Rapid high-throughput analysis of DNaseI hypersensitive sites using a modified Multiplex Ligation-dependent Probe Amplification approach.
BACKGROUND
Mapping DNaseI hypersensitive sites is commonly used to identify regulatory regions in the genome. However, currently available methods are either time consuming and laborious, expensive or require large numbers of cells. We aimed to develop a quick and straightforward method for the analysis of DNaseI hypersensitive sites that overcomes these problems.


RESULTS
We have developed a modified Multiplex Ligation-dependent Probe Amplification (MLPA) approach for the identification and analysis of genomic regulatory regions. The utility of this approach was demonstrated by simultaneously analysing 20 loci from the ENCODE project for DNaseI hypersensitivity in a range of different cell lines. We were able to obtain reproducible results with as little as 5 x 10(4) cells per DNaseI treatment. Our results broadly matched those previously reported by the ENCODE project, and both technical and biological replicates showed high correlations, indicating the sensitivity and reproducibility of this method.


CONCLUSIONS
This new method will considerably facilitate the identification and analysis of DNaseI hypersensitive sites. Due to the multiplexing potential of MLPA (up to 50 loci can be examined) it is possible to analyse dozens of DNaseI hypersensitive sites in a single reaction. Furthermore, the high sensitivity of MLPA means that fewer than 10(5) cells per DNaseI treatment can be used, allowing the discovery and analysis of tissue specific regulatory regions without the need for pooling. This method is quick and easy and results can be obtained within 48 hours after harvesting of cells or tissues. As no special equipment is required, this method can be applied by any laboratory interested in the analysis of DNaseI hypersensitive regions.
Ohnesorg
Thomas
T
Murdoch Children's Research Institute and Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville, VIC, Australia.
Eggers
Stefanie
S
Leonhard
Wouter N
WN
Sinclair
Andrew H
AH
White
Stefan J
SJ
D002452:Cell Count; D002460:Cell Line; D002874:Chromosome Mapping / Q000379:methods; D015342:DNA Probes; D003850:Deoxyribonuclease I / Q000737:chemistry*; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015203:Reproducibility of Results
D015342:DNA Probes; D003850:Deoxyribonuclease I