Table 2. PCR primers forward (F), reverse (R) and sequencing primers (S) used for the PCFT gene Pyrosequencing methylation assays.
| CpG site No. | Nucleotide No. * | Primer sequence, 5′-3′ | Ta (°C) | Size (bp) |
|---|---|---|---|---|
| 1–5 5′CGI shore |
-4604, -4600,-4598 -4560, -4551 |
F: GGAAAAGAAAATTTGTATTTAGAGTGAGA R: biotin-ATCAAACTACTAACCTCAAATAATCC |
57.5 |
170 |
| |
|
S: ATTTGTAAAAATAATAATATTTGGT |
|
|
| 6–8 |
-2650, -2616, -2594 |
F:TTTGGGTTTTGGATTTTATAGTGTAG |
55 |
177 |
| 5′CGI shore |
|
R: biotin-ACTACCATATCCCCAACATCTAAAATAAT |
|
|
| |
|
S: TTTTTTTTGAGATTGAGAGTT |
|
|
|
9–31
CGI 1 |
-55, -50, -46, -44,
-42, -37, -34, -30 -23, -14, -11, -9 11, 13, 22, 33, 35 39, 46, 53, 63, 70 79 |
F:GGTGGTTTTAGGTTATAGG R: biotin-ATTACTACAACCCCCCCTTTA S1: GTATATGGAGGGGAG S2: GTTGGTTTTAGGTAG |
57.3 |
366 |
|
32–36
CGI 1 |
276, 287, 295, 304
318 |
F: GAGTTTGGTAGGTGGAGGGTT R: biotin-ATCAAACCCCTTTACTCTAATCCC |
60 |
136 |
| |
|
S: AGGGTTTTGGTTTGG |
|
|
|
38–44
CGI 2 |
687, 693, 716, 741
754, 757, 761, 779 |
F: TTTTTTATTGGATTTTTTATATGAA R: biotin-ACACTAAAACCTAAAACAACAAACC |
55 |
147 |
| |
|
S1: TTTATTGGATTTTTTATATG |
|
|
| |
|
S2: TGTTGGGAGTTTGGA |
|
|
| 45–48 3′CGI shore |
138, 1148, 1153 1162 |
F:TATGTAGTTTTTTGTTTTGGTGAGAT R: biotin-CCCAAAATACACAATAATCACCAC |
60 |
177 |
| |
|
S: GGTGAGATTTTAAAGGAGTTA |
|
|
| 49–50 |
2373, 2379 |
F:GTGTTGGGATTATAGGTATGAGT |
60 |
170 |
| 3′CGI shore |
|
R: biotin-AAAATTCCCTTCTACTTAATTATCAAAC |
|
|
| |
|
S: GGGATTATAGGTATGAGTTAT |
|
|
| 51–52 |
3926, 3931 |
F: AGATAGAGTAGGGTGAGTGTTAG |
59 |
100 |
| 3′CGI shore |
|
R: biotin-ACTATTCCCCCAACCATAAC |
|
|
| |
|
S: GGTTTTTTATGTTTAGTGTTGT |
|
|
*
Nucleotide number is relative to the ATG start site.