Crude biological derivatives competent for nucleic acid detection

Abstract

The invention relates to methods for the detection of a specific sequence of RNA in a cell or tissue sample. The invention also relates to methods to enzymatically manipulate the RNA in a crude cell lysate in a number of applications.

What is claimed is:  
     
         1 . A method comprising: 
 obtaining at least one biological unit containing RNA;    obtaining at least one catabolic enzyme;    preparing an admixture of the biological unit and the catabolic enzyme; and    incubating the admixture under conditions where the catabolic enzyme is active.    
     
     
         2 . The method of  claim 1 , further comprising obtaining at least two catabolic enzymes.  
     
     
         3 . The method of  claim 2 , wherein the at least two catabolic enzymes are a protease and a nuclease.  
     
     
         4 . The method of  claim 3 , wherein the protease is Proteinase K and the nuclease is DNase I.  
     
     
         5 . The method of  claim 1 , wherein the biological unit is a cell.  
     
     
         6 . The method of  claim 5 , wherein the admixture contains a concentration of cells from about 5 to about 50,000 cells/μl.  
     
     
         7 . The method of  claim 6 , wherein the concentration of cells is about 10,000 cells/μl.  
     
     
         8 . The method of  claim 5 , wherein the cell is in obtained from a cell culture.  
     
     
         9 . The method of  claim 5 , wherein the cell is a prokaryotic cell.  
     
     
         10 . The method of  claim 5 , wherein the cell is a fungal cell.  
     
     
         11 . The method of  claim 5 , wherein the cell is a eukaryotic cell.  
     
     
         12 . The method of  claim 11 , wherein the cell is a human cell.  
     
     
         13 . The method of  claim 5 , wherein the biological unit is obtained from a subject.  
     
     
         14 . The method of  claim 5 , wherein the biological unit is obtained from a sample of body fluid.  
     
     
         15 . The method of  claim 14 , wherein the body fluid is whole blood, plasma, serum, cerebral spinal fluid or urine.  
     
     
         16 . The method of  claim 13 , wherein the biological unit is in a tissue sample.  
     
     
         17 . The method of  claim 1 , wherein the biological unit is a virus.  
     
     
         18 . The method of  claim 1 , wherein the catabolic enzyme is a protease.  
     
     
         19 . The method of  claim 18 , further defined as a method of inactivating ribonucleases in the admixture.  
     
     
         20 . The method of  claim 18 , wherein the protease is proteinase K.  
     
     
         21 . The method of  claim 1 , wherein the catabolic enzyme degrades carbohydrates.  
     
     
         22 . The method of  claim 21 , wherein the catabolic enzyme is amylase or cellulase.  
     
     
         23 . The method of  claim 1 , wherein the catabolic enzyme degrades lipids.  
     
     
         24 . The method of  claim 23 , wherein the catabolic enzyme is lipase.  
     
     
         25 . The method of  claim 1 , wherein the catabolic enzyme degrades DNA.  
     
     
         26 . The method of  claim 1 , wherein the catabolic enzyme is bovine pancreatic DNase I.  
     
     
         27 . The method of  claim 1 , further comprising adding an RNase inhibitor to the admixture.  
     
     
         28 . The method of  claim 28 , wherein the RNase inhibitor is a non-proteinaceous RNase inhibitor  
     
     
         29 . The method of  claim 28 , wherein the RNase inhibitor is ADP or a vanadyl complex.  
     
     
         30 . The method of  claim 27 , wherein in the RNase inhibitor is a proteinaceous inhibitor.  
     
     
         31 . The method of  claim 30 , wherein the proteinaceous inhibitor is placental ribonuclease inhibitor or an anti-RNase antibody.  
     
     
         32 . The method of  claim 1 , wherein preparing an admixture of the biological unit and the catabolic enzyme is further defined as comprising preparing an extract of the biological unit and preparing an admixture of the extract of the biological unit and the catabolic enzyme.  
     
     
         33 . The method of  claim 32 , wherein preparing an admixture of the extract of the biological unit and the catabolic enzyme comprises: 
 first preparing the extract; and    then mixing the extract with the catabolic enzyme.    
     
     
         34 . The method of  claim 32 , wherein preparing an admixture of the extract of the biological unit and the catabolic enzyme comprises: 
 first mixing the biological unit and the catabolic enzyme; and    then preparing the extract from the biological unit in the presence of the catabolic enzyme.    
     
     
         35 . The method of  claim 1 , further defined as a method for producing cDNA from one or more biological units and further comprising incubating the admixture with reverse transcriptase under conditions to promote reverse transcription.  
     
     
         36 . The method of  claim 35 , further comprising amplifying the products of the reverse transcription