Supplemental information

METHYLATION OF THE NON-HOMOLOGOUS END JOINING REPAIR PATHWAY GENES DOES NOT EXPLAIN THE INCREASE OF TRANSLOCATIONS WITH AGING

Idoia Martín-Guerrero,1 Elena de Prado,1 Elixabet Lopez-Lopez1 Maite Ardanaz,3 Juan Carlos Vitoria,4 Luis Parada,5 Cristina García-Orad,6 Africa García-Orad1,2

1Department of Genetics, Physical Anthropology and Animal Physiology, Faculty of Medicine and Odontology, University of the Basque Country UPV/EHU, Leioa, Spain 2BioCruces Health Research Institute 3Hospital Txagorritxu, Vitoria, Spain 4Hospital de Cruces, Barakaldo, Spain 5Institute of Experimental Pathology, UNSa-CONICET, Salta, Argentina 6Assistance to primary health care center -Torrent 1, Hospital General Valencia, Spain

Table S1. Sequences of primers used for the detection of Bcl2-JH translocations
Gene / Sequence (5´-3´) / Reference
1st PCR
Bcl2_F / CAGCCTTGAAACATTGATGG / Yasukawa et al.,2001
TATGGTGGTTTGACCTTTAG
JH_R consensus / CACCTGAGGAGACGGTGACC / Modified from Van Dongen et al., 2003
2nd PCR
Bcl2_FI / CTTTAGAGAGTTGCTTTACGTGGCC / Schmitt et al., 2006
JH_RI consensus / GGAGACGGTGACCAGGGT / Schmitt et al., 2006

F, forward; R, reverse; FI, forward internal; RI, reverse internal

Table S2.Sequences of primers used for the detection of BCR-ABL, MLL-AF4 and TEL-AML1 translocations.
Gene / Sequence (5´-3´) / Size (bp)
External primers / NA
BCR / P1-F: CGCTCTCCCTCGCAGAACT
P2-F: GAGTCACTGCTGCTGCTTATGTC
ABL / P1-R: TTTTGGTTTGGGCTTCACAC
MLL / P1-F: CCGCCTCAGCCACCTACTAC
P2-F: AGCACTCTCTCCAATGGCAATAGT
AF4 / P1-R: GAATTTGAGTGAGTTTTTGAAGATGTATC
TEL / P-S: CACTCCGTGGATTTCAAACAGTC
AML / P-R: AGCCGAGTAGTTTTCATCATTGC
Internal primers
BCR / N1-F: ACT GCCCGGTTGTCGTGTC / 320
N2-F: CAC GTTCCTGATCTCCTCTGAC / 397
ABL / N1-R 6-FAM: ACACCATTCCCCATTGTGATTAT / 472
MLL / N1-F: GGACCGCCAAGAAAAGAAGT / 72-475
N2-F: AGCAGATGGAGTCCACAGGATCAG / 475
AF4 / N1-R HEX: GTTTTTGGTTTTGGGTTACAGAACT
TEL / N-F CTCATCGGGAAGACCTGGCTTAC / 293
AML1 / N-R TET-AGCACGGAGCAGAGGAAGTTG / 332
bp: base pairs

Table S3. Primers used for methylation-specific PCR

Gen / Sequence (5’-3’) / Annealing Tª / Size (bp)
ACTB_F / TGGTGATGGAGGAGGTTTAGTAAGT / 59ºC / 133
ACTB_R / AACCAATAAAACCTACTCCTCCCTTAA / 59ºC
ATM_M_F / ATTCGTTTTCGTTCGTTTTC / 59ºC / 151
ATM_M_R / GACTTAACGTTTACGACTCGC / 59ºC
ATR_M_F / TGGTCGTTTAAAACGAAAGTTTTAC / 59ºC / 125
ATR_M_R / CGAAAAACCGTTAAAAAACGA / 59ºC
DCLRE1C_M_F / GTAGTAGATAAGTAGGAAGCGGAAC / 55ºC / 133
DCLRE1C_M_R / ATATCTATAAAAATACAATCGCGAA / 55ºC
LIG4_M_F / CGTACGATTTTTAGGGGAAGTC / 58ºC / 174
LIG4_M_R / CGAATCTAAATAAATACGACTCGAA / 58ºC
LIG4CORTA_M_F / CGTAGTTTATAGCGTTGTGGATTC / 58ºC / 241
LIG4CORTA_M_R / TCGAAAAATCGTCAAACGAA / 58ºC
NHEJ1_M_F / TTTTTATTTTGTTAAAAAGAGATCGG / 57ºC / 265
NHEJ1_M_R / AAAAACGAAACCTATACCTAACGTA / 57ºC
PRKDC_M_F / TAGGTTTTTTCGTAGGGGTTTTC / 59ºC / 235
PRKDC_M_R / CGCGAATCAATTAATAACCGA / 59ºC
XRCC4_M_F / TAATTTTTTTTCGAAGATTAATCGC / 58ºC / 183
XRCC4_M_R / CCGTCTTAATAAACGATACCGTA / 58ºC
XRCC5_M_F / AGAATTTGCGTATGTTCGGTC / 59ºC / 154
XRCC5_M_R / GACAAATAACGAAAAAAACCGC / 59ºC
XRCC6A_M_F / GTTTGGATTTTTTCGTATTCGAC / 58ºC / 141
XRCC6A_M_R / CACAATAATCGTTACCGATAACG / 58ºC
XRCC6B_M_F / TGTTATTACGAGGGGTATCGC / 58ºC / 138
XRCC6B_M_R / GACGAAAATATAAAATAAACCAACG / 58ºC

Note: Primer design and optimization in our lab