Sensitive non-radioactive dot-blot hybridization using DNA probes labelled with chelate group substituted psoralen and quantitative detection by europium ion fluorescence.

AUTOR(ES)

Oser, A

RESUMO

A new labelling method for cloned DNA probes used in hybridization assays is described. The DNA insert of recombinant plasmid DNA was made partially single-stranded for the labelling reaction by a restriction enzyme digest, followed by a controlled exonuclease III incubation. A thiol-containing psoralen derivative was covalently bound through irradiation with UV-light to the remaining double-stranded region of the plasmid DNA. The psoralen-SH groups were labelled with a large number of metal chelators (diethylentriamine pentaacetic acid, DTPA) using poly-L-lysine as a macromolecular carrier. The main advantage of the labelling procedure is that a high degree of labelling is achieved without modification of the single-stranded DNA hybridizing sequences. The specific hybrids were labelled after filter hybridization with europium ions through the chelating groups of DTPA. The europium ions were quantitatively detected by time-resolved fluorometry. The sensitivity of the assay for target DNA detection was in the low picogram range, comparable to radioactively labelled DNA probes.

Documentos Relacionados

• Non-radioactive chemical sequencing of biotin labelled DNA.
• Optimization of non-radioactive Southern blot hybridization: single copy detection and reuse of blots.
• DNA fingerprinting using non-radioactive oligonucleotide probes specific for simple repeats.
• Comparison of genomic homologies in the coxsackievirus B group by use of cDNA:RNA dot-blot hybridization.
• Simple and efficient system for synthesis of non-radioactive nucleic acid hybridization probes using PCR.