Rapid identification of clones using the same degenerate oligonucleotide mixture for both screening and sequencing

Journal Article · Fri Apr 01 04:00:00 EST 1988 · Anal. Biochem.; (United States)

DOI:https://doi.org/10.1016/0003-2697(88)90096-6· OSTI ID:6804964

Nichols, R; Dixon, J E

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 /sup 32/P-labeled 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.

• Research Organization: Purdue Univ., West Lafayette, IN (USA)
• OSTI ID: 6804964
• Journal Information: Anal. Biochem.; (United States), Journal Name: Anal. Biochem.; (United States) Vol. 170:1; ISSN ANBCA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ATP
BACTERIOPHAGES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMISTRY
CHEMISTRY
CLONING
DAYS LIVING RADIOISOTOPES
DNA
DNA SEQUENCING
DNA-CLONING
HYBRIDIZATION
ISOTOPES
LABELLED COMPOUNDS
LIGHT NUCLEI
MICROORGANISMS
NUCLEI
NUCLEIC ACIDS
NUCLEOTIDES
ODD-ODD NUCLEI
OLIGONUCLEOTIDES
ORGANIC COMPOUNDS
PARASITES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
RADIOISOTOPES
RECOMBINANT DNA
STRUCTURAL CHEMICAL ANALYSIS
VIRUSES