Processes for incorporating nucleic acid sequences into an analyte or library of analytes
First Claim
1. 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 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 wherein said chimeric nucleic acid is incapable of strand extension; and
(iii) reagents for carrying out hybridization and template-dependent strand extension;
b) binding or hybridizing said chimeric nucleic acid (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.
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Accused Products
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.
60 Citations
62 Claims
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1. A process for incorporating a desirable nucleic acid sequence into an analyte or a library of analytes, said process comprising the steps of:
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a) providing (i) said analyte or library of analytes; (ii) a chimeric nucleic acid 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 wherein said chimeric nucleic acid is incapable of strand extension; and (iii) reagents for carrying out hybridization and template-dependent strand extension; b) binding or hybridizing said chimeric nucleic acid (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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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22. A process for incorporating a desirable nucleic acid sequence into an analyte or a library of analytes, said process comprising the steps of:
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a) providing (i) said analyte or library of analytes; (ii) a set of chimeric nucleic acids, each such nucleic acid 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)nwherein N independently comprises G, A, T or C, and wherein n is an integer from one to eight and wherein said chimeric nucleic acid is or has been rendered incapable of strand extension; and (iii) reagents for carrying out hybridization and template-dependent strand extension; b) binding or hybridizing said chimeric nucleic acids (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. - View Dependent Claims (23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44)
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45. A process for incorporating a desirable nucleic acid sequence into an analyte or a library of analytes, said process comprising the steps of:
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(a) providing; (i) said analyte or library of analytes; (ii) a chimeric nucleic acid comprising at least two segments, wherein the first of said two segments is complementary to a first portion of said analyte nucleic acid sequence, wherein the second of said two segments is complementary to a second portion of said analyte nucleic acid sequence and wherein said chimeric nucleic acid is or has been rendered incapable of strand extension; (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 (ii) with said analyte or library of analytes (i), thereby forming a first complex between said first segment and said first portion of said analyte, and a second complex between said second segment and said second portion of said analyte; wherein said first portion is resistant to digestion by said endonuclease, and wherein said second portion comprises one or more sites sensitive to endonucleolytic digestion; (d) digesting said second portion with said endonuclease (iv); and (e) extending the 3′
-end of said second portion, said 3′
-end having been generated by said endonuclease (iv),wherein said extending step (e) is carried out by template dependent polymerization or ligation. - View Dependent Claims (46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62)
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Specification