Clonal amplification of nucleic acid on solid surface with template walking

Abstract

Novel methods of generating a localized population of immobilized clonal amplicons on a support are provided.

1 . A method of primer extension, comprising:
 a) hybridizing a first primer molecule (“first forward primer”) to a complementary primer-binding sequence (“reverse-strand PBS”) on a nucleic acid strand (“reverse strand”); wherein at least 60% of nucleotide bases of the first forward primer are adenine, thymine or uracil or are complementary to adenine, thymine or uracil;   b) generating an extended first forward strand that is a full-length complement of the reverse strand and is hybridized thereto, by extending the first forward primer molecule using the reverse strand as template; and   c) hybridizing a second primer molecule (“second forward primer”) to the reverse-strand PBS where the reverse strand is also hybridized to the first forward strand.   
     
     
         2 . The method of  claim 1 , comprising amplifying the forward strand by one or more amplification cycles comprising steps (b) and (c), wherein the second forward primer of step (c) of a first amplification cycle is the first forward primer of step (b) of a subsequent amplification cycle; and wherein a substantial proportion of reverse strands are hybridized to forward strands at all times during or between said one or more repetitions. 
     
     
         3 . The method of  claim 1 , wherein the substantial proportion of reverse strands is at least 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of reverse strands. 
     
     
         4 . The method of  claim 1 , comprising amplifying the reverse strand by:
 a) hybridizing a first reverse primer molecule to a complementary reverse-primer-binding sequence (“forward-strand PBS”) on an extended forward strand;   b) generating an extended first reverse strand that is a full-length complement of the forward strand and hybridized thereto, by extending the first reverse primer molecule in template-dependent fashion using the forward strand as template; and   c) hybridizing a second primer (“second reverse primer”) to the forward-strand PBS where the forward strand is also hybridized to the first reverse strand.   
     
     
         5 . The method of  claim 4 , comprising amplifying the reverse strand by one or more repetitions of steps (b)-(c), wherein the second reverse primer of step (c) is the first reverse primer of repeated step (b); and wherein a substantial proportion of forward strands are hybridized to reverse strands at all times during or between said one or more repetitions. 
     
     
         6 . The method of  claim 1 , wherein the substantial proportion of reverse strands is at least 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of reverse strands. 
     
     
         7 . The method of  claim 1 , wherein the first and/or second forward primers are immobilized to a single support, or the first and/or second reverse primers are immobilized to a single support 
     
     
         8 . The method of  claim 1 , further comprising completely separating the extended forward strands from reverse strands after performing the desired number of amplification cycles, and optionally removing separated forward strands from the presence of separated reverse strands, or vice versa. 
     
     
         9 . The method of  claim 1 , wherein during one or more amplification cycles all nucleic acid reagents are not in contact with a recombinase or reverse transcriptase or helicase or nicking enzyme or any other enzyme that is not a polymerase at any time. 
     
     
         10 . The method of  claim 1 , wherein template-dependent extension of a forward primer using a reverse strand as template results in displacement of another forward strand that was already hybridized to the reverse strand. 
     
     
         11 . The method of  claim 1 , wherein the Tm of all forward primers is not more than 50° C., 55° C., 60° C. or 65° C., and wherein the Tm of the reverse strands is not less than 95° C., 90° C., 85° C., 80° C. or 75° C. 
     
     
         12 . The method of  claim 1 , wherein amplification is performed under isothermal conditions. 
     
     
         13 . The method of  claim 1 , wherein the isothermal conditions are adjusted to a temperature that is higher than the Tm of all forward primers, but lower than the Tm of the reverse strands, wherein the Tm of a reverse strand is the temperature at which half of the reverse strands in a clonal population of identical reverse strands are fully denatured from a perfectly complementary strand that is fully hybridized to the reverse strand across its entire length. 
     
     
         14 . The method of  claim 1 , wherein said first and second forward primers are adjacently immobilized to the same support, whereby amplification generates an immobilized clonal populations of extended forward strands. 
     
     
         15 . The method of  claim 1 , wherein said first and second forward primers are adjacently immobilized to the same support, whereby amplification generates an immobilized clonal population of extended forward strands. 
     
     
         16 . The method of  claim 1 , wherein a plurality of template nucleic acids are individually hybridized to spatially-separated immobilization sites, whereby amplification generates spatially-separated clonal populations corresponding to individual template nucleic acids. 
     
     
         17 - 25 . (canceled) 
     
     
         26 . A method of generating separated and immobilized clonal populations of a first template sequence (“template 1”) and a second template sequence (“template 2”), comprising amplifying the first and second template sequence, wherein:
 a) both templates are in single-stranded form and are both contained within the same continuous liquid phase, where a first and second immobilization site (respectively, “IS1” and “IS2”) are in contact with said continuous liquid phase, and where IS1 and IS2 are spatially separated, 
 b) template 1 comprises a first subsequence (“T1-FOR”) at its 3′ end, and a second subsequence (“T1-REV”) that is non-overlapping with T1-FOR and at its 5′ end, 
 c) template 2 comprises a first subsequence (“T2-FOR”) at its 3′ end, and a second subsequence (“T2-REV”) that is non-overlapping with T2-FOR and at its 5′ end, 
 d) IS1 comprises an immobilized primer (“IS1 primer”) that can hybridize to both T1-FOR and T2-FOR, 
 e) IS2 comprises an immobilized primer (“IS2 primer”) that can hybridize to both T1-FOR and T2-FOR, 
 f) the reverse complement of T1-REV cannot hybridize substantially to primers on IS1, and/or the reverse complement of T2-REV cannot hybridize substantially to primers on IS2, but can each hybridize substantially to a non-immobilized primer in the continuous liquid phase; 
 whereby amplification results in a population of clonal amplicons of template 1 substantially attached to IS1 and not to IS2, and/or a population of clonal amplicons of template 2 substantially attached to IS2 and not to IS1. 
 
     
     
         27 . The method of  claim 26 , wherein intermixing of non-immobilized nucleic acid molecules is substantially unretarded in the continuous liquid phase at some timepoint during amplification. 
     
     
         28 . The method of  claim 27 , wherein intermixing is substantially unretarded for a period of time during amplification, and optionally is substantially unretarded during the entire duration of amplification. 
     
     
         29 . The method of  claim 27 , wherein any nucleic acid that has dissociated from one immobilization site is capable of substantially hybridizing to both immobilization sites and any movement (e.g., movement by diffusion, convection) of said dissociated nucleic acid to another immobilization site is not substantially retarded in the continuous liquid phase. 
     
     
         30 . The method of  claim 26 , wherein the continuous liquid phase is in simultaneous contact with IS1 and IS2. 
     
     
         31 - 103 . (canceled)