SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 7, 2017, is named 46846702301SL.txt and is 8,071 bytes in size.

BACKGROUND

Nucleic acid amplification methods permit amplification of nucleic acid molecules in a sample, such as a biological sample. A nucleic acid molecule can be amplified, via, for example, thermal cycling based approaches (e.g., polymerase chain reaction (PCR)) or via isothermal approaches. Nucleic acid amplification may also be useful in preparing a nucleic acid molecule for subsequent analysis in numerous applications related to nucleic acid analysis such as, for example in detecting target nucleic acid sequences, detecting rare nucleic acid molecules/sequences in a sample and/or preparing a nucleic acid molecule for a sequencing reaction. Thus, due to the applicability of nucleic acid amplification to a wide range of applications, there exists a need for compositions and methods useful for amplifying nucleic acid molecules and/or for analyzing amplified nucleic acid molecules that are generated from nucleic acid amplification.

SUMMARY

The disclosure provides probes, devices, priming oligonucleotides, compositions and methods for the amplification and analysis of nucleic acids.

An aspect of the disclosure provides a probe having a nucleic acid sequence that is complementary to a single-stranded target nucleic acid molecule. The nucleic acid sequence can have (i) at least one cleavable moiety, (ii) a detectable moiety, and (iii) a quencher. The quencher can quench the detectable moiety when the probe is not hybridized to the single-stranded target nucleic acid molecule.

In some embodiments, the probe may be single-stranded. In some embodiments, the probe may not capable of intramolecular hybridization. In some embodiments the probe may comprise a plurality of cleavable moieties. In some embodiments, the detectable moiety may be a fluorophore. In some embodiments, the detectable moiety may be adapted to provide a detectable signal upon hybridization of the probe to the single-stranded target nucleic acid molecule.

In some embodiments, the at least one cleavable moiety may be a ribose moiety. Additionally, a solution may contain the probe and may, for example comprise an alkaline buffer.

In some embodiments, the probe may comprise a nucleotide sequence A and a nucleotide sequence B flanking the at least one cleavable moiety. The nucleotide sequence A and nucleotide sequence B can each be complementary to a portion of the single-stranded target nucleic acid molecule. In some embodiments, the cleavable moiety may be cleavable upon hybridization of the probe to the single-stranded target nucleic acid molecule. In some embodiments, the cleavable moiety may be cleavable at alkaline conditions and/or cleavable without the aid of an enzyme.

In some embodiments, the probe may be adapted to hybridize to the single-stranded target nucleic acid molecule such that the at least one cleavable moiety is situated at a single nucleotide polymorphism (SNP) site of the single-stranded target nucleic acid molecule. In some embodiments, the at least one cleavable moiety is complementary with respect to a nucleotide at the SNP site.

Additionally, the probe may be immobilized on an array that comprises a substrate, such as, for example, a solid substrate. The solid substrate may comprise a material selected from the group consisting of a metal, a semi-metal, a glass, a polymer, a metal oxide, a silicon oxide and a silicon nitride. In some embodiments, the probe may be immobilized in a well of the array.

An additional aspect of the disclosure provides a method for detecting a single nucleotide polymorphism (SNP). The method can comprise providing a reaction mixture comprising a single-stranded target nucleic acid molecule and probe. The probe can have a nucleic acid sequence with (i) at least one cleavable moiety, (ii) a detectable moiety, and (iii) a quencher. Moreover, the probe can be complementary to the single-stranded target nucleic acid molecule and the quencher can quench the detectable moiety when the probe is not hybridized to the single-stranded target nucleic acid molecule. The method can further comprise hybridizing the probe to the single-stranded target nucleic acid molecule, where upon hybridization, the cleavable moiety is disposed adjacent to a nucleotide in a single nucleotide polymorphism (SNP) site of the single-stranded target nucleic acid molecule. The method can further comprise detecting a signal from the probe that is indicative of the presence of the nucleotide in the SNP site.

In some embodiments, providing the reaction mixture and hybridizing the probe to the single-stranded target nucleic acid molecule such that the cleavable moiety is disposed adjacent to a nucleotide in a SNP site of the single-stranded target nucleic acid molecule may be performed without the aid of an enzyme and/or without cycling a temperature of the reaction mixture. In some embodiments, the method further comprises maintaining a pH of the reaction mixture at above 7 such as, for example, from 8 to 11.

