Single nucleotide polymorphisms (SNPs) result from a single nucleotide mutation in the genome. Many SNPs are closely linked to human diseases and drug efficiency. Single-nucleotide variations (SNVs) are biomarkers allowing the detection of drug resistance in cancer and bacterial infection. Unfortunately, the nonspecific binding of DNA probes limits their specific detection.
In 2016, Chen et al. developed a universal low-cost assay for the colorimetric discrimination of drug-resistance-related point mutation. The assay utilizes a universal DNA probe and a split G-quadruplex allowing for recognition with the naked eye at room temperature. Using the DNA probe as a signal reporter improves the universality and enables a high specificity during probe hybridization.
Chen et al. applied the assay for the detection of cancer-related SNVs in the following genes: the epidermal growth factor receptor (EGFR) gene, Kirsten rat sarcoma viral oncogene homolog (KRAS), and tuberculosis drug-resistance related point mutations in the RNA polymerase beta subunit gene (rpoB).
The researchers suggested that this method is simple, rapid, effective, and enables high-throughput detection suitable for point-of-care applications.
A similar method called MERFISH enables multiplexed fluorescence in situ hybridization.
Table 1: Signal report strand for colorimetric detection.
A | GTTAAATCGTGGATAGTAGACGCACATGGGT |
B | TGGGTAGGGCGGGTGTGCCAGGTACATTTGCTCGTCCTT |
Table 2: Signal report strand for fluorescence detection.
A | BHQ1-GTGCGAACAGGTACATTTGCTCGTCCTT |
B | GTTAAATCGTGGATAGTAGACTTCGCAC-FAM'6 |
Table 3: Sequences of different signal probe for optimization of G-quadruplex split modes.
1:1 | A | CCAAGGTGGTGTGTGTATAGTGAGGGCAGGG |
B | GGGAGGTGCTCACTATACACACACCACCAACC | |
1:3+s | A | CCAAGGTGGTGTGTGTATAGTGATGGGTAGGGCGGG |
B | AGTCAGTCAGTCACTCACTATACACACACCACCAACC | |
S | TGGGTGACTGACTGACT | |
1:3 | A | CCAAGGTGGTGTGTGTATAGTGAATGGGT |
| B | TGGGTAGGGCGGGTCTCACTATACACACACCACCAACC |
Table 4: Sequences for the optimization of the number of complement bases between A and B.
5’- TCG CAC | A | GTTAAATCGTGGATAGTAGACTCGCACATGGGT |
B | TGGGTAGGGCGGGTGTGCGACAGGTACATTTGCTCGTCCTT | |
5’- CG CAC | A | GTTAAATCGTGGATAGTAGACCGCACATGGGT |
B | TGGGTAGGGCGGGTGTGCGCAGGTACATTTGCTCGTCCTT | |
5’- G CAC | A | GTTAAATCGTGGATAGTAGACGCACATGGGT |
B | TGGGTAGGGCGGGTGTGCCAGGTACATTTGCTCGTCCTT | |
5’- CAC | A | GTTAAATCGTGGATAGTAGACCACATGGGT |
B | TGGGTAGGGCGGGTGTGCAGGTACATTTGCTCGTCCTT | |
5’- AC | A | GTTAAATCGTGGATAGTAGACACATGGGT |
B | TGGGTAGGGCGGGTGTCAGGTACATTTGCTCGTCCTT |
Table 5: Sequences of SNV, WT, and target-specific X-probe components for EGFR mutations.
EGFR-G719A | SNV | TTCAAAAAGATCAAAGTGCTGGCCTCCGGT |
WT | TTCAAAAAGATCAAAGTGCTGGGCTCCGGT | |
P | AAGGACGAGCAAATGTACCTGCACAAAAAGATCAAAGTGCTGG | |
C | CGGAGGCCAGCACTTTGATCTTTTTGTGGTCTACTATCCACGATTTAAC | |
EGFR-S768I | SNV | GCCTACGTGATGGCCATCGTGGACAACCCC |
WT | GCCTACGTGATGGCCAGCGTGGACAACCCC | |
P | AAGGACGAGCAAATGTACCTGCACTACGTGATGGCCATCGT | |
C | GGTTGTCCACGATGGCCATCACGTAGTGGTCTACTATCCACGATTTAAC | |
EGFR-T790M | SNV | GTGCAGCTCATCATGCAGCTCATGCCCTTC |
WT | GTGCAGCTCATCACGCAGCTCATGCCCTTC | |
P | AAGGACGAGCAAATGTACCTGCAGCAGCTCATCATGCAGCTC | |
C | AGGGCATGAGCTGCATGATGAGCTGCTGGTCTACTATCCACGATTTAAC | |
EGFR-L858R | SNV | ATGTCAAGATCACAGATTTTGGGCGGGCCA |
WT | ATGTCAAGATCACAGATTTTGGGCTGGCCA | |
P | AAGGACGAGCAAATGTACCTGCAGTCAAGATCACAGATTTTGG | |
C | GCCCGCCCAAAATCTGTGATCTTGACTGGTCTACTATCCACGATTTAAC | |
EGFR-L861Q | SNV | TGGCCAAACAGCTGGGTGCGGAAGAGAAAG |
WT | TGGCCAAACTGCTGGGTGCGGAAGAGAAAG | |
P | AAGGACGAGCAAATGTACCTG CAGCCAAACAGCTGGGTGCG | |
C | TTTCTCTTCCGCACCCAGCTGTTTGGCTG GTCTACTATCCACGATTTAAC |
Table 6: Sequences of SNV, WT, and target-specific X-probe components for KARAS mutations.
