Nucleic acid sequencing method

Abstract

The present invention provides a method for sequencing a nucleic acid using an immersion reaction protocol. The immersion reaction protocol comprises sequentially immersing a solid support having nucleic acid molecules immobilized thereon in different reaction containers to realize nucleic acid sequencing.

1 - 22 . (canceled) 
     
     
         23 . A method for sequencing a nucleic acid using a contact reaction protocol, the contact reaction protocol comprising the following steps:
 a) providing a first batch of solid supports having nucleic acid molecules immobilized thereon,   b) contacting the nucleic acid molecules immobilized on the solid supports with one or more reaction solutions to generate a signal representing one or more nucleotides that bind to the nucleic acid molecules on the solid supports, wherein each of the one or more reaction solutions is placed in a separate reaction container,   wherein, after removing the contact between the solid supports and the reaction solutions, contacting the solid supports with one or more washing solutions to remove the reaction solutions remaining on the solid supports,   c) detecting the signal on the solid supports, and   f) optionally, repeating the steps b) to c),   wherein the solid supports are unencapsulated,   wherein the first batch of solid supports consists of one or more solid supports.   
     
     
         24 . The method according to  claim 23 , wherein the method further comprises the following steps after step c) and before step f):
 d) contacting the solid supports with one or more regeneration solutions each placed in a separate reaction container to eliminate the signal on the solid supports, and   e) removing the contact between the solid supports and the regeneration solutions, and contacting the solid supports with one or more washing solutions to remove the regeneration solutions remaining on the solid supports,   and wherein in step f), optionally, repeating the steps b) to e) or the steps b) to c).   
     
     
         25 . The method according to  claim 23 , wherein the solid supports can be selected from beads, chips, glasses, sensors, electrodes, or silicon wafers. 
     
     
         26 . The method according to  claim 23 , which further comprises: after contacting the first batch of solid supports with the one or more reaction solutions of the step b) and before the end of the step c) or e), contacting a second batch of solid supports having nucleic acid molecules immobilized thereon with the one or more reaction solutions of the step b), and then operating the second batch of solid supports according to the steps b) to f);
 optionally iteratively repeating this process for N−1 times,   wherein, 1≤N≤t cycle /t speed-limit , and N is an integer value,   wherein, t cycle  is a total time from the step b) to the step c) or from the step b) to the step e), and t speed-limit  is the time of the longest procedure in each contacting and washing in the step b), detecting in the step c), contacting in the step d), and washing in the step e).   
     
     
         27 . The method according to  claim 26 , wherein the time interval, tN, between contacting a N th  batch of solid supports with the one or more reaction solutions of the step b) and contacting a (N+1) th  batch of solid supports with the one or more reaction solutions of the step b) meets the following conditions: 
       
         
           
             
               
                 
                   t 
                   N 
                 
                 ≥ 
                 
                   t 
                   
                     speed 
                     - 
                     limit 
                   
                 
               
               , 
               
                 
                   and 
                   ⁢ 
                       
                   
                     
                       ∑ 
                       
                         N 
                         = 
                         1 
                       
                       N 
                     
                     
                       t 
                       N 
                     
                   
                 
                 ≤ 
                 
                   
                     t 
                     cycle 
                   
                   - 
                   
                     
                       t 
                       
                         speed 
                         - 
                         limit 
                       
                     
                     . 
                   
                 
               
             
           
         
       
     
     
         28 . The method according to  claim 27 , wherein each batch of solid supports from the (N+1) th  batch of solid supports consists of one or more solid supports. 
     
     
         29 . The method according to  claim 23 , wherein the signal comprises a fluorescence signal. 
     
     
         30 . The method according to  claim 23 , wherein the sequencing comprises sequencing-by-ligation, sequencing-by-synthesis or sequencing by combinatorial probe-anchor synthesis (cPAS). 
     
     
         31 . The method according to  claim 30 , wherein the sequencing is sequencing-by-ligation, and wherein the one or more reaction solutions of the step b) comprise a solution containing anchor probes, labeled sequencing probes, ligase, or a mixture thereof, provided that the solid supports are in contact with each of the anchor probes, the labeled sequencing probes and the ligase, and wherein the signal on the solid supports is generated by the labeled sequencing probe that complementarily bind to the nucleic acid molecules on the solid supports, the labeled sequencing probes are linked via the ligase to the anchor probes that complementarily bind to the same nucleic acid molecules,
 optionally, the regeneration solutions of the step d) comprise reagents that are capable of removing labels from the labeled sequencing probes or reagents that are capable of removing the labeled sequencing probes from the nucleic acid molecule.   
     
     
         32 . The method according to  claim 30 , wherein the sequencing is sequencing-by-ligation, further comprising: before the step b), contacting the solid supports having the nucleic acid molecules immobilized thereon with the anchor probes, so that the anchor probes hybridize to the nucleic acid molecule on the solid supports. 
     