In some embodiments, the single-stranded target nucleic acid molecule may not contain a detectable moiety. In some embodiments, the cleavable moiety can be cleaved upon hybridization of the probe to the single-stranded target nucleic acid molecule. In some embodiments, the detectable moiety can be adapted to provide the signal upon hybridization of the probe to the single-stranded target nucleic acid molecule. The signal may be, for example, an optical signal, electrostatic signal, or electrochemical signal.

In some embodiments, the probe comprises a plurality of cleavable moieties. In some embodiments, the probe can comprise a nucleotide sequence A and a nucleotide sequence B flanking the at least one cleavable moiety. The nucleotide sequence A and nucleotide sequence B can each be complementary to a portion of the single-stranded target nucleic acid molecule. In some embodiments, the cleavable moiety may be complementary with respect to the nucleotide in the SNP site. In some embodiments, the probe may be immobilized on a substrate of an array. In some embodiments, the probe may be immobilized in a well of the array.

An additional aspect of the disclosure provides a method for detecting a single nucleotide polymorphism (SNP). The method can comprise providing a first reaction mixture comprising (i) a first strand of a target nucleic acid molecule and (ii) a first primer having a purine derivative at a nucleotide site of the first primer that corresponds to a nucleotide site of the first strand that is suspected of comprising a first nucleotide site of a single nucleotide polymorphism (SNP). The purine derivative can preferentially hybridize with a pyrimidine base of the first strand over other types of bases of the first strand. The method can further comprise providing a second reaction mixture comprising (i) a complementary strand of the first strand of the target nucleic acid molecule and (ii) a second primer having a pyrimidine derivative at a nucleotide site of the second primer that corresponds to a nucleotide site of the complementary strand that is suspected of comprising a second nucleotide site of the SNP. The pyrimidine derivative can preferentially hybridize with a purine base of the complementary strand over other types of bases of the complementary strand. The method can further comprise subjecting the first and second reaction mixtures to conditions such that the first primer is extendable upon hybridization to the first strand to yield a first nucleic acid product and the second primer is extendable upon hybridization to the complementary strand to yield a second nucleic acid product and determining relative quantities of the first and second nucleic acid products.

In some embodiments, the pyrimidine base of the first strand may be a cytosine base. In some embodiments, the purine base of the complementary strand may be an adenine base. In some embodiments, the purine derivative may not be a guanine. In some embodiments, the purine derivative can be hypoxanthine or a derivative thereof. In some embodiments, the pyrimidine derivative may not be thymine. In some embodiments, the pyrimidine derivative may be uracil or a derivative thereof. In some embodiments, the purine derivative may be replaceable by an adenine base with the aid of an enzyme having 3′ to 5′ exonuclease activity. In some embodiments, the pyrimidine derivative may be replaceable by a cytosine base with the aid of an enzyme having 3′ to 5′ exonuclease activity.

In some embodiments, the first and second reaction mixtures can be in separate containers. In some embodiments, each of the first and second reaction mixtures can comprise a detectable moiety such as, for example, an optically detectable moiety (e.g., SYBR green, EvaGeen, BEBO, BOXTO, an intercalating dye).

In some embodiments, determining relative quantities of the first and second nucleic acid products may be completed with the aid of a melting curve analysis. In some embodiments, determining relative quantities of the first and second nucleic acid products may be completed with the aid of a change in cycle threshold (ΔCt). In some embodiments, determining relative quantities of the first and second nucleic acid products may be completed with the aid of gel electrophoresis. In some embodiments, determining relative quantities of the first and second nucleic acid products may be completed with the aid of a computer processor that is programmed to compare the relative quantities of the first and second nucleic acid products. In some embodiments, a greater quantity of the first nucleic acid product in relation to the second nucleic acid product can be indicative of the presence of a purine base at the first nucleotide site of the SNP. In some embodiments, a greater quantity of the second nucleic acid product in relation to the first nucleic acid product can be indicative of the presence of a pyrimidine base at the second nucleotide site of the SNP.

An additional aspect of the disclosure provides an inactivatable priming oligonucleotide. The inactivatable priming oligonucleotide can comprise a priming region having (i) a nucleic acid sequence A that can be hybridizable to a single-stranded target nucleic acid molecule, where a 3′ end of the nucleic acid sequence A is adapted to be extended in a primer extension reaction to form a complement nucleic acid strand of the single-stranded target nucleic acid molecule, and (ii) at least one cleavable moiety. The inactivatable priming oligonucleotide can further comprise a nucleic acid sequence B adjacent to the priming region that can exhibit sequence complementarity to the priming region.

