Methods for nucleotide sequencing and high fidelity polynucleotide synthesis

Abstract

Methods of obtaining sequence information about target polynucleotide having a predefined sequence are disclosed. The methods include sequencing by ligation and sequencing by polymerase.

1 .- 20 . (canceled) 
     
     
         21 . A method for identifying a target polynucleotide having a predefined sequence, the method comprising:
 a. providing a support comprising a plurality of anchor oligonucleotides, wherein each plurality of anchor oligonucleotides comprises a terminal region having a predefined sequence complementary to a predetermined region of a target polynucleotide having a predefined sequence;   b. contacting the anchor oligonucleotides with the target polynucleotide under conditions suitable to hybridize the predetermined region of the target polynucleotide to the terminal region of the anchor oligonucleotides;   c. contacting the target polynucleotide with a plurality of sequencing oligonucleotide probes, wherein each plurality of oligonucleotide probes comprises an interrogating RNA nucleotide at an interrogation position and a label;   d. ligating the plurality of sequencing oligonucleotide probes to the plurality of anchor oligonucleotides;   e. detecting the presence or absence of the label wherein the presence of label is correlated to a polynucleotide sequence having the predefined sequence;   f. cleaving the plurality of sequencing oligonucleotide 3′ to the RNA base;   g. repeating steps c) through f) if the presence of the label is detected, until the target polynucleotide having predefined sequence is identified.   
     
     
         22 . The method of  claim 21  wherein the RNA base is adjacent to the 3′ end nucleotide of the plurality of sequencing oligonucleotides and cleaving the plurality of sequencing oligonucleotide 3′ to the RNA base such as the anchor oligonucleotides are extended by a single nucleotide. 
     
     
         23 . The method of  claim 21  wherein the RNA base is two bases upstream of the 3′ end nucleotide of the plurality of sequencing oligonucleotides, and cleaving the plurality of sequencing oligonucleotide 3′ to the RNA base such as the anchor oligonucleotides are extended by a dinucleotide. 
     
     
         24 . The method of  claim 23  wherein the 3′ end nucleotide is an interrogating nucleotide. 
     
     
         25 . A method for identifying a target polynucleotide having a predefined sequence, the method comprising:
 a. providing a support comprising a plurality of anchor oligonucleotides, wherein each plurality of anchor oligonucleotides comprises a terminal region having a predefined sequence complementary to a predetermined region of a target polynucleotide having a predefined sequence;   b. contacting the anchor oligonucleotides with the target polynucleotide under conditions suitable to hybridize the predetermined region of the target polynucleotide to the terminal region of the anchor oligonucleotides;   c. contacting the target polynucleotide with a plurality of sequencing oligonucleotide probes, wherein each plurality of oligonucleotide probes comprises a single RNA nucleotide at the 3′end of the plurality of sequencing oligonucleotide probes and a label;   d. ligating the plurality of sequencing oligonucleotide probes to the plurality of anchor oligonucleotides;   e. detecting the absence of the label wherein the absence of label is correlated to a polynucleotide sequence not having the predefined sequence; and   f. cleaving the plurality of sequencing oligonucleotides 3′ to the RNA base, thereby regenerating the support comprising the plurality of anchor oligonucleotides.   
     
     
         26 . A method for identifying a target polynucleotide having a predefined sequence, the method comprising:
 a. providing a support comprising a plurality of anchor oligonucleotides, wherein each plurality of anchor oligonucleotides comprises a terminal region having a predefined sequence complementary to a predetermined region of a target polynucleotide having a predefined sequence;   b. contacting the anchor oligonucleotides with the target polynucleotide under conditions suitable to hybridize the predetermined region of the target polynucleotide to the terminal region of the anchor oligonucleotides thereby forming at least one target polynucleotide-anchor oligonucleotide duplex;   c. contacting the at least one target polynucleotide-anchor oligonucleotide duplex with at least one labeled nucleotide;   d. extending the anchor oligonucleotide in a template-dependent manner in presence of a polymerase; and   e. detecting the incorporation of the labeled nucleotide into the at least one target polynucleotide-anchor oligonucleotide duplex.   
     
     
         27 . The method of  claim 26  optionally repeating steps c), d) and e). 
     
     
         28 . The method of  claim 26  wherein the at least one target polynucleotide-anchor oligonucleotide duplex is contacted with four different labeled nucleotides and wherein each nucleotide is differently labeled. 
     
     
         29 . The method of  claim 26  wherein each plurality of anchor oligonucleotides has a different length and differs from each other from at least one additional predefined nucleotide. 
     
     
         30 . The method of  claim 26  wherein each plurality of anchor oligonucleotides comprises at least three degenerate bases and differs from each other from at least one additional predefined nucleotide. 
     
     
         31 . The method of  claim 26  wherein the target polynucleotide comprises a first primer binding site at its 5′ end and a second primer binding site at its 3′ end. 
     
     
         32 . The method of  claim 26  wherein the target polynucleotide is first amplified before being hybridized to the plurality of anchor oligonucleotides. 
     
     
         33 . The method of  claim 26  wherein the support comprises a plurality of discrete features and wherein each plurality of anchor oligonucleotide is immobilized on a distinct discrete feature. 
     
     
         34 . A support comprising a plurality of oligonucleotides, wherein each plurality of oligonucleotides is immobilized at a distinct feature of the support, wherein each plurality of oligonucleotides has a different length and comprises a sequence having a terminal region having a predefined sequence, the predefined sequence being complementary to a predetermined region of a target polynucleotide. 
     
     
         35 . The method of  claim 21 , wherein in step (a), the plurality of anchor oligonucleotides include a descending staircase of anchor oligonucleotides. 
     
     
         36 . The method of  claim 25 , wherein in step (a), the plurality of anchor oligonucleotides include a descending staircase of anchor oligonucleotides.