Optimized real time nucleic acid detection processes
First Claim
1. A process for detecting qualitatively or quantitatively the presence of more than one single-stranded or double-stranded nucleic acid analyte in a sample, said process comprising the steps of:
- (a) providing(i) the sample for analysis;
(ii) for each analyte, a first nucleic acid primer having a 5′
end and a 3′
end and comprising a nucleic acid sequence complementary to at least a portion of one strand of the nucleic acid analyte;
(iii) for each analyte, a second nucleic acid primer having a 5′
end and a 3′
end and comprising a nucleic acid sequence identical to at least a portion of said one strand for the analyte;
(iv) labeled nucleotides comprising a first energy transfer element; and
(v) reagents for carrying out nucleic acid strand extension,wherein the first nucleic acid primer, the second nucleic acid primer, or both the first nucleic acid primer and the second nucleic acid primer comprise a second energy transfer element, andwherein said first energy transfer element is an energy transfer donor and said second energy transfer element is an energy transfer acceptor, or said first energy transfer element is an energy transfer acceptor and said second energy transfer element is an energy transfer donor;
(b) for each analyte, forming a reaction mixture comprising (i), (ii), (iii), (iv) and (v) above;
(c) for each analyte, contacting under hybridization conditions said first nucleic acid primer with one strand of said nucleic acid analyte and contacting under hybridization conditions said second nucleic acid primer with the complementary strand of said nucleic acid analyte if present;
(d) for each analyte, extending said first nucleic acid primer by more than one nucleotide to form a first primer-extended nucleic acid sequence and, if said complementary strand is present, extending said second nucleic acid primer by more than one nucleotide to form a second primer-extended nucleic acid sequence, thereby incorporating more than one labeled nucleotide into (i) the first primer-extended nucleic acid sequence and (ii) the second primer-extended nucleic acid sequence if said complementary strand is present;
(e) for each analyte, separating said first primer-extended nucleic acid sequence from said nucleic acid analyte and separating said second primer-extended nucleic acid sequence from said complementary strand of said nucleic acid analyte if present;
(f) for each analyte, contacting under hybridization conditions said first nucleic acid primer with said nucleic acid analyte or said second primer-extended nucleic acid sequence from step (e), and contacting under hybridization conditions said second nucleic acid primer with said first primer-extended nucleic acid sequence from step (e); and
(g) for each analyte, detecting the presence or quantity of said nucleic acid analyte by detecting energy transfer between a second energy transfer element of said first nucleic acid primer, said second nucleic acid primer, or both, and a first energy element of incorporated labeled nucleotides,wherein the same sample is used for the detection of each analyte, andwherein the sample comprises nucleic acid purified from a tissue preparation or a serum preparation.
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Abstract
This invention provides for compositions for use in real time nucleic acid detection processes. Such real time nucleic acid detection processes are carried out with energy transfer elements attached to nucleic acid primers, nucleotides, nucleic acid probes or nucleic acid binding agents. Real time nucleic acid detection allows for the qualitative or quantitative detection or determination of single-stranded or double-stranded nucleic acids of interest in a sample. Other processes are provided by this invention including processes for removing a portion of a homopolymeric sequence, e.g., poly A sequence or tail, from an analyte or library of analytes. Compositions useful in carrying out such removal processes are also described and provided. Paneling and multiplex analyses of more than one nucleic acid analyte using one sample are also provided.
126 Citations
14 Claims
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1. A process for detecting qualitatively or quantitatively the presence of more than one single-stranded or double-stranded nucleic acid analyte in a sample, said process comprising the steps of:
-
(a) providing (i) the sample for analysis; (ii) for each analyte, a first nucleic acid primer having a 5′
end and a 3′
end and comprising a nucleic acid sequence complementary to at least a portion of one strand of the nucleic acid analyte;(iii) for each analyte, a second nucleic acid primer having a 5′
end and a 3′
end and comprising a nucleic acid sequence identical to at least a portion of said one strand for the analyte;(iv) labeled nucleotides comprising a first energy transfer element; and (v) reagents for carrying out nucleic acid strand extension, wherein the first nucleic acid primer, the second nucleic acid primer, or both the first nucleic acid primer and the second nucleic acid primer comprise a second energy transfer element, and wherein said first energy transfer element is an energy transfer donor and said second energy transfer element is an energy transfer acceptor, or said first energy transfer element is an energy transfer acceptor and said second energy transfer element is an energy transfer donor; (b) for each analyte, forming a reaction mixture comprising (i), (ii), (iii), (iv) and (v) above; (c) for each analyte, contacting under hybridization conditions said first nucleic acid primer with one strand of said nucleic acid analyte and contacting under hybridization conditions said second nucleic acid primer with the complementary strand of said nucleic acid analyte if present; (d) for each analyte, extending said first nucleic acid primer by more than one nucleotide to form a first primer-extended nucleic acid sequence and, if said complementary strand is present, extending said second nucleic acid primer by more than one nucleotide to form a second primer-extended nucleic acid sequence, thereby incorporating more than one labeled nucleotide into (i) the first primer-extended nucleic acid sequence and (ii) the second primer-extended nucleic acid sequence if said complementary strand is present; (e) for each analyte, separating said first primer-extended nucleic acid sequence from said nucleic acid analyte and separating said second primer-extended nucleic acid sequence from said complementary strand of said nucleic acid analyte if present; (f) for each analyte, contacting under hybridization conditions said first nucleic acid primer with said nucleic acid analyte or said second primer-extended nucleic acid sequence from step (e), and contacting under hybridization conditions said second nucleic acid primer with said first primer-extended nucleic acid sequence from step (e); and (g) for each analyte, detecting the presence or quantity of said nucleic acid analyte by detecting energy transfer between a second energy transfer element of said first nucleic acid primer, said second nucleic acid primer, or both, and a first energy element of incorporated labeled nucleotides, wherein the same sample is used for the detection of each analyte, and wherein the sample comprises nucleic acid purified from a tissue preparation or a serum preparation. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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Specification