Analysis of chromatin in limited numbers of cells: a PCR-SSCP based assay of allele-specific nuclease sensitivity.
Chromatin can be analysed by assaying its sensitivity to DNase I or other nucleases in purified nuclei. Usually, this is performed by Southern analysis of genomic DNA extracted from nuclease-treated nuclei, a methodology that requires many cells. Applying restriction fragment length polymorphisms (RFLPs), this methodology has been used for parental allele-specific chromatin studies on imprinted mammalian genes. However, such allelic studies are limited by the availability of suitable RFLPs. We therefore developed an alternative, PCR and single strand conformation polymorphism (SSCP)-based assay with which allelic sensitivity to nucleases can be determined in virtually all localised regions that have nucleotide polymorphisms. We also demonstrate that analysis of DNase I sensitivity can be performed on permeabilised cells. Combining the two approaches, in the imprinted mouse U2af1-rs1 gene we analysed parental allele-specific chromatin conformation in limited numbers of cultured cells. We also applied the PCR-SSCP approach to assay allelic DNA methylation at specific restriction enzyme sites. In summary, we developed an allele-specific assay that should be useful for biochemical and developmental investigation of chromatin, in particular for studies on genomic imprinting and X-chromosome inactivation.
Gregory
R I
RI
Programme in Developmental Genetics, The Babraham Institute, Cambridge CB2 4AT, UK.
Feil
R
R
D000483:Alleles; D000818:Animals; D001681:Biological Assay / Q000379:methods*; D015139:Blotting, Southern; D002460:Cell Line; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D047108:Embryonic Development; D005260:Female; D018392:Genomic Imprinting; D051379:Mice; D008810:Mice, Inbred C57BL; D009115:Muridae; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D018807:Polymorphism, Single-Stranded Conformational; D011247:Pregnancy; D011487:Protein Conformation; D011506:Proteins / Q000235:genetics; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF
D002843:Chromatin; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D011506:Proteins; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C000607495:Zrsr1 protein, mouse; D003850:Deoxyribonuclease I