Processes for detection of nucleic acids

• US 9,068,948 B2
• Filed: 11/26/2012
• Issued: 06/30/2015
• Est. Priority Date: 03/12/2002
• Status: Expired due to Fees

• Current Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Original Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Inventors: Stavrianopoulos, Jannis G., Donegan, James J., Coleman, Jack, Liu, Dakai, Rabbani, Elazar
• Primary Examiner(s): Chunduru, Prabha
• Application Number:
  US13/684,958
• Publication Number:
  US 20130078640A1
• Time in Patent Office: 946 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: G01N 21/64 Fluorescence; Phosphorescence
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence

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.

Claims

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) a nucleic acid primer that comprises;

(A) a nucleic acid sequence complementary to at least a portion of said nucleic acid of interest and

(B) a first energy transfer element;

(iii) labeled nucleotide or labeled 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 comprising (i), (ii), (iii) and (iv) above, wherein said reaction mixture lacks the presence of any nucleotides that are strand terminators;

(c) contacting under hybridization conditions said nucleic acid primer with said nucleic acid of interest;

(d) extending said nucleic acid primer by more than one nucleotide, thereby incorporating more than one labeled nucleotide into the extended primer; and

(e) detecting the presence or quantity of said nucleic acid of interest 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.

2. The process of claim 1, wherein said nucleic acid of interest has been rendered single stranded before or during step (b).

3. The process of claim 1, wherein prior to step (e), said process further comprises the step of (b′

) separating said extended nucleic acid from the nucleic acid of interest and step (c) step (d).

4. The process of claim 3, wherein said step (b′

), step (c) and step (d) are repeated one or more times.

5. The process of claim 1, wherein in step (a), said reagents comprise a thermostable DNA polymerase.

6. The process of claim 1, wherein said nucleic acid primer comprises more than one first energy transfer element.

7. The process of claim 1, wherein 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), a cyanine dye, Cy3, Cy5, or a derivative of any of the foregoing.

8. The process of claim 1, wherein any or all of said steps are carried out in a closed container system.

9. 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) a first nucleic acid primer that comprises a nucleic acid sequence complementary to at least a portion of one strand of said nucleic acid of interest;

(iii) a second nucleic acid primer that comprises a nucleic acid sequence identical to at least a portion of said one strand;

(iv) labeled nucleotide or 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

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 comprising (i), (ii), (iii), (iv) and (v) above, wherein said reaction mixture lacks the presence of any nucleotides that are strand terminators;

(c) contacting under hybridization conditions said first nucleic acid primer with one strand of said nucleic acid of interest and contacting under hybridization conditions said second nucleic acid primer with the complementary strand of said nucleic acid of interest if present;

(d) 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 more than one labeled nucleotides into each of the extended primers;

(e) separating said first primer-extended nucleic acid sequence from said nucleic acid of interest and separating said second primer-extended nucleic acid sequence from said complementary strand of said nucleic acid of interest if present;

(f) contacting under hybridization conditions said first nucleic acid primer with said nucleic acid of interest 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 of said nucleic acid of interest 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.

10. The process of claim 9, wherein after said contacting step (f), said process further comprises repeating said extending step (d).

11. The process of claim 9, said process further comprising the step (e′

) of separating said first primer-extended nucleic acid sequence from said second primer-extended nucleic acid sequence, and repeating step (f) thereafter.

12. The process of claim 9, wherein said first nucleic acid primer comprises more than one first energy transfer element and/or said second nucleic acid primer comprises more than one second energy transfer element.

13. The process of claim 9, wherein at least one of said first or second nucleic acid primers is fixed or immobilized to a solid support.

14. The process of claim 13, wherein said solid support comprises a bead, a tube, a microtiter plate, a glass slide, a plastic slide, a microchip array, a well or a depression.

15. 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) a first nucleic acid primer that comprises a nucleic acid sequence complementary to at least a portion of one strand of said nucleic acid of interest;

(iii) a second nucleic acid primer that comprises a nucleic acid sequence identical to at least a portion of said one strand;

(iv) one or more first nucleotides, said first nucleotides comprising a first energy transfer element, and one or more second nucleotides, said second nucleotides comprising 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; and

(v) reagents for carrying out nucleic acid strand extension;

(b) forming a reaction mixture comprising (i), (ii), (iii), (iv) and (v) above, wherein said reaction mixture lacks the presence of any nucleotides that are strand terminators;

(c) contacting under hybridization conditions said first nucleic acid primer with one strand of said nucleic acid of interest and contacting under hybridization conditions said second nucleic acid primer with the complementary strand of said nucleic acid of interest if present;

(d) extending said first nucleic acid primer and said second nucleic acid primer in a PCR reaction 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 first and second nucleotides; and

(e) detecting the presence or quantity of said nucleic acid of interest by detecting energy transfer between said first energy transfer element and said second energy transfer element during said PCR reaction.

16. The process of claim 15, further comprising:

(b′

) separating said first primer-extended nucleic acid sequence from said nucleic acid of interest and separating said second primer-extended nucleic acid sequence from said complementary strand of said nucleic acid of interest if present;

(c′

) contacting under hybridization conditions said first nucleic acid primer with said second primer-extended nucleic acid sequence from step (b′

), if said second strand is present, and contacting under hybridization conditions said second nucleic acid primer with said first primer-extended nucleic acid sequence from step (b′

); and

repeating step (d) thereafter.

17. The process of claim 16, further comprising the step (b″

) of separating said first primer-extended nucleic acid sequence from said second primer-extended nucleic acid sequence, and the step (c″

) of contacting under hybridization conditions said first nucleic acid primer with said second primer-extended nucleic acid sequence from step (b″

), and contacting under hybridization conditions said second nucleic acid primer with said first primer-extended nucleic acid sequence from step (b″

); and

repeating step (d) thereafter.

18. The process of claim 17, wherein said separating step (b″

), said contacting step (c″

) and said extending step (d) are repeated.

19. The process of claim 15, wherein said detecting step (e) is carried out at specific intervals during or after any contacting step, extending step, or separating step.

20. The process of claim 15, wherein at least one of said first or second nucleic acid primers or any primer-extended nucleic acid sequence is fixed or immobilized to a solid support.