Detection of target nucleic acid and variants

Abstract

The present invention relates to highly sensitive and specific methods for detection of nucleic acids, which for example are useful for detection of rare mutations, or for detection of low-abundance variants in nucleic acids sequences. The methods involve an asymmetric incremental polymerase reaction (AIPR) followed by an exponential polymerase chain reaction (PCR).

1 - 20 . (canceled) 
     
     
         21 : A method of predicting the presence of a clinical condition in an individual, wherein said clinical condition is linked to the presence of a variant sequence in a target nucleic acid sequence or said clinical condition is linked to the presence of a target nucleic acid sequence, said method comprising the steps of
 a) providing a sample from said individual comprising template nucleic acids   b) providing a set of primers comprising at least a pair of primers specifically capable of amplification of the target nucleic acid sequence, wherein the set of primers at least comprises a primer-H and a primer-L, wherein the melting temperature of primer-H is at least 16° C. higher than the melting temperature of primer-L, and wherein primer-L contains a sequence complementary to a fragment of the elongation product of primer-H,   c) providing a nucleic acid polymerase having polymerase activity at an elongation temperature,   d) preparing partitioned PCR reactions each comprising a part of the sample, the set of primers, the nucleic acid polymerase, PCR reagents and optionally detection reagents,   e) performing an asymmetric incremental polymerase reaction (AIPR) comprising the steps of:
 i) incubating the partitioned PCR reactions at a denaturation temperature, thereby denaturing DNA to single-stranded molecules, 
 ii) incubating the partitioned PCR reactions at a high annealing temperature allowing annealing of primer-H, but not of primer-L, 
 iii) optionally incubating the partitioned PCR reactions at the elongation temperature, 
 iv) optionally repeating steps i to iii, 
   f) performing a polymerase chain reaction (PCR) comprising the steps of:
 1) incubating the partitioned PCR reactions from step e) at a denaturation temperature, thereby denaturing DNA to single-stranded molecules, 
 2) incubating the PCR at a low annealing temperature allowing annealing of both primer-H and primer-L, 
 3) incubating the PCR at the elongation temperature thereby allowing extension of all annealed primers, 
 4) repeating steps 1 to 3, 
   g) detecting whether the PCR product comprises the target nucleic acid sequence or the variant sequence in the target nucleic acid sequence,   wherein the presence of said variant sequence in said target nucleic acid or the presence of said target nucleic acid sequence is indicative of the presence of said clinical condition.   
     
     
         22 : The method according to  claim 21 , wherein the clinical condition is cancer. 
     
     
         23 : The method according to  claim 21 , wherein the variant sequence is a mutation associated with cancer. 
     
     
         24 : The method according to  claim 22 , wherein the cancer is selected from the group consisting of carcinoma of the breast, colorectal, pancreas, stomach, GIST, hepatocellular, lung, small cell lung, ovarian, uterine, cervix, bladder, renal, prostate, testis, thyroid carcinoma, malignant melanoma, osteosarcoma, chondrosarcoma, myosarcoma, glioblastoma or other brain tumors, head/neck, other gastrointestinal and germ cell tumors, and haematologic malignancies. 
     
     
         25 : The method according to  claim 21 , wherein the sample comprises DNA from cancer cells, optionally wherein the sample comprises cancer cells comprising DNA and/or free DNA derived from cancer cells. 
     
     
         26 : The method according to  claim 21 , wherein the sample is a blood sample or a fraction thereof, and the template nucleic acids are selected from the group consisting of cell-free DNA, nucleosomal DNA and circulating tumor DNA. 
     
     
         27 : The method according to  claim 21 , wherein the sample is selected from the group consisting of saliva samples, urine samples, vaginal fluid samples, ascites fluid sample, cerebrospinal fluid samples and tissue exudate samples. 
     