In some embodiments, the 3′ end of the nucleic acid sequence A may only be extendable at a temperature equal to or above a melting temperature of the nucleic acid sequence B. In some embodiments, the priming region may include, from 3′ to 5′, the nucleic acid sequence A, the cleavable moiety and a nucleic acid sequence A′ that can be complementary to the single-stranded target nucleic acid molecule. In some embodiments, the nucleic acid sequence B may not complementary to the single-stranded target nucleic acid molecule. In some embodiments, the nucleic acid sequence B can be at least partially complementary to a complement of the single-stranded target nucleic acid molecule. In some embodiments, the nucleic acid sequence B can comprise a spacer that is not complementary to the priming region and/or the single-stranded target nucleic acid molecule. In some embodiments, the spacer cannot be copied by a polymerase. In some embodiments, the spacer may be a sequence of one or more nucleotides.

In some embodiments, the priming region can have a first melting temperature and the nucleic acid sequence B can have a second melting temperature that is less than the first melting temperature. In some embodiments, the first melting temperature may be at least about 50° C., 55° C., 60° C., 65° C., 70° C., 75° C. or 80° C. In some embodiments, the second melting temperature may be less than or equal to about 80° C., 75° C., 70° C., 65° C., 60° C., 55° C. or 50° C. In some embodiments, the second melting temperature may be less than first melting temperature by about 1° C. to 80° C., 5° C. to 30° C., or 10° C. to 20° C.

In some embodiments, the priming region may further comprise a nucleic acid sequence A′. In some embodiments, the nucleic acid sequence A can have a third melting temperature that is less than the first melting temperature upon cleavage of the cleavable moiety. In some embodiments, the third melting temperature may be less than the second melting temperature. In some embodiments, the third melting temperature may be less than or equal to about 80° C., 75° C., 70° C., 65° C., 60° C., 55° C. or 50° C.

In some embodiments, the at least one cleavable moiety may be a ribose. In some embodiments, the cleavable moiety may be photo cleavable. In some embodiments, the cleavable moiety may be enzymatically cleavable such as, for example, cleavable by an enzyme having endonuclease activity. In some embodiments, the cleavable moiety may be chemically cleavable. In some embodiments, the inactivatable priming oligonucleotide may comprise a plurality of cleavable moieties. In some embodiments, the inactivatable priming oligonucleotide may be a loop inactivatable priming oligonucleotide.

In some embodiments, the inactivatable priming oligonucleotide may be contained in a solution that may, in some embodiments, comprise an alkaline buffer.

An additional aspect of the disclosure provides an inactivatable priming oligonucleotide. The inactivatable priming oligonucleotide can comprise a priming region having (i) a nucleic acid sequence A that is hybridizable to a single-stranded target nucleic acid molecule, where the nucleic acid sequence A has a 3′ end that is adapted to be extended in a primer extension reaction to form a complement nucleic acid strand of the single-stranded target nucleic acid molecule, and (ii) at least one cleavable moiety. The inactivatable priming oligonucleotide can also comprise a nucleic acid sequence B adjacent to the priming region, where the priming region has a first melting temperature and the nucleic acid sequence B has a second melting temperature that is less than the first melting temperature.

In some embodiments, the 3′ end of the nucleic acid sequence A may only extendable at a temperature equal to or above the second melting temperature. In some embodiments, the priming region may include, 3′ to 5′, the nucleic acid sequence A, the cleavable moiety adjacent to the nucleic acid sequence A, and a nucleic acid sequence A′ that is adjacent to the cleavable moiety. In some embodiments, the nucleic acid sequence A′ may be complementary to the single-stranded target nucleic acid molecule. In some embodiments, the nucleic acid sequence B may not be complementary to the single-stranded target nucleic acid molecule. In some embodiments, the nucleic acid sequence B may be at least partially complementary to a complement of the single-stranded target nucleic acid molecule. In some embodiments, the nucleic acid sequence B may comprise a spacer that is not complementary to the priming region and/or the single-stranded target nucleic acid molecule. In some embodiments, the spacer may be a sequence of one or more nucleotides.

In some embodiments, the first melting temperature may be at least about 50° C., 55° C., 60° C., 65° C., 70° C., 75° C. or 80° C. In some embodiments, the second melting temperature may be less than or equal to about 80° C., 75° C., 70° C., 65° C., 60° C., 55° C. or 50° C. In some embodiments, the second melting temperature may be less than the first melting temperature by at least about 1° C. to 80° C., 5° C. to 30° C., or 10° C. to 20° C. In some embodiments, the nucleic acid sequence A can have a third melting temperature that is less than the first melting temperature upon cleavage of the cleavable moiety. In some embodiments, the third melting temperature may be less than the second melting temperature upon cleavage of the cleavable moiety. In some embodiments, the third melting temperature may be less than or equal to about 80° C., 75° C., 70° C., 65° C., 60° C., 55° C. or 50° C.

