Processes for quantitative or qualitative detection of single-stranded or double-stranded nucleic acids
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.
1. A process for detecting qualitatively or quantitatively the presence of a single-stranded or double-stranded nucleic acid of interest in a sample, said process comprising the steps of:
(a) providing
(i) a sample suspected of containing said nucleic acid of interest;
(ii) either at least (a) a first oligonucleotide primer wherein said first oligonucleotide primer comprises a sequence for a phage RNA promoter or (b) a first oligonucleotide primer and a second oligonucleotide primer, wherein either the first oligonucleotide primer, or said second oligonucleotide primer comprises a phage RNA promoter sequence;
(iii) one or more first ribonucleotides, said first ribonucleotides comprising a first energy transfer element, and one or more second ribonucleotides, said second ribonucleotides comprising a second energy transfer element;
(iv) reagents for carrying out nucleic acid strand extension and RNA transcription;
(b) forming a reaction mixture comprising (i), (ii) and (iii) above;
(c) contacting under hybridization conditions said first nucleic acid primer with one strand of said nucleic acid of interest;
(d) extending said first oligonucleotide primer to form a primer-extended nucleic acid sequence;
(e) synthesizing a second nucleic acid strand complementary to said primer-extended nucleic acid sequence or a portion thereof, thereby forming a double-stranded nucleic acid that comprises said phage RNA promoter;
transcribing from said RNA promoter in said double-stranded nucleic acid formed in step (e) with a cognate phage RNA polymerase to incorporate said first and second ribonucleotides (iii) into transcripts;
(g) detecting the presence or quantity of said nucleic acid of interest by means of energy transfer between said first energy transfer element and said second energy transfer element;
wherein said transcribing step (f) is carried out by said RNA promoter sequence in either the primer-extended nucleic acid sequence synthesized in step (d) or the second nucleic acid strand synthesized in step (e).
2. The process of claim 1 , wherein said first oligonucleotide primer (a)(ii)(a) is provided, and wherein in said synthesizing step (e), the second nucleic acid strand is synthesized by primer extension of RNA fragments produced by the action of RNase H.
3. The process of claim 1 , wherein said first oligonucleotide primer and said second oligonucleotide primer (a)(ii)(b) are provided, and wherein in said synthesizing step (e), the second nucleic acid strand is synthesized by primer extension of said second oligonucleotide primer.
4. The process of claim 1 , wherein said first oligonucleotide primer and said second oligonucleotide primer (a)(ii)(b) are provided, and wherein in said synthesizing step (e), the second nucleic acid strand is synthesized by terminal addition or terminal ligation to said primer-extended nucleic acid sequence, which terminal addition or ligation is followed by contacting said terminally added or ligated sequences with one or more of said second oligonucleotide primers complementary to said terminally added or ligated sequences, followed by extension of said second oligonucleotide primers.
5. The process of claim 1 , wherein said RNA promoter comprises a T3 promoter, a T7 promoter or an SP6 promoter.
6. The process of claim 1 , wherein said nucleic acid of interest (i) has been rendered single stranded before or during said reaction mixture forming step (b).
7. The process of claim 1 , wherein said detecting step (e) is carried out one or more times or at specific intervals.
8. The process of claim 1 , wherein in said providing step (a), either said first energy transfer element is an energy donor and said second energy transfer element is an energy acceptor, or said first energy transfer element is an energy acceptor and said second energy transfer element is an energy donor.
9. The process of claim 1 , wherein in said providing step (a), said first energy transfer element and said second energy transfer element independently comprise fluorescein, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (6-FAM), naphthofluorescein, rhodamine, rhodamine 6G, rhodamine X, rhodol, sulforhodamine 101, tetramethylrhodamine (TAMRA), tetramethylrhodamineisothiocyanate (TRITC), 4,7-dichlororhodamine, eosin, eosinisothiocyanate (EITC), dansyl, hydroxycoumarin, methoxycoumarin or p-(Dimethyl aminophenylazo) benzoic acid (DABCYL), cyanine dyes, or derivatives of any of the foregoing.
10. The process of claim 9 , wherein said first energy transfer element and/or said second energy transfer element comprise the cyanine dye Cy3 or Cy5.
11. The process of claim 1 , wherein said transcripts obtained step (f) are fixed or immobilized to a solid support.
12. The process of claim 11 , wherein said solid support comprises beads, tubes, microtiter plates, glass slides, plastic slides, microchip arrays, wells or depressions.
13. The process of claim 11 , wherein said fixation or immobilization is direct or indirect.
14. The process of claim 1 , wherein any or all of said steps are carried out in a closed container system.
15. The process of claim 14 , wherein said closed container system comprises an illumination source and a detection device or unit.