Nucleic acid amplification

Abstract

Methods and compositions for the amplification of nucleic acids are disclosed. Amplification methods provided herein may be performed under isothermal conditions. Methods and compositions may include reagents such as restriction enzymes, polymerases, ligases, primers, and polynucleotide adaptors.

1 . A method for amplifying a linear double-stranded nucleic acid template comprising two separate complementary strands, comprising:
 (A) generating a circular strand from the linear double-stranded nucleic acid template, wherein the circular strand comprises the single-strand component of at least one restriction enzyme recognition sequence and the sequences of each of the two separate complementary strands of the template;   (B) treating the circular strand with a first oligonucleotide primer and a polymerase, under conditions such that an extension product of the first primer is synthesized;   (C) treating the extension product of the first primer of step (B) with a second oligonucleotide primer and a polymerase, under conditions such that an extension product of the second primer is synthesized which is complementary to the extension product of the first primer of step (B), to produce a new double-stranded nucleic acid comprising at least a portion of the extension product of the first primer and at least a portion of the extension product of the second primer; and   (D) treating the new double-stranded nucleic acid of step (C) with a restriction enzyme that recognizes a full double-stranded restriction enzyme recognition sequence corresponding to the at least one restriction enzyme recognition sequence of step (A), under conditions such that the new double-stranded nucleic acid of step (C) is cleaved to form two or more shorter double-stranded nucleic acids, at least two of which shorter double-stranded nucleic acids comprise at least a portion of a copy of the linear double-stranded nucleic acid template of step (A), thereby amplifying the linear double-stranded nucleic acid template.   
     
     
         2 . (canceled) 
     
     
         3 . The method of  claim 1 , further comprising:
 treating the linear double-stranded nucleic acid template comprising the two complementary strands and a first and second end with a first and a second polynucleotide adaptor, under conditions such that: i) the first adaptor is ligated to the termini of both complementary strands present at the first end of the linear double-stranded nucleic acid template, ii) the second adaptor is ligated to the termini of both complementary strands present at the second end of the linear double-stranded nucleic acid, and iii) the circular strand comprising the two adaptors and the two complementary strands is formed, wherein each adaptor: i) comprises a single nucleic acid strand which contains the single strand component of a restriction enzyme recognition sequence and ii) is configured to, under certain conditions, adopt a stem-loop structure.   
     
     
         4 . The method of  claim 1 , further comprising repeating steps (A)-(D) for one or more additional cycles, using a shorter double-stranded nucleic acid of step (D) of a first cycle as the linear double-stranded nucleic acid template of step (A) of a second cycle. 
     
     
         5 . (canceled) 
     
     
         6 . The method of  claim 1 , further comprising repeating steps (A)-(D) for one or more additional cycles, using a shorter double-stranded nucleic acid of step (D) of a first cycle as the linear double-stranded nucleic acid template of step (A) of a second cycle, and wherein step (B) of the second cycle comprises treating at least two different circular strands with the first oligonucleotide primer and polymerase, wherein the at least two different circular strands comprise: i) a circular strand formed in the first cycle and ii) a circular strand formed in the second cycle. 
     
     
         7 . A method for amplifying a linear double-stranded nucleic acid template comprising two separate complementary strands, comprising:
 (A) ligating an adaptor comprising a single nucleic acid strand to each end of a linear double-stranded nucleic acid template, to yield a circular strand containing the general formula in the 5′ to 3′ direction: -A1-S1-A2-S2-, wherein
 each adaptor contains at least four nucleotide bases and a single strand component of a restriction enzyme recognition sequence, 
 A1 and A2 denote separate adaptors, 
 S1 denotes a first complementary strand of the linear double-stranded nucleic acid template, 
 S2 denotes a second complementary strand of the linear double-stranded nucleic acid template, 
 the 3′ terminus of A1 is linked to the 5′ terminus of S1, 
 the 3′ terminus of S1 is linked to the 5′ terminus of A2, 
 the 3′ terminus of A2 is linked to the 5′ terminus of S2, and 
 the 3′ terminus of S2 is linked to the 5′ terminus of A1; 
   (B) annealing a first oligonucleotide primer to the circular strand;   (C) extending the first oligonucleotide primer along the circular strand by using a polymerase, to form an extension product of the first primer;   (D) annealing a second oligonucleotide primer to the extension product of the first primer;   (E) extending the second oligonucleotide primer along at least a portion of the extension product of the first primer by using a polymerase, to produce a new double-stranded nucleic acid comprising at least a portion of the extension product of the first primer and at least a portion of the extension product of the second primer; and   (F) cleaving the new double-stranded nucleic acid of step with a restriction enzyme that recognizes a full double-stranded restriction enzyme recognition sequence corresponding to the single strand component of a restriction enzyme sequence of at least one of the adaptors, to form two or more shorter double-stranded nucleic acids, at least two of which comprise at least a portion of a copy of the linear double-stranded nucleic acid template of step (A), thereby amplifying the linear double-stranded nucleic acid template.   
     
