Sequencing systems with two slow-step polymerase enzymes

Abstract

Systems for nucleotide sequencing comprising producing polymerase reactions that exhibit two kinetically observable steps within an observable phase of the polymerase reaction. Two slow step systems can be produced, for example, by selecting the appropriate polymerase enzyme, polymerase reaction conditions including cofactors, and polymerase reaction substrates including the primed template and nucleotides.

1 . A system for single-molecule nucleotide sequencing comprising:
 a) a reaction mixture having: (i) a polymerase enzyme, (ii) polymerase reaction conditions including cofactors, and (iii) polymerase reaction substrates including a primed template and nucleotides comprising observable labels, such that a reaction comprising incorporation of the nucleotides into a growing nucleic acid occurs, wherein the polymerase enzyme, the polymerase reaction conditions, and the polymerase reaction substrates are selected such that the reaction exhibits two kinetically observable steps, each of which kinetically observable steps proceeds from an intermediate in which a nucleotide or a polyphosphate product is bound to the polymerase enzyme or each of which kinetically observable steps proceeds from an intermediate in which the nucleotide and the polyphosphate product are not bound to the polymerase enzyme; and   (b) a detector to detect the observable label to measure the sequential incorporation of nucleotides into the growing nucleic acid thereby obtaining a sequence of the template   
     
     
         2 . The system of  claim 1  wherein the detector comprises a CCD, ICCD, EMCCD or CMOS detector. 
     
     
         3 . The system of  claim 1  wherein the observable labels comprise optical labels. 
     
     
         4 . The system of  claim 3  wherein the optical labels comprise fluorescent labels. 
     
     
         5 . The system of  claim 1  further comprising an illumination source. 
     
     
         6 . The system of  claim 1  wherein the two kinetically observable steps are each steps which proceed from an intermediate in which a nucleotide or a polyphosphate product is bound to the polymerase enzyme. 
     
     
         7 . The system of  claim 1  wherein the two kinetically observable steps are each steps which proceed from an intermediate in which the nucleotide and the polyphosphate product are not bound to the polymerase enzyme. 
     
     
         8 . The system of  claim 1  wherein the two kinetically observable steps are selected from a group consisting of enzyme isomerization, nucleotide incorporation, and product release. 
     
     
         9 . The system of  claim 1  wherein the two kinetically observable steps are template translocation and nucleotide binding. 
     
     
         10 . The system of  claim 1  wherein the ratio of the rate constants of the kinetically observable steps is from 2:1 to 1:2. 
     
     
         11 . The system of  claim 1  wherein the rate constant for one of the kinetically observable steps is less than about 100 per second. 
     
     
         12 . The system of  claim 1  wherein the rate constant for one of the kinetically observable steps is between about 0.1 per second and about 60 per second. 
     
     
         13 . The system of  claim 1  wherein the rate constant for one of the kinetically observable steps is between about 1 per second and about 20 per second. 
     
     
         14 . The system of  claim 1  wherein the reaction exhibits more than two kinetically observable steps in an observable phase. 
     
     
         15 . The system of  claim 1  wherein the polymerase enzyme comprises a modified recombinant Φ29-type polymerase. 
     
     
         16 . The system of  claim 15  wherein the polymerase enzyme comprises a modified recombinant Φ29, B103, GA-1, PZA, Φ15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, or L-17 polymerase. 
     
     
         17 . The system of  claim 15  wherein the polymerase enzyme comprises a modified recombinant Φ29 DNA polymerase having at least one amino acid substitution or combination of substitutions selected from the group consisting of: an amino acid substitution at position 484, an amino acid substitution at position 198, and an amino acid substitution at position 381. 
     
     
         18 . The system of  claim 15  wherein the polymerase enzyme comprises a modified recombinant Φ29 DNA polymerase having at least one amino acid substitution or combination of substitutions selected from the group consisting of E375Y, K512Y, T368F, A484E, A484Y, N387L, T372Q, T372L, K478Y, 1370W, F198W, and L381 A. 
     
     
         19 . The system of  claim 1  wherein the polymerase reaction conditions comprise one or more of metal cofactor concentration, pH, temperature, an enzyme activity modulator, D 2 O, an organic solvent, and buffer. 
     
     
         20 . The system of  claim 19  wherein the polymerase reaction conditions comprise a mixture of divalent metal ions comprising at least one catalytic metal ion and at least one non-catalytic metal ion. 
     
     
         21 . The system of  claim 20  wherein the catalytic metal is selected from Mg2+, Mn2+ and mixtures thereof, and the non-catalytic metal is selected from Zn2+, Co2+, Ni2+, Eu2+, Sr2+, Ba2+, Fe2+, Eu2+ and mixtures thereof. 
     
     
         22 . The system of  claim 20  wherein a ratio of catalytic metal to non-catalytic metal in the reaction mixture is from about 10:1 to about 1:10. 
     
     
         23 . The system of  claim 19  wherein the conditions comprise an organic solvent selected from the group consisting of ethanol, methanol, THE, dioxane, DMA, DMF, and DMSO.