In some embodiments, the at least one cleavable moiety can be a ribose. In some embodiments, the cleavable moiety may be photo cleavable. In some embodiments, the cleavable moiety is enzymatically cleavable, such as, for example, cleavable by an enzyme having endonuclease activity. In some embodiments, the cleavable moiety may be chemically cleavable. In some embodiments, the inactivatable priming oligonucleotide comprises a plurality of cleavable moieties. In some embodiments, the inactivatable priming oligonucleotide can be a loop inactivatable priming oligonucleotide.

In some embodiments, the inactivatable priming oligonucleotide may be contained in a solution. Such a solution may comprise an alkaline buffer.

An additional aspect of the disclosure provides a method for inactivating an inactivatable priming oligonucleotide. The method can comprise providing a solution containing an inactivatable priming oligonucleotide comprising (a) a priming region having (i) a nucleic acid sequence A that is hybridizable to a single-stranded target nucleic acid molecule, where a 3′ end of the nucleic acid sequence A is adapted to be extended in a primer extension reaction to form a complement nucleic acid strand of the single-stranded target nucleic acid molecule, and (ii) at least one cleavable moiety; and providing a nucleic acid sequence B adjacent to the priming region that exhibits sequence complementarity to the priming region. The method can further comprise cleaving the cleavable moiety, thereby inactivating the priming region of the inactivatable priming oligonucleotide.

In some embodiments, cleaving the cleavable moiety can be with the use of an enzyme such as, for example, with an endonuclease. In some embodiments, cleaving the cleavable moiety can be with the use of light. In some embodiments, cleaving the cleavable moiety can be via a pH of the solution that is greater than 7.

An additional aspect of the disclosure provides a method for inactivating an inactivatable priming oligonucleotide. The method can comprise providing a solution containing an inactivatable priming oligonucleotide comprising (a) a priming region having (i) a nucleic acid sequence A that is hybridizable to a single-stranded target nucleic acid molecule, where a 3′ end of the nucleic acid sequence A is adapted to be extended in a primer extension reaction to form a complement nucleic acid strand of the single-stranded target nucleic acid molecule, and (ii) at least one cleavable moiety; and providing a nucleic acid sequence B adjacent to the priming region, where the priming region has a first melting temperature and the nucleic acid sequence B has a second melting temperature that is less than the first melting temperature. The method can further comprise cleaving the cleavable moiety, thereby inactivating the priming region of the inactivatable priming oligonucleotide.

In some embodiments, the priming region can comprise, from 3′ to 5′, the nucleic acid sequence A, the cleavable moiety adjacent to the nucleic acid sequence A, and a nucleic acid sequence A′ that is adjacent to the cleavable moiety. In some embodiments, the method further comprises decreasing a temperature of the solution to equal to or below the second melting temperature. In some embodiments, the cleavable moiety can be cleaved at a temperature of the solution that is equal to or below the second melting temperature. In some embodiments, the method further comprises increasing the temperature of the solution to equal to or above the second melting temperature after cleaving the cleavable moiety.

In some embodiments, upon cleavage of the cleavable moiety, the nucleic acid sequence A may not stably hybridize with the single-stranded target nucleic acid molecule at a temperature equal to or above its melting temperature and/or the first melting temperature. In some embodiments, the first melting temperature may be at least about 50° C., 55° C., 60° C., 65° C., 70° C., 75° C. or 80° C. In some embodiments, the second melting temperature may be less than or equal to about 80° C., 75° C., 70° C., 65° C., 60° C., 55° C. or 50° C. In some embodiments, the second melting temperature may be less than the first melting temperature by about 1° C. to 80° C., 5° C. to 30° C., or 10° C. to 20° C. In some embodiments, the nucleic acid sequence A can have a third melting temperature that is less than the first melting temperature upon cleavage of the cleavable moiety. In some embodiments, the third melting temperature may be less than the second melting temperature. In some embodiments, the third melting temperature may be less than or equal to about 80° C., 75° C., 70° C., 65° C., 60° C., 55° C. or 50° C. In some embodiments, the method further comprises increasing the temperature of the solution to equal to or above the third melting temperature.

