More efficient DNA isolation techniques

1. Mice mated a/a dam X Avy/a sire while the dam is on control or 3SZM diet produce Avy/a and a/a offspring. Avy/a offspring have different distributions of epigenetic phenotypes in the population due to maternal diet.

2. Methyl metabolismshowing some major metabolic intermediates, cofactors and dietary sources of methyl groups and an intersection with antioxidant metabolism through homocysteine, cysteine and glutathione.

3. Survival declines over a considerable range in adult humans (40 to 100 y of age) and in some inbred rodent strains (18 to 33 mo of age, scales are approximate). (Lower panel) Epigenetic traits are probably distributed unevenly and over a considerable range in populations.

4. Agouti gene expression in the Avy allele. TOP: When the LTR is active, it drives expression of the agouti gene in Avy mice. Transcription is constant, rather than cyclical (indicated by the bold arrow) and is ectopic (i.e., in all tissues studied). BOTTOM: When the LTR is suppressed in Avy mice, expression of the agouti gene is regulated by its normal promoters, is cyclic and occurs only in the hair follicles (indicated by the narrow arrow). LTR, long term repeat.

5. Map of the Avy allele showing the LTR, agouti exons, primer positions and HaeII (H) and HinP1I (P) sites. IAP, intracisternal A particle; LTR, long terminal repeat.

6. More efficient DNA isolation techniques • Based on magnetic silica beads • High throughput manually • 96 well plates and multichannel pipets • No ethanol precipitation • Takes about 30 minutes • Can be automated

7. DNA Methylation of Specific Sequences • Methylation Specific PCR Safar et al. (2005) Methylation Profiling of Archived Non-Small Cell Lung Cancer: A Promising Prognostic System. Clinical Cancer Research 11, 4400-4405. • Methylation Sensitive PCR Kurmasheva et al. (2005) Upstream CpG Island Methylation of the PAX3 Gene in Human Rhabdomyosarcomas. Pediatric Blood and Cancer 44, 328-337 Cooney et al. (2002) Maternal Methyl Supplements in Mice Affect Epigenetic Variation and DNA Methylation of Offspring. Journal of Nutrition 132, 2393S-2400S • Bisulfite Genomic Sequencing Grunau et al. (2001) Bisulfite genomic sequencing: systematic investigation of critical experimental parameters. Nucleic Acids Res. 29, e65

8. Bisulfite Modification of DNA Unmodified sequence: GTCmGAACCGTTCATGTTGCmGAGCTG Bisulfite Modification GTCmGAAUUGTTUATGTTGCmGAGUTG PCR GT CG AATTGTTTATGTTG CG AGTTG

9. Bisulfite Genomic Sequencing

10. Solvent percentage does not affect measurement of percent methylation using completely volatilebuffer.

11. Methylation of locus 224 in several tissues (A-E) in two types of epigenetically different mice

12. Global DNA Methylation • HPLC Cooney et al. (1997) An Improved Sample Preparation Method for the Quantitative HPLC Determination of 5-Methyldeoxycytidine in Animal Tissue DNA. Journal of Liquid Chromatography 20, 1279-1293 • Methylation Levels on Repetitive Elements Yang et al. (2004) A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements. Nucleic Acids Res. 32, e38

13. Typical Chromatogram for Global Methylation by HPLC Deoxycytidine 5-methyldeoxycytidine

14. Global DNA Methylation in a Range of Eukaryotes Lyko et al 2000 Cooney et al unpubl

15. Metabolic Efficiency • Respirometry (Core B). • MRI of metabolites. • Relate to ongoing diabetes studies. • Studies of genotype (Avy/a versus A/a as surrogates for epigenetic phenotypes) already done. These relate AL and CR to body weight and to liver gene expression (Kaput et al. 2004).

16. TOP: Y2 (heavily mottled Avy/a) mouse. BOTTOM: black (a/a) mouse

17. TOP: black (a/a) mouse BOTTOM: Y0 (pseudoagouti Avy/a) mouse.

18. LEFT: Y5 (clear yellow Avy/a) mouse. RIGHT: Y4 (slightly mottled Avy/a) mouse.