     
         28 : The method according to  claim 21 , wherein the variant sequence is mutation is selected from the group consisting of:
 A. a mutation in PIK3CA, such as any one of the mutations H1047R or E542K or E545K;   B. a mutation in BRAF, such as V600E;   C. a mutation in KRAS, such as any one of the mutations G12D, G12V, G12C or G13D; and   D. a mutation in EGFR, such as any one of the mutations L858R or T790M.   
     
     
         29 : The method according to  claim 21 , wherein the variant sequence is a mutation selected from the group consisting of one of the following the mutations:
 a) an A to G mutation of nucleotide 3140 of SEQ ID NO: 69;   b) a G to A mutation of nucleotide 1624 of SEQ ID NO: 69;   c) a G to A mutation of nucleotide 1633 of SEQ ID NO: 69;   d) a T to A mutation of nucleotide 1799 of SEQ ID NO: 70;   e) a T to G mutation of nucleotide 2573 of SEQ ID NO: 71;   f) a C to T mutation of nucleotide 2369 of SEQ ID NO: 71;   g) a G to A mutation of nucleotide 38 of SEQ ID NO: 72;   h) a G to T mutation of nucleotide 34 of SEQ ID NO: 72;   i) a G to C mutation of nucleotide 34 of SEQ ID NO: 72;   j) a G to A mutation of nucleotide 34 of SEQ ID NO: 72;   k) a G to T mutation of nucleotide 35 of SEQ ID NO: 72;   l) a G to C mutation of nucleotide 35 of SEQ ID NO: 72; and/or   m) a G to A mutation of nucleotide 35 of SEQ ID NO: 72.   
     
     
         30 : The method according to  claim 21  wherein:
 A) the variant sequence is at least one of the nucleotides 3140, 1624 or 1633 of SEQ ID NO:69, and wherein primer-H and primer-L flank at least one of the nucleotides 3140, 1624 or 1633 of SEQ ID NO:69, and 
 
       wherein primer-H and primer-L anneal to SEQ ID NO: 69 on opposite strands and in opposite orientations so that together primer-H and primer-L amplify a target sequence comprising at least one of the nucleotides 3140, 1624 or 1633 of SEQ ID NO:69;
 B) the variant sequence is the nucleotide 1799 of SEQ ID NO: 70, and wherein primer-H and primer-L flank nucleotide 1799 of SEQ ID NO: 70, and wherein primer-H and primer-L anneal to SEQ ID NO: 70 on opposite strands and in opposite orientations so that together primer-H and primer-L amplify a target sequence comprising nucleotide 1799 of SEQ ID NO:70; 
 C) the variant sequence is at least one of the nucleotides 2573 or 2369 of SEQ ID NO: 71, and wherein primer-H and primer-L flank at least one of nucleotides 2573 or 2369 of SEQ ID NO: 71, and wherein primer-H and primer-L anneal to SEQ ID NO: 71 on opposite strands and in opposite orientations so that together primer-H and primer-L amplify a target sequence comprising at least one of nucleotides 2573 or 2369 of SEQ ID NO:71; or 
 D) the variant sequence is at least one of the nucleotides 34, 35 or 38 of SEQ ID NO: 72, and wherein primer-H and primer-L flank at least one of nucleotides 34, 35 or 38 of SEQ ID NO: 72, and wherein primer-H and primer-L anneal to SEQ ID NO: 72 on opposite strands and in opposite orientations so that together primer-H and primer-L amplify a target sequence comprising at least one of nucleotides 34, 35 or 38 of SEQ ID NO:72. 
 
