Site- or sequence-specific process for cleaving analytes and library of analytes
View Patent ↗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 cleaving an analyte or a library of analytes at selected sites or sequences, said process comprising the steps of:
(a) providing:
(i) said analyte or library of analytes;
(ii) a chimeric nucleic acid construct comprising at least a first segment comprised of four or more universal bases, and a second segment wherein the 5′ end of said first segment is attached to the 3′ end of said second segment;
(iii) a restriction endonuclease;
(iv) reagents for hybridization and endonucleolytic digestion;
(b) hybridizing said composition (ii) with said analyte or library of analytes (i) to form hybrids between said second segment and said selected sites or sequences in said analyte or library of analyte (i); and
(c) cleaving endonucleolytically said hybrids.
2. The process of claim 1 , wherein in said providing step (a), said universal bases comprise inosine, 5-nitroindole or 3-nitropyrrole.
3. The process of claim 1 , wherein in said providing step (a), said second segment comprises a recognition sequence for said restriction enzyme.
4. The process of claim 3 , wherein in said providing step (a), said second segment comprises modified nucleotides that render said second segment resistant to said restriction enzyme.
5. The process of claim 1 , wherein in said providing step (a), said chimeric nucleic acid construct further comprises a third segment comprising a desirable nucleic acid sequence.
6. The process of claim 5 , wherein said desirable nucleic acid sequence comprises at least one primer binding sequence or promoter sequence.
7. The process of claim 1 , wherein in said providing step (a), said chimeric nucleic acid construct is or has been rendered incapable of strand extension.
8. The process of claim 1 , further comprising the step (d) of treating with a phosphatase.
9. The process of claim 1 , wherein said providing step (a) reagents for strand extension are also provided.
10. The process of claim 9 , further comprising the step of extending a 3′ end in the remaining analyte or library of analytes, said 3′ end having been produced by said endonucleolytic cleaving step (c).
11. The process of claim 10 , wherein said extending step is carried out in the presence of one or more labeled nucleotides or oligonucleotides.