Process for the enzymatic synthesis and amplification of nucleic acids

Abstract

A method for amplification of nucleic acids in which substantially use is made of the fact that a pre-defined nucleic acid chain (target sequence) can be multiplied/amplified in the presence of a target sequence-specific activator oligonucleotide. The target sequence-specific activator oligonucleotide causes the separation of re-synthesized complementary primer extension products by strand displacement, so that a new primer oligonucleotide can attach to the respective template strand. The thus formed complex of a primer oligonucleotide and a template strand can initiate a new primer extension reaction. The thus formed primer extension products in turn function as templates, so that an exponential amplification reaction results. Amplification of a particular target sequence takes place more efficiently in case of perfect match complemetary base pair formation between the activator oligonucleotide and the corresponding target sequence. Mismatches between the activator oligonucleotide and a particular target sequence can result in less efficient amplification. The efficiency of synthesis of perfect match target sequences and mismatch sequences can be measured and compared.

1 . A reaction mixture for the amplification of nucleic acid comprising:
 at least one polymerase which is a nucleic acid synthesis inducing agent causing the synthesis of the primer extension products as soon as a starter nucleic acid chain is added to the reaction mixture as a template, with   at least one first primer oligonucleotide (P1) comprising:   i. a first primer region in the 3′ segment of the first primer oligonucleotide that can sequence-specifically bind to a strand of a nucleic acid chain to be amplified,   ii. a second region that is directly or via a linker linked to the 5′ end of the first primer region of the first primer oligonucleotide and that comprises a polynucleotide tail which is suitable for binding an activator oligonucleotide and supporting strand displacement by the activator oligonucleotide, wherein the polynucleotide tail remains substantially uncopied by polymerase,   
       at least one activator oligonucleotide comprising in 3′-5′ direction:
 i. a first single-stranded region that can bind to the polynucleotide tail of the second region of the first primer oligonucleotide, 
 ii. a second single-stranded region that is substantially complementary and can bind to the first region of the first primer oligonucleotide, 
 iii. a third single-stranded region that is substantially complementary to at least a segment of the extension product, which has been synthesized by polymerase, of the first primer extension product, whereby the length of this segment comprises at least 5 nucleotides,
 wherein the activator oligonucleotide comprises at least one nucleotide modification, whereby it does not serve as a template for a primer extension of the first primer oligonucleotide, 
 
 and 
 at least one second primer oligonucleotide comprising: 
 
       a 3-segment of the second oligonucleotide primer comprising a sequence which can hybridize to the first primer extension product, 
       for carrying out an amplification reaction for the reproduction of a nucleic acid chain to be amplified, wherein the amplification method comprises:
 a) hybridizing a first primer oligonucleotide to the 3′ segment of a strand of a nucleic acid chain to be amplified, wherein the nucleic acid chain to be amplified comprises a target sequence; 
 b) extending the first primer oligonucleotide by means of a polymerase to form a first primer extension product comprising a sequence that is complementary to the target sequence of the nucleic acid chain to be amplified; 
 c) binding the activator oligonucleotide to the polynucleotide tail of the second region of the first extended primer oligonucleotide; 
 d) binding the activator oligonucleotide to the first primer region of the first extended primer oligonucleotide by displacing the strand of the nucleic acid chain to be amplified that is complementary to said first primer region; 
 e) binding the activator oligonucleotide to the complementary segment of the extension product of the first extended primer oligonucleotide by displacing the strand of the nucleic acid chain to be amplified that is complementary to said extension product, wherein the 3′ segment of the first primer extension product becomes single-stranded; 
 f) hybridizing a second oligonucleotide primer to the first primer extension product, wherein the 3′ segment of the second oligonucleotide primer comprises a sequence that can hybridize to the first primer extension product; 
 g) extending the second oligonucleotide primer with polymerase to form a second primer extension product, wherein the extension takes place up to and including the first primer region of the first primer oligonucleotide and said first primer region is copied by the polymerase, wherein the polynucleotide tail of the second region remains uncopied; and 
 h) repeating steps a)-g) until the desired degree of amplification has been achieved. 
 
     
     
         2 . The reaction mixture according to  claim 1 , wherein the nucleic acid chain to be amplified comprises a sequence complementary to the target sequence. 
     
     
         3 . The reaction mixture according to  claim 1  for amplification of a nucleic acid chain to be amplified comprising at least a first and/or a second primer extension product, wherein the length of the extension product of a specific primer extension product is between 10 and 300 nucleotides. 
     
     
         4 . The reaction mixture according to  claim 1  further comprising at least one starter nucleic acid chain wherein said starter nucleic acid chain comprises at least one target sequence. 
     
