General colorimetric method for DNA diagnostics allowing direct solid-phase genomic sequencing of the positive samples.
A system for rapid colorimetric detection of specific genome DNA fragments amplified by the polymerase chain reaction (PCR) is described that has been designed to allow direct solid-phase sequencing of positive samples. The amplified material is immobilized on magnetic beads by using the biotin streptavidin system. An Escherichia coli lac operator DNA sequence is incorporated in the amplified material during the second step of a nested primer procedure. This 21-base-pair sequence is used for a general colorimetric detection with a fusion protein consisting of the E. coli Lac repressor and beta-galactosidase. Positive samples can be treated subsequently with alkali to obtain a single-stranded DNA template suitable for direct genomic sequencing. This method to detect immobilized amplified nucleic acids (DIANA) is well adapted for automated or semiautomated clinical assays. Here, we show that it can be used to detect and sequence Chlamydia trachomatis genomic DNA in clinical samples.
Wahlberg
J
J
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Lundeberg
J
J
Hultman
T
T
Uhlén
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002690:Chlamydia Infections / Q000175:diagnosis*; D002692:Chlamydia trachomatis / Q000235:genetics*; D003124:Colorimetry / Q000379:methods; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006801:Humans; D007763:Lac Operon; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000138:chemical synthesis; D016133:Polymerase Chain Reaction / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence / Q000379:methods; D001616:beta-Galactosidase / Q000235:genetics / Q000302:isolation & purification
D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase