Processes for incorporating nucleic acid sequences into an analyte or library of analytes

• US 20060035264A1
• Filed: 09/27/2005
• Published: 02/16/2006
• Est. Priority Date: 03/12/2002
• Status: Active Grant

• 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., Rabbani, Elazar, Coleman, Jack, Liu, Dakai
• Granted Patent: US 7,858,302 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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

1-413. -413. (canceled)

414. A process for incorporating a desirable nucleic acid sequence into an analyte or a library of analytes, said process comprising the steps of:

a) providing

(i) said analyte or library of analytes;

(ii) a chimeric nucleic acid construct comprising at least two segments, the first segment comprising five or more universal bases and the second segment comprising a template for synthesizing said desirable nucleic acid sequence; and

(iii) reagents for carrying out hybridization and template-dependent strand extension;

b) binding or hybridizing said chimeric nucleic acid construct (ii) to said analyte or library of analytes (i); and

c) extending the 3′

end of said analyte or library of analytes;

wherein said second segment is a template for said extending step (c) when said second segment is in proximity to the 3′

end of said analyte or library of analytes.

415. The process of claim 414, wherein the 5′

end of said first segment is attached to the 3′

end of said second segment.

416. The process of claim 414, wherein said desirable nucleic acid sequence comprises at least one primer binding sequence or promoter sequence.

417. The process of claim 416, wherein said primer binding sequence is homopolymeric or heteropolymeric.

418. The process of claim 417, wherein said homopolymeric sequence comprises poly T, poly U, poly G, poly C or poly A.

419. The process of claim 417, wherein said heteropolymeric sequence comprises an RNA promoter sequence.

420. The process of claim 419, wherein said RNA promoter sequence comprises a T3 promoter, a T7 promoter or an SP6 promoter.

421. The process of claim 414, wherein in said providing step (a), the analyte or library of analytes (i) comprise DNA or RNA.

422. The process of claim 421, wherein said DNA comprises chromosomal DNA, episomal DNA or viral DNA.

423. The process of claim 421, wherein said RNA comprises mRNA, hRNA, snRNA, rRNA, tRNA or viral RNA.

424. The process of claim 414, wherein in said providing step (a), the DNA or RNA is or has been fragmented.

425. The process of claim 424, wherein said fragmentation has been carried out by a chemical, physical or enzymatic process.

426. The process of claim 425, wherein said enzymatic process is carried out by an endonuclease.

427. The process of claim 414, wherein in said providing step (a) the first segment comprises from about eight to about fifteen universal bases.

428. The process of claim 414, wherein in said providing step (a) the universal bases comprise inosine, 5-nitroindole or 3-nitropyrrole.

429. The process of claim 414, wherein in said providing step (a) the reagents (iii) comprise polymerases or ligases.

430. The process of claim 429, wherein said polymerases comprise reverse transcriptases, DNA Pol I or Klenow fragment of Pol I.

431. The process of claim 429, wherein said ligases comprise T4 DNA ligase.

432. The process of claim 414, wherein said reagents (iii) comprise one or more labeled nucleotides or oligonucleotides.

433. The process of claim 414, wherein said binding or hybridizing step (b) is carried at or near a temperature and under conditions sufficient to form a stable complex or hybrid between said chimeric nucleic acid construct (ii) and said analyte or library of analytes (i).

434. The process of claim 414, further comprising the step (a′

) of pretreating said analyte or library of analytes with a phosphatase.

435. The process of claim 414, wherein said providing step (a), the chimeric nucleic acid construct (ii) is incapable of strand extension.

436-447. -447. (canceled)

448. A process for incorporating a desirable nucleic acid sequence into an analyte or a library of analytes, said process comprising the steps of:

a) providing

(i) said analyte or library of analytes;

(ii) a set of chimeric nucleic acid constructs, each such construct comprising at least three segments, the first segment comprising four or more universal bases, the second segment comprising permutations of from one to eight nucleotides, the third segment comprising a template for synthesizing said desirable nucleic acid sequence,

wherein in said second segment, the permutations of from one to eight nucleotides comprise

(N)_n

wherein N independently comprises G, A, T or C, and wherein n is an integer from one to eight; and

(iii) reagents for carrying out hybridization and template-dependent strand extension;

b) binding or hybridizing said chimeric nucleic acid construct (ii) to said analyte or library of analytes (i); and

c) extending the 3′

end of said analyte or library of analytes;

wherein said third segment is a template for said extending step (c) when said second segment is hybridized to the 3′

end of said analyte or library of analytes.

449. The process of claim 448, wherein in said providing step (a), each such construct, the 5′

end of said first segment is attached to the 3′

end of said second segment, and the 5′

end of said second segment is attached to the 3′

end of said third segment.

450. The process of claim 448, wherein said desirable nucleic acid sequence comprises at least one primer binding sequence or promoter sequence.

451. The process of claim 450, wherein said primer binding sequence is homopolymeric or heteropolymeric.

452. The process of claim 451, wherein said homopolymeric sequence comprises poly T, poly U, poly G, poly C or poly A.

453. The process of claim 451, wherein said heteropolymeric sequence comprises an RNA promoter sequence.

454. The process of claim 453, wherein said RNA promoter sequence comprises a T3 promoter, a T7 promoter or an SP6 promoter.

455. The process of claim 448, wherein in said providing step (a), the analyte or library of analytes (i) comprise DNA or RNA.

