Cleaving DNA with DNA.
A DNA construction is described that may cleave single-stranded DNA oligonucleotides within the presence of ionic copper. This “deoxyribozyme” can self-cleave or can function as a bimolecular advanced that concurrently makes use of duplex and triplex interactions to bind and cleave separate DNA substrates.
Bimolecular deoxyribozyme-mediated strand scission proceeds with a kobs of 0.2 min-1, whereas the corresponding uncatalyzed response couldn’t be detected.
The duplex and triplex recognition domains might be altered, making doable the focused cleavage of single-stranded DNAs with totally different nucleotide sequences. Several small artificial DNAs had been made to perform as easy “restriction enzymes” for the site-specific cleavage of single-stranded DNA.
Isolation and characterization of DNA–DNA and DNA-RNA.
A easy technique for the isolation and characterization of DNA–DNA and DNA-RNA hybrid molecules fashioned in resolution was developed.
It was based mostly on the truth that, in acceptable salt focus, corresponding to 5% Na2HPO4, DNA in both double-stranded (DNA–DNA or DNA-RNA) or single-stranded varieties, however not free nucleotides, can bind to diethylaminoethylcellulose disc filters (DE81).
Thus examined samples had been handled with the single-strand-specific nuclease S1 after which utilized to DE81 filters. The free nucleotides, ensuing from degrading the single-stranded molecules, had been eliminated by intensive washing with 5% Na2HPO4, leaving solely the hybrid molecules on the filters.
The usefulness of this technique was illustrated in dissociation and reassociation research of viral (SV40) or mobile (NIH/3T3) DNAs and DNA-RNA hybrid molecules.
Using this method the reassociation of denatured SV40 DNA was discovered to be a really fast course of. Dissociation research revealed that the melting curves of examined DNAs had been depending on salt focus.
Thus the melting temperatures ™ obtained for SV40 DNA had been 76 levels C at 1 X SSC (0.15 M NaCl-0.015 M sodium citrate) and 65 levels C at 0.1 X SSC, and for NIH/3T3 DNA 82 levels C at 1 X SSC and 68 levels C at 0.1 X SSC. MuLV DNA-RNA hybrid molecules had been fashioned by annealing in vitro synthesized MuLV DNA with 70S MuLV RNA at 68 levels C.
The melting temperature of this hybrid within the annealing resolution was 87 levels C. Another necessary characteristic of this process was that, after being selectively sure to the filters, the hybrid molecules might effectively be recovered by heating the filters for five min at 60 levels C in 1.5-1.7 M KCl.
The recovered molecules had been intact hybrids as they had been discovered to be utterly proof against S1 nuclease.