KRAS-G12A | SNV | CTTGTGGTAGTTGGAGCTGCTGGC |
WT | CTTGTGGTAGTTGGAGCTGGTGGC | |
P | AAGGACGAGCAAATGTACCTGCAACTTGTGGTAGTTGGAG | |
C | GCCAGCAGCTCCAACTACCACAAGTTGGTCTACTATCCACGATTTAAC | |
KARAS-G12R | SNV | CTTGTGGTAGTTGGAGCTCGTGGC |
WT | CTTGTGGTAGTTGGAGCTGGTGGC | |
P | AAGGACGAGCAAATGTACCTGCAACTTGTGGTAGTTGGAGC | |
C | GCCACGAGCTCCAACTACCACAAGTTGGTCTACTATCCACGATTTAAC | |
KARAS-G13D | SNV | CTTGTGGTAGTTGGAGCTGGTGACGTAGGC |
WT | CTTGTGGTAGTTGGAGCTGGTGGCGTAGGC | |
P | AAGGACGAGCAAATGTACCTGCATGTGGTAGTTGGAGCTGG | |
C | CTACGTCACCAGCTCCAACTACCACATGGTCTACTATCCACGATTTAAC | |
KARAS-G13V | SNV | CTTGTGGTAGTTGGAGCTGGTGTCGTAGGC |
WT | CTTGTGGTAGTTGGAGCTGGTGGCGTAGGC | |
P | AAGGACGAGCAAATGTACCTG CATGTGGTAGTTGGAGCTGG | |
C | CTACGACACCAGCTCCAACTACCACATGGTCTACTATCCACGATTTAAC | |
KARAS-Q61H | SNV | GCAGGTCACGAGGAGTACAGTGCAATGAGG |
WT | GCAGGTCAAGAGGAGTACAGTGCAATGAGG | |
P | AAGGACGAGCAAATGTACCTG CAAGGTCACGAGGAGTACAG | |
C | TCATTGCACTGTACTCCTCGTGACCTTG GTCTACTATCCACGATTTAAC |
Table 7: Sequences of SNV, WT, and target-specific X-probe components for EGFR mutations.
rpoB-531 | SNV | ACCCACAAGCGCCGACTGTTG |
WT | ACCCACAAGCGCCGACTGTCG | |
P | AAGGACGAGCAAATGTACCTGCA ACCCACAAGCGCCGA | |
C | CAACAGTCGGCGCTTGTGGGTTGGTCTACTATCCACGATTTAAC |
Table 8: Sequences for the mismatched detection.
rpoB-531 | Target DNA | ACCCACAAGCGCCGACTGTTG |
Single-base mismatch DNA | ACCCACAAGCGCCGACTGTCG | |
Three-base mismatch DNA | ACCCACAAGCGCCGACTCACG | |
Non-complementary DNA | TAGTGGTCTCATGTCCACGTA | |
EGFR-T790M | Target DNA | GTGCAGCTCATCATGCAGCTCATGCCCTTC |
Single-base mismatch DNA | GTGCAGCTCATCACGCAGCTCATGCCCTTC | |
Three-base mismatch DNA | GTGCAGCTCATCTCACAGCTCATGCCCTTC | |
Non-complementary DNA | TACTGATGACCAGTCGACGAACATGATCGT | |
KARAS-G12R | Target DNA | CTTGTGGTAGTTGGAGCTCGTGGC |
Single-base mismatch DNA | CTTGTGGTAGTTGGAGCTGGTGGC | |
Three-base mismatch DNA | CTTGTGGTAGTTGGAGCAGCTGGC | |
Non-complementary DNA | TACTGATGTCCACTCTAGGAACTA |
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Reference
Chen X, Zhou D, Shen H, Chen H, Feng W, Xie G. A universal probe design for colorimetric detection of single-nucleotide variation with visible readout and high specificity. Sci Rep. 2016 Feb 2;6:20257. [ PMC ]
Read out probes for MERFISH
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