Late-PCR
First Claim
1. A non-symmetric polymerase chain reaction (PCR) amplification method comprising thermally cycling a PCR reaction mixture containing a deoxyribonucleic acid (DNA) amplification target sequence, a pair of PCR primers, dNTP'"'"'s and a thermostable polymerase repeatedly through PCR steps of strand melting, primer annealing and primer extension, wherein, at the outset of amplification (a) the reaction mixture contains up to 1,000,000 copies of the amplification target sequence, (b) the PCR primer pair comprises a limiting primer and an excess primer, the limiting primer being present at a concentration of up to 200 nM and the excess primer being present at an initial concentration at least five times higher than the limiting primer (c) an initial, concentration-adjusted melting temperature of the limiting primer that is equal to or greater than an initial, concentration-adjusted melting temperature of the excess primer when the initial, concentration-adjusted melting temperature is determined using a formula that takes into account initial primer concentration and composition, (d) if the limiting primer is not fully complementary to the target sequences the initial, concentration-adjusted melting temperature of that portion of the limiting primer which hybridizes to the target sequence is not more than 5°
- C. below the initial, concentration-adjusted melting temperature of the excess primer when the initial, concentration-adjusted melting temperature is determined using a formula that takes into account initial primer concentration and composition, (e) the melting temperature of an amplicon produced by extension of the excess primer exceeds the initial, concentration-adjusted melting temperature of the excess primer by not more than 18°
C., and (f) thermal cycling is repeated a number of times sufficient to include multiple cycles of linear amplification using the excess primer following exhaustion of the limiting primer.
1 Assignment
0 Petitions
Accused Products
Abstract
A non-symmetric polymerase chain reaction (PCR) amplification method employing a limiting primer in low concentration whose concentration-adjusted melting point at least equals, and preferably exceeds, that of the excess primer, the latter in turn not being more than 25° C. below the melting temperature of the amplicon. Assays employing such amplification and labeled hybridization probes, including assays that include a detection step following primer extension or a low-temperature probe, or both. Kits for performing such assays and primer or primer-and-probe sets for performing the foregoing amplifications and assays.
85 Citations
84 Claims
-
1. A non-symmetric polymerase chain reaction (PCR) amplification method comprising thermally cycling a PCR reaction mixture containing a deoxyribonucleic acid (DNA) amplification target sequence, a pair of PCR primers, dNTP'"'"'s and a thermostable polymerase repeatedly through PCR steps of strand melting, primer annealing and primer extension, wherein, at the outset of amplification (a) the reaction mixture contains up to 1,000,000 copies of the amplification target sequence, (b) the PCR primer pair comprises a limiting primer and an excess primer, the limiting primer being present at a concentration of up to 200 nM and the excess primer being present at an initial concentration at least five times higher than the limiting primer (c) an initial, concentration-adjusted melting temperature of the limiting primer that is equal to or greater than an initial, concentration-adjusted melting temperature of the excess primer when the initial, concentration-adjusted melting temperature is determined using a formula that takes into account initial primer concentration and composition, (d) if the limiting primer is not fully complementary to the target sequences the initial, concentration-adjusted melting temperature of that portion of the limiting primer which hybridizes to the target sequence is not more than 5°
- C. below the initial, concentration-adjusted melting temperature of the excess primer when the initial, concentration-adjusted melting temperature is determined using a formula that takes into account initial primer concentration and composition, (e) the melting temperature of an amplicon produced by extension of the excess primer exceeds the initial, concentration-adjusted melting temperature of the excess primer by not more than 18°
C., and (f) thermal cycling is repeated a number of times sufficient to include multiple cycles of linear amplification using the excess primer following exhaustion of the limiting primer. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 70, 75)
- C. below the initial, concentration-adjusted melting temperature of the excess primer when the initial, concentration-adjusted melting temperature is determined using a formula that takes into account initial primer concentration and composition, (e) the melting temperature of an amplicon produced by extension of the excess primer exceeds the initial, concentration-adjusted melting temperature of the excess primer by not more than 18°
-
19. A non-symmetric polymerase chain reaction (PCR) method comprising thermally cycling a PCR reaction mixture containing a deoxyribonucleic acid (DNA) amplification target sequence, a pair of matched limiting PCR primers, an excess primer, dNTP'"'"'s and a thermostable polymerase repeatedly through PCR steps of strand melting, primer annealing and primer extension, wherein the matched limiting PCR primers are present in approximately equimolar initial concentration of up to 200 nM, the excess primer is present at an initial concentration at least five times higher than the limiting primers, an initial, concentration-adjusted melting temperatures of the excess primer is at least 5°