     
         5 . The reaction mixture according to  claim 4 , wherein the starter nucleic acid chain comprises the sequence segments shown in  FIG. 23B , whereby:
 the sequence segment-1 comprises a sequence which is homologous to the sequence of the second primer or is identical to the copyable portion of the 3′-segment of the second primer and said sequence segment-1 is located in the 5′-segment of the starter nucleic acid chain;   the sequence segment-3 comprises a sequence which is suitable to complementarily bind to the first region of the first primer or its 3′-segment by forming an extendable primer template complex, and wherein particularly the sequence segment-3 is located downstream of the sequence segment-1; and   whereby segment-2 is a target sequence which is located partially or completely between segment-1 and segment-3.   
     
     
         6 . The reaction mixture according to  claim 4 , wherein the starter nucleic acid chain comprises the sequence elements shown in  FIG. 23C , whereby:
 the sequence segment-5 comprises a sequence which is homologous to the sequence of the first region of the first primer or is identical with the copyable portion of the first region of the first primer and is located in the 5′-segment of the starter nucleic acid chain;   the sequence fragment-7 comprises a sequence which is suitable to the complementary binding of a second primer or its 3′-segment by forming an extendable primer template complex which is located downstream of segment-5; as well as   a target sequence designated as sequence-6 which is located partially or completely between segment-5 and segment-7.   
     
     
         7 . The reaction mixture according to  claim 1  comprising a DNA polymerase which is heat resistant. 
     
     
         8 . The reaction mixture according to  claim 1  comprising a polymerase which is able to support strand displacement. 
     
     
         9 . The reaction mixture according to  claim 1 , wherein the first primer oligonucleotide comprises a first primer region in the 3-segment of the first primer oligonucleotide, wherein the sequence length comprises between 3 and 30 nucleotides. 
     
     
         10 . The reaction mixture according to  claim 1 , wherein the first primer oligonucleotide comprises a second region and the length of the second region comprises between 3 and 60 nucleotides. 
     
     
         11 . The reaction mixture according to  claim 1 , wherein the second region of the first primer oligonucleotide comprises at least one modification which inhibits the copying of the polynucleotide tail by the polymerase. 
     
     
         12 . The reaction mixture according to  claim 11 , wherein the second region of the first primer oligonucleotide comprises nucleotide modifications selected from the group consisting of: 2′-O-alkyl modifications, including 2′-O-methyl, 2′-O-(2-methoxyethyl), 2′-O-propyl, 2′-O-propargyl nucleotide modifications, PNA, morpholino modifications, iso-dG, iso-dC. 
     
     
         13 . The reaction mixture according to  claim 11 , wherein the second region of the first primer oligonucleotide comprises linker selected from the group consisting of: C3, C6, HEG linker. 
     
     
         14 . The reaction mixture according to  claim 1 , wherein the activator oligonucleotide comprises sequence segments corresponding to the target sequence. 
     
     
         15 . The reaction mixture according to  claim 1 , wherein the sequence of the activator oligonucleotide comprises nucleotide modifications, wherein the nucleotide modifications comprise base modifications and/or sugar-phosphate-residue modifications, so that the first primer oligonucleotide, if adjacent to the activator oligonucleotide under reaction conditions, cannot be extended by the polymerase, and the nucleotide modifications are selected from the group consisting of: 2′-O-alkyl modifications (including 2′-O-methyl, 2′-O-(2-methoxyethyl), 2′-O-propyl, 2′-O-propargyl nucleotide modifications), 2′-amino-2′-deoxy nucleotide modifications, 2′-amino-alkyl-2′-deoxy nucleotide modifications, PNA, morpholino modifications. 
     
     
         16 . The reaction mixture according to  claim 1 , wherein the activator oligonucleotide comprises:
 a first single-stranded region that can bind to the polynucleotide tail of the second region of the first primer oligonucleotide, whereby the length of this first region comprises 3-80 nucleotides;   and the number of complementary bases that can complementarily bind to the second region of the first primer oligonucleotide comprises between 1 and 40;   and/or   the extent of complementarity between the second region of the first primer oligonucleotide and the first region of the activator oligonucleotide is between 20% and 100%.   
     
     
         17 . The reaction mixture according to  claim 1  wherein the activator oligonucleotide comprises a second single-stranded region whereby the length of the second region of the activator oligonucleotide is adapted to the length of the first region of the first primer oligonucleotide and is in accordance therewith;
 and 
 the sequence of the first region of the first primer oligonucleotide and the sequence of the second region of the activator oligonucleotide are complementary to each other 
 
       whereby:
 the second region of activator oligonucleotide encompasses at least one nucleotide modification which inhibits the extension of the first primer oligonucleotide, however does not block or not substantially inhibit the formation of complementary double strands selected from the group consisting of: 2′-O-alkyl RNA analogs, such as 2′-O-Me, 2′-O-(2-methoxyethyl), 2′-O-propyl, 2′-O-propargyl nucleotide modifications, LNA, PNA or morpholino nucleotide modifications. 
 