In some embodiments, cleaving of the cleavable moiety may be with the use of an enzyme. In some embodiments, cleaving of the cleavable moiety may be with the use of light. In some embodiments, the cleaving of the cleavable moiety is via a pH of the solution that is greater than 7.

An additional aspect of the disclosure provides an activatable priming oligonucleotide. The activatable priming oligonucleotide can comprise a priming region having (i) a nucleic acid sequence A that is hybridizable to a single-stranded target nucleic acid molecule and (ii) at least one cleavable moiety, where the nucleic acid sequence A has a 3′ end that is adapted to be extended in a primer extension reaction to form a complement nucleic acid strand of the single-stranded target nucleic acid molecule only upon cleavage of the cleavable moiety. The activatable priming oligonucleotide can also comprise a nucleic acid sequence B that is 3′ to the priming region and exhibits sequence complementarity to the priming region.

In some embodiments, the priming region can include, from 5′ to 3′, the nucleic acid sequence A, the cleavable moiety, and a nucleic acid sequence A′. In some embodiments, the nucleic acid sequence A′ may not be complementary to the single-stranded target nucleic acid molecule. In some embodiments, the nucleic acid sequence B may not be complementary to the single-stranded target nucleic acid molecule. In some embodiments, the nucleic acid sequence B may be at least partially complementary to a complement of the single-stranded target nucleic acid molecule. In some embodiments, the priming region can have a first melting temperature and the nucleic acid sequence B has a second melting temperature that is less than the first melting temperature. In some embodiments, the first melting temperature may be at least about 50° C., 55° C., 60° C., 65° C., 70° C., 75° C. or 80° C. In some embodiments, the second melting temperature may be less than or equal to about 80° C., 75° C., 70° C., 65° C., 60° C., 55° C. or 50° C. In some embodiments, the second melting temperature may be less than the first melting temperature by about 1° C. to 80° C., 5° C. to 30° C., or 10° C. to 20° C.

In some embodiments, the priming region can further comprise a nucleic acid sequence A′. In some embodiments, upon cleavage of the cleavable moiety, the nucleic acid sequence A can have a third melting temperature that is less than the first melting temperature. In some embodiments, the third melting temperature can be less than or greater than the second melting temperature. In some embodiments, the third melting temperature can be less than or equal to about 80° C., 75° C., 70° C., 65° C., 60° C., 55° C. or 50° C.

In some embodiments, the at least one cleavable moiety may be a ribose. In some embodiments, the cleavable moiety may be photo cleavable. In some embodiments, the cleavable moiety may be enzymatically cleavable such as, for example, cleavable by an endonuclease. In some embodiments, the cleavable moiety may be chemically cleavable. In some embodiments, the activatable priming oligonucleotide may comprise a plurality of cleavable moieties. In some embodiments, the activatable priming oligonucleotide may be a loop activatable priming oligonucleotide.

In some embodiments, the activatable priming oligonucleotide may be contained within a solution. Such a solution may comprise an alkaline buffer.

An additional aspect of the disclosure provides an activatable priming oligonucleotide. The activatable priming oligonucleotide can comprise a priming region having (i) a nucleic acid sequence A that is hybridizable to a single-stranded target nucleic acid molecule and (ii) at least one cleavable moiety, where the nucleic acid sequence A has a 3′ end that is adapted to be extended in a primer extension reaction to form a complement nucleic acid strand of the single-stranded target nucleic acid molecule only upon cleavage of the cleavable moiety. The activatable priming oligonucleotide can further comprise a nucleic acid sequence B that is 3′ to the priming region, where the priming region has a first melting temperature and the nucleic acid sequence B has a second melting temperature that is less than the first melting temperature.

In some embodiments, the priming region can include, from 5′ to 3′, the nucleic acid sequence A, the cleavable moiety, and a nucleic acid sequence A′. In some embodiments, the priming region nucleic acid sequence A′ may not be complementary to the single-stranded target nucleic acid molecule. In some embodiments, the nucleic acid sequence B may not be complementary to the single-stranded target nucleic acid molecule. In some embodiments, the nucleic acid sequence B may be at least partially complementary to a complement of the single-stranded target nucleic acid molecule.