- C. below an initial, concentration-adjusted melting temperature of the limiting primers when the initial, concentration-adjusted melting temperature is determined using a formula that takes into account initial primer concentration and composition, and wherein the method comprises a first phase wherein an annealing temperature is higher than the initial, concentration-adjusted melting temperature of the excess primer and the matched limiting primers generate a first amplicon, and a second phase wherein the annealing temperature is lowered and the excess primer generates a second amplicon, shorter than the first amplicon, utilizing the first amplicon as a template strand, and wherein the melting temperature of the second amplicon exceeds the initial, concentration-adjusted melting temperature of the excess primer by not more than 25°
C. - View Dependent Claims (20, 21, 71, 76)
- C. below an initial, concentration-adjusted melting temperature of the limiting primers when the initial, concentration-adjusted melting temperature is determined using a formula that takes into account initial primer concentration and composition, and wherein the method comprises a first phase wherein an annealing temperature is higher than the initial, concentration-adjusted melting temperature of the excess primer and the matched limiting primers generate a first amplicon, and a second phase wherein the annealing temperature is lowered and the excess primer generates a second amplicon, shorter than the first amplicon, utilizing the first amplicon as a template strand, and wherein the melting temperature of the second amplicon exceeds the initial, concentration-adjusted melting temperature of the excess primer by not more than 25°
-
22. A non-symmetric polymerase chain reaction (PCR) method with removal of single-stranded amplicon comprising a) thermally cycling a PCR reaction mixture containing a DNA amplification target sequence, a pair of PCR primers for the amplification target sequence, dNTP'"'"'s and a thermostable DNA polymerase through repeated cycles of strand melting, primer annealing and primer extension, wherein (i) the PCR primer pair comprises a limiting primer and an excess primer, (ii) the limiting primer is present at an initial concentration of up to 200 nM, and the excess primer is present at an initial concentration at least five times higher than the limiting primer, (iii) an initial, concentration-adjusted melting temperature of the limiting primer is at least equal to an initial, concentration-adjusted melting temperature of the excess primer when in initial, concentration-adjusted melting temperature is determined using a formula that takes into account initial primer concentration and composition, and (iv) thermal cycling is repeated a number of times sufficient to include multiple cycles of linear amplification using the excess primer following exhaustion of the limiting primer;
- and b) during at least some cycles of linear amplification, following the step of primer extension, removing a single-stranded extension product of the excess primer from the reaction mixture by hybridizing the single-stranded extension product to capture probes.
- View Dependent Claims (23, 24, 25, 26, 27, 72, 77)
-
28. A homogeneous detection assay for at least one DNA amplification target sequence employing non-symmetric polymerase chain reaction (PCR) amplification, comprising (a) thermally cycling through multiple cycles of PCR steps of strand melting, primer annealing and primer extension a PCR reaction mixture containing the at least one DNA amplification target sequence, a thermostable DNA polymerase, dNTP'"'"'s, and for each amplification target sequence a pair of PCR primers for amplifying the amplification target sequence and at least one labeled hybridization probe that hybridizes to an amplicon produced by the primers and (b) detecting signal produced by the at least one probe as an indication of the presence of the at least one amplification target sequence, wherein (i) each PCR primer pair comprises a limiting primer and an excess primer, (ii) the limiting primer is present at an initial concentration of up to 200 nM, and for each primer pair the excess primer is present at an initial concentration of at least five times higher than the limiting primer concentration, (iii) an initial, concentration-adjusted melting temperature of the limiting primer of each primer pair is at least equal to an initial, concentration-adjusted melting temperature the excess primer of the same pair when the initial, concentration-adjusted melting temperature is determined using a formula that takes into account initial primer concentration and composition, (iv) for each primer pair the melting temperature of the amplicon exceeds the initial, concentration-adjusted melting temperature of the excess primer by not more than 25°
- C., (v) thermal cycling is repeated a number of times sufficient to include multiple cycles of linear amplification using the excess primers following exhaustion of the limiting primers, and (vi) the probes is selected from the group consisting of probes that hybridize to an extension product of the limiting primer, and probes that hybridize to an extension product of the excess primer and signal upon hybridization, and (viii) the probes hybridize to the amplicons during the PCR step of primer annealing.
