Electrophoresis is a powerful technique which can be used not only for the size-based separation of DNA in slab gels and sieving liquid polymers, but also for the analysis of sequence-dependent variations in DNA conformation and structure. Polyacrylamide gels are useful for conformational analysis, because bent or curved DNA molecules migrate anomalously slowly in this gel medium. Bending is often (but not always) associated with runs of adenine residues (A-tracts) that occur in phase with the helix repeat. The unique structure responsible for DNA bending "melts out" at a temperature considerably below that of strand separation. The circular permutation assay is another polyacrylamide gel-based method of detecting bending. It has usually been applied to small restriction fragments, but can also be used to detect bending in plasmid-sized DNA molecules. The apparent bends in plasmid DNAs tend to be located near biologically important regions of the sequence, such as the origin of replication, the start site of transcription, and the promoters of certain genes. Finally, capillary electrophoresis in free solution (without sieving liquid polymers) can be used to determine the diffusion coefficients of small DNA molecules, detect DNA-buffer interactions, and analyze the sequence dependence of counterion binding. Counterions appear to be preferentially bound to DNA oligomers containing A-tracts, especially the A(n)T(n) sequence motif. Typical examples of these applications of gel and capillary electrophoresis to the study of DNA conformation and structure are described
The use of gel and capillary electrophoresis to investigate some of the fundamental physical properties of DNA
Gelfi C;
2002
Abstract
Electrophoresis is a powerful technique which can be used not only for the size-based separation of DNA in slab gels and sieving liquid polymers, but also for the analysis of sequence-dependent variations in DNA conformation and structure. Polyacrylamide gels are useful for conformational analysis, because bent or curved DNA molecules migrate anomalously slowly in this gel medium. Bending is often (but not always) associated with runs of adenine residues (A-tracts) that occur in phase with the helix repeat. The unique structure responsible for DNA bending "melts out" at a temperature considerably below that of strand separation. The circular permutation assay is another polyacrylamide gel-based method of detecting bending. It has usually been applied to small restriction fragments, but can also be used to detect bending in plasmid-sized DNA molecules. The apparent bends in plasmid DNAs tend to be located near biologically important regions of the sequence, such as the origin of replication, the start site of transcription, and the promoters of certain genes. Finally, capillary electrophoresis in free solution (without sieving liquid polymers) can be used to determine the diffusion coefficients of small DNA molecules, detect DNA-buffer interactions, and analyze the sequence dependence of counterion binding. Counterions appear to be preferentially bound to DNA oligomers containing A-tracts, especially the A(n)T(n) sequence motif. Typical examples of these applications of gel and capillary electrophoresis to the study of DNA conformation and structure are describedI documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