In some embodiments, the first melting temperature may be at least about 50° C., 55° C., 60° C., 65° C., 70° C., 75° C. or 80° C. In some embodiments, the second melting temperature may be less than or equal to about 80° C., 75° C., 70° C., 65° C., 60° C., 55° C. or 50° C. In some embodiments, the second melting temperature may be less than the first melting temperature by about 1° C. to 80° C., 5° C. to 30° C., or 10° C. to 20° C. In some embodiments, upon cleavage of the cleavable moiety, the nucleic acid sequence A can have a third melting temperature that is less than the first melting temperature. In some embodiments, the third melting temperature may be less than or greater than the second melting temperature. In some embodiments, the third melting temperature may be less than or equal to about 80° C., 75° C., 70° C., 65° C., 60° C., 55° C. or 50° C.

In some embodiments, the at least one cleavable moiety may be a ribose. In some embodiments, the cleavable moiety may be photo cleavable. In some embodiments, the cleavable moiety may be enzymatically cleavable such as, for example, cleavable by an endonuclease. In some embodiments, the cleavable moiety may be chemically cleavable. In some embodiments, the activatable priming oligonucleotide can comprise a plurality of cleavable moieties. In some embodiments, the activatable priming oligonucleotide can be a loop activatable priming oligonucleotide.

The activatable priming oligonucleotide can be contained in a solution. Such a solution may comprise an alkaline buffer.

An additional aspect of the disclosure provides a method for nucleic acid amplification. The method can comprise providing a solution containing an activatable priming oligonucleotide comprising a priming region having (i) a nucleic acid sequence A that is hybridizable to a single-stranded target nucleic acid molecule, (ii) at least one cleavable moiety and a nucleic acid sequence B that is 3′ to the priming region and exhibits sequence complementarity to the priming region. The nucleic acid sequence A can have a 3′ end that is adapted to be extended in a primer extension reaction to form a complement nucleic acid strand of the single-stranded target nucleic acid molecule only upon cleavage of the cleavable moiety. The method further comprises cleaving the cleavable moiety and performing the primer extension reaction using the nucleic acid sequence A to form the complement nucleic acid strand.

In some embodiments, cleaving the cleavable moiety can be with the use of an enzyme such as, for example, an endonuclease. In some embodiments, cleaving the cleavable moiety can be with the use of light. In some embodiments, cleaving the cleavable moiety can be via a pH of the solution that is greater than 7.

In some embodiments, the priming region can include, from 5′ to 3′, the nucleic acid sequence A, the cleavable moiety and a nucleic acid sequence A′. In some embodiments, the nucleic acid sequence A′ may not be complementary to the single-stranded target nucleic acid molecule. In some embodiments, the nucleic acid sequence B may not be complementary to the single-stranded target nucleic acid molecule. In some embodiments, the nucleic acid sequence B may be at least partially complementary to a complement of the single-stranded target nucleic acid molecule.

In some embodiments, the priming region can have a first melting temperature and the nucleic acid sequence B can have a second melting temperature that is less than the first melting temperature. In some embodiments, the first melting temperature may be at least about 50° C., 55° C., 60° C., 65° C., 70° C., 75° C. or 80° C. In some embodiments, the second melting temperature can be less than or equal to about 80° C., 75° C., 70° C., 65° C., 60° C., 55° C. or 50° C. In some embodiments, the second melting temperature can be less than the first melting temperature by about 1° C. to 80° C., 5° C. to 30° C., or 10° C. to 20° C. In some embodiments, the priming region further comprises a nucleic acid sequence A′. In some embodiments, upon cleavage of the cleavable moiety, the nucleic acid sequence A can have a third melting temperature that is less than the first melting temperature. In some embodiments, the third melting temperature may be less than or greater than the second melting temperature. In some embodiments, the third melting temperature may be less than or equal to about 80° C., 75° C., 70° C., 65° C., 60° C., 55° C. or 50° C. In some embodiments, the method can further comprise, prior to performing the primer extension reaction, reducing the temperature of the solution to equal to or below the third melting temperature.

In some embodiments, the at least one cleavable moiety may be a ribose. In some embodiments, the activatable priming oligonucleotide may comprise a plurality of cleavable moieties. In some embodiments, the activatable priming oligonucleotide may be a loop activatable priming oligonucleotide.

An additional aspect of the disclosure provides a method for nucleic acid amplification. The method can comprise providing a solution containing an activatable priming oligonucleotide comprising a priming region having (i) a nucleic acid sequence A that is hybridizable to a single-stranded target nucleic acid molecule, (ii) at least one cleavable moiety and a nucleic acid sequence B that is 3′ to the priming region. The priming region can have a first melting temperature and the nucleic acid sequence B can have a second melting temperature that is less than the first melting temperature. Moreover, the nucleic acid sequence A can have a 3′ end that is adapted to be extended in a primer extension reaction to form a complement nucleic acid strand of the single-stranded target nucleic acid molecule only upon cleavage of the cleavable moiety. The method can further comprise cleaving the cleavable moiety and performing the primer extension reaction using the nucleic acid sequence A to form the complement nucleic acid strand.