     
     
         31 : The method according to  claim 21  wherein:
 A) the variant sequence is at least one of the nucleotides 3140, 1624 or 1633 of SEQ ID NO:69, and primer-H and primer-L comprise or consist of a sequence identical to a part of the target sequence, and wherein the target sequence comprises at least one of the nucleotides 3140, 1624 or 1633 of SEQ ID NO:69; 
 B) the variant sequence is the nucleotide 1799 of SEQ ID NO:70, and primer-H and primer-L comprise or consist of a sequence identical to a part of the target sequence, and wherein the target sequence comprises the nucleotide 1799 of SEQ ID NO:70; 
 C) the variant sequence is at least one of the nucleotides 2573 or 2369 of SEQ ID NO:71, and primer-H and primer-L comprise or consist of a sequence identical to a part of the target sequence, and wherein the target sequence comprises at least one of the nucleotides 2573 or 2369 of SEQ ID NO:71; or 
 D) the variant sequence is at least one of the nucleotides 34, 35 or 38 of SEQ ID NO:72, and primer-H and primer-L comprise or consist of a sequence identical to a part of the target sequence, and wherein the target sequence comprises at least one of the nucleotides 34, 35 or 38 of SEQ ID NO:72. 
 
     
     
         32 : The method according to  claim 21  wherein
 A) primer-H anneals to a sequence within SEQ ID NO: 69 and primer-L anneals to a sequence complementary to a sequence within SEQ ID NO: 69; or primer-H anneals to sequence complementary to a sequence within SEQ ID NO: 69 and primer-L anneals to a sequence within SEQ ID NO: 69; and wherein primer-H and primer-L are together capable of amplifying at least one of the nucleotides 3140, 1624 or 1633 of SEQ ID NO:69; 
 B) primer-H anneals to a sequence within SEQ ID NO: 70 and primer-L anneals to a sequence complementary to a sequence within SEQ ID NO: 70; or primer-H anneals to sequence complementary to a sequence within SEQ ID NO: 70 and primer-L anneals to a sequence within SEQ ID NO: 70; and wherein primer-H and primer-L are together capable of amplifying at least the nucleotide 1799 of SEQ ID NO:70; 
 C) primer-H anneals to a sequence within SEQ ID NO: 71 and primer-L anneals to a sequence complementary to a sequence within SEQ ID NO: 71; or primer-H anneals to sequence complementary to a sequence within SEQ ID NO: 71 and primer-L anneals to a sequence within SEQ ID NO: 71; and wherein primer-H and primer-L are together capable of amplifying at least one of the nucleotides 2573 or 2369 of SEQ ID NO:71; 
 D) primer-H anneals to a sequence within SEQ ID NO: 72 and primer-L anneals to a sequence complementary to a sequence within SEQ ID NO: 72; or primer-H anneals to sequence complementary to a sequence within SEQ ID NO: 72 and primer-L anneals to a sequence within SEQ ID NO: 72; and wherein primer-H and primer-L are together capable of amplifying at least one of the nucleotides 34, 35 or 38 of SEQ ID NO:72. 
 
     
     
         33 : The method according to  claim 21 , wherein:
 a) the primer-H comprises a consecutive sequence of in the range of 50 to 100 nucleotides of SEQ ID NO:69 and primer-L comprises a consecutive sequence of in the range of 10 to 20 nucleotide of the sequence complementary to SEQ ID NO:69; or   b) the primer-H comprises a consecutive sequence of in the range of 50 to 100 nucleotides of the sequence complementary to SEQ ID NO:69, and primer-L comprises a consecutive sequence of in the range of 10 to 20 nucleotide of SEQ ID NO:69;   and wherein primer-H and primer-L together are capable of amplifying a target sequence comprising at least one of the nucleotides 3140, 1624 or 1633 of SEQ ID NO:69.   
     
     
         34 : The method according to  claim 21 , wherein:
 a) the primer-H comprises a consecutive sequence of in the range of 50 to 100 nucleotides of SEQ ID NO:70 and primer-L comprises a consecutive sequence of in the range of 10 to 20 nucleotide of the sequence complementary to SEQ ID NO:70; or   b) the primer-H comprises a consecutive sequence of in the range of 50 to 100 nucleotides of the sequence complementary to SEQ ID NO:70, and primer-L comprises a consecutive sequence of in the range of 10 to 20 nucleotide of SEQ ID NO:70;   and wherein primer-H and primer-L together are capable of amplifying a target sequence comprising nucleotide 1799 of SEQ ID NO:70.   
     