     
         33 . The method according to  claim 32 , wherein the one or more reaction solutions of the step b) comprise a solution containing the labeled sequencing probes, the ligase, or a mixture thereof, provided that the solid supports are in contact with each of the labeled sequencing probes and the ligase, and wherein the signal on the solid supports is generated by the labeled sequencing probes that complementarily bind to the nucleic acid molecules on the solid supports, the labeled sequencing probes are linked via the ligase to the anchor probes that complementarily bind to the same nucleic acid molecule,
 optionally, the regeneration solutions of the step d) comprise reagents that are capable of removing labels from the labeled sequencing probes or reagents that are capable of removing the labeled sequencing probes from the nucleic acid molecule.   
     
     
         34 . The method according to  claim 30 , wherein the sequencing is sequencing-by-synthesis, and wherein the one or more reaction solutions of the step b) comprise a solution containing a polymerase, sequencing primers, a labeled nucleotide, or a mixture thereof, provided that the solid supports are in contact with each of the polymerase, the sequencing primers, and the labeled nucleotide, and wherein the signal on the solid supports is generated by the labeled nucleotide that complementarily binds to the nucleic acid molecules on the solid supports, the labeled nucleotide is polymerized to a 3′ end of the sequencing primers via the polymerase using the nucleic acid molecule on the solid supports as a template,
 optionally, the regeneration solutions of the step d) contain reagents capable of removing a label from the labeled nucleotide, 
 optionally, the labeled nucleotide further comprises a 3′ blocking group. 
 
     
     
         35 . The method according to  claim 30 , wherein the sequencing is sequencing-by-synthesis, and wherein the nucleic acid molecules are immobilized on the solid supports by hybridization of the sequencing primers immobilized on the solid supports. 
     
     
         36 . The method according to  claim 30 , wherein the sequencing is sequencing-by-synthesis, further comprising: before the step b), contacting the solid supports having the nucleic acid molecules immobilized thereon with the sequencing primers, so that the sequencing primers hybridize to the nucleic acid molecules on the solid supports. 
     
     
         37 . The method according to  claim 35 , wherein the one or more reaction solutions of the step b) comprise a solution containing a polymerase, a labeled nucleotide, or a mixture thereof, provided that the solid supports are in contact with each of the polymerase and the labeled nucleotide, and wherein the signal on the solid supports is generated by the labeled nucleotide that complementarily binds to the nucleic acid molecule on the solid supports, the labeled nucleotide is polymerized to a 3′ end of the sequencing primers via the polymerase using the nucleic acid molecule on the solid supports as a template,
 optionally, the regeneration solutions of the step d) comprise reagents capable of removing a label from the labeled nucleotide, 
 optionally, the labeled nucleotide further comprises a 3′ blocking group. 
 
     
     
         38 . The method according to  claim 24 , further comprising:
 adding humectants to the one or more reaction solutions of the step b), the regeneration solutions of the step d) and/or the washing solutions of the step e), and/or   adding reagents to the one or more reaction solutions in the step b), the regeneration solutions in the step d) and/or the washing solutions in the step e) so as to retain the reaction solutions and/or the washing solutions remaining on the solid supports thereon when removing the contact between the solid supports and the reaction solutions and/or the washing solutions.   
     
     
         39 . The method according to  claim 23 , further comprising: optimizing a motion speed of the solid supports between the reaction containers so as to minimize the time that the solid supports are exposed to air. 
     
     
         40 . The method according to  claim 23 , further comprising: using a temperature control device to adjust the temperature in each of the reaction containers. 
     
     
         41 . The method according to  claim 24 , wherein during optionally repeating the steps b) to c) or the steps b) to e), the one or more reaction solutions of the step b) and/or the regeneration solutions of the step d) are replaced or not upon each repeating,
 optionally, when the one or more reaction solutions in the step b) have a reaction temperature of below about 55° C., the time interval for replacing the one or more reaction solutions in the step b) is less than about 8 hours,   optionally, when the one or more reaction solutions in the step b) have a reaction temperature of below about 45° C., the time interval for replacing the one or more reaction solutions in the step b) is less than about 24 hours.   
     
     
         42 . An apparatus for sequencing a nucleic acid molecule by using the contact reaction protocol according to  claim 23 , the apparatus comprising:
 a) one or more reaction containers, each containing one or more reaction solutions for contacting a nucleic acid molecule to generate a signal representing one or more nucleotides that bind to the nucleic acid molecules;   b) one or more reaction containers containing one or more washing reagents;   c) one or more reaction containers containing one or more reaction solutions for eliminating the signal from solid supports;   d) device for detecting the signal; and   e) temperature control device for controlling the temperature of the reaction container in above a) to c).