Reverse Southern hybridization.

AUTOR(ES)

Gamper, H B

RESUMO

A DNA oligomer 25 nucleotides long which contained an HMT (4'-hydroxymethyl-4,5', 8-trimethylpsoralen) furan side monoadduct to thymidine at a 5'-TpA-3' site was used as a probe for the polylinker sequence present in single-stranded M13 mp19 DNA and in double-stranded pUC 19 DNA. Hybridization and photofixation were carried out simultaneously in solution under conditions approximating the melting temperature of the probe-target hybrid. Use of probe concentrations greater than 10(-8) M permitted hybridization times of a few minutes. Irradiation with near ultraviolet light converted the HMT monoadduct present in hybrid complexes into an interstrand crosslink. Efficient photofixation removed hybrid from the equilibrium distribution and resulted in the formation of additional probe-target complex. After removal of excess probe by centrifugation through a semi-permeable membrane (Centricon-30), samples were electrophoresed through an alkaline agarose gel which was analyzed by autoradiography. When using an HMT-modified 25-mer probe end-labeled with 3,000 Ci/mmole 32P, 0.015 ng (3.8 X 10(6) copies) of M13 DNA could be detected. With this same probe 10 micrograms of denatured human DNA (corresponding to 3.0 X 10(6) copies) did not give a signal.

Documentos Relacionados

• Detection and differentiation of antigenically distinct small round-structured viruses (Norwalk-like viruses) by reverse transcription-PCR and southern hybridization.
• Characterization of Mycobacterium tuberculosis strains from Vietnamese patients by Southern blot hybridization.
• Detection of mutations and DNA polymorphisms using whole genome Southern Cross hybridization.
• Detection of naturally occurring enteroviruses in waters by reverse transcription, polymerase chain reaction, and hybridization.
• Identification of DNA homologies among H incompatibility group plasmids by restriction enzyme digestion and Southern transfer hybridization.