Improved method of amplifying target nucleic acids applicable to both polymerase and ligase chain reactions

Claim 1

A method of amplifying a target nucleic acid sequence enzymatically to yield amplification products, wherein an enzyme utilizes: a nucleic acid initiator; the target sequence or amplification product to which it hybridizes as a template; and at least one additional nucleoside-containing reactant which can be enzymatically assembled to form amplification products complementary to the target, the amplification products themselves serving as further templates; said method characterized in that it comprises: (a) providing requisite initiators capable of hybridizing with the target, wherein at least one of the initiators is modified such that, when the initiator is hybridized, the enzyme is substantially incapable of acting on the initiator as its substrate, so that amplification product is not assembled; (b) hybridizing the initiator to the target, if present, to form an initiator-template complex; (c) correcting the modification in a target dependent manner to allow the initiator-template complex to be acted upon by the enzyme; (d) enzymatically assembling an amplification product; and (e) dissociating the amplification product from the target and repeating the hybridization, correction and assembling steps to amplify the desired target sequence.

Claim 21

A method for distinguishing a first, target nucleic acid sequence from a second, nontarget sequence which differs from the target by the identity of at least one base, said method comprising: a. providing an excess of each of four probes, a first probe complementary to a first segment of the target, a second probe complementary to a second segment of the target, a third probe complementary to the first probe and a fourth probe complementary to the second probe, (i) wherein the 5' end of the first segment of the target is spaced from the 3' end of the second segment by X n bases, each X being independently selected from a set N consisting of from one to any three of the four bases, and n being any integer greater than or equal to 1; (ii) wherein the third probe hybridizes to the first probe, such that the base on the first probe complementary to the 5' end of the third probe is spaced from the 3' end of the first probe by Y m bases, each Y being independently selected from a set M consisting of from zero to any three of the four bases, m being zero or any integer greater than or equal to 1, provided that at least one base remains unused in the X n and Y m sequences, and that the sequence of X n bases is not complementary to the sequence of Y m bases; (iii) wherein the base adjacent the 5' end of X n and the base adjacent the 5' end of Y m are selected from the set Q, which consists of not N and not M; and (iv) wherein the nontarget sequence differs by the identity of at least one base, Z, in the X n region or the Y m region; b. combining said four probes under hybridizing conditions with single stranded target or double stranded target or double stranded target separated from its complementary strand; c. while hybridized to target, extending the first probe to fill the X n gap, and optionally the fourth probe to fill the Y m gap, using an excess of deoxy X' triphosphates and deoxy Y' triphosphates, wherein X' and Y' represent the complements of X and Y, respectively, on the target strand but omitting deoxy Z' triphosphates complementary to the single different base in the nontarget gap; d. while hybridized to target, ligating the extended first probe to the second probe, and optionally ligating the extended fourth probe to the third probe, to form at least one double stranded complex of ligated probe hybridized to the target sequence; and e. separating and detecting the properly extended and ligated probes from the improperly extended, unligated probes.

Claim 22

A method for distinguishing a first, target nucleic acid sequence from a second, nontarget sequence which differs from the target by the identity of at least one base, said method comprising: a. providing an excess of each of four probes, a first probe complementary to a first segment of the target, a second probe complementary to a second segment of the target, a third probe complementary to the first probe and a fourth probe complementary to the second probe, (i) wherein the 5' end of the first segment of the target is spaced from the 3' end of the second segment by X n bases, each X being independently selected from a set N consisting of from one to any three of the four bases, and n being any integer greater than or equal to 1; (ii) wherein the third probe hybridizes to the first probe, such that the base on the first probe complementary to the 5' end of the third probe is spaced from the 3' end of the first probe by Y m bases, each Y being independently selected from a set M consisting of from zero to any three of the four bases, m being zero or any integer greater than or equal to 1, provided that at least one base remains unused in the X n and Y m sequences, and that the sequence of X n bases is not complementary to the sequence of Y m bases; (iii) wherein the base adjacent the 5' end of X n and the base adjacent the 5' end of Y m are selected from the set Q, which consists of not N and not M; and (iv) wherein the nontarget sequence differs by the identity of at least one base, Z, in the Q region, such that Z is a base contained in the set N, the set M or both; b. combining said four probes under hybridizing conditions with single stranded target or double stranded target or double stranded target separated from its complementary strand; c. while hybridized to target, extending the first probe to fill the X n gap, and optionally the fourth probe to fill the Y m gap, using an excess of deoxy X' triphosphates and deoxy Y' triphosphates, wherein X' and Y' represent the complements of X and Y, respectively, whereby Z fails to terminate extension of the first and optional fourth probes; d. while hybridized to target, ligating the properly extended first probe to the second probe, and optionally ligating the properly extended fourth probe to the third probe, to form at least one double stranded complex of ligated probe hybridized to the target sequence; and e. separating and detecting the properly extended and ligated probes from the improperly extended, unligated probes.