     
     
         18 . The reaction mixture according to  claim 1 , wherein the activator oligonucleotide comprises:
 a third single-stranded region substantially complementary to at least a segment of the extension product, which has been synthesized by polymerase, of the first primer product, wherein the total length of the third region of the activator oligonucleotide comprises 2 to 100 nucleotides.   
     
     
         19 . The reaction mixture according to  claim 18 , wherein the activator oligonucleotide comprises a sequence wherein more than 5 nucleotides of the third region of the activator oligonucleotide complementarily can bind to the first primer extension product. 
     
     
         20 . The reaction mixture according to  claim 15 , wherein the sequence of the activator oligonucleotide comprises nucleotide modifications whereby at least 2 of such nucleotide modifications are coupled side by side in the activator oligonucleotide. 
     
     
         21 . The reaction mixture according to  claim 1 , wherein the activator oligonucleotide comprises:
 a third single-stranded region which is complementary to at least a segment of the extension product, which has been synthesized by polymerase, of the first primer extension product, and the length of the third region is adapted such that the third region binds to the 5′-primed segment of the extension product of the first primer oligonucleotide, and on the other hand does not bind to the 3-primed segment of the extension product and the length of the 3′-primed segment of the extension product of the first primer oligonucleotide, which is not bound by the activator oligonucleotide, comprises regions having 5 to 60 nucleotides.   
     
     
         22 . The reaction mixture according to  claim 1 , wherein the second primer oligonucleotide has a length of between 15 and 100 nucleotides, and the second primer oligonucleotide is able to bind to the 3′-segment of a first specific primer extension product of the first primer oligonucleotide and to initiate a polymerase-dependent synthesis of a second primer extension product. 
     
     
         23 . The reaction mixture according to  claim 22 , wherein the second primer oligonucleotide in its 3′-segment contains sequence portion which can complementarily and sequence-specifically bind to a sequence segment of a target sequence and initiate or support a successful primer extension reaction by polymerase, and the length of such a sequence segment comprises regions of 6 to 40 nucleotides. 
     
     
         24 . A kit comprising components of the reaction mixture according to  claim 1 , characterized in that the kit comprises:
 at least a first primer oligonucleotide (P1) comprising:   i. a first primer region in the 3′ segment of the first primer oligonucleotide that can sequence-specifically bind to a strand of a nucleic acid chain to be amplified;   ii. a second region that is directly or via a linker linked to the 5′ end of the first primer region of the first primer oligonucleotide and that comprises a polynucleotide tail which is suitable for binding an activator oligonucleotide and supporting strand displacement by the activator oligonucleotide, wherein the polynucleotide tail remains substantially uncopied by polymerase;   at least one activator oligonucleotide comprising in 3′-5′ direction:   i. a first single-stranded region that can bind to the polynucleotide tail of the second region of the first primer oligonucleotide;   ii. a second single-stranded region that is substantially complementary and can bind to the first region of the first primer oligonucleotide;   iii. a third single-stranded region that is substantially complementary to at least a segment of the extension product, which has been synthesized by polymerase, of the first primer extension product, and the length of this segment comprises at least 5 nucleotides;
 whereby the activator oligonucleotide comprises at least one nucleotide modification, whereby it does not serve as a template for a primer extension of the first primer oligonucleotide; 
   and   contains at least one polymerase for amplification that is a nucleic acid synthesis inducing agent causing the synthesis of the primer extension products, as soon as a starter nucleic acid chain is added to the reaction mixture as a template.   
     
     
         25 . A kit comprising components of the reaction mixture according to  claim 2  characterized in that it further contains a second oligonucleotide primer comprising:
 a 3-segment of the second oligonucleotide primer comprising a sequence that can hybridize to the first primer extension product. 
 
