Sample preparation method and apparatus

Abstract

A method of preparing a nucleic acid sample with target enrichment uses a reaction vessel, within which is added a chelating agent to a sample with heating to about 99° C. to provide a crude lysate. A PNA probe is provided at a concentration sufficient for binding and capture of discernible levels of target nucleic acid. The PNA probe may be attached to beads which are initially embedded in a wax body and are released during the heating so that the y are free to move and come into contact with the PNA probe and target DNA. After binding has occurred, the heads are magnetically attracted back into a pocket along with the wax, which is allowed to solidify before they are removed from the reaction vessel.

1 . A method of preparing a nucleic acid sample for analysis with target enrichment, the method comprising the steps of:
 a. in a reaction vessel ( 7 ), adding a chelating agent to the sample until cells are lysed, to provide a crude lysate, and   b. providing a PNA probe at a concentration sufficient for binding and capture of discernible levels of target nucleic acid.   
     
     
         2 . A method as claimed in  claim 1 , wherein the sample is heated with the chelating agent. 
     
     
         3 . A method as claimed in  claim 1 , comprising the step of providing beads ( 26 ) to which the PNA probes attach. 
     
     
         4 . A method as claimed in  claim 3 , wherein the beads are provided after the cells are lysed and the PNA probes have had time to bind to the target nucleic acid. 
     
     
         5 . A method as claimed in  claim 3 , wherein the beads ( 26 ) are paramagnetic or magnetic. 
     
     
         6 . A method as claimed in  claim 3 , wherein the beads are in the size range of 50 nm to 5 μm. 
     
     
         7 . (canceled) 
     
     
         8 . A method as claimed in  claim 1 , wherein the PNA probe and the crude lysate are brought into contact after the crude lysate cools to below a lysate annealing temperature. 
     
     
         9 . A method as claimed in  claim 1 , wherein the step (a) is performed so that the target nucleic acid is sheared into fragments. 
     
     
         10 . A method as claimed in  claim 1 , wherein the sample is partially or fully caused to flow by magnetic ( 19 ) attraction. 
     
     
         11 . A method as claimed in  claim 1 , wherein the chelating agent concentration is in the range of 1% w/v to 20% w/v with respect to the sample. 
     
     
         12 . A method as claimed in  claim 1 , wherein the chelating agent is a styrene-divinylbenzene co-polymer containing iminodiacetic acid groups. 
     
     
         13 . A method as claimed in  claim 2 , wherein the heating is at a temperature of 70° C. to 99.5° C. for at least 5 minutes to provide a sample pH in the range of 10 to 11. 
     
     
         14 . A method as claimed in  claim 3 , wherein the beads are magnetically removed after target nucleic acid has attached to the PNA probes. 
     
     
         15 . A method as claimed in  claim 14 , wherein the beads are removed on a surface ( 16 ) in the magnetic field of the magnet. 
     
     
         16 . A method as claimed in  claim 15 , wherein the beads are removed in a recess ( 16 ) or pocket of a body in the magnetic field of the magnet. 
     
     
         17 . A method as claimed in  claim 16 , wherein the pocket includes a carrier of oil or a wax ( 25 ). 
     
     
         18 . A method as claimed in  claim 17 , wherein the oil or wax ( 25 ) is in liquid form during the reaction without application of a magnetic field, and is caused to solidify after the magnetic field has been applied and the beads have been attracted into the pocket. 
     
     
         19 . A method as claimed in  claim 18 , wherein the oil or wax is molten due to applied heating for lysing.