Methods for sorting polynucleotides using oligonucleotide tags
First Claim
1. A method of sorting polynucleotides from a population of polynucleotides onto one or more solid phase supports, the method comprising the steps of:
- (a) attaching an oligonucleotide tag from a repertoire of tags to each polynucleotide in a population of polynucleotides (i) such that substantially all different polynucleotides in the population 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 and(b) sorting the polynucleotides from the population by specifically hybridizing the oligonucleotide tags with their respective complements, the respective complements being attached as uniform populations of substantially identical complements in spacially discrete regions on the one or more solid phase supports.
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Abstract
The invention provides a method of tracking, identifying, and/or sorting classes or subpopulations of molecules by the use of oligonucleotide tags. Oligonucleotide tags of the invention each consist of a plurality of subunits 3 to 6 nucleotides in length selected from a minimally cross-hybridizing set. A subunit of a minimally cross-hybridizing set forms a duplex or triplex having two or more mismatches with the complement of any other subunit of the same set. The number of oligonucleotide tags available in a particular embodiment depends on the number of subunits per tag and on the length of the subunit. 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, and the like, wherein many target polynucleotides or many segments of a single target polynucleotide are sequenced simultaneously.
722 Citations
31 Claims
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1. A method of sorting polynucleotides from a population of polynucleotides onto one or more solid phase supports, the method comprising the steps of:
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(a) attaching an oligonucleotide tag from a repertoire of tags to each polynucleotide in a population of polynucleotides (i) such that substantially all different polynucleotides in the population 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 and (b) sorting the polynucleotides from the population by specifically hybridizing the oligonucleotide tags with their respective complements, the respective complements being attached as uniform populations of substantially identical complements in spacially discrete regions on the one or more solid phase supports. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. 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; 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 (12, 13, 14, 15, 16, 17, 18, 19, 20)
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21. A method of identifying a population of mRNA molecules, the method comprising the steps of:
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forming a population of cDNA molecules from the population of mRNA molecules, the cDNA molecules being complementary to the mRNA molecules and each cDNA molecule having an oligonucleotide tag attached, (i) such that substantially all different cDNA molecules have different oligonucleotide tags attached and (ii) such that each oligonucleotide tag comprises a plurality of subunits and each subunit of the plurality consists of an oligonucleotide having a length from three to six nucleotides, 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 cDNA molecules 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; determining the nucleotide sequence of a portion of each of the sorted cDNA molecules; and identifying the population of mRNA molecules by the frequency distribution of the portions of sequences of the cDNA molecules. - View Dependent Claims (22, 23, 24)
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25. A method of sorting a mixture of polynucleotides, the method comprising the steps of:
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(a) providing a solution containing a mixture of polynucleotides, each polynucleotide of the mixture having attached an oligonucleotide tag from a repertoire of tags (i) such that substantially all polynucleotides of different sequences 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; and (b) contacting the solution with one or more solid phase supports having attached thereto the complements of the oligonucleotide tags under conditions that promote the formation of perfectly matched duplexes between the oligonucleotide tags and their respective complements. - View Dependent Claims (26, 27, 28, 29, 30, 31)
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Specification