SYNTHESIS AND ENRICHMENT OF NUCLEIC ACID SEQUENCES
The present disclosure relates to the enrichment of target nucleic acid sequences present in low-abundance relative to corresponding non-target or reference nucleic acid sequence in a sample. In particular, the methods allow for a substantially greater level of detection sensitivity of target sequence by orders of magnitude enrichment of a low-abundance sequence.
1 . A method for enriching a target nucleic acid sequence in a nucleic acid sample suspected of containing one or more low abundance target sequence comprising:
a. preparing a reaction mixture comprising: a reference sequence, an excess of blocking sequence relative to the amount of reference sequence in the mixture, and suspected of containing one or more target sequence, wherein:
the target sequence is at least 50% homologous to the reference sequence;
the blocking sequence is fully complementary with region of the reference sequence, the region of the reference sequence being between or overlapping the target sequence
b. subjecting the reaction mixture to two or more cycles of:
i. heating the temperature of the reaction mixture to a preselected denaturation temperature (Tsd) allowing but not requiring denaturation of blocker sequences annealed to reference sequence, wherein the Tsd is above a calculated melting temperature of the reference sequence-blocker sequence duplex, and
ii. lowering the temperature of the reaction mixture to an elongation temperature allowing primer anneal and elongation of the one or more primer from its complementary target sequence to form enriched target sequence.
2 . A method for enriching a target nucleic acid sequence in a sample suspected of having one or more low abundance target sequence comprising:
a) preparing a reaction mixture including a reference sequence, an excess of blocking sequence that is fully complementary to the reference sequence, and a primer pair that is fully complementary with a region of the target sequence wherein the target sequence has at least 50% complementarity to the reference sequence and subjecting the reaction mixture to two or more cycles of:
i) heating the reaction mixture to a first temperature sufficient to allow denaturation of homoduplexed reference and target sequences;
ii) cooling the reaction mixture to a temperature that allows preferential formation of reference-blocker duplexed sequences;
iii) heating the reaction mixture to a selective denaturation temperature (Ts) so as to allow denaturation of the blocker-target duplexed sequences; and
iv) cooling the reaction mixture to a temperature that is below the melting temperatures of the blocker-reference sequence, the primer pair, and the blocker-mutant sequence so as to allow elongation of primer sequences annealed to the target sequence and substantial enrichment of target sequence relative to reference sequence in the reaction mixture.
3 . The method of claim 2 wherein the 3′ end on the reference blocking sequence is blocked to prevent extension.
4 . The method of claim 2 wherein the 5′ end on the reference blocking sequence comprises a nucleotide that prevents 5′ to 3′ exonucleolysis by Taq DNA polymerase.
5 . The method of claim 2 wherein the reference blocking sequence is fully complementary with one of the strands of the reference sequence between its primer binding sites, or overlapping at either of the primer binding sites.
6 . The method of claim 2 wherein the reference blocking sequence is a short blocker consisting of 40 base pairs or less in length.
7 . The method of claim 2 wherein the primer sequence has a lower annealing temperature than the reference sequence-blocker sequence melting temperature.
8 . The method of claim 2 wherein the selective temperature is about 5° C. or more above the melting temperature of the reference sequence-blocker sequence melting temperature.
9 . The method of claim 1 wherein the target sequence is a EGFR Exon 19 deletion and the selective denaturation temperature is about 5° C. or more above the melting temperature (Tm) of the reference sequence-blocker sequence melting temperature.
10 . The method of claim 9 wherein the selective denaturation temperature is about 5.5° C. above the melting temperature of the reference sequence-blocker sequence melting temperature and about 15.5° C. above the Tm of the primer sequence.
11 . The method of claim 9 wherein the selective denaturation temperature is lower than the melting temperature of the reference sequence-blocker sequence melting temperature and about 15.5° C. above the Tm of the primer sequence.
12 . The method of claim 9 wherein the selective denaturation temperature is about 5.5° C. above the melting temperature of the reference sequence-blocker sequence or melting temperature and above the Tm of the primer sequence.
13 . The method of claim 10 wherein the elongation temperature is at about 62.4° C. or between about 61° C. and about 64° C.
14 . The method of claim 2 wherein a difference in melting temperature between reference:blocker sequence and blocker:target sequence is between about 5 to 14 degrees Celsius.
15 . The method of claim 2 wherein the reference sequence is a KRAS and a difference in melting temperature between a reference:blocker sequence and a blocker:target is −1.1° C.
16 . The method of claim 2 wherein the target sequence is a EGFR Exon 20 T790M selective denaturation temperature is about 14.3° C. or more above the melting temperature of the reference sequence-blocker sequence melting temperature.
17 . The method of claim 12 wherein the selective annealing temperature is about 2° C. below the melting temperature of the reference sequence-blocker sequence melting temperature.
18 . The method of claim 12 wherein the elongation temperature is at about 62.4° C. or between about 61° C. and about 64° C.
19 . The method of claim 2 wherein the mutant allele is selected from the group consisting of: BRAF; KRAS; EGFR; NRAS; C-MET; HER-2; HER-3; PIK3CA; AKT-1; MAP2PK; ER; AR; FGFR1; FGFR2; FGFR3; KIT; PDGFR1; PDFGR2; PDGFR3; TP53; and SMAD1.
20 . The method of claim 2 wherein after cycling, a sample of the reaction mixture is analyzed using one or more of the methods selected from the group consisting of: MALDI-TOF, HR-Melting, Di-deoxy-sequencing, Single-molecule sequencing, pyrosequencing. Second generation high-throughput sequencing, SSCP, RFLP, dHPLC, CCM, digital PCR and quantitative-PCR.