Nucleotide sequencing via repetitive single molecule hybridization

Abstract

Methods of obtaining sequence information about target oligonucleotides by repetitive single molecule hybridization are disclosed. The methods include exposing a target oligonucleotide to one or more copies of a test oligonucleotide; measuring hybridization; dehybridizing the test oligonucleotide; and repeating until the information content from the hybridization trials equals or exceeds the information content of the target oligonucleotide.

1 . A method of synthesizing de novo a nucleic acid sequence, the method comprising the steps of:
 a) synthesizing a nucleic acid sequence de novo;   b) obtaining information about the nucleic acid sequence by repetitive single molecule hybridization, thereby to detect an error in the nucleic acid sequence; and   c) correcting the error in the nucleic acid sequence by site-directed mutagenesis, thereby synthesizing de novo the nucleic acid sequence.   
     
     
         2 . The method of  claim 1 , wherein step b) is effected by:
 (a) hybridizing one or more copies of a test oligonucleotide comprising at least two informative region sequences to a single molecule of target oligonucleotides under conditions permitting hybridization of the test oligonucleotide to a perfectly complementary sequence;   (b) measuring a signal of said hybridization of the test oligonucleotide to the target oligonucleotide, thereby completing a first hybridization trial;   (c) dehybridizing the test oligonucleotide;   (d) exposing the target oligonucleotide to one or more copies of at least one additional test oligonucleotide, said additional test oligonucleotide comprising at least two informative regions;   (e) measuring a signal from the hybridization of the additional test oligonucleotide to the target oligonucleotide, thereby completing a further hybridization trial;   (f) repeating steps c) to e) until the information content from the hybridization trials exceeds the information content of the target oligonucleotide; and   (g) analyzing said signals.   
     
     
         3 . The method of  claim 2 , wherein said signal of said hybridization comprises a fluorescent tag. 
     
     
         4 . The method of  claim 2 , wherein said signal of said hybridization comprises a quantum dot or nanoparticle tag. 
     
     
         5 . The method of  claim 2 , wherein said measuring is effected mechanically. 
     
     
         6 . The method of  claim 2 , wherein said measuring is effected by sensing a change in charge associated with hybridization. 
     
     
         7 . The method of  claim 2 , wherein said measuring is effected electronically. 
     
     
         8 . The method of  claim 2 , wherein said target oligonucleotide is immobilized on a solid support. 
     
     
         9 . The method of  claim 2 , wherein a plurality of said target oligonucleotides are immobilized on a solid support. 
     
     
         10 . The method of  claim 1 , wherein step (b) is effected by:
 (a) hybridizing one or more copies of a test oligonucleotide to a single molecule of target oligonucleotides under conditions permitting hybridization of the test oligonucleotide to a perfectly complementary sequence;   (b) measuring a signal of said hybridization of the test oligonucleotide to the target oligonucleotide, thereby completing a first hybridization trial;   (c) dehybridizing the test oligonucleotide;   (d) exposing the target oligonucleotide to one or more copies of at least one additional test oligonucleotide;   (e) measuring a signal from the hybridization of the additional test oligonucleotide to the target oligonucleotide, thereby completing a further hybridization trial;   (f) repeating steps c) to e) until the information content from the hybridization trials exceeds the information content of the target oligonucleotide; and   (g) analyzing said signals; and   (h) analyzing an absence of said signals to eliminate oligonucleotide test members.   
     
     
         11 . The method of  claim 10 , wherein said signal of said hybridization comprises a fluorescent tag. 
     
     
         12 . The method of  claim 10 , wherein said signal of said hybridization comprises a quantum dot or nanoparticle tag. 
     
     
         13 . The method of  claim 10 , wherein said measuring is effected mechanically. 
     
     
         14 . The method of  claim 10 , wherein said measuring is effected by sensing a change in charge associated with hybridization. 
     
     
         15 . The method of  claim 10 , wherein said measuring is effected electronically. 
     
     
         16 . The method of  claim 10 , wherein said target oligonucleotide is immobilized on a solid support. 
     
     
         17 . The method of  claim 10 , wherein a plurality of said target oligonucleotides are immobilized on a solid support.