Nucleic acid detection methods
First Claim
1. A method for detecting a target sequence within a nucleic acid comprising the steps of:
- a) hybridizing the nucleic acid to an array of probes wherein each probe comprises a 5'"'"'-region complementary to the 3'"'"'-region of said nucleic acid, a 3'"'"'-region complementary to the 5'"'"'-region of said nucleic acid and an internal variable region which is unique to each probe to form a hybridized array;
b) digesting the hybridized array with a single-strand specific nuclease;
c) treating said array with a nucleic acid polymerase; and
d) identifying a probe with an internal variable region complementary to the target sequence.
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Abstract
The invention relates to methods for rapidly determining the sequence and/or length a target sequence. The target sequence may be a series of known or unknown repeat sequences which are hybridized to an array of probes. The hybridized array is digested with a single-strand nuclease and free 3'"'"'-hydroxyl groups extended with a nucleic acid polymerase. Nuclease cleaved heteroduplexes can be easily distinguish from nuclease uncleaved heteroduplexes by differential labeling. Probes and target can be differentially labeled with detectable labels. Matched target can be detected by cleaving resulting loops from the hybridized target and creating free 3-hydroxyl groups. These groups are recognized and extended by polymerases added into the reaction system which also adds or releases one label into solution. Analysis of the resulting products using either solid phase or solution. These methods can be used to detect characteristic nucleic acid sequences, to determine target sequence and to screen for genetic defects and disorders. Assays can be conducted on solid surfaces allowing for multiple reactions to be conducted in parallel and, if desired, automated.
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Citations
58 Claims
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1. A method for detecting a target sequence within a nucleic acid comprising the steps of:
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a) hybridizing the nucleic acid to an array of probes wherein each probe comprises a 5'"'"'-region complementary to the 3'"'"'-region of said nucleic acid, a 3'"'"'-region complementary to the 5'"'"'-region of said nucleic acid and an internal variable region which is unique to each probe to form a hybridized array; b) digesting the hybridized array with a single-strand specific nuclease; c) treating said array with a nucleic acid polymerase; and d) identifying a probe with an internal variable region complementary to the target sequence. - 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, 56)
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28. A method for determining a length of a target sequence within a nucleic acid comprising the steps of:
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a) hybridizing the nucleic acid to an array of probes wherein each probe comprises a 5'"'"'-region complementary to the 3'"'"'-region of said nucleic acid, a 3'"'"'-region complementary to the 5'"'"'-region of said nucleic acid, and an internal variable region which is unique to each probe to form a hybridized array; b) digesting the hybridized array with a single-strand specific nuclease; c) treating said array with a nucleic acid polymerase; and d) determining the length of the target sequence. - View Dependent Claims (29, 30, 31)
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32. A method for determining the number of repeat sequences within a target sequence of a nucleic acid comprising the steps of:
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a) hybridizing the nucleic acid to an array of probes wherein each probe comprises a 5'"'"'-region complementary to the 3'"'"'-region of said nucleic acid, a 3'"'"'-region complementary to the 5'"'"'-region of said nucleic acid and an internal region containing one or more repeat sequences which is unique to each probe to form a hybridized array; b) digesting the hybridized array with a single-strand specific nuclease; c) treating said array with a nucleic acid polymerase; and d) identifying a probe containing an internal variable region complementary to the target sequence and determining the number of repeat sequences within the target sequence. - View Dependent Claims (33, 34, 35, 36, 37, 38, 39, 40, 41, 57, 58)
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42. A method for screening a patient suspected of having a genetic disorder characterized by the presence or absence of a target repeat sequence comprising the steps of:
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a) amplifying a nucleic acid from a patient sample; b) hybridizing said nucleic acid to an array of probes wherein each probe comprises a 5'"'"'-region complementary to the 3'"'"'-region of said nucleic acid, a 3'"'"'-region complementary to the 5'"'"'-region of said nucleic acid and an internal variable region which is unique to each probe to form a hybridized array; c) digesting the hybridized array with a single-strand specific nuclease; d) treating said array with a nucleic acid polymerase; and e) identifying a probe containing an internal variable region complementary to the target repeat sequence and detecting the presence or absence of the genetic disorder. - View Dependent Claims (43, 44, 45, 46, 47)
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- 48. An array of at least 10 probes wherein each probe comprises an identical 5'"'"'-region, an identical 3'"'"'-region and a variable internal region wherein said variable region comprises one or more repeat sequences which is unique to each probe.
Specification