Methods for identifying nucleotides in target sequences

Abstract

The present invention is directed to methods and compositions for acquiring nucleotide sequence information of target sequences. In particular, the present invention provides methods and compositions for improving the efficiency of sequencing reactions by using fewer labels to distinguish between nucleotides and by detecting nucleotides at multiple detection positions in a target sequence.

1 .- 21 . (canceled) 
     
     
         22 . A method for obtaining sequence information for each of a plurality of different target sequences, comprising
 i) providing an array comprising single stranded polynucleotides immobilized at different locations on a support, wherein each of said single stranded polynucleotide comprises a target sequence, each target sequence comprises a detection position, each detection position is characterized by a first nucleotide at the detection position, and at a plurality of the different locations copies of a single target sequence are immobilized thereon;   ii) contacting the support and the immobilized single stranded polynucleotides at the plurality of different locations on the support with four different types of fluorescently labeled nucleotides, wherein the four different types of fluorescently labeled nucleotides comprise fluorescently labeled nucleotides with an adenine (A) nucleobase, fluorescently labeled nucleotides with a thymine (T) nucleobase, fluorescently labeled nucleotides with a cytosine (C) nucleobase, and fluorescently labeled nucleotides with a guanine (G) nucleobase,   wherein the contacting occurs under conditions in which, at individual locations of the plurality of different locations on the support, one of the four different types of fluorescently labeled nucleotides forms a base pair with the first nucleotide at the detection position of the target sequence at that location on the support, wherein base pairs are formed based on base-pairing complementarity such that A basepairs with T and G basepairs with C;   iii) illuminating the array to cause emission of light from fluorescent dyes associated with the four different types of fluorescently labeled nucleotides; and   iv) determining for each of the plurality of the locations on the support which one of the four different types of fluorescently labeled nucleotides is base-paired to the first nucleotide at the detection position of the target sequence at the location on the support,   wherein the step of determining comprises detecting
 (a) an absence, presence or brightness of emitted light detectable at a first wavelength from fluorescent dyes associated with the four different types of fluorescently labeled nucleotides, and 
 (b) an absence, presence or brightness of emitted light detectable at a second wavelength from fluorescent dyes associated with the four different types of fluorescently labeled nucleotides, wherein the second wavelength is different from the first wavelength, 
 wherein each of the four different types of fluorescently labeled nucleotides has a predefined and different pattern of light emission detectable at the first and second wavelength, 
   thereby obtaining sequence information for each of the plurality of different target sequences.   
     
     
         23 . A method for obtaining sequence information for each of a plurality of different target sequences, comprising
 i) providing an array comprising single stranded polynucleotides immobilized at different locations on a support, wherein each of said single stranded polynucleotide comprises a target sequence, each target sequence comprises a detection position, each detection position is characterized by a first nucleotide at the detection position, and at a plurality of the different locations copies of a single target sequence are immobilized thereon;   ii) contacting the support and the immobilized single stranded polynucleotides at the plurality of different locations on the support with four different types of fluorescently labeled probe sets each of which comprises a set of probes with a specific base at an interrogation position of the probes and degenerate bases at other positions of the probe, wherein the four different types of fluorescently labeled probe sets comprise fluorescently labeled probes with a nucleotide comprising an adenine (A) nucleobase at the interrogation position, fluorescently labeled probes with a nucleotide comprising thymine (T) nucleobase at the interrogation position, fluorescently labeled probes sets with a nucleotide comprising cytosine (C) nucleobase at the interrogation position, and fluorescently labeled probe sets with a nucleotide comprising guanine (G) nucleobase at the interrogation position,   wherein the contacting occurs under conditions in which, at individual positions of the plurality of different positions on the support, probes from one of the four different types of fluorescently labeled probe sets is perfectly complementary to a sequence of the target sequence such that hybridization of a portion of the target sequence and the probes occur and the nucleotide at the interrogation position forms a base pair with the first nucleotide at the detection position of the target sequence at that location on the support, wherein base pairs are formed based on base-pairing complementarity such that A basepairs with T and G basepairs with C;   iii) illuminating the array to cause emission of light from fluorescent dyes associated with the four different types of fluorescently labeled probe sets;   iv) determining for each of the plurality of the locations on the support which one of the four different types of fluorescently labeled probe sets comprises nucleotides at the interrogation position base-paired to the first nucleotide at the detection position of the target sequence at the location on the support,   wherein the step of determining comprises detecting
 (a) an absence, presence or brightness of emitted light detectable at a first wavelength from fluorescent dyes associated with the four different types of fluorescently labeled probe sets, and 
 (b) an absence, presence or brightness of emitted light detectable at a second wavelength from fluorescent dyes associated with the four different types of fluorescently labeled probe sets, wherein the second wavelength is different from the first wavelength, and 
   wherein each of the four different types of fluorescently labeled probe sets has a predefined and different pattern of light emission detectable at the first and second wavelength,   thereby obtaining sequence information for each of the plurality of different target sequences.