Rapid identification of clones using the same degenerate oligonucleotide mixture for both screening and sequencing.
A simple and rapid strategy for distinguishing between positively hybridizing colonies and false positive-hybridization signals is described. The isolation of a specific DNA sequence depends on the ability to distinguish between a clone that contains the correct sequence and a false hybridization-positive or background signal. This procedure utilizes the same oligonucleotide mixture both as a screening probe and as a sequencing primer. The mixture of oligonucleotides is used as a primer to obtain sequence information directly from double-stranded DNA. Conditions for sequencing with oligonucleotides having up to 64-fold degeneracy are described. Since the sequence information obtained is directly adjacent to the site of oligonucleotide:DNA hybridization, it is necessary to know only a minimal length of DNA or peptide sequence to both design oligonucleotide probes and confirm the authenticity of the hybridization positives. The advantages of the degenerate oligonucleotide sequencing method include the rapid, reliable identification of authentic versus false hybridization positives made directly without subcloning into single-stranded M13 phage, without sequencing large regions of DNA, or without synthesizing sequence-specific primers.
Nichols
R
R
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
Dixon
J E
JE
D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000032:analysis*; D010455:Peptides / Q000032:analysis; D010957:Plasmids
D009841:Oligonucleotides; D010455:Peptides; D004247:DNA