Reversed-phase ion-pair liquid chromatography method for purification of duplex DNA with single base pair resolution.
Nucleic Acids Res
; 41(20): e194, 2013 Nov.
Article
en En
| MEDLINE
| ID: mdl-24013567
Obtaining quantities of highly pure duplex DNA is a bottleneck in the biophysical analysis of protein-DNA complexes. In traditional DNA purification methods, the individual cognate DNA strands are purified separately before annealing to form DNA duplexes. This approach works well for palindromic sequences, in which top and bottom strands are identical and duplex formation is typically complete. However, in cases where the DNA is non-palindromic, excess of single-stranded DNA must be removed through additional purification steps to prevent it from interfering in further experiments. Here we describe and apply a novel reversed-phase ion-pair liquid chromatography purification method for double-stranded DNA ranging in lengths from 17 to 51 bp. Both palindromic and non-palindromic DNA can be readily purified. This method has the unique ability to separate blunt double-stranded DNA from pre-attenuated (n-1, n-2, etc) synthesis products, and from DNA duplexes with single base pair overhangs. Additionally, palindromic DNA sequences with only minor differences in the central spacer sequence of the DNA can be separated, and the purified DNA is suitable for co-crystallization of protein-DNA complexes. Thus, double-stranded ion-pair liquid chromatography is a useful approach for duplex DNA purification for many applications.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
ADN
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Cromatografía de Fase Inversa
Idioma:
En
Revista:
Nucleic Acids Res
Año:
2013
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
Reino Unido