     
         35 : The method according to  claim 21 , wherein:
 a) the primer-H comprises a consecutive sequence of in the range of 50 to 100 nucleotides of SEQ ID NO:71 and primer-L comprises a consecutive sequence of in the range of 10 to 20 nucleotide of the sequence complementary to SEQ ID NO:71; or   b) the primer-H comprises a consecutive sequence of in the range of 50 to 100 nucleotides of the sequence complementary to SEQ ID NO:71, and primer-L comprises a consecutive sequence of in the range of 10 to 20 nucleotide of SEQ ID NO:71;   and wherein primer-H and primer-L together are capable of amplifying a target sequence comprising at least one of nucleotides 2573 or 2369 of SEQ ID NO:71.   
     
     
         36 : The method according to  claim 21 , wherein:
 a) the primer-H comprises a consecutive sequence of in the range of 50 to 100 nucleotides of SEQ ID NO:72 and primer-L comprises a consecutive sequence of in the range of 10 to 20 nucleotide of the sequence complementary to SEQ ID NO:72; OR   b) the primer-H comprises a consecutive sequence of in the range of 50 to 100 nucleotides of the sequence complementary to SEQ ID NO:72, and primer-L comprises a consecutive sequence of in the range of 10 to 20 nucleotide of SEQ ID NO:72;   and wherein primer-H and primer-L together are capable of amplifying a target sequence comprising at least nucleotide 34, 35 or 38 of SEQ ID NO:72.   
     
     
         37 : A method of predicting the presence of a clinical condition in an individual, wherein said clinical condition is linked to the presence of a target nucleic acid sequence, said method comprising the steps of
 a. providing a sample from said individual comprising template nucleic acids   b. providing a set of primers comprising at least a pair of primers specifically capable of amplification of the target nucleic acid sequence, wherein the set of primers at least comprises a primer-H and a primer-L, wherein the melting temperature of primer-H is at least 16° C. higher than the melting temperature of primer-L, and wherein primer-L contains a sequence complementary to the elongation product of primer-H,   c. providing a nucleic acid polymerase having polymerase activity at an elongation temperature, which is higher than the melting temperature of primer-H,   d. preparing partitioned PCR reactions each comprising a part of the sample, the set of primers, the nucleic acid polymerase, PCR reagents and optionally detection reagents   e. performing an asymmetric incremental polymerase reaction (AIPR) comprising the steps of:
 i. incubating the partitioned PCR reactions at a denaturation temperature, thereby denaturing DNA to single-stranded molecules 
 ii. incubating the partitioned PCR reactions at a high annealing temperature allowing annealing of primer-H, but not of primer-L, 
 iii. optionally incubating the partitioned PCR reactions at the elongation temperature, 
 iv. optionally repeating steps i to iii, 
   f. performing a polymerase chain reaction (PCR) comprising the steps of:
 1. incubating the partitioned PCR reactions at a denaturation temperature, thereby denaturing DNA to single-stranded molecules 
 2. incubating the PCR at a low annealing temperature allowing annealing of both primer-H and primer-L, 
 3. incubating the PCR at the elongation temperature thereby allowing extension of all annealed primers 
 4. optionally repeating steps 1 to 3, thereby obtaining a PCR product 
   g. detecting whether the PCR product comprises the target nucleic acid sequence   wherein the presence of said target nucleic acid is indicative of the presence of said clinical condition.   
     
     
         38 : The method according to  claim 37 , wherein the clinical condition is infection by an infectious pathogen. 
     
     
         39 : The method according to  claim 38 , wherein the target nucleic acid is a nucleic acid sequence from the genome of said pathogen.