MICROARRAY SYSTEM WITH IMPROVED SEQUENCE SPECIFICITY
First Claim
1. A method of preparing a detectably labeled single-stranded polynucleotide target from a double stranded DNA comprising the detectably labeled polynucleotide target hybridized to a complementary polynucleotide comprising:
- contacting the double stranded DNA with a 5′
to 3′
exonuclease under suitable conditions to degrade at least a portion of the complementary polynucleotide to form a detectably labeled single-stranded polynucleotide target, the polynucleotide target comprising at least one of;
(1) a cyanine dye moiety positioned between the second and third nucleotides from the 5′
end of the polynucleotide target;
or(2) a cyanine dye moiety positioned between first and second nucleotides from the 5′
end of the polynucleotide target and having a modified linkage between the second and third nucleotides from the 5′
end of the polynucleotide target;
or(3) a cyanine dye moiety attached at the 5′
end of the polynucleotide target, with the complementary strand modified with;
(a) a 5′
-phosphate group;
or(b) a primary amine with an aliphatic linker arm connected to the polynucleotide via a phosphate linkage and the first 5′
-residue of said polynucleotide being an A or a T base;
or(4) a dye moiety attached at the 5′
end of the polynucleotide target and the first 5′
-residue of said polynucleotide being a G or C base, with the complementary strand modified with;
(a) a 5′
-phosphate group;
or(b) a primary amine with an aliphatic linker arm connected to the polynucleotide via a phosphate linkage and the first 5′
-residue of said polynucleotide being an A or a T base.
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Abstract
The invention provides a novel array method for nucleic acid sequence detection with improved specificity which allows for detection of genetic variation, from simple SNPs (where the variation occurs at a fixed position and is of limited allelic number) to more complex sequence variation patterns (such as with multigene families or multiple genetic strains of an organism where the sequence variation between the individual members is neither fixed nor consistent). The array is comprised of short, synthetic oligonucleotide probes attached to a solid surface which are hybridized to single-stranded targets. Single stranded targets can be produced using a method that employs primers modified on the 5′ end to prohibit degradation by a 5′-exonuclease that is introduced to degrade the unprotected strand. The invention further provides for printing buffers/solutions for the immobilization of oligonucleotide probes to an array surface. The invention also provides hybridization and wash buffers and conditions to maximize hybridization specificity and signal intensity, and reduce hybridization times.
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Citations
42 Claims
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1. A method of preparing a detectably labeled single-stranded polynucleotide target from a double stranded DNA comprising the detectably labeled polynucleotide target hybridized to a complementary polynucleotide comprising:
contacting the double stranded DNA with a 5′
to 3′
exonuclease under suitable conditions to degrade at least a portion of the complementary polynucleotide to form a detectably labeled single-stranded polynucleotide target, the polynucleotide target comprising at least one of;(1) a cyanine dye moiety positioned between the second and third nucleotides from the 5′
end of the polynucleotide target;
or(2) a cyanine dye moiety positioned between first and second nucleotides from the 5′
end of the polynucleotide target and having a modified linkage between the second and third nucleotides from the 5′
end of the polynucleotide target;
or(3) a cyanine dye moiety attached at the 5′
end of the polynucleotide target, with the complementary strand modified with;(a) a 5′
-phosphate group;
or(b) a primary amine with an aliphatic linker arm connected to the polynucleotide via a phosphate linkage and the first 5′
-residue of said polynucleotide being an A or a T base;
or(4) a dye moiety attached at the 5′
end of the polynucleotide target and the first 5′
-residue of said polynucleotide being a G or C base, with the complementary strand modified with;(a) a 5′
-phosphate group;
or(b) a primary amine with an aliphatic linker arm connected to the polynucleotide via a phosphate linkage and the first 5′
-residue of said polynucleotide being an A or a T base.- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42)
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9. A method of generating a double stranded DNA comprising a detectably labeled polynucleotide target hybridized to a complementary polynucleotide, the detectably labeled the polynucleotide target comprising at least one of the following modifications:
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(1) a cyanine dye moiety positioned between the second and third nucleotides from the 5′
end of the polynucleotide target;
or(2) a cyanine dye moiety positioned between first and second nucleotides from the 5′
end of the polynucleotide target and having a modified linkage between the second and third nucleotides from the 5′
end of the polynucleotide target;
or(3) a cyanine dye moiety attached at the 5′
end of the polynucleotide target, with the complementary strand modified with;(a) a 5′
-phosphate group;
or(b) a primary amine with an aliphatic linker arm connected to the polynucleotide via a phosphate linkage and the first 5′
-residue of said polynucleotide being an A or a T base;
or(4) a dye moiety attached at the 5′
end of the polynucleotide target and the first 5′
-residue of said polynucleotide being a G or C base, with the complementary strand modified with;(a) a 5′
-phosphate group;
or(b) a primary amine with an aliphatic linker arm connected to the polynucleotide via a phosphate linkage and the first 5′
-residue of said polynucleotide being an A or a T base;the method comprising; amplifying the polynucleotide target and the complementary polynucleotide from a sample comprising the polynucleotide target and complementary polynucleotides using oligonucleotide primers comprising the modification or modifications. - View Dependent Claims (10, 11, 12, 13, 14, 15)
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16. An array comprising a surface comprising epoxide moieties and a plurality of oligonucleotides each comprising a 5′
- hydrazide linker attached to the surface of the array through a bond formed between the linker and an epoxide moiety.
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17. A method of forming a microarray on a surface comprising epoxide moieties, comprising:
depositing a plurality of samples onto the surface in discrete domains, each sample comprising a spotting buffer and an oligonucleotide comprising a 5′
hydrazide linker, under conditions that allow formation of a bond between the hydrazide linker and the epoxide moiety.- View Dependent Claims (18, 19, 20)
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21. The method of 20, wherein the pH is in the range of from about 4.5 to about 5.5.
- 22. A buffer having a pH in the range of from about 4.0 to about 8.0 and comprising sodium phosphate (monobasic) in a concentration in the range of from about 1 mM to about 1M, an ethylene oxide based nonionic detergent in a concentration in the range of from about 0.001% to about 1% (v/v), and ethylene glycol in a concentration in the range of from about 10% to about 90% (v/v).
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26. A method of detecting the presence of a specific nucleic acid sequence within a pool of detectably labeled target polynucleotides by hybridization to one or more oligonucleotide probes attached to a support comprising:
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contacting the support with the polynucleotide under high stringency conditions in a buffer comprising a tertiary alkyl ammonium salt and formamide for a period of about 6 hours or less; removing unhybridized target polynucleotides from the support by washing the support under conditions similar to those used during the hybridization for a period about 30 minutes or less; and detecting the presence or absence of labeled target polynucleotides on the washed support. - View Dependent Claims (27, 28, 29)
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Specification