Transcript mapping using [35S]DNA probes, trichloroacetate solvent and dideoxy sequencing ladders: a rapid method for identification of transcriptional start points.
A simple method for RNA transcript mapping has been developed that combines the use of 35S-labeled M13 DNA probes and the presence of high concentrations of sodium trichloroacetate in the hybridization buffer. These hybridization conditions permit the use of M13 probes without purification from the template. The dideoxy sequencing ladders used for sizing the protected DNA fragments are obtained from the same M13 templates utilized to synthesize the DNA probes. The method was tested by analyzing the transcripts controlled by lac, ptr and trxA promoters. Comparison of the results with previously published data obtained with the conventional S1 nuclease mapping technique indicated that the present method is just as precise and at least 50 times more sensitive. Clones constructed for sequencing a gene of interest can be used directly to identify transcriptional start points.
Aldea
M
M
Department of Genetics, University of Georgia, Athens 30602.
Claverie-Martín
F
F
Díaz-Torres
M R
MR
Kushner
S R
SR
D001483:Base Sequence; D003090:Coliphages / Q000235:genetics*; D004279:DNA, Viral / Q000187:drug effects / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000187:drug effects*; D009710:Nucleotide Mapping; D011401:Promoter Regions, Genetic; D013462:Sulfur Radioisotopes; D014158:Transcription, Genetic; D014238:Trichloroacetic Acid / Q000494:pharmacology*
D004279:DNA, Viral; D013462:Sulfur Radioisotopes; D014238:Trichloroacetic Acid