RT-PCR analysis of DNMT1 expression in oocytes
Detailed descriptions of the harvesting of human oocyte material and cDNA preparation will be published elsewhere (Huntriss et al., in preparation). Briefly, follicle samples were obtained from an ovarian cortex biopsy donated for research (22 year-old woman), and were measured and staged microscopically. Metaphase II oocytes and preimplantation embryos were surplus to clinical requirements under IVF or ICSI procedures. All samples were obtained with informed consent under ethically approved protocols including a licence from the Human Fertilisation and Embryo Authority (HFEA). RNA was prepared by binding of lysates to oligo(dT)25 magnetic beads (Dynal Biotech, Oslo, Norway), according to the manufacturer's protocols. cDNA was synthesized using a SMART™ protocol (Clontech, Palo Alto, U.S.A.) and pre-amplified using PCR primers matching the SMART IV and CDS III/3' sequences in the cDNA (SMART cDNA library construction kit).
Individual PCR reactions were then performed on this cDNA, using the following cycling conditions: 5 min at 94°C followed by 9 cycles of 94°C for 30 s, 70°C (reduced by 1°C each cycle) for 30 s, 72°C for 60 s and then 32 cycles of 94°C for 30 s, 61°C for 30 s, 72°C for 60 s. The DNMT1s-specific upstream primer (exon 1) was dTCTGCTGAAGCCTCCGAGATG and the DNMT1o-specific primer (Figure 1b) was dCTGGATGAAAGACAGCTGGATTC.
The common downstream primer (exon 5) was dCCTGGTGCTTTTCCTTGTAATC.
Mutation analysis was performed by direct cycle-sequencing of PCR products, using either 33P-ddNTP labelling (Amersham Biosciences, Uppsala, Sweden) or BigDye fluorescent terminators and an ABI377 sequencer (Applied Biosystems, Foster City, U.S.A.).
Primers for amplification and sequencing of DNMT3L :
Exon 1: dGCTGACGGCTTTCTCACATCT dTGGGATCAGCATCCTAAGTGA
Exon 2: dTTTGGAAAACGAATTGTGATGC dTTGTCTGGTGTGGGCCAGTAT
Exons 3–4: dTTTGACAGCACTGGGAAAACA dGAATGAGTAGCTGAAAGGATGG
Exon 5: dCCAAAACAAGGCTCAATCAGG dGTAAGTCAGGGCCTGCTGCT
Exon 6: dGGCACAATGTCCTGTTTTGGT dTCCATGTGTGTCTTTGGGACA
Exon 7: dACAATCCACGCTCACCCTCT dGTCTGAGAGTTCACGGCGGTA
Exon 8: dACTGCTGCTGCCTCTCAGTG dGACAGGCTGCCAGAACTGAA
Exon 9: dCTCTCAAGCCCCAGTTTCCTT dACTGAGCTCCACCAGAAAAGC
Exon 10: dTTACCGTCCAGGTCCCCTGA dATGTTCCCCTTGACCCTGAAC
Exon 11: dCTGCGCTTGCTCACAACCTT dACGTGAGGAAAGTTGCCTTTG
Exon 4 sequencing primer: dGGATGACACAAGCCAGCCT
Primers for amplification and sequencing of DNMT3B :
For exon 1b, PCR primers were dGACAGGCCAGCAGACCAATG and dCAGGATGCATTCTTAGGGTGAT. Other PCR primers were as previously described http://humgen.med.uu.nl/research/ICF/default.html. To amplify a polymorphic (GT)n sequence upstream of exon 1a, dTCACCTGGTAAGAGATAC and dTGCCCAGATCTTTGCAGG were used.
Primers for amplification and sequencing of DNMT2 (for each exon, the first two primers given were used for PCR, others for sequencing):
Exon 1: dGAGACGGGGGAACTGAAACG dGAGAAGGGAGTCAGCGTCTG dGCAGGCCTAGCTCCGGGGCT
Exon 2: dCCACTGACAGTCTGAGATGAGG dCAATCACATTTCCCTTGCTACA dTCTATAATGAACTTAAGGAC
Exon 3: dACCAAAGTTGATTTGACAAGCA dGCATGAAAATGGACCAATACAA dTCCCCATTCTATCCATGTTT
Exon 4: dTTTTTGTGAGGACCAACTTATTTTT dTTCCAGACCTGGAGTTTCAAAT dAGTGATTTGACATCTTTTTATC
Exon 5: dTCTCCAGCCTTATAAAATTCCA dTGTCAGAATGTATGCACTGGTTT dCTTAGTAATGAGATATTTAGAAT
Exon 6: dCCTGGGCAACAGAGTGAGACT dAAGCCATTCTGCTTATCGAATC dAATAAGAACTGCCTTTCCAAG
Exon 7: dAAGCTAAATGTAGGCTCGTTTGA dAACAGCCTTGAAGATGGACCTA dTGCTGTTTAACTTGGTGCTAA
Exon 8: dTTTAATGGCAACCCCATAAAG dGGCAATTAAGGGAAAATACCTCA dCGTAATGTTGACTGGACATTG dTGGATACAGGATCAATAGATC
Exon 9: dTTCAGAAGGATGTAACTGTGTAGATTG dCATGGCCAGTTGAATGCATAA dACACATACATCAGGTGCTGCA
Exon 10: dCTTTGCCACTGTGTGTTACAGC dGCAAGTCCTAGACACACAAGTCC dAATCTAGTTGCACATTACTT
Exon 11: dTGGCTCCAAATTCTCTTTTGTT dTGCCTGTTATGACACTTCACAAG dAAGGATACATCCTTTCATCTC dTACTCATGTAGACACTAAAGC