Table 2.
Primer sequences and conditions for MS-HRM analysis
| Primer sequence | Primer concentration [nM] | Additional MgCl2 concentration [mM] | Ta [°C]a | Amplicon length [bp] | No. of CpGsb | LOD/LOQ [%] | Reference | |
|---|---|---|---|---|---|---|---|---|
| APC | f: 5′ AAGTAGTTGTGTAATTCGTTGGAT 3′ r: 5′ CACCTCCATTCTATCTCCAATA 3′ |
500 | - | 53 | 149 | 10 | 0.4/1.4 | [32] |
| BRCA1 | f: 5′ TTGTTGTTTAGCGGTAGTTTTTTGGTT 3′ r: 5′ AACCTATCCCCCGTCCAAAAA 3′ |
250 | 2 | 61 | 122 | 9 | 0.4/1.6 | [33] |
| CDKN2A | f: 5′ CGGAGGAAGAAAGAGGAGGGGT 3′ r: 5′ CGCTACCTACTCTCCCCCTCT 3′ |
400 | - | 62 | 93 | 7 | 1.0/3.3 | [34] |
| CDKN2A (exon 2) | f: 5′ GGCGGAGTTGTTGTTGTTTTATG 3′ r: 5′ ACAACACCACCAACGTATCCAA 3′ |
250 | 1.5 | 52 | 116 | 10 | 3.5/10.7 | In-house [NG_007485.1] |
| ESR1 | f: 5′ TTAAAGTTGGAGGTTCGGGAGT 3′ r: 5′ CTACCCCGAAAACCTACGAATC 3′ |
250 | - | 52 | 112 | 8 | 0.1/0.5 | In-house [NG_008493] |
| HER2/neu | f: 5′ GGATTGGAGAAATTAGGGGAGT 3′ r: 5′ ACTCCGACTAAACCCGACTAAA 3′ |
250 | 2 | 52 | 137 | 15 | 0.4/1.4 | In-house [NG_007503.1] |
| LINE-1 | f: 5′ TGTTAGATAGTGGGTGTAGGTT 3′ r: 5′ AAATACATCCGTCACCCCTTT 3′ |
250 | - | 57 | 139 | 9 | 11.7/22.3 | In-house [X58075.1] |
| PTEN | f: 5′ TCGGTTGGGTTTTTGGGTAGAGG 3′ r: 5′ CGCAAACTCTACTAAACATACCCAATAT 3′ |
250 | - | 60 | 156 | 9 | 0.1/0.5 | [35] |
f forward primer, r reverse primer, T a annealing temperature, bp base pairs, LOD limit of detection, LOQ limit of quantification
aTouchdown in 1 °C increments in cycles 1-7
bWithout CpGs in the primers