Restriction nuclease digestions driven to completion by Escherichia coli RNA polymerase and T4 gene 32 protein.

D F Dombroski,A R Morgan

doi:10.1016/s0021-9258(18)89747-2

Abstract

Restriction enzyme digestions of large scale DNA preparations often do not go to completion. This is due to product inhibition by the newly generated ends of the digested DNA. The addition of exogenous proteins that bind tightly to the free ends of DNA or to single-stranded DNA will relieve this inhibition. We show that a considerable savings on restriction nucleases can be attained by the addition of RNA polymerase or T4 gene 32 protein in stoichiometric amounts to the newly produced DNA ends.

Highlights

Restriction enzyme digestions of large scale DNA preparations often do not go to completion
We looked at restriction fragments with overhanging 5’ ends (HindIII), overhanging 3’ ends (PstI), or blunt ends (HueIII)
Lune 1 shows a digestion with PstI followed by a HindIII digestion which would not go to completion even after an overnight incubation (“Materials and Methods”)

Summary

THEJOURNAOLF BIOLOGICACHLEMISTRY

Vol 260, No 1, Issue of January 10, pp. 415-417, 1985 Printed in U.S.A. Restriction Nuclease Digestions Driven to Completion byEscherichia coli RNA Polymerase and T4 Gene 32 Protein*. We show that a considerable savings on restriction nucleases can be attained by the addition of RNA polymerase or T4 gene 32 protein in stoichiometric amountsto the newly produced DNAends It has been observed in many laboratories that restriction enzymes often do not digest DNA samples to completion even though the enzyme is probably still active. In attempts tcoircumvent this product inhibition, we tested proteins which bind either to the ends of DNA or to singlestranded DNA. Of these Escherichia coli RNA polymerase and the single-stranded DNA binding protein coded by gene 32of T4 phage werevery effective in driving restriction nuclease digests to completion

MATERIALS ANDMETHODS

RESULTS

Completion of Restriction Nuclease Digestions

HedlO was selected by its binding to denaturedcalf thymus

DISCUSSION

TIME MINUTES