US5604097A — Methods for sorting polynucleotides using oligonu…

Claim 1

Independent Claim 168 words

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.

Claim 2

Independent Claim 168 words

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.

Claim 3

Dependent Claim 27 words

The method of claim 1 wherein said solid phase supports are a plurality of microparticles, each having attached thereto a uniform population of one of said complements.

Claim 4

Dependent Claim 16 words

The method of claim 2 wherein said oligonucleotide tag and said complement are single stranded oligonucleotides.

Claim 5

Dependent Claim 24 words

The method of claim 3 wherein said polynucleotide or subpopulation of polynucleotides have a length in the range of from 50 to 5000 nucleotides.

Claim 6

Dependent Claim 22 words

The method of claim 3 wherein said microparticle is selected from the group consisting of glass microparticles, magnetic beads, and polystyrene microparticles.

Claim 7

Dependent Claim 22 words

The method of claim 1 wherein said solid phase support is a planar substrate having a plurality of spacially discrete surface regions.

Claim 8

Dependent Claim 21 words

The method of claim 6 wherein different said spacially discrete surface regions have different uniform populations of substantially identical said complements.

Claim 9

Dependent Claim 11 words

The method of claim 7 wherein said planar substrate is glass.

Claim 10

Dependent Claim 11 words

The method of claim 7 wherein said planar substrate is silicon.

Claim 11

Dependent Claim 11 words

The method of claim 7 wherein said planar substrate is plastic.

Claim 12

Independent Claim 195 words

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; 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.

Claim 13

Independent Claim 195 words

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; 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.

Claim 14

Dependent Claim 16 words

The method of claim 11 wherein said oligonucleotide tags and said complements are single stranded oligonucleotides.

Claim 15

Dependent Claim 18 words

The method of claim 12 wherein said step of generating produces randomly overlapping fragments of said target polynucleotide.

Claim 16

Dependent Claim 31 words

The method of claim 12 wherein said step of determining said nucleotide sequence of said fragments are carried out simultaneously for said plurality of fragments by a single base sequencing method.

Claim 17

Dependent Claim 19 words

The method of claim 14 wherein said portions of each of said fragments includes from 12 to 50 nucleotides.

Claim 18

Dependent Claim 19 words

The method of claim 15 wherein said portion of each of said fragments includes from 12 to 25 nucleotides.

Claim 19

Dependent Claim 17 words

The method of claim 16 wherein said target polynucleotide is between one and fifty kilobases in length.

Claim 20

Dependent Claim 15 words

The method of claim 14 wherein said solid phase supports are a plurality of microparticles.

Claim 21

Dependent Claim 21 words

The method of claim 18 wherein after said step of sorting said plurality of microparticles are fixed to a planar substrate.

Claim 22

Dependent Claim 36 words

The method of claim 19 wherein said plurality of microparticles are disposed randomly on the surface of said planar substrate at a density of between about 1000 microparticles to about 100 thousand microparticles per square centimeter.

Claim 23

Independent Claim 190 words

A method of identifying a population of mRNA molecules, the method comprising the steps of: 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.

Claim 24

Independent Claim 190 words

A method of identifying a population of mRNA molecules, the method comprising the steps of: 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.

Claim 25

Dependent Claim 34 words

The method of claim 21 wherein said solid phase supports are a plurality of microparticles and wherein a uniform population of one of said complements is attached to each of said plurality of microparticles.

Claim 26

Dependent Claim 22 words

The method of claim 22 wherein said portion of said cDNA molecules is in the range of from 12 to 50 nucleotides.

Claim 27

Dependent Claim 22 words

The method of claim 23 wherein said portion of said cDNA molecules is in the range of from 12 to 25 nucleotides.

Claim 28

Independent Claim 161 words

A method of sorting a mixture of polynucleotides, the method comprising the steps of: (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.

Claim 29

Independent Claim 161 words

A method of sorting a mixture of polynucleotides, the method comprising the steps of: (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.

Claim 30

Dependent Claim 27 words

The method of claim 25 wherein said solid phase supports are a plurality of microparticles, each having attached thereto a uniform population of substantially identical said complements.

Claim 31

Dependent Claim 16 words

The method of claim 26 wherein said oligonucleotide tag and said complement are single stranded oligonucleotides.

Claim 32

Dependent Claim 19 words

The method of claim 27 wherein said polynucleotides have lengths in the range of from 50 to 5000 nucleotides.

Claim 33

Dependent Claim 22 words

The method of claim 28 wherein said microparticle is selected from the group consisting of glass microparticles, magnetic beads, and polystyrene microparticles.

Claim 34

Dependent Claim 32 words

The method of claim 25 wherein said solid phase support is a planar substrate having a plurality of spacially discrete surface regions having uniform populations of substantially identical said complements attached thereto.

Claim 35

Dependent Claim 21 words

The method of claim 30 wherein different said spacially discrete surface regions have different uniform populations of substantially identical said complements.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	2000-02-18	2000-08-19	2001-02-18	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	2004-02-18	2004-08-19	2005-02-18	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2008-02-18	2008-08-19	2009-02-18	$7700.00	Window Closed / Lapsed

Methods for sorting polynucleotides using oligonucleotide tags

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