Hypermethylation of CDKN2A exon 2 in tumor, tumor-adjacent and tumor-distant tissues from breast cancer patients

Table 2 Primer sequences and conditions for MS-HRM analysis

	Primer sequence	Primer concentration [nM]	Additional MgCl₂ concentration [mM]	T_a [°C]^a	Amplicon length [bp]	No. of CpGs^b	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

ISSN: 1471-2407