Table 1 PCR and Pyrosequencing primers and conditions.

Forward

5′-TTTTTTTTTTGAAGTTTTTTTA-3’

95 °C

(14′30″)

Reverse

5′-BIOTIN-CCCCCAACTCCCCAAAAA-3’

94 °C

(30″)

50 °C

(30″)

45 cycles

72 °C

(30″)

(410 bp)

72 °C

(10′)

4 °C

Indefinitely

Sequencing primers

40 to 42 CpG

5′-AGAAAAGAAATTGGAGAAATT-3′

43 to 47 CpG

5′-GTTTTAGAGAGATTAGGT-3′

Analyzed sequences

Seq 1

YGGTGGTTTTTTTAAYGTYGTTTTAATCGTGTTGATCAGTCGCTTA

Seq 2

YGGTTTTYGTYGTTGTYGTYGTTAGTCAGTTCAGTCGTAGTCAGTCGTA