Table 3. PCR primers forward (F), reverse (R) and sequencing primers (S) used for the RFC1gene Pyrosequencing methylation assays.
| CpG site No. | Nucleotide No. * | Primer sequence, 5′-3′ | Ta (°C) | Size (bp) |
|---|---|---|---|---|
| 1–3 5′CGI shore |
-8067,-8042,-8025 |
F: GTTAGTGTTGGGAGGTTTGA R: biotin - TACAACTACCCCTCTCTCCA |
59 |
181 |
| |
|
S: GTTTTTGTGATGGGTTAT |
|
|
| 4–7 5′CGI shore |
-7784, -7779, -7687,-7773 |
F: TGGAAATGTGGGAGGAAAAAT R: biotin-ACACCCCATATACAAAAAAAC |
58 |
107 |
| |
|
S: ATTGTATATGTTGGGGTA |
|
|
| 8–14 5′CGI shore |
-6224, -6222,-6218 -6212, -6206,-6202 -6188 |
F: TGTGTGGTTGGGGAATTTT R: biotin-AAACCAATCCCTCACCTATCTC S: AGGGTAGTTTGGGTAGGTTTTT |
57 |
131 |
| 15–17 5′CGI shore |
-5456, -6030, -6010 |
F: GGAAGGGGGTGGGAGTTAT R: CCAAAACAACCTACTCCCTTTAC |
57 |
119 |
| |
|
S: TTTGTGGGAAGGGGTTTA |
|
|
|
18–23
CGI 1 |
-5205, -5199, -5193
-5184, -5182, -5171 |
F: TGGGAGAGTGGTTTAGGT R: biotin-AACCACCAATCCCCATCC |
57 |
107 |
| |
|
S: GTTTTTTTTTTTGAGTGTGAT |
|
|
|
24–30
CGI 1 |
-3713,-3704,-3693
-3688,-3664,-3649 -3629 |
F: GTGTAATTTTTTTTTGGAGTAGTTGTGG R: biotin-AACCCTAAAAAAATAACTTTCCTACTA |
57 |
147 |
| |
|
S1: GTGTAATTTTTTTTTGGAGTAGTTGTGG |
|
|
| |
|
S2: AGAGAGAGTTTGGATAG |
|
|
| 31–33 3′CGI shore |
-2989,-2974,-2953 |
F: TTTGGTTAGTTTTTTAGAGTAGGAGTTG R: biotin-ACCCCACTATCCACAATAAC |
56 |
145 |
| |
|
S: GTAGGAGTTGGTTTAAAT |
|
|
| 34–37 3′CGI shore |
-2065,-2079,-2083 -2092 |
F: biotin-ATAGGTTTTGAGGAGGTATGG R: ACAACCAAAACCCTAAAAATTCTC |
58 |
148 |
| |
|
S: AACCACTACCAAACC |
|
|
| 38–42 5′CGI2 shore |
-1082,-1067,-1049 -1037,-1032 |
F: GTGGTATTTGGAGATATGATTTTGATAAGT R: biotin-ACCCCAAACCACAAAACTTC |
57.3 |
271 |
| |
|
S: ATTTTGATAAGTGGATAGGT |
|
|
| 43–49 5′CGI2 shore |
-971,-982,-988 -950,-939,-930 -918 |
F: biotin-TTGGGTAGAATTAGGATTGAAAG R: ACCCCAAACCACAAAACTT |
57 |
159 |
| |
|
S1: AATAAAACAACAACAACAC |
|
|
| |
|
S2: ACCCAACCTTAAAAACC |
|
|
|
50–55
|
38,59,66,75,78,83
|
F: AGATTATTTTTTAAGGTGTTTTGATTTTAT |
57.3 |
240 |
|
CGI 2
|
|
R: biotin-AAAATAATAAAACTCTCCCCTAACC |
|
|
| S: GGTGGAGAAGTAGGTG |
*
Nucleotide number is relative to the ATG start site.