In some embodiments, cleaving the cleavable moiety is with the use of an enzyme such as, for example, an endonuclease. In some embodiments, cleaving the cleavable moiety is with the use of light. In some embodiments, cleaving the cleavable moiety is via a pH of the solution that is greater than 7.

In some embodiments, the priming region can include, from 5′ to 3′, the nucleic acid sequence A, the cleavable moiety and a nucleic acid sequence A′. In some embodiments, the nucleic acid sequence A′ may not be complementary to the single-stranded target nucleic acid molecule. In some embodiments, the nucleic acid sequence B may not be complementary to the single-stranded target nucleic acid molecule. In some embodiments, the nucleic acid sequence B may be at least partially complementary to a complement of the single-stranded target nucleic acid molecule.

In some embodiments, the first melting temperature may be at least about 50° C., 55° C., 60° C., 65° C., 70° C., 75° C. or 80° C. In some embodiments, the second melting temperature may be less than or equal to about 80° C., 75° C., 70° C., 65° C., 60° C., 55° C. or 50° C. In some embodiments, the second melting temperature may be less than the first melting temperature by about 1° C. to 80° C., 5° C. to 30° C., or 10° C. to 20° C. In some embodiments, upon cleavage of the cleavable moiety, the nucleic acid sequence A can have a third melting temperature that is less than the first melting temperature. In some embodiments, the third melting temperature can be less than or greater than the second melting temperature. In some embodiments, the third melting temperature may be less than or equal to about 80° C., 75° C., 70° C., 65° C., 60° C., 55° C. or 50° C. In some embodiments, prior to performing the primer extension reaction, the method further comprises reducing the temperature of the solution to equal to or below the third melting temperature.

In some embodiments, the at least one cleavable moiety may be a ribose. In some embodiments, the activatable priming oligonucleotide may comprise a plurality of cleavable moieties. In some embodiments, the activatable priming oligonucleotide may be a loop activatable priming oligonucleotide.

An additional aspect of the disclosure provides a reaction mixture that can comprise an activatable priming oligonucleotide and an inactivatable priming oligonucleotide. The activatable priming oligonucleotide can comprises a first priming region having a nucleic acid sequence A′ that is hybridizable to a target nucleic acid molecule and at least one first cleavable moiety. The activatable priming oligonucleotide can become activated only upon cleavage of the first cleavable moiety, such that a 3′ end of the nucleic acid sequence A′ becomes extendable in a primer extension reaction to form a complement nucleic acid strand of the target nucleic acid molecule. Additionally, the inactivatable priming oligonucleotide can comprise a second priming region having a nucleic acid sequence A that is hybridizable to the target nucleic acid molecule and at least one second cleavable moiety. The inactivatable priming oligonucleotide can become inactivated upon cleavage of the second cleavable moiety, such that the nucleic sequence A does not form a stable complex with the target nucleic acid molecule to facilitate a primer extension reaction based on the target nucleic acid molecule sequence.

In some embodiments, the reaction mixture can further comprise the target nucleic acid molecule. In some embodiments, the activatable priming oligonucleotide can further comprise a nucleic acid sequence B′ that is 3′ to the priming region of the activatable priming oligonucleotide and exhibits sequence complementarity to the priming region of the activatable priming oligonucleotide. In some embodiments, the inactivatable priming oligonucleotide can further comprise a nucleic acid sequence B adjacent to the priming region of the inactivatable priming oligonucleotide that exhibits sequence complementarity to the priming region of the inactivatable priming oligonucleotide.

In some embodiments, the first cleavable moiety and/or the second cleavable moiety may be ribose. In some embodiments, the first cleavable moiety and/or second cleavable moiety are cleavable by an enzyme such as, for example, an endonuclease. In some embodiments, the first cleavable moiety and the second cleavable moiety may be cleavable by the same enzyme. In some embodiments, the inactivatable oligonucleotide may be inactivatable upon cleavage of the first cleavable moiety of the activatable oligonucleotide. In some embodiments, the activatable oligonucleotide may be activatable upon cleavage of the second cleavable moiety of the inactivatable oligonucleotide.

