Rapid and specific detection of PCR products using light-up probes.
Newly developed light-up probes offer an attractive tool for PCR product detection. The light-up probe, which consists of a thiazole orange derivative linked to a peptide nucleic acid oligomer, hybridizes specifically to complementary nucleic acids. Upon hybridization the thiazole orange moiety interacts with the nucleic acid bases and the probe becomes brightly fluorescent. This eliminates the need to separate bound from unbound probes and reduces the risk of cross contamination during sample handling. We demonstrate here the applicability of light-up probes in two different PCR assays, one directed towards the human beta-actin gene and the other towards the invA gene of Salmonella. The probes do not interfere with the PCR reaction and can either be included in the sample mixture or added after completed amplification. The specificity of the probe is found to be excellent: a single-base mismatch in the target sequence is sufficient to prevent probe binding as indicated by the lack of fluorescence increase. Furthermore, a clear correlation is found between the intensity of gel bands and the measured probe fluorescence in solution, which suggests that the amount of PCR products can be quantified using light-up probes.
Isacsson
J
J
Department of Molecular Biotechnology, Chalmers University of Technology, S-405 30 Göteborg, Sweden.
Cao
H
H
Ohlsson
L
L
Nordgren
S
S
Svanvik
N
N
Westman
G
G
Kubista
M
M
Sjöback
R
R
Sehlstedt
U
U
D000199:Actins / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D020029:Base Pairing; D052160:Benzothiazoles; D015342:DNA Probes / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D020135:Peptide Nucleic Acids / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012475:Salmonella / Q000235:genetics; D012680:Sensitivity and Specificity; D013844:Thiazoles / Q000737:chemistry
D000199:Actins; D001426:Bacterial Proteins; D052160:Benzothiazoles; D015342:DNA Probes; D005456:Fluorescent Dyes; D020135:Peptide Nucleic Acids; D011804:Quinolines; D013844:Thiazoles; C051000:thiazole orange; C075518:invA protein, Bacteria