Real-time PCR mapping of DNaseI-hypersensitive sites using a novel ligation-mediated amplification technique.
Mapping sites within the genome that are hypersensitive to digestion with DNaseI is an important method for identifying DNA elements that regulate transcription. The standard approach to locating these DNaseI-hypersensitive sites (DHSs) has been to use Southern blotting techniques, although we, and others, have recently published alternative methods using a range of technologies including high-throughput sequencing and genomic array tiling paths. In this article, we describe a novel protocol to use real-time PCR to map DHS. Advantages of the technique reported here include the small cell numbers required for each analysis, rapid, relatively low-cost experiments with minimal need for specialist equipment. Presented examples include comparative DHS mapping of known TAL1/SCL regulatory elements between human embryonic stem cells and K562 cells.
Follows
George A
GA
Department of Haematology, Cambridge Institute for Medical Research, Addenbrooke's Hospital, University of Cambridge, Cambridge CB2 2XY, UK. gf246@cam.ac.uk
Janes
Mary E
ME
Vallier
Ludovic
L
Green
Anthony R
AR
Gottgens
Berthold
B
D015139:Blotting, Southern; D002478:Cells, Cultured; D002874:Chromosome Mapping / Q000379:methods*; D004247:DNA / Q000032:analysis; D003850:Deoxyribonuclease I; D053595:Embryonic Stem Cells / Q000737:chemistry; D015698:Genomic Library; D006801:Humans; D020014:K562 Cells; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012045:Regulatory Sequences, Nucleic Acid
D004247:DNA; D003850:Deoxyribonuclease I