Optimized real time nucleic acid detection processes
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 analyzes of more than one nucleic acid analyte using one sample are also provided.
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,
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 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.
2. The process of claim 1 , wherein a separate aliquot of the sample is used for detection of each analyte.
3. The process of claim 1 , wherein the same aliquot of the sample is used for detection of each analyte.
4. The process of claim 1 , wherein the sample is nucleic acid purified from a tissue preparation.
5. The process of claim 1 , wherein the sample is nucleic acid purified from a serum preparation.
6. The process of claim 1 , wherein the sample is RNA purified from a tissue or serum preparation.
7. The process of claim 1 , wherein the sample is DNA purified from a tissue or serum preparation.