- View Dependent Claims (29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 73, 78)
-
52. A homogeneous detection assay for at least one DNA amplification target sequence employing non-symmetric polymerase chain reaction (PCR) amplification, comprising (a) thermally cycling through multiple cycles of PCR steps of strand melting, primer annealing and primer extension a PCR reaction mixture containing the at least one amplification target sequence, a thermostable DNA polymerase, dNTP'"'"'s, and for each amplification target sequence a pair of PCR primers for amplifying the amplification target sequence and at least one labeled low-temperature hybridization probe that hybridizes to an amplicon produced by the primers and (b) detecting signal produced by the at least one probe as an indication of the presence of the at least one amplification target sequence, wherein (i) each PCR primer pair comprises a limiting primer and an excess primer, (ii) each limiting primer is present at an initial concentration of up to 200 nM, and for each primer pair the excess primer is present at an initial concentration at least five times higher than the limiting primer concentration, (iii) for each amplification target sequence, the low-temperature hybridization probe binds to the extension product of the excess primer and emits a detectable signal upon hybridization, (iv) for each amplification target sequence an initial, concentration-adjusted melting temperature of the low-temperature hybridization probe is at least 5°
- C. below an initial, concentration-adjusted melting temperature of the limiting primer when the initial, concentration-adjusted melting temperature is determined using a formula that takes into account initial primer concentration and composition, (v) thermal cycling is repeated a number of times sufficient to include multiple cycles of linear amplification using the excess primers following exhaustion of the limiting primers, and (vi) detection is performed at a temperature sufficiently low for the low-temperature probes to hybridize and signal.
- View Dependent Claims (53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 74, 79)
-
65. A non-symmetric polymerase chain reaction (PCR) amplification method comprising thermally cycling a PCR reaction mixture containing a deoxyribonucleic acid (DNA) amplification target sequence, a pair of PCR primers, dNTP'"'"'s and a thermostable polymerase repeatedly through PCR steps of strand melting, primer annealing and primer extension, wherein, at the outset of amplification (a) the reaction mixture contains up to 1,000,000 copies of the amplification target sequence, (b) the PCR primer pair comprises a limiting primer and an excess primer, the limiting primer being present at an initial concentration of up to 200 nM and the excess primer being present at an initial concentration at least five times higher than the limiting primer (c) an initial, concentration-adjusted melting temperature of the limiting primer that is equal to or greater than an initial, concentration-adjusted melting temperature of the excess primer when the initial, concentration-adjusted melting temperature is determined using a Nearest Neighbor formula, (d) if the limiting primer is not fully complementary to the target sequences the initial, concentration-adjusted melting temperature of that portion of the limiting primer which hybridizes to the target sequence is not more than 5°
- C. below the initial, concentration-adjusted melting temperature of the excess primer when the initial, concentration-adjusted melting temperature is determined using a Nearest Neighbor formula, (e) the melting temperature of the amplicon produced by extension of the excess primer exceeds the initial, concentration-adjusted melting temperature of the excess primer by not more than 18°
C., and (f) thermal cycling is repeated a number of times sufficient to include multiple cycles of linear amplification using the excess primer following exhaustion of the limiting primer. - View Dependent Claims (80)
- C. below the initial, concentration-adjusted melting temperature of the excess primer when the initial, concentration-adjusted melting temperature is determined using a Nearest Neighbor formula, (e) the melting temperature of the amplicon produced by extension of the excess primer exceeds the initial, concentration-adjusted melting temperature of the excess primer by not more than 18°
-
66. A non-symmetric polymerase chain reaction (PCR) method comprising thermally cycling a PCR reaction mixture containing a deoxyribonucleic acid (DNA) amplification target sequence, a pair of matched limiting PCR primers, an excess primer, dNTP'"'"'s and a thermostable polymerase repeatedly through PCR steps of strand melting, primer annealing and primer extension, wherein the matched limiting PCR primers are present in approximately equimolar initial concentrations of up to 200 nM, the excess primer is present at an initial concentration at least five times higher than the limiting primers, an initial, concentration-adjusted melting temperatures of the excess primer is at least 5°
- C. below an initial, concentration-adjusted melting temperature of the limiting primers when the initial, concentration-adjusted melting temperature is determined using a Nearest Neighbor formula, and wherein the reaction comprises a first phase wherein the annealing temperature is higher than the initial, concentration-adjusted melting temperature of the excess primer and the matched limiting primers generate a first amplicon, and a second phase wherein the annealing temperature is lowered and the excess primer generates a second amplicon, shorter than the first amplicon, utilizing the first amplicon as a template strand, and wherein the melting temperature of the second amplicon exceeds the initial, concentration-adjusted melting temperature of the excess primer by not more than 25°
C. - View Dependent Claims (81)
- C. below an initial, concentration-adjusted melting temperature of the limiting primers when the initial, concentration-adjusted melting temperature is determined using a Nearest Neighbor formula, and wherein the reaction comprises a first phase wherein the annealing temperature is higher than the initial, concentration-adjusted melting temperature of the excess primer and the matched limiting primers generate a first amplicon, and a second phase wherein the annealing temperature is lowered and the excess primer generates a second amplicon, shorter than the first amplicon, utilizing the first amplicon as a template strand, and wherein the melting temperature of the second amplicon exceeds the initial, concentration-adjusted melting temperature of the excess primer by not more than 25°
-
67. A non-symmetric polymerase chain reaction (PCR) method with removal of single-stranded amplicon comprising a) thermally cycling a PCR reaction mixture containing a DNA amplification target sequence, a pair of PCR primers for the amplification target sequence, dNTP'"'"'s and a thermostable DNA polymerase through repeated cycles of strand melting, primer annealing and primer extension, wherein (i) the PCR primer pair comprises a limiting primer and an excess primer, (ii) the limiting primer is present at an initial concentration of up to 200 nM, and the excess primer is present at an initial concentration at least five times higher than the limiting primer, (iii) an initial, concentration-adjusted melting temperature of the limiting primer is at least equal to an initial, concentration-adjusted melting temperature of the excess primer when the initial, concentration-adjusted melting temperature is determined using a Nearest Neighbor formula, and (iv) thermal cycling is repeated a number of times sufficient to include multiple cycles of linear amplification using the excess primer following exhaustion of the limiting primer;
- and b) during at least some cycles of linear amplification, following the step of primer extension, removing single-stranded extension product of the excess primer from the reaction mixture by hybridizing the product to capture probes.
