Long-range and highly sensitive DNase I footprinting by an automated infrared DNA sequencer.
We have shown that an automated DNA sequencer is applicable to fluorescence-based detection of fragments in DNase I footprinting. We demonstrated the potential of long-range and highly sensitive DNase I footprinting taking advantage of an infrared-fluorescence automated DNA sequencer. Footprints of human transcription factor SpI were reproducibly detected ranging approximately between 100 and 750 bp on both strands of an 895-bp DNA fragment in a single electrophoresis run. We developed techniques in data collection and subsequent image processing for highly sensitive detection. Less than 0.1 footprinting unit (fpu: approximately 4.5 ng) of SpI was detected using 3.1 fmol of a 512-bp DNA fragment. This is greater than 10-fold increase in sensitivity over what has previously been reported by visible dye fluorescence DNA sequencers. This method will be very important in systematic analysis of transcription regulatory regions and in large-scale analysis of the transcription process.
Machida
M
M
Department of Molecular Biology, National Institute of Bioscience and Human-Technology, Ibaraki, Japan.
Kamio
H
H
Sorensen
D
D
D004247:DNA / Q000032:analysis; D018983:DNA Footprinting / Q000379:methods*; D003850:Deoxyribonuclease I; D006801:Humans; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*; D016329:Sp1 Transcription Factor / Q000235:genetics; D013055:Spectrophotometry, Infrared
D016329:Sp1 Transcription Factor; D004247:DNA; D003850:Deoxyribonuclease I