456. The process of claim 455, wherein said DNA comprises chromosomal DNA, episomal DNA or viral DNA.

457. The process of claim 455, wherein said RNA comprises mRNA, hRNA, snRNA, rRNA, tRNA or viral RNA.

458. The process of claim 448, wherein in said providing step (a), the DNA or RNA is or has been fragmented.

459. The process of claim 458, wherein said fragmentation has been carried out by a chemical, physical or enzymatic process.

460. The process of claim 459, wherein said enzymatic process is carried out by an endonuclease.

461. The process of claim 448, wherein in said providing step (a) the first segment comprises from about six to about fifteen universal bases.

462. The process of claim 448, wherein in said providing step (a) the first segment comprises four universal bases and n is 4.

464. The process of claim 448, wherein in said providing step (a) the universal bases comprise inosine, 5-nitroindole or 3-nitropyrrole.

465. The process of claim 448, wherein in said providing step (a) the reagents (iii) comprise polymerases or ligases.

466. The process of claim 465, wherein said polymerases comprise reverse transcriptases, DNA Pol I or Klenow fragment of Pol I.

467. The process of claim 466, wherein said ligases comprise T4 DNA ligase.

468. The process of claim 448, wherein said reagents (iii) comprise one or more labeled nucleotides or oligonucleotides.

469. The process of claim 448, wherein said binding or hybridizing step (b) is carried at or near a temperature and under conditions sufficient to form a stable complex or hybrid between said chimeric nucleic acid construct (ii) and said analyte or library of analytes (i).

470. The process of claim 448, further comprising the step (a′

) of pretreating said analyte or library of analytes with a phosphatase.

471. The process of claim 448, wherein said providing step (a), the chimeric nucleic acid construct (ii) is or has been rendered incapable of strand extension.

472-496. -496. (canceled)

497. A process for incorporating a desirable nucleic acid sequence into an analyte or a library of analytes, said process comprising the steps of:

(a) providing;

(i) said analyte or library of analytes;

(ii) a chimeric nucleic acid construct comprising at least two segments, wherein the first of said two segments is complementary to a first analyte nucleic acid sequence and wherein the second of said two segments is complementary to a second analyte nucleic acid sequence;

(iii) reagents for hybridization and endonucleolytic digestion;

(iv) an endonuclease; and

(v) reagents for strand extension;

(b) forming a mixture comprising (i), (ii), (iii) and (iv);

(c) hybridizing said chimeric nucleic acid construct (ii) with said analyte or library of analytes (i), thereby forming a first complex between said first segment and said first analyte nucleic acid sequence, and a second complex between said second segment and said second analyte nucleic acid sequence;

wherein said first complex is resistant to digestion by said endonuclease, and wherein said second complex comprises one or more sites sensitive to endonucleolytic digestion;

(d) digesting said second complex with said endonuclease (iv); and

(e) extending the 3′

-end of said second analyte nucleic acid sequence, said 3′

-end having been generated by said endonuclease (iv),

wherein said extending step (e) is carried out by template dependent polymerization or ligation.

498. The process of claim 497, wherein in the providing step (a), said analyte or library of analytes (i) comprise DNA, RNA, or a mixture of DNA and RNA.

499. The process of claim 497, wherein in the providing step (a), said first segment, said first analyte nucleic acid sequence comprises RNA, said second segment comprises DNA, said second analyte nucleic acid sequence comprise RNA and said endonuclease (iv) comprises RNase H.

500. The process of claim 497, wherein in the providing step (a), said second segment and said second analyte nucleic acid sequence comprise a recognition sequence for an asymmetric restriction enzyme.

501. The process of claim 500, wherein said asymmetric restriction enzyme comprises N. BstN B I.

502. The process of claim 497, wherein in the providing step (a), said first segment comprises modified nucleotides or nucleotide analogs that confer resistance to digestion by said endonuclease (iv).

503. The process of claim 497, wherein in the forming step (b), (i), (ii) and (iii) are mixed together, and then (iv) and (v) are added after said hybridizing step (c).

504. The process of claim 497, wherein in the forming step (b), (i), (ii), (iii) and (v) are mixed together, and then (iv) is added after said hybridizing step (c).

505. The process of claim 497, wherein said strand extending step (e) also comprises strand displacement.

506. The process of claim 497, further comprising the step (f) of carrying out a dephosphorylation step.

507. The process of claim 497, wherein said chimeric nucleic acid construct (ii) further comprises a third segment complementary to a third analyte nucleic acid sequence, and wherein said hybridizing step (c) further comprises forming a third complex between said third segment and said third analyte nucleic acid sequence, wherein said third complex is resistant to digestion by said endonuclease.

508. The process of claim 507, wherein in the providing step (a) said third segment comprises modified nucleotides or nucleotide analogs which confer resistance to digestion by said endonuclease (iv).

509. The process of claim 497, wherein said chimeric nucleic acid construct (ii) further comprises a third segment non-complementary to said analyte or library of analytes.

510. The process of claim 509, wherein said third segment comprises a primer binding site or an RNA promoter sequence.

511. The process of claim 497, wherein the providing step (a) said analyte or library of analytes (i) have been fragmented.

512. The process of claim 511, wherein said analyte or library of analytes (i) have been fragmented by an endonuclease.

513. The process of claim 512, wherein said endonuclease comprises RNase, DNase, S1 nuclease, mung bean nuclease or a restriction enzyme.

514. The process of claim 497, wherein said reagents for strand extension comprise one or more labeled nucleotides or labeled oligonucleotides.

515. The process of claim 497, wherein said chimeric nucleic acid construct (ii) is or has been rendered incapable of strand extension.

516-543. -543. (canceled)