Identification of the DNA bases of a DNase I footprint by the use of dye primer sequencing on an automated capillary DNA analysis instrument.
We have adapted the techniques of DNA footprint analysis to an Applied Biosystems 3730 DNA Analyzer. The use of fluorescently labeled primers eliminates the need for radioactively labeled nucleotides, as well as slab gel electrophoresis, and takes advantage of commonly available automated fluorescent capillary electrophoresis instruments. With fluorescently labeled primers and dideoxynucleotide DNA sequencing, we have shown that the terminal base of each digested fragment may be accurately identified with a capillary-based instrument. Polymerase chain reaction (PCR) was performed with a 6FAM-labeled primer to amplify a typical target promoter region. This PCR product was then incubated with a transcriptional activator protein, or bovine serum albumin as a control, and then partially digested with DNase I. A clone of the promoter was sequenced with the Thermo Sequenase Dye Primer Manual Cycle Sequencing kit (USB) and the FAM-labeled primer. Through the use of Genemapper software, the Thermo sequenase and DNasei digestion products were accurately aligned, providing a ready means to assign correct nucleotides to each peak from the DNA footprint. This method was used to characterize the binding of two different transcriptional activator proteins to their respective promoter regions.
Zianni
Michael
M
Plant-Microbe Genomics Facility, The Ohio State University, Columbus, OH 43210-1292, USA. zianni.1@osu.edu
Tessanne
Kimberly
K
Merighi
Massimo
M
Laguna
Rick
R
Tabita
F R
FR
D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry; D003720:Densitometry; D003850:Deoxyribonuclease I / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000494:pharmacology*; D007202:Indicators and Reagents / Q000494:pharmacology; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000737:chemistry; D010944:Plants / Q000382:microbiology; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D015203:Reproducibility of Results; D016992:Rhodobacter / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000295:instrumentation* / Q000379:methods*
D017931:DNA Primers; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D009711:Nucleotides; D004247:DNA; D003850:Deoxyribonuclease I