Kits for amplifying and detecting nucleic acid sequences
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Abstract
This invention provides novel processes for amplifying nucleic acid sequences of interest, including linear and non-linear amplification. In linear amplification, a single initial primer or nucleic acid construct is utilized to carry out the amplification process. In non-linear amplification, a first initial primer or nucleic acid construct is employed with a subsequent initial primer or nucleic acid construct. In other non-linear amplification processes provided by this invention, a first initial primer or nucleic acid construct is deployed with a second initial primer or nucleic acid construct to amplify the specific nucleic acid sequence of interest and its complement that are provided. A singular primer or a singular nucleic acid construct capable of non-linear amplification can also be used to carry out non-linear amplification in accordance with this invention. Post-termination labeling process for nucleic acid sequencing is also disclosed in this invention that is based upon the detection of tagged molecules that are covalently bound to chemically reactive groups provided for chain terminators. A process for producing nucleic acid sequences having decreased thermodynamic stability to complementary sequences is also provided and achieved by this invention. Unique nucleic acid polymers are also disclosed and provided in addition to other novel compositions, kits and the like.
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Citations
72 Claims
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1-59. -59. (canceled)
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60. A kit comprising:
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a first oligonucleotide primer comprising (i) a 3′
terminal nucleotide sequence that anneals to a sample single-stranded nucleic acid molecule and serves as the origin of synthesis for synthesizing a first single-stranded nucleic acid molecule complementary at least in part to the sample single-stranded nucleic acid molecule, and (ii) a 5′
terminal nucleotide sequence that is complementary to an arbitrary region of the first single-stranded nucleic acid molecule;a second oligonucleotide primer comprising a nucleotide sequence which anneals to a region of the sample single-stranded nucleic acid molecule located 3′
to where the first oligonucleotide primer anneals thereto;a third oligonucleotide primer comprising (i) a 3′
terminal nucleotide sequence that anneals to the first single-stranded nucleic acid molecule prepared using the first oligonucleotide primer and serves as the origin of synthesis for synthesizing a second single-stranded nucleic acid molecule complementary at least in part to the first single-stranded nucleic acid molecule, and (ii) a 5′
terminal nucleotide sequence that is complementary to an arbitrary region of the second single-stranded nucleic acid molecule;a polymerizing enzyme having strand displacement activity; and one or more nucleotides which are used by the polymerizing enzyme to extend the primers. - View Dependent Claims (61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72)
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Specification