KITS AND PROCESSES FOR REMOVING CONTAMINANTS FROM NUCLEIC ACIDS IN ENVIRONMENTAL AND BIOLOGICAL SAMPLES

• US 20090111159A1
• Filed: 12/01/2008
• Published: 04/30/2009
• Est. Priority Date: 05/21/2004
• Status: Abandoned Application

• Current Assignee: MO BIO Laboratories, Inc. (Qiagen NV)
• Original Assignee: MO BIO Laboratories, Inc. (Qiagen NV)
• Inventors: Stolow, David, Venugopal, Raveendran J., BROLASKI, Mark N.
• Application Number:
  US12/326,018
• Publication Number:
  US 20090111159A1
• Time in Patent Office: Days
• US Class Current: 435/193
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2565/113 based on agglutination/precipitation

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.

Claims

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 (MgCl₂), ferric chloride (FeCl₃), an iron salt or an aluminum salt, calcium chloride (CaCl₂), 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.