Enhanced sequencing by hybridization using pools of probes
First Claim
1. A nucleic acid assay comprising the steps of contacting a nucleic acid target with a set of probes of known informative sequence arranged in a plurality of probe pools, wherein probes with minimal relatedness in the informative sequence are included in the same pool and detecting pools with a probe that hybridizes to the target.
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Accused Products
Abstract
The invention provides methods for sequencing by hybridization (SBH) using pools of probes that allow greater efficiency in conducting SBH by reducing the number of separate measurements of hybridization signals required to identify each particular nucleotide in a target nucleic acid sequence. The invention also provides pools and sets of pools of probes, as well as methods of generating pools of probes.
10 Citations
26 Claims
- 1. A nucleic acid assay comprising the steps of contacting a nucleic acid target with a set of probes of known informative sequence arranged in a plurality of probe pools, wherein probes with minimal relatedness in the informative sequence are included in the same pool and detecting pools with a probe that hybridizes to the target.
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6. A method of analyzing a target nucleic acid sequence comprising a) providing a set of individually prepared oligonucleotides of a given number of informative bases, wherein at least two probes that differ at more than one position and that are either not labeled or labeled with the same label are pooled wherein each probe is present only in one pool reducing the number of measurements in testing the said set of oligonucleotides compared to testing each oligonucleotide individually;
- and b) hybridizing said pools to a target nucleic acid attached to a support or in solution thus analyzing some target bases multiple times with a number of oligonucleotide close or equal to said number of informative bases.
- View Dependent Claims (7, 8, 9, 10)
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11. A method of analyzing a target nucleic acid sequence comprising the steps of a) providing a set of oligonucleotides of a given number of informative bases;
- wherein at least two probes that differ at more than one position and that are either not labeled or labeled with the same label are synthesized together as a pool reducing the number of measurements in testing the said set of oligonucleotides compared to testing each oligonucleotide individually; and
b) hybridizing said pools to a target nucleic acid thus analyzing some target bases multiple times with a number of oligonucleotide close or equal to said number of informative bases. - View Dependent Claims (12, 13, 14)
- wherein at least two probes that differ at more than one position and that are either not labeled or labeled with the same label are synthesized together as a pool reducing the number of measurements in testing the said set of oligonucleotides compared to testing each oligonucleotide individually; and
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15. A method of detecting one or more sequences in a nucleic acid target comprising:
- performing ligation of two sets of oligonucleotides of known informative sequence using a target nucleic acid as template, wherein at least one of said oligonucleotide sets is in the form of a predefined number of pools, at least some pools containing two or more oligonucleotides having different informative sequences and the same label, wherein none, some or all of said pools are combined if oligonucleotides in different pools are labeled with different labels, and comparing ligation results of at least some of said pools.
- View Dependent Claims (16, 17, 18, 19, 20, 21, 22)
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23. A method of detecting one or more sequences in a plurality of nucleic acid targets comprising:
- performing ligation of a set of oligonucleotide pools to oligonucleotide molecules present in support bound hybridization complexes of said oligonucleotide molecules and said nucleic acid targets, wherein each oligonucleotide pool contains two or more oligonucleotides not labeled or labeled with the same label and having different known informative sequence, and wherein none, some or all of said oligonucleotide pools are combined if oligonucleotides in different pools are labeled with different labels, and comparing ligation results of at least some of said pools.
- View Dependent Claims (24, 25, 26)
Specification