Simultaneous sequencing of tagged polynucleotides

Claim 1

A method for simultaneously determining the nucleotide sequences of a population of polynucleotides, the method comprising the steps of: (a) attaching an oligonucleotide tag from a repertoire of tags to each polynucleotide of the population to form tag-polynucleotide conjugates such that substantially all different polynucleotides have different oligonucleotide tags attached; (b) selectively amplifying tag-polynucleotide conjugates with primers whose 3' ends form perfectly matched duplexes with one or more terminal nucleotides of the polynucleotides of the population; (c) labeling each tag of the selectively amplified tag-polynucleotide conjugates according to the identity of the one or more terminal nucleotides of its associated polynucleotide; (d) cleaving the tags from the selectively amplified tag-polynucleotide conjugates; and (e) sorting the labeled tags onto a spatially addressable array of tag complements for detection of the labeled tags and identification of the one or more nucleotides of each polynucleotide.

Claim 2

A method for simultaneously determining the nucleotide sequences of a population of polynucleotides, the method comprising the steps of: (a) attaching an oligonucleotide tag from a repertoire of tags to each polynucleotide of the population to form tag-polynucleotide conjugates such that substantially all different polynucleotides have different oligonucleotide tags attached; (b) selectively amplifying tag-polynucleotide conjugates with primers whose 3' ends form perfectly matched duplexes with one or more terminal nucleotides of the polynucleotides of the population; (c) labeling each tag of the selectively amplified tag-polynucleotide conjugates according to the identity of the one or more terminal nucleotides of its associated polynucleotide; (d) cleaving the tags from the selectively amplified tag-polynucleotide conjugates; and (e) sorting the labeled tags onto a spatially addressable array of tag complements for detection of the labeled tags and identification of the one or more nucleotides of each polynucleotide.

Claim 5

A method for simultaneously determining the nucleotide sequences of a population of polynucleotides, the method comprising the steps of: (a) attaching an oligonucleotide tag from a repertoire of tags to each polynucleotide of the population to form tag-polynucleotide conjugates such that substantially all different polynucleotides have different oligonucleotide tags attached; (b) amplifying the tag-polynucleotide conjugates by a polymerase chain reaction using a first primer and a second primer, the second primer having a 3' terminal nucleotide and forming a duplex with a primer binding site and one or more nucleotides at one end of the tag-polynucleotide conjugate, such that a tag-polynucleotide conjugate is amplified only if the defined 3' terminal nucleotide basepairs with a nucleotide of the tag-polynucleotide conjugate; (c) cleaving the tags from the tag-polynucleotide conjugates; (d) labeling the tags according to the identity of the defined 3' terminal nucleotide of the second primer; and (e) sorting the labeled tags onto a spatially addressable array of tag complements for detection of the labeled tags and identification of the one or more nucleotides of each polynucleotide.

Claim 6

A method for simultaneously determining the nucleotide sequences of a population of polynucleotides, the method comprising the steps of: (a) attaching an oligonucleotide tag from a repertoire of tags to each polynucleotide of the population to form tag-polynucleotide conjugates such that substantially all different polynucleotides have different oligonucleotide tags attached; (b) amplifying the tag-polynucleotide conjugates by a polymerase chain reaction using a first primer and a second primer, the second primer having a 3' terminal nucleotide and forming a duplex with a primer binding site and one or more nucleotides at one end of the tag-polynucleotide conjugate, such that a tag-polynucleotide conjugate is amplified only if the defined 3' terminal nucleotide basepairs with a nucleotide of the tag-polynucleotide conjugate; (c) cleaving the tags from the tag-polynucleotide conjugates; (d) labeling the tags according to the identity of the defined 3' terminal nucleotide of the second primer; and (e) sorting the labeled tags onto a spatially addressable array of tag complements for detection of the labeled tags and identification of the one or more nucleotides of each polynucleotide.

Claim 12

A method for simultaneously determining the nucleotide sequences of a population of polynucleotides, the method comprising the steps of: (a) attaching an oligonucleotide tag from a repertoire of tags to each polynucleotide of the population; (b) sampling the population of polynucleotides such that substantially all different polynucleotides in the population have different oligonucleotide tags attached; (c) amplifying the sampled polynucleotides by a polymerase chain reaction using a first primer and a second primer, the second primer having a 3' terminal nucleotide and forming a duplex with a primer binding site and one or more nucleotides at one end of the polynucleotide, such that a polynucleotide is amplified only if the defined 3' terminal nucleotide basepairs with a nucleotide of the polynucleotide; (d) copying the tags from the polynucleotides; (e) labeling the copied tags according to the identity of the defined 3' terminal nucleotide of the second primer; and (f) sorting the labeled tags onto a spatially addressable array of tag complements for detection of the labeled tags and identification of the one or more nucleotides of each polynucleotide.

Claim 13

A method for simultaneously determining the nucleotide sequences of a population of polynucleotides, the method comprising the steps of: (a) attaching an oligonucleotide tag from a repertoire of tags to each polynucleotide of the population; (b) sampling the population of polynucleotides such that substantially all different polynucleotides in the population have different oligonucleotide tags attached; (c) amplifying the sampled polynucleotides by a polymerase chain reaction using a first primer and a second primer, the second primer having a 3' terminal nucleotide and forming a duplex with a primer binding site and one or more nucleotides at one end of the polynucleotide, such that a polynucleotide is amplified only if the defined 3' terminal nucleotide basepairs with a nucleotide of the polynucleotide; (d) copying the tags from the polynucleotides; (e) labeling the copied tags according to the identity of the defined 3' terminal nucleotide of the second primer; and (f) sorting the labeled tags onto a spatially addressable array of tag complements for detection of the labeled tags and identification of the one or more nucleotides of each polynucleotide.