An additional aspect of the disclosure provides a method for nucleic acid amplification. The method can comprise providing a solution comprising a target nucleic acid molecule, an activatable priming oligonucleotide and an inactivatable priming oligonucleotide. The method further comprises performing successive nucleic acid amplification reactions. The successive nucleic acid amplification reactions can comprise at least a first phase followed by a second phase, where the first phase comprises amplification of a target nucleic acid molecule using at least a portion of the inactivatable priming oligonucleotide as a primer, where the second phase comprises amplification of the target nucleic acid molecule or a region thereof using at least a portion of the activatable priming oligonucleotide as another primer. The activatable priming oligonucleotide can be activated and the inactivatable priming oligonucleotide can be inactivated after the first phase. Moreover, the successive amplification reactions can yield amplified products of the target nucleic acid molecule.

In some embodiments, the activatable priming oligonucleotide and the inactivatable priming oligonucleotide can be activated and inactivated, respectively, by an enzyme having endonuclease activity. In some embodiments, the enzyme is thermal stable and/or activatable. In some embodiments, the enzyme is an RNase such as, for example, RNase HI, RNase HII or RNase HIII. In some embodiments, the successive nucleic acid amplification reactions can take place in a single reaction mixture. In some embodiments, the successive nucleic acid amplification reactions can take place in more than one reaction mixture. In some embodiments, the solution can further comprise at least one reverse primer.

In some embodiments, the activatable priming oligonucleotide can comprise a priming region having (i) a nucleic acid sequence A′ that is hybridizable to the target nucleic acid molecule, (ii) at least one cleavable moiety and a nucleic acid sequence B′ that is 3′ to the priming region and exhibits sequence complementarity to the priming region. The nucleic acid sequence A′ can have a 3′ end that is adapted to be extended in a primer extension reaction to form a complement nucleic acid strand of the target nucleic acid molecule only upon cleavage of the cleavable moiety.

In some embodiments, the inactivatable priming oligonucleotide can comprise a priming region having (i) a nucleic acid sequence A that is hybridizable to the target nucleic acid molecule, (ii) at least one cleavable moiety and a nucleic acid sequence B adjacent to the priming region that exhibits sequence complementarity to the priming region. The nucleic acid sequence A can have a 3′ end that is adapted to be extended in a primer extension reaction to form a complement nucleic acid strand of the target nucleic acid molecule.

In some embodiments, the inactivatable priming oligonucleotide can be inactivatable upon cleavage of the cleavable moiety of the activatable priming oligonucleotide. In some embodiments, the activatable priming oligonucleotide can be activatable upon cleavage of the cleavable moiety of the inactivatable priming oligonucleotide. In some embodiments, the method further comprises detecting the amplified products of the target nucleic acid molecule. The detecting can be completed via, for example, optical detection, electrostatic detection, or electrochemical detection.

An additional aspect of the disclosure provides a method for nucleic acid amplification. The method can comprise providing a reaction mixture comprising a target nucleic acid molecule, an activatable priming oligonucleotide and an inactivatable priming oligonucleotide, each of which is hybridizable to a different region of the target nucleic acid molecule. The method can further comprise, in the reaction mixture, selectively priming the target nucleic acid molecule with at least a portion of the activatable priming oligonucleotide or at least a portion of the inactivatable priming oligonucleotide. The method can further comprise performing a nucleic amplification reaction using the target nucleic acid molecule selectively primed with at least a portion of the activatable priming oligonucleotide or at least a portion of the inactivatable priming oligonucleotide to yield an amplified product(s) of the target nucleic acid molecule.

In some embodiments, the target nucleic acid molecule can be selectively primed in the reaction mixture and without removal of contents thereof. In some embodiments, the selectively priming the target nucleic acid molecule with at least a portion of the activatable priming oligonucleotide can be effectuated by an enzyme that activates the activatable priming oligonucleotide to permit a primer extension reaction to yield the amplified product(s). In some embodiments, the enzyme can have endonuclease activity.

In some embodiments, the selectively priming the target nucleic acid molecule with at least a portion of the activatable priming oligonucleotide can be effectuated by an enzyme that inactivates the inactivatable priming oligonucleotide such that it does not form a stable complex with the target nucleic acid molecule. In some embodiments, the enzyme can have endonuclease activity.

In some embodiments, the selectively priming the target nucleic acid molecule with the inactivatable priming oligonucleotide can be effectuated in the absence of an active enzyme having endonuclease activity.

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

