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) a separate composition of matter for each analyte comprising at least two parts;
a first part comprising at least one first nucleic acid primer, wherein the first nucleic acid primer comprises(A) at least one first energy transfer element; and
(B) a nucleic acid sequence that is complementary to a nucleotide sequence in at least a portion of the nucleic acid analyte; and
a second part comprising at least one second nucleic acid primer, where the second nucleic acid primer comprises;
(A′
) at least one second energy transfer element; and
(B′
) a nucleic acid sequence that is identical to a nucleotide sequence in at least a portion of the nucleic acid analyte;
whereinsaid first nucleic acid primer for each analyte does not comprise said second energy transfer element, and wherein said second nucleic acid primer for each analyte does not comprise said first energy transfer element,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, andneither the first nucleic acid primer nor the second nucleic acid primer for is fixed or immobilized to a solid support;
(ii) a sample suspected of containing each nucleic acid analyte; and
(iii) reagents for carrying out nucleic acid strand extension;
(b) forming a reaction mixture for each analyte comprising (i), (ii) and (iii) above;
(c) for each analyte, contacting under hybridization conditions said first nucleic acid primer with one strand of the 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 and said second nucleic acid primer to form a first primer-extended nucleic acid sequence and a 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 the nucleic acid analyte and separating said second primer-extended nucleic acid sequence from said complementary strand of the 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) detecting the presence or quantity for each nucleic acid analyte by detecting energy transfer between said first and second energy transfer elements,wherein the same sample is used for detection of each analyte.
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Accused Products
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.
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Citations
20 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:
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(a) providing (i) a separate composition of matter for each analyte comprising at least two parts; a first part comprising at least one first nucleic acid primer, wherein the first nucleic acid primer comprises (A) at least one first energy transfer element; and (B) a nucleic acid sequence that is complementary to a nucleotide sequence in at least a portion of the nucleic acid analyte; and a second part comprising at least one second nucleic acid primer, where the second nucleic acid primer comprises; (A′
) at least one second energy transfer element; and(B′
) a nucleic acid sequence that is identical to a nucleotide sequence in at least a portion of the nucleic acid analyte;wherein said first nucleic acid primer for each analyte does not comprise said second energy transfer element, and wherein said second nucleic acid primer for each analyte does not comprise said first energy transfer element, 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, and neither the first nucleic acid primer nor the second nucleic acid primer for is fixed or immobilized to a solid support; (ii) a sample suspected of containing each nucleic acid analyte; and (iii) reagents for carrying out nucleic acid strand extension; (b) forming a reaction mixture for each analyte comprising (i), (ii) and (iii) above; (c) for each analyte, contacting under hybridization conditions said first nucleic acid primer with one strand of the 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 and said second nucleic acid primer to form a first primer-extended nucleic acid sequence and a 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 the nucleic acid analyte and separating said second primer-extended nucleic acid sequence from said complementary strand of the 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) detecting the presence or quantity for each nucleic acid analyte by detecting energy transfer between said first and second energy transfer elements, wherein the same sample is used for detection of each analyte. - View Dependent Claims (15, 16, 17, 18, 19, 20)
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2. A process for detecting qualitatively or quantitatively the presence of a more than one single-stranded or double-stranded nucleic acid analyte in a sample, said process comprising the steps of:
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(a) providing (i) a sample suspected of containing said nucleic acid analyte; (ii) a nucleic acid primer for each analyte that comprises; (A) a nucleic acid sequence complementary to at least a portion of said nucleic acid analyte and (B) a first energy transfer element; (iii) labeled nucleotide or nucleotides comprising a second energy transfer element; and (iv) reagents for carrying out nucleic acid strand extension, wherein the nucleic acid primer is not fixed or immobilized to a solid support; and 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) forming a reaction mixture for each analyte comprising (i), (ii), (iii) and (iv) above; (c) for each analyte, contacting under hybridization conditions said nucleic acid primer with said nucleic acid analyte; (d) for each analyte, extending said nucleic acid primer by more than one nucleotide, thereby incorporating said labeled nucleotide or nucleotides; and (e) for each analyte, detecting the presence or quantity of said nucleic acid analyte by detecting energy transfer between said first energy transfer element in said nucleic acid primer and said second energy transfer element in an incorporated labeled nucleotide, wherein the same sample is used for the detection of each analyte.
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3. 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:
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(a) providing (i) a sample suspected of containing each nucleic acid analyte; (ii) a first nucleic acid primer that comprises a nucleic acid sequence complementary to at least a portion of one strand of each nucleic acid analyte; (iii) a second nucleic acid primer that comprises a nucleic acid sequence identical to at least a portion of said one strand for each analyte; (iv) labeled nucleotide or 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, 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 and said second nucleic acid primer to form a first primer-extended nucleic acid sequence and a second primer-extended nucleic acid sequence if said complementary strand is present, thereby incorporating said labeled nucleotide or nucleotides; (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 in said first nucleic acid primer, said second nucleic acid primer, or both, and a first energy element in an incorporated nucleotide, wherein the same sample is used for the detection of each analyte.
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4-14. -14. (canceled)
Specification