Massively parallel sequencing of sorted polynucleotides
First Claim
1. A method for determining the nucleotide sequence of a target polynucleotide, the method comprising the steps of:
- generating from the target polynucleotide a plurality of fragments that cover the target polynucleotide;
attaching an oligonucleotide tag from a repertoire of tags to each fragment of the plurality (i) such that substantially all different fragments have different oligonucleotide tags attached and (ii) such that each oligonucleotide tag from the repertoire comprises a plurality of subunits and each subunit of the plurality consists of an oligonucleotide having a length from three to six nucleotides or from three to six basepairs, the subunits being selected from a minimally cross-hybridizing set wherein a subunit of the set and a complement of any other subunit of the set would have at least two mismatches;
sorting the fragments by specifically hybridizing the oligonucleotide tags with their respective complements, the respective complements being attached as uniform populations of substantially identical complements in spatially discrete regions on one or more solid phase supports and the respective complements being oligodeoxyribonucleotide N3'"'"'→
P5'"'"' phosphoramidates or peptide nucleic acids;
determining the nucleotide sequence of a portion of each of the fragments of the plurality; and
determining the nucleotide sequence of the target polynucleotide by collating the sequences of the fragments.
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Abstract
The invention provides a method and materials for sorting polynucleotides with oligonucleotide tags. Oligonucleotide tags of the invention are capable of hybridizing to complementary oligomeric compounds consisting of subunits having enhanced binding strength and specificity as compared to natural oligonucleotides. Such complementary oligomeric compounds are referred to herein as "tag complements." Subunits of tag complements may consist of monomers of non-natural nucleotide analogs, referred to herein as "antisense monomers" or they may comprise oligomers having lengths in the range of 3 to 6 nucleotides or analogs thereof, including antisense monomers, the oligomers being selected from a minimally cross-hybridizing set. In such a set, a duplex made up of an oligomer of the set and the complement of any other oligomer of the set contains at least two mismatches. Preferred antisense monomers include peptide nucleic acid monomers and nucleoside phosphoramidates having a 3'"'"'-NHP(O)(O--)O-5'"'"' linkage with its adjacent nucleoside. An important aspect of the invention is the use of the oligonucleotide tags for sorting polynucleotides by specifically hybridizing tags attached to the polynucleotides to their complements on solid phase supports. This embodiment provides a readily automated system for manipulating and sorting polynucleotides, particularly useful in large-scale parallel operations, such as large-scale DNA sequencing, mRNA fingerprinting, or the like, wherein many target polynucleotides or many segments of a single target polynucleotide are sequenced simultaneously.
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Citations
23 Claims
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1. A method for determining the nucleotide sequence of a target polynucleotide, the method comprising the steps of:
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generating from the target polynucleotide a plurality of fragments that cover the target polynucleotide; attaching an oligonucleotide tag from a repertoire of tags to each fragment of the plurality (i) such that substantially all different fragments have different oligonucleotide tags attached and (ii) such that each oligonucleotide tag from the repertoire comprises a plurality of subunits and each subunit of the plurality consists of an oligonucleotide having a length from three to six nucleotides or from three to six basepairs, the subunits being selected from a minimally cross-hybridizing set wherein a subunit of the set and a complement of any other subunit of the set would have at least two mismatches; sorting the fragments by specifically hybridizing the oligonucleotide tags with their respective complements, the respective complements being attached as uniform populations of substantially identical complements in spatially discrete regions on one or more solid phase supports and the respective complements being oligodeoxyribonucleotide N3'"'"'→
P5'"'"' phosphoramidates or peptide nucleic acids;determining the nucleotide sequence of a portion of each of the fragments of the plurality; and determining the nucleotide sequence of the target polynucleotide by collating the sequences of the fragments. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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12. A method for determining the nucleotide sequence of a target polynucleotide, the method comprising the steps of:
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generating from the target polynucleotide a plurality of fragments that cover the target polynucleotide; attaching an oligonucleotide tag from a repertoire of tags to each fragment of the plurality such that substantially all different fragments have different oligonucleotide tags attached; sorting the fragments by specifically hybridizing the oligonucleotide tags with their respective complements, the respective complements being attached as uniform populations of substantially identical complements in spatially discrete regions on one or more solid phase supports and the respective complements being oligodeoxyribonucleotide N3'"'"'→
P5'"'"' phosphoramidates or peptide nucleic acids;determining the nucleotide sequence of a portion of each of the fragments of the plurality; and determining the nucleotide sequence of the target polynucleotide by collating the sequences of the fragments. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)
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Specification