Kits and methods for isolating high molecular weight DNA from prokaryotic cells in stool

Abstract

A method for extracting high molecular weight DNA from prokaryotic cells in stool samples, comprising preparing the biological sample comprising prokaryotes in a prechilled buffer solution; homogenizing the biological sample through first mechanical means; partially disrupting cell walls by treating the sample with at least one lytic enzyme; treating with a chaotropic agent; disrupting the sample by a second mechanical means and applying an adsorbent to the sample. Preferably the buffer comprises a pH stabilizing agent and an ionic strength modifier. The first mechanical means comprises vortexing the sample or bead beating. The lytic enzyme is lysozyme, mutanolysin or lysostaphin and the chaotropic agent comprises guanidinium thiocyanate. Also disclosed is a reagent system for extracting high molecular weight DNA comprising a buffer solution comprising a pH-stabilizing agent and an ionic strength modifier, at least one mechanical homogenization tool, at least one lytic enzyme, a chaotropic agent and an adsorbent. Further disclosed is a kit comprising a first container comprising a buffer solution comprising a pH-stabilizing agent and an ionic strength modifier, one or more beads, at least one receptacle comprising at least one lytic enzyme, a second container comprising a chaotropic agent and a third container comprising an adsorbent. [Fig. 1]