Kits and processes for removing contaminants from nucleic acids in environmental and biological samples

Abstract

The invention provides methods and compositions, e.g., kits, for removing contaminants from nucleic acids in a sample, e.g., environmental or biological samples such as soil, food, plant, animal, microorganism or water samples. The invention provides methods and compositions for isolating nucleic acids from environmental and biological samples in a scaleable process free of contaminating substances that inhibit PCR and other downstream applications. Exemplary sample types include soil, water, plant and food. The methods and compositions of the invention can be used for isolating and/or detecting nucleic acids from prokaryotic and eukaryotic organisms and for detecting multiple types of organisms in a sample. Thus, the methods and compositions of the invention are useful for detecting organisms pertaining to agriculture, forensics biology and/or combating bioterrorism.

1 . A method for releasing RNA from a sample comprising:
 (a) releasing an RNA from the sample comprising a step of adding a first flocculant comprising a quaternary ammonium or tertiary amine containing polymer to an processed, unprocessed, preserved, freshly isolated, crude or unrefined sample medium, to generate a first flocculant precipitate and a first RNA-comprising supernatant,   wherein optionally the quaternary ammonium or tertiary amine comprises an ammonium acetate;   (b) contacting the first RNA-comprising supernatant with a second flocculant comprising a quaternary ammonium or tertiary amine to generate a second flocculant precipitate and a second RNA-comprising supernatant,   wherein optionally the method further comprises after step (b) contacting the nucleic acid with a buffer comprising phenol.   
   
   
       2 . A kit for isolating a nucleic acid from a samples comprising at least one flocculant and instructions describing a method for use according to  claim 1 . 
   
   
       3 . The kit of  claim 2 , wherein the flocculant comprises an anionic, cationic, zwitterionic or uncharged chemical substance or combination thereof, wherein optionally the cationic substance comprises a quaternary ammonium or tertiary amine containing polymer. 
   
   
       4 . The kit of  claim 2 , wherein the flocculant is selected from the group consisting of ammonium acetate, magnesium chloride (MgCl 2 ), ferric chloride (FeCl 3 ), an iron salt or an aluminum salt, calcium chloride (CaCl 2 ), a polyacrylamide, aluminum ammonium sulfate and derivatives thereof. 
   
   
       5 . The kit of  claim 2 , further comprising a detergent or a surfactant. 
   
   
       6 . The kit of  claim 2 , wherein the detergent is selected from the group consisting of sodium dodecyl sulfate (SDS), sarkosyl, sodium lauryl sarcosinate, cetyltrimethyl ammonium bromide (CTAB), cholic acid, deoxycholic acid, benzamidotaurocholate (BATC), octyl phenol polyethoxylate, polyoxyethylene sorbitan monolaurate, tert-octylphenoxy poly(oxyethylene)ethanol, polyethylene glycoltert-octylphenyl ether (Triton® X-100), (1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol (Triton®X-114) and a combination thereof. 
   
   
       7 . The kit of  claim 2 , further comprising a homogenizing material. 
   
   
       8 . The kit of  claim 2 , further comprising a bead, wherein optionally the bead is a homogenizing bead. 
   
   
       9 . The kit of  claim 2 , further comprising one or more solutions or buffers for performing a method according to any of the  claims 1  to  50 . 
   
   
       10 . The kit of  claim 2 , wherein the instructions describing a method for obtaining a sample for processing. 
   
   
       11 . The kit of  claim 2 , further comprising one or more tube vessels useful for performing the method of use. 
   
   
       12 . The kit of  claim 2 , further comprising one or more oligonucleotides, and optionally free nucleotides, and optionally sufficient free nucleotides to carry out a PCR reaction, a rolling circle replication, a ligase-chain reaction, a reverse transcription or derivative methods thereof. 
   
   
       13 . The kit of  claim 2 , further comprising at least one enzyme, wherein optionally the enzyme is a polymerase. 
   
   
       14 . The kit of  claim 2 , further comprising one or more oligonucleotides, free nucleotides and at least one polymerase or enzyme capable of amplifying a nucleic acid in a PCR reaction, a rolling circle replication, a ligase-chain reaction, a reverse transcription or derivative methods thereof. 
   
   
       15 . The kit of  claim 12  or  claim 14 , wherein the one or more oligonucleotides specifically hybridizes to a nucleic acid from a microorganism, an animal, a plant, an insect, a yeast, a virus, a phage, a nematode, a bacteria or a fungi.