     
     
         26 . A homogenous amplification system comprising components of the reaction mixture according to  claim 1  characterized in that the amplification system comprises:
 at least a first primer oligonucleotide (P1) comprising:
 i. a first primer region in the 3′ segment of the first primer oligonucleotide that can sequence-specifically bind to a strand of a nucleic acid chain to be amplified, 
 ii. a second region that is directly or via a linker linked to the 5′ end of the first primer region of the first primer oligonucleotide and that comprises a polynucleotide tail which is suitable for binding an activator oligonucleotide and supporting strand displacement by the activator oligonucleotide, wherein the polynucleotide tail remains substantially uncopied by polymerase, 
 
 
       and
 at least a second primer oligonucleotide comprising: 
 a 3′-segment of the second oligonucleotide primer comprising a sequence that can hybridize to the first primer extension product; 
 at least one activator oligonucleotide comprising in 3′-5′ direction: 
 i. a first single-stranded region that can bind to the polynucleotide tail of the second region of the first primer oligonucleotide; 
 ii. a second single-stranded region that is substantially complementary and can bind to the first region of the first primer oligonucleotide; 
 iii. a third single-stranded region that is substantially complementary to at least a segment of the extension product, which has been synthesized by polymerase, of the first primer extension product, and the length of this segment comprises at least 5 nucleotides;
 wherein the activator oligonucleotide comprises at least one nucleotide modification, whereby it does not serve as a template for a primer extension of the first primer oligonucleotide; 
 and contains at least one polymerase which is a nucleic acid synthesis inducing agent causing the synthesis of the primer extension products, as soon as a starter nucleic acid chain is added to the reaction mixture as a template. 
 
 
     
     
         27 . The reaction mixture according to  claim 3 , wherein the length of the extension product is between 10 and 180 nucleotides. 
     
     
         28 . The reaction mixture according to  claim 3 , wherein the length of the extension product is between 20 and 120 nucleotides. 
     
     
         29 . The reaction mixture according to  claim 3 , wherein the length of the extension product is between 30 and 80 nucleotides. 
     
     
         30 . The reaction mixture according to  claim 9 , wherein the sequence length comprises between 5 and 20 nucleotides. 
     
     
         31 . The reaction mixture according to  claim 10 , wherein the length of the second region comprises between 5 and 40 nucleotides. 
     
     
         32 . The reaction mixture according to  claim 10 , wherein the length of the second region comprises between 6 and 15 nucleotides. 
     
     
         33 . The reaction mixture according to  claim 16 , wherein the length of the first region comprises 4-40 nucleotides. 
     
     
         34 . The reaction mixture according to  claim 16 , wherein the length of the first region comprises 6-20 nucleotides. 
     
     
         35 . The reaction mixture according to  claim 16 , wherein the number of complementary bases that can complementarily bind to the second region of the first primer oligonucleotide comprises between 3 and 20. 
     
     
         36 . The reaction mixture according to  claim 16 , wherein the number of complementary bases that can complementarily bind to the second region of the first primer oligonucleotide comprises between 6 and 15. 
     
     
         37 . The reaction mixture according to  claim 16 , wherein the extent of complementarity between the second region of the first primer oligonucleotide and the first region of the activator oligonucleotide is between 50% and 100%. 
     
     
         38 . The reaction mixture according to  claim 16 , wherein the extent of complementarity between the second region of the first primer oligonucleotide and the first region of the activator oligonucleotide is between 80% and 100%. 
     
     
         39 . The reaction mixture according to  claim 18 , wherein the length of the third region of the activator oligonucleotide comprises 6 to 60 nucleotides. 
     
     
         40 . The reaction mixture according to  claim 18 , wherein the length of the third region of the activator oligonucleotide comprises 10 to 40 nucleotides. 
     
     
         41 . The reaction mixture according to  claim 19 , wherein the activator oligonucleotide comprises a sequence wherein more than 10 nucleotides of the third region of the activator oligonucleotide complementarily can bind to the first primer extension product. 
     
     
         42 . The reaction mixture according to  claim 19 , wherein the activator oligonucleotide comprises a sequence wherein more than 20 nucleotides of the third region of the activator oligonucleotide complementarily can bind to the first primer extension product. 
     
     
         43 . The reaction mixture according to  claim 20 , wherein the sequence of the activator oligonucleotide comprises nucleotide modifications whereby at least 5 of such nucleotide modifications are coupled side by side in the activator oligonucleotide. 
     
     
         44 . The reaction mixture according to  claim 20 , wherein the sequence of the activator oligonucleotide comprises nucleotide modifications whereby at least 10 of such nucleotide modifications are coupled side by side in the activator oligonucleotide. 
     
     
         45 . The reaction mixture according to  claim 21 , wherein the length of the 3′-primed segment of the extension product of the first primer oligonucleotide comprises regions having 15 to 30 nucleotides. 
     
     
         46 . The reaction mixture according to  claim 22 , wherein the second primer oligonucleotide has a length of between 30 and 50 nucleotides. 
     
     
         47 . The reaction mixture according to  claim 23 , wherein the length of the sequence segment comprises regions of 10 to 25 nucleotides.