Dear Alexander,

S1 nuclease will remove single-stranded DNA and RNA. Its value in examining strains for large plasmids by PFGE is that it will remove a lot of the background nucleic acid that might otherwise obscure plasmid bands. There is no reason why you should not be able to look at plasmid profiles without S1 nuclease, though many protocols (e.g. for agar-embedded cells) a RNAse is often included.

Regards,

Andrew

Dear Andrew,

We are getting bands between 150 and 50 KB. In some cases we can see bands without S1 in this range.

It is hard to blieve that RNA will interfere. I suppose that Plasmid DNA might attach to the Chromosome by single stranded DNA fragments. Or may be by RNA?

Do you know the answer?

Dear Alexander,

RNA will not interfere with such large plasmids - but RNA might obscure bands if the gels are not run very far in the first instance. Damaged ss DNA might interfere with plasmid DNA and prevent it separating from the chromosomal DNA during isolation, or prevent it from migrating from the chromosoal DNA in an agarose plug, but this is obviously not problem for you as you can see the plasmid bands. I would not worry about using or not using S1 niuclease!

Regards,

Andrew

To accurately determine the size of large plasmids, it is necessary to relieve supercoiling and linearize the plasmids in order to produce molecules that migrate similarly to a set of molecular weight markers. Partial digestion by brief treatment with low concentrations of S1 nuclease has been shown to be an effective means of linearizing large plasmids for PFGE (1). S1 nuclease is a non-sequence-specific ssDNA endonuclease that cleaves supercoiled dsDNA plasmids in sites of ssDNA caused by loops, mismatches, and other localized features. S1 nuclease has high selectivity for ssDNA and prefers ssDNA as the substrate over RNA and 3’AMP. The use of  S1 nuclease in PFGE-based plasmid analysis is based on the fact that this enzyme also showed rapid conversion of the Form I DNA (supercoiled DNA) to a more relaxed state of Form II (circular) DNA in a 1973 paper (2).

1. Barton, B.M., G.P. Harding, and A.J. Zuccarelli. 1995. A general method for detecting and sizing large plasmids. Anal. Biochem. 226:235–240.

2. Méchali, M., A.-M. de Recondo, and M. Girard. 1973. Action of the S1 endonuclease from Aspergillus oryzae on simian virus 40 supercoiled component I DNA. Biochem. Biophys. Res. Commun. 54:1306–1320. 

Thank you very much for your answers and all that sonds quite logical.

However in our hands we can see that some pleamid bands show up without S1 treatment, however some appear only ater S1 treatment. Apparently there shoud ne some SS junctions with chromosomal DNA