DNA Methylation Validation Methods: a Coherent Review with Practical Comparison

Table 1 Primer sequences and characteristics

Primer type	Forward/ sequencing primer	Reverse primer	T_ann [°C]	Product length [bp]
M pyrosequencing	GGTAGGAGGATGGTTTGAATT/ GGTGGAAATGAAGTAGGTGTGTTTG	GTGCCGAGGCTCAGGCAACACTACTCTTACCAAAACAACC	60	373/ 227
IM pyrosequencing	GTTAAGGGGGTGTATTTTAGAGA/ GGTAGAGAGAAGTTTTTTTTGTAGG	GTGCCGAGGCTCAGGCCTTAACTACTTTCCCAAACTACCT	58	399/ 339
U pyrosequencing	GGGGGGGTGTTAGTATTTG/ TTAGTATTTGYGTTGTGGAGTG	GTGCCGAGGCTCAGGCCCAAACTAACCTAATAAAACC	58	300/ 290
Universal biotinylated primer	5’biotin-TCTGTGCCGAGGCTCAGGC
M MSRE	TTTTCTGTGACCTCCTTTGG	CAGTGTGACTGCTGGTGAAG	60	243
N MSRE	GCAATAGGCGTTAATGTCGT	AGGAGTGGCAAAAGAGGACT	60	199
U MSRE	CGCTTAGCAATCATCGACTT	GAAACAGGCCGCATCCTC	60	265
M MSP Met	GTATATTCGGAATTATTTCGTTTTC	AATTAACAACCGACAACCG	56	72
M MSP Unm	GATGTATATTTGGAATTATTTTGTTTTT	AATTAACAACCAACAACCA	56	75
IM MSP Met	CGGTTTTTATAGTTTTGAATTAGATC	TTATTTATTATCACATCAACTACTTCCG	58	166
IM MSP Unm	ATTGGTTTTTATAGTTTTGAATTAGATT	TTATTTATTATCACATCAACTACTTCCA	58	168
U MSP Met	CGTTGTGGAGTGAAGTGAATC	ACCGAACGAACAATAAACGAA	54	210
U MSP Unm	TGTGTTGTGGAGTGAAGTGAATT	ACCAAACAAACAATAAACAAAAAA	54	212
M HRM	TTGGGTGGAAATGAAGTAGGTGTG	CCAAACCATTAACCATAACAATA	54–58^*	94
IM HRM	TTTGGGGAAAAAATATATGGAGTT	CTACTAATAAAACCCTTTACTCCCA	54–58^*	90
U HRM	TTAGTATTTGYGTTGTGGAGTG	CCRACACTTACTCTTATTAACRATC	54–58^*	93
M HRM Wojdacz	CGGGGGGGTGTTAGTATTTG	CCCGACACTTACTCTTATTAACRATC	55	110
U HRM Wojdacz	TCGTGTTTTTTTTTGGGTGGAAATG	GCGACCAAACCATTAACCATAACA	55	104

^*For MS-HRM experiments T_ann was 55 °C, in qMSP experiments T_ann of MSP primers was used
M methylated locus, IM intermediately methylated locus, U unmethylated locus, MSP methylation specific PCR, Met primers for methylated DNA sequence, Unm primers for unmethylated DNA sequence, HRM high resolution melting analysis, T_ann annealing temperature

ISSN: 1480-9222