Rapid detection and sequencing of specific in vitro amplified DNA sequences using solid phase methods.
We describe a rapid solid phase assay for detection and sequencing of DNA sequences based on selective introduction of biotin and isotope into the specific DNA fragment amplified by the polymerase chain reaction (PCR). A two-step PCR procedure is used to lower the background signal. The in vitro amplified material is immobilized on magnetic beads with covalently coupled streptavidin and the amount of bound label is measured. Samples identified as positive can be analysed by direct solid phase DNA sequencing. A strategy is also described to use general primers for detection, capturing and sequencing, which are not homologous to the specific sequence to be detected. The concept has been optimized using oligonucleotides specific for Staphylococci and Streptococci, respectively. Here, we show that the assay can be used for detection of Plasmodium falciparum in clinical samples.
Wahlberg
J
J
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm.
Lundeberg
J
J
Hultman
T
T
Holmberg
M
M
Uhlén
M
M
D000818:Animals; D001412:Bacillus subtilis / Q000235:genetics; D001426:Bacterial Proteins; D001483:Base Sequence; D001710:Biotin; D004247:DNA / Q000097:blood*; D004269:DNA, Bacterial / Q000032:analysis; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D015345:Oligonucleotide Probes; D010963:Plasmodium falciparum / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D013205:Staphylococcal Protein A / Q000235:genetics; D013211:Staphylococcus aureus / Q000235:genetics / Q000302:isolation & purification; D019809:Streptavidin; D013291:Streptococcus / Q000235:genetics; D013698:Templates, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D013205:Staphylococcal Protein A; D001710:Biotin; D004247:DNA; D019809:Streptavidin