Method for sequencing nucleic acid molecules

Abstract

The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

1 - 61 . (canceled) 
     
     
         62 . A method of sequencing a target nucleic acid molecule, comprising:
 subjecting a target nucleic acid molecule that is attached to a support to a polymerization reaction whereby a first polymerase enzyme forms a complex with the target nucleic acid molecule, and polymerization occurs to yield a growing nucleic acid strand complementary to the target nucleic acid molecule in the presence of a plurality of types of nucleotides or nucleotide analogs; and detecting a time sequence of incorporation of said plurality of types of nucleotides or nucleotide analogs into the growing nucleotide strand at a polymerase enzyme active site,   wherein the first polymerase enzyme dissociates from the target nucleic acid during the polymerization reaction, and a second polymerase enzyme forms a complex with the target nucleic acid, whereby the optically detecting of said time sequence of incorporation continues, thereby sequencing said target nucleic acid.   
     
     
         63 . The method according to  claim 62 , wherein the polymerization reaction is performed by a nucleic acid polymerizing enzyme selected from the group consisting of a DNA polymerase, an RNA polymerase, a reverse transcriptase, and a mixture thereof 
     
     
         64 . The method according to  claim 62 , wherein the target nucleic acid molecule is selected from the group consisting of double-stranded DNA, single-stranded DNA, single stranded DNA hairpins, DNA/RNA hybrids, RNA with a recognition site for binding of the polymerase, and RNA hairpins. 
     
     
         65 . The method according to  claim 62 , wherein the nucleotide analogs are selected from the group consisting of a ribonucleotide, a deoxyribonucleotide, a modified ribonulcleotide, a modified deoxyribonucleotide, a piptide nucleotide, a modified peptide nucleotide, and a modified phosphate-sugar backbone nucleotide. 
     
     
         66 . The method according to  claim 62 , wherein the nucleotides or nucleotide analogs further comprise a label. 
     
     
         67 . The method according to  claim 66 , wherein the label is selected from the group consisting of chromophores, fluorescent moieties, enzymes, antigens, dyes, phosphorescent groups, radioactive materials, chemiluminescent moieties, scattering or fluorescent nanoparticles, and Raman signal generating moieties. 
     
     
         68 . The method according to  claim 66 , wherein the label is attached to the nucleotides or nucleotide analogs at their base, sugar moiety, alpha phosphate, beta phosphate, or gamma phosphate groups. 
     
     
         69 . The method according to  claim 66 , wherein each of the plurality of types of nucleotides or nucleotide analogs have different labels which are distinguished from one another. 
     
     
         70 . The method according to  claim 66 , wherein each of the three or less of the plurality of types of nucleotides or nucleotide analogs have a different label. 
     
     
         71 . The method according to  claim 66 , wherein the different types of nucleotides or nucleotide analogs have the same label but are distinguished by different properties due to a presence of base fluorophores, quenched fluorophores, or fluorogenic nucleotide analogs. 
     
     
         72 . The method according to  claim 62 , wherein the nucleic acid polymerizing enzyme carries a label and said identifying is carried out by detecting interaction between the label and the nucleotide or nucleotide analogs. 
     
     
         73 . The method according to  claim 72 , wherein the label is a fluorescence resonance energy transfer donor or acceptor. 
     
     
         74 . The method according to  claim 62 , wherein said identifying is achieved by spectral wavelength discrimination, measurement and separation of fluorescence lifetimes, fluorophore identification, background suppression, or a combination thereof. 
     
     
         75 . The method of  claim 62 , wherein the target nucleic acid is attached to the support by hybridization to an oligonucleotide primer. 
     
     
         76 . The method of  claim 62 , wherein the target nucleic acid comprises a single-stranded or a double-stranded target nucleic acid molecule directly bound to the support. 
     
     
         77 . A method of sequencing comprising:
 providing a sequencing reaction mixture comprising a target nucleic acid attached to a support, a first polymerase enzyme, a second polymerase enzyme, and a plurality of types of nucleotides or nucleotide analogs under conditions whereby polymerization occurs to yield a growing nucleic acid strand that is complementary to the target nucleic acid molecule, and   optically detecting a time sequence of incorporation of said plurality of types of nucleotides or nucleotide analogs at an active site complementary to the target nucleic acid under conditions whereby the first polymerase enzyme forms a complex with the target nucleic acid to produce a first time sequence of incorporation, the first polymerase enzyme dissociates, and the second polymerase enzyme forms a complex with the target nucleic acid to produce a second time sequence of incorporation.   
     
     
         78 . The method according to  claims 77 , wherein the polymerization reaction is performed by a nucleic acid polymerizing enzyme selected from the group consisting of a DNA polymerase, an RNA polymerase, a reverse transcriptase, and a mixture thereof. 
     
     
         79 . The method according to  claim 77 , wherein the target nucleic acid molecule is selected from the group consisting of double-stranded DNA, single-stranded DNA, single stranded DNA hairpins, DNA/RNA hybrids, RNA with a recognition site for binding of the polymerase, and RNA hairpins. 
     
     
         80 . The method according to  claim 77 , wherein the nucleotide analogs are selected from the group consisting of a ribonucleotide, a deoxyribonucleotide, a modified ribonulcleotide, a modified deoxyribonucleotide, a piptide nucleotide, a modified peptide nucleotide, and a modified phosphate-sugar backbone nucleotide.