- View Dependent Claims (82)
-
68. A homogeneous detection assay for at least one DNA amplification target sequence employing non-symmetric polymerase chain reaction (PCR) amplification, comprising (a) thermally cycling through multiple cycles of PCR steps of strand melting, primer annealing and primer extension a PCR reaction mixture containing the at least one amplification target sequence, a thermostable DNA polymerase, dNTP'"'"'s, and for each amplification target sequence a pair of PCR primers for amplifying the amplification target sequence and at least one labeled hybridization probe that hybridizes to the amplicon produced by the primers and (b) detecting signal produced by the at least one probe as an indication of the presence of the at least one amplification target sequence, wherein (i) each PCR primer pair comprises a limiting primer and an excess primer, (ii) the limiting primer is present at an initial concentration of up to 200 nM, and for each primer pair the excess primer is present at an initial concentration of at least five times higher than the limiting primer concentration, (iii) an initial, concentration-adjusted melting temperature of the limiting primer of each primer pair is equal to or greater than the initial, concentration-adjusted melting temperature of the excess primer of the same primer pair when the initial, concentration-adjusted melting temperature is determined using a Nearest Neighbor formula, (iv) for each primer pair the melting temperature of the amplicon exceeds the initial, concentration-adjusted melting temperature of the excess primer by not more than 25°
- C., (v) thermal cycling is repeated a number of times sufficient to include multiple cycles of linear amplification using the excess primers following exhaustion of the limiting primers, and (vi) the probes are selected from the group consisting of probes that hybridize to the extension product of the limiting primer, and probes that hybridize to the extension product of the excess primer and signal upon hybridization, and (viii) the probes hybridize to the amplicons during the PCR step of primer annealing.
- View Dependent Claims (83)
-
69. A homogeneous detection assay for at least one DNA amplification target sequence employing non-symmetric polymerase chain reaction (PCR) amplification, comprising (a) thermally cycling through multiple cycles of PCR steps of strand melting, primer annealing and primer extension a PCR reaction mixture containing the at least one amplification target sequence, a thermostable DNA polymerase, dNTP'"'"'s, and for each amplification target sequence a pair of PCR primers for amplifying the amplification target sequence and at least one labeled low-temperature hybridization probe that hybridizes to the amplicon produced by the primers and (b) detecting signal produced by the at least one probe as an indication of the presence of the at least one amplification target sequence, wherein (i) each PCR primer pair comprises a limiting primer and an excess primer, (ii) each limiting primer is present at an initial concentration of up to 200 nM, and for each primer pair the excess primer is present at an initial concentration at least five times higher than the limiting primer concentration, (iii) for each amplification target sequence, the low-temperature hybridization probe binds to the extension product of the excess primer and emits a detectable signal upon hybridization, (iv) for each amplification target sequence an initial, concentration-adjusted melting temperature of the low-temperature hybridization probe is at least 5°
- C. below an initial, concentration-adjusted melting temperature of the limiting primer when the initial, concentration-adjusted melting temperature is determined using a Nearest Neighbor formula, (v) thermal cycling is repeated a number of times sufficient to include multiple cycles of linear amplification using the excess primers following exhaustion of the limiting primers, and (vi) detection is performed at a temperature sufficiently low for the low-temperature probes to hybridize and signal.
- View Dependent Claims (84)
Specification