Process for detecting or quantifying nucleic acids in a library
First Claim
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1. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
- a) providing;
(i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of said nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified;
(iii) a first set of primers and a second set of primers,wherein primers of said first set of primers are fixed or immobilized to a solid support,wherein primers of said second set of primers comprise a 3′
end capable of hybridizing to a homopolymeric nucleotide sequence; and
wherein primers of said second set of primers comprise at least one production center;
(iv) polymerizing means for synthesizing nucleic acid copies of said nucleic acid analytes using said first and second sets of primers;
(v) synthesizing means for synthesizing nucleic acid copies from a production center under isothermal or isostatic conditions; and
(vi) extending means for extending a nucleic acid copy by adding a non-inherent homopolymeric sequence of at least four nucleotides to the 3′
end thereof;
b) contacting said library of nucleic acid analytes with said first set of primers to form more than one first bound entity, each comprising a first set primer hybridized to a nucleic acid analyte;
c) using said polymerizing means, extending said bound first set primers of the first bound entities using said nucleic acid analytes of the first bound entities as templates to form first copies of said analytes;
d) using said extending means, extending said first copies by adding a non-inherent homopolymeric nucleotides sequence of at least four nucleotides thereto to form extended first copies;
e) contacting said extended first copies with said second set of primers to form more than one second bound entity, each comprising a second set primer hybridized to the added non-inherent homopolymeric sequence of an extended first copy;
f) using said polymerizing means, extending said bound second set primers of the second bound entities using the extended first copies of the second bound entities as templates to form more than one complex, each comprising an extended first copy of a second bound entity and an extended second set primer of said second bound entity;
g) synthesizing from a production center in said second set of primers in said complexes one or more nucleic acid copies under isothermal or isostatic conditions;
h) hybridizing said nucleic acid copies formed in step g) to said array of nucleic acids provided in step a) (i); and
i) detecting or quantifying any of said hybridized copies obtained in step h).
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Abstract
This invention provides novel compositions and processes for analyte detection, quantification and amplification. Nucleic acid arrays and libraries of analytes are usefully incorporated into such compositions and processes. Universal detection elements, signaling entities and the like are employed to detect and if necessary or desirable, to quantify analytes. Amplification of target analytes are also provided by the compositions and processes of this invention.
84 Citations
33 Claims
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1. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
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a) providing; (i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of said nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; (iii) a first set of primers and a second set of primers, wherein primers of said first set of primers are fixed or immobilized to a solid support, wherein primers of said second set of primers comprise a 3′
end capable of hybridizing to a homopolymeric nucleotide sequence; andwherein primers of said second set of primers comprise at least one production center; (iv) polymerizing means for synthesizing nucleic acid copies of said nucleic acid analytes using said first and second sets of primers; (v) synthesizing means for synthesizing nucleic acid copies from a production center under isothermal or isostatic conditions; and (vi) extending means for extending a nucleic acid copy by adding a non-inherent homopolymeric sequence of at least four nucleotides to the 3′
end thereof;b) contacting said library of nucleic acid analytes with said first set of primers to form more than one first bound entity, each comprising a first set primer hybridized to a nucleic acid analyte; c) using said polymerizing means, extending said bound first set primers of the first bound entities using said nucleic acid analytes of the first bound entities as templates to form first copies of said analytes; d) using said extending means, extending said first copies by adding a non-inherent homopolymeric nucleotides sequence of at least four nucleotides thereto to form extended first copies; e) contacting said extended first copies with said second set of primers to form more than one second bound entity, each comprising a second set primer hybridized to the added non-inherent homopolymeric sequence of an extended first copy; f) using said polymerizing means, extending said bound second set primers of the second bound entities using the extended first copies of the second bound entities as templates to form more than one complex, each comprising an extended first copy of a second bound entity and an extended second set primer of said second bound entity; g) synthesizing from a production center in said second set of primers in said complexes one or more nucleic acid copies under isothermal or isostatic conditions; h) hybridizing said nucleic acid copies formed in step g) to said array of nucleic acids provided in step a) (i); and i) detecting or quantifying any of said hybridized copies obtained in step h). - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33)
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Specification