Versatile method employing basic techniques of genetic engineering to study the ability of low-molecular-weight compounds to bind covalently with DNA in cell-free systems - Publikacja - MOST Wiedzy

Wyszukiwarka

Versatile method employing basic techniques of genetic engineering to study the ability of low-molecular-weight compounds to bind covalently with DNA in cell-free systems

Abstrakt

Numerous antitumor and carcinogenic compounds and free radicals are able to modify DNA by forming covalent bonds, mainly with nucleophilic centers in nucleobases. Such a binding is usually of utmost importance for the biological outcome. The level of DNA adducts formed by a given agent is in most cases extremely low; hence their detection is very difficult. Here we propose a simple approach, exploiting techniques widely used in genetic engineering, to demonstrate and characterize the covalent modification of a DNA fragment by any low-molecular-weight compound of interest in a cell-free system. The specifically designed, several-hundred-base-pairs-long double-stranded deoxyoligonucleotide (PCR amplified)—subject to modification—includes two restriction sites: one containing only GC base pairs recognized by restriction endonuclease MspI and the other including only AT base pairs recognized by restriction endonuclease Tru1I. The covalent modification of the restriction sites abolishes their recognition and thus cleavage by the endonucleases applied. The formation of DNA adducts is induced by incubating the oligonucleotide with increasing concentrations of a studied compound, in the appropriate activating system if required. Then, the modified oligonucleotide is submitted to digestion by the above-mentioned restriction endonucleases and the DNA fragments are separated by polyacrylamide gel electrophoresis. The inhibition of cleavage indicates the occurrence of covalent modification of the restriction site(s) while simultaneously pointing at the kind of base pairs involved in DNA adduct formation. The validation of the method was performed for two DNA binding antitumor compounds, cisplatin and CC-1065, which form adducts preferentially with guanine and adenine, respectively.

Cytowania

  • 4

    CrossRef

  • 4

    Web of Science

  • 4

    Scopus

Cytuj jako

Pełna treść

pełna treść publikacji nie jest dostępna w portalu

Informacje szczegółowe

Kategoria:
Publikacja w czasopiśmie
Typ:
artykuły w czasopismach
Opublikowano w:
ANALYTICAL BIOCHEMISTRY nr 313, strony 53 - 59,
ISSN: 0003-2697
Język:
angielski
Rok wydania:
2003
Opis bibliograficzny:
Bartoszek-Pączkowska A., Sielenko A., Węsiora M.: Versatile method employing basic techniques of genetic engineering to study the ability of low-molecular-weight compounds to bind covalently with DNA in cell-free systems// ANALYTICAL BIOCHEMISTRY -Vol. 313,iss. 1 (2003), s.53-59
DOI:
Cyfrowy identyfikator dokumentu elektronicznego (otwiera się w nowej karcie) 10.1016/s0003-2697(02)00564-x
Weryfikacja:
Politechnika Gdańska

wyświetlono 4 razy

Publikacje, które mogą cię zainteresować

Meta Tagi