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 DNA sequence in a sample comprising cell-free DNA from a bodily fluid suspected of containing a low abundance target DNA sequence, wherein
the target DNA sequence differs by one nucleotide from a reference DNA sequence present in the sample, and
the reference DNA sequence is more prevalent in the sample than the target DNA sequence, the method comprising
(a) prepare a reaction mixture comprising the sample, a polymerase, two primers, and a blocker, wherein
the two primers, comprising a first primer hybridizing to the reference DNA sequence and a second primer hybridizing to the complement of the reference DNA sequence, each has a melting temperature (T m ) that is (i) above the T m of the blocker hybridized to the target DNA sequence, and (ii) below the T m of the blocker hybridized to the reference DNA sequence;
the blocker is fully complementary to the reference DNA sequence but not the target DNA sequence;
the two primers are fully complementary to the reference DNA sequence or its complement and the target DNA sequence or its complement;
(b) subjecting the reaction mixture to two or more cycles of
(1) heating the reaction mixture to a temperature above the T m of the blocker sequence hybridized to the reference DNA sequence; then
(2) cooling the reaction mixture to a temperature below the T m of the blocker sequence hybridized to the reference DNA sequence, allowing hybridization of the blocker, but not the primers, to the reference DNA sequence; then
(3) heating the reaction mixture to a temperature above the Tm of the blocker sequence hybridized to the reference DNA sequence; then
(4) cooling the reaction mixture to a temperature below the Tm of the primer sequences hybridized to the reference DNA sequence; then
(5) allowing extension of the primers by the polymerase,
thereby enriching the target DNA sequence in the sample.
2. The method of claim 1 , wherein the target DNA sequence is in EGFR Exon 20 and encodes a T790M mutation.
3. The method of claim 1 , wherein at least one primer overlaps the blocker.
4. The method of claim 1 , wherein the blocker consists of 40 base pairs or less.
5. The method of claim 1 , wherein the 3′ end of the blocker is blocked to prevent extension.
6. The method of claim 1 , wherein the 5′ end of the reference blocking sequence comprises a nucleotide that prevents 5′ to 3′ exonucleolysis by Taq DNA polymerase.
7. The method of claim 1 , wherein at least one primer further comprises an adapter sequence that is not complementary to the reference sequence, target sequence, or complements thereof.
8. The method of claim 1 , wherein the target sequence is a mutant sequence of BRAF, EGFR, c-MET, HER-2, HER-3, NRAS, PIK3CA, KRAS, AKT-1, MAP2PK, ER, AR, FGFR1, FGFR2, FGFR3, KIT, PDGFR1, PDFGR2, PDGFR3, TP53, or SMAD1.
9. The method of claim 1 , wherein the target sequence is a mutant sequence of KRAS, EGFR, PIK3CA, TP53, or BRAF.
10. The method of claim 1 , 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, high-throughput sequencing, SSCP, RFLP, dHPLC, CCM, digital PCR and quantitative-PCR.
11. The method of claim 1 , wherein an amplicon of less than 50 bp is amplified using the method.
12. The method of claim 1 , wherein the bodily fluid is plasma, blood, serum or urine.
13. The method of claim 1 , wherein the bodily fluid is urine.
14. The method of claim 1 , wherein the extension of the primers by the polymerase is at the temperature below the Tm of the primer sequences hybridized to the reference DNA sequence.
15. A method for enriching a target DNA sequence in a sample comprising cell-free DNA from a bodily fluid suspected of containing a low abundance target DNA sequence, wherein
the target DNA sequence differs by one nucleotide from a reference DNA sequence present in the sample, and
the reference DNA sequence is more prevalent in the sample than the target DNA sequence, the method comprising
(a) prepare a reaction mixture comprising the sample, a polymerase, two primers, and a blocker, wherein
the two primers, comprising a first primer hybridizing to the reference DNA sequence and a second primer hybridizing to the complement of the reference DNA sequence, each has a melting temperature (T m ) that is (i) above the T m of the blocker hybridized to the target DNA sequence, and (ii) below the T m of the blocker hybridized to the reference DNA sequence;
the blocker is fully complementary to the reference DNA sequence but not the target DNA sequence;
the two primers are fully complementary to the reference DNA sequence or its complement and the target DNA sequence or its complement;
(b) subjecting the reaction mixture to two or more cycles of
(1) heating the reaction mixture to a temperature above the T m of the blocker sequence hybridized to the reference DNA sequence; then
(2) cooling the reaction mixture to a temperature below the T m of the blocker sequence hybridized to the reference DNA sequence, allowing hybridization of the blocker, but not the primers, to the reference DNA sequence; then
(3) heating the reaction mixture to a higher temperature below the T m of the blocker sequence hybridized to the reference DNA sequence; then
(4) cooling the reaction mixture to a temperature below the Tm of the primer sequences hybridized to the reference DNA sequence; then
(5) allowing extension of the primers by the polymerase, thereby enriching the target DNA sequence in the sample.
16. The method of claim 15 , wherein the target DNA sequence is in KRAS Exon 2 and is a single base substitution from the reference sequence.
17. The method of claim 15 , wherein the extension of the primers by the polymerase is at the temperature below the Tm of the primer sequences hybridized to the reference DNA sequence.
18. The method of claim 15 , wherein at least one primer overlaps the blocker.
19. The method of claim 15 , wherein an amplicon of less than 50 bp is amplified using the method.
20. The method of claim 15 , wherein the bodily fluid is plasma, blood, serum or urine.