     
         8 . The method of  claim 7 , further comprising repeating steps (A)-(F) for one or more additional cycles, using a shorter double-stranded nucleic acid of step (F) of a first cycle as the linear double-stranded nucleic acid template of step (A) of a second cycle. 
     
     
         9 . (canceled) 
     
     
         10 . The method of  claim 7 , further comprising repeating steps (A)-(F) for one or more additional cycles, using a shorter double-stranded nucleic acid of step (F) of a first cycle as the linear double-stranded nucleic acid template of step (A) of a second cycle, and wherein steps (B) and (C) of the second cycle comprises treating at least two different circular strands with the first oligonucleotide primer and polymerase, wherein the at least two different circular strands comprise: i) a circular strand formed in the first cycle and ii) a circular strand formed in the second cycle. 
     
     
         11 . The method of  claim 1 , wherein the first oligonucleotide primer is complementary to a complementary strand of the linear double-stranded nucleic acid template. 
     
     
         12 . The method of  claim 1 , wherein the second oligonucleotide primer is complementary to a complementary strand of the linear double-stranded nucleic acid template. 
     
     
         13 . The method of  claim 1 , wherein both the first oligonucleotide primer and the second oligonucleotide primer are complementary to a complementary strand of the linear double-stranded nucleic acid template, and wherein the first oligonucleotide primer and the second oligonucleotide primer are complementary to different strands of the linear double-stranded nucleic acid template. 
     
     
         14 . The method of  claim 3 , wherein at least one of the first oligonucleotide primer or the second oligonucleotide primer is complementary to at least one of the adaptors. 
     
     
         15 - 18 . (canceled) 
     
     
         19 . The method of  claim 3 , wherein at least one of the adaptors contains a nucleotide sequence comprising a 5′ region, a middle region, and a 3′ region, wherein the 5′ region and 3′ region of the sequence are complementary to each other such that under certain conditions they anneal to each other and form the stem of a stem-loop structure, and wherein the outermost part of the stem contains a blunt end. 
     
     
         20 . The method of  claim 3 , wherein at least one of the adaptors contains a nucleotide sequence comprising a 5′ region, a middle region, and a 3′ region, wherein the 5′ region and 3′ region of the sequence are complementary to each other such that under certain conditions they anneal to each other and form the stem of a stem-loop structure, and wherein the outermost part of the stem contains a sticky end. 
     
     
         21 . The method of  claim 3 , wherein at least one of the adaptors contains a nucleotide sequence comprising a 5′ region, a middle region, and a 3′ region, wherein the 5′ region and 3′ region of the sequence are complementary to each other such that under certain conditions they anneal to each other and form the stem of a stem-loop structure, and wherein the outermost part of the stem contains half of a full double-stranded restriction enzyme recognition sequence. 
     
     
         22 - 26 . (canceled) 
     
     
         27 . The method of  claim 1 , wherein all steps of the method are performed at a temperature of no greater than 70 C. 
     
     
         28 - 31 . (canceled) 
     
     
         32 . The method of  claim 1 , wherein the linear double-stranded nucleic acid template is amplified at least 10-fold within 60 minutes of initiation of the method. 
     
     
         33 . A method for amplifying a linear double-stranded nucleic acid template, comprising:
 (A) preparing a reaction mixture comprising:
 (i) the linear double-stranded nucleic acid template comprising a first complementary strand and a second complementary strand, 
 (ii) an isolated nucleic acid ligase, 
 (iii) an isolated nucleic acid polymerase, and 
 (iv) an isolated restriction enzyme; and 
   (B) incubating the reaction mixture for at least 1 minute.   
     
     
         34 . The method of  claim 33 , wherein the reaction mixture further comprises two polynucleotide adaptors, wherein each adaptor comprises a single nucleic acid strand which contains the single strand component of a restriction enzyme recognition sequence and which is configured to, under certain conditions, adopt a stem-loop structure. 
     
     
         35 - 50 . (canceled) 
     
     
         51 . The method of  claim 33 , wherein all steps of the method are performed at a temperature of no greater than 70 C. 
     
     
         52 - 54 . (canceled) 
     
     
         55 . The method of  claim 33 , wherein the linear double-stranded nucleic acid template is amplified at least 10-fold within 60 minutes of initiation of the method. 
     
     
         56 - 97 . (canceled)