Fluorescently labeled oligonucleotide extension: a rapid and quantitative protocol for primer extension.
Identification of nucleotides used for RNA chain initiation or for contacting DNA binding proteins is basic to our understanding of gene regulation. Normally, a radioactive primer is used to copy RNA or DNA. The polymerase extension stops at free ends of mRNA (as in promoter mapping) or at the position of template cleavage or modification (as in footprinting). The locations of these positions are then analyzed by polyacrylamide gel electrophoresis. These analyses have been improved using fluorescently labeled primers and commonly available DNA sequencing machines. The protocol, which we call fluorescently labeled oligonucleotide extension (FLOE), eliminates the need for handling radioactivity and polyacrylamide. The DNA sequencer delivers data as a "trace" that is ready for quantification, which eliminates the need to trace gels separately. The data analysis is further improved by new software, Scanalyze, which we present here. We demonstrate that by using promoter mapping and footprinting, FLOE shortens experimental time, extends the stretch of analyzable sequence, and simplifies quantification compared to radioactive methods and is as sensitive in terms of detecting templates.
Fekete
Richard A
RA
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA. feketer@mail.nih.gov
Miller
Mark J
MJ
Chattoraj
Dhruba K
DK
D000465:Algorithms; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes; D020869:Gene Expression Profiling; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D015345:Oligonucleotide Probes; D017422:Sequence Analysis, DNA / Q000379:methods*; D012984:Software; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*; D014158:Transcription, Genetic / Q000235:genetics
D017931:DNA Primers; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes