primer sequence flanked by at the 3' end with the reverse complement of the forward primer binding site; and e. generating a single stranded primer from the double stranded primer precursor molecule; andAttorney Docket No. : Ventana-0298 WO (PCT Application) wherein a first member of the pair of single stranded primers includes an affinity label; and wherein a second member of the pair of single stranded primers does not include an affinity label.

57. The method of claim 56, wherein the affinity label is biotin.

58. The method of any one of claims 56 - 57, wherein the affinity label is included within the forward primer binding site.

59. A kit comprising (i) an uracil competent polymerase; (ii) a forward primer; and (iii) a reverse primer, the reverse primer including one or more uracil bases.

60. The kit of claim 59, wherein the forward primer includes one or more phosphorothioate bonds.

61. The kit of claim 59, wherein the forward primer includes an affinity label.

62. The kit of claim 60, wherein the affinity label is biotin.

63. The kit of any one of claims 59 - 62, further comprising an uracil DNA glycosylase and an endonuclease.

64. The kit of claim 63, wherein the uracil DNA glycosylase is selected from the group consisting ofUGl, SUG1, and hSMUGl.

65. The kit of claim 63, wherein the endonuclease is selected from the group consisting of Endonuclease III, Endonuclease IV, and Endonuclease VIII.

66. The kit of any one of claims 59 - 65, further comprising a 3' single stranded DNA exonuclease, a single stranded endonuclease, or a polymerase that has 3' to 5' exonuclease activity, and which lacks 5' to 3' exonuclease activity.

67. The kit of claim 66, wherein the 3' single stranded DNA exonuclease is selected from the group consisting of exonuclease I, thermolabile exonuclease I, Msz exonuclease, and exonuclease T.

68. The kit of claim 66, wherein the single stranded endonuclease is mung bean nuclease or nuclease Pl.

69. The kit of claim 66, wherein the polymerase that has 3' to 5' exonuclease activity and which lacks 5' to 3' exonuclease activity is a T4 DNA polymerase.

70. The kit of any one of claims 59 - 69, further comprising a 5' double stranded DNA exonuclease.

71. The kit of claim 70, wherein the 5' double stranded DNA exonuclease is selected from the group consisting of a T7 exonuclease, a T5 exonuclease, lambda exonuclease, and Exonuclease VIII.

72. The kit of any one of claims 59 - 71, further comprising Recjf.Attorney Docket No. : Ventana-0298 WO (PCT Application)T*>. A kit comprising (i) an uracil competent polymerase; (ii) a forward primer; (iii) a reverse primer, the reverse primer including one or more uracil bases and a recognition site for a nick enzyme; and (iv) a nicking enzyme specific to the recognition site.

74. The kit of claim 73, further comprising a 3' single stranded DNA exonuclease, a single stranded endonuclease, or a polymerase that has 3' to 5' exonuclease activity, and which lacks 5' to 3' exonuclease activity.

75. The kit of claim 74, wherein the 3' single stranded DNA exonuclease is selected from the group consisting of exonuclease I, thermolabile exonuclease I, Msz exonuclease, and exonuclease T.

76. The kit of claim 74, wherein the single stranded endonuclease is mung bean nuclease or nuclease Pl.

77. The kit of claim 74, wherein the polymerase that has 3' to 5' exonuclease activity and which lacks 5' to 3' exonuclease activity is a T4 DNA polymerase.

78. The kit of any one of claims 73 - 77, wherein the kit further includes a 5' double stranded DNA exonuclease.

79. The kit of claim 78, wherein the 5' double stranded DNA exonuclease is selected from the group consisting of a T7 exonuclease, a T5 exonuclease, a lambda exonuclease, and an Exonuclease VIII.

80. The kit of any one of claims 73 - 79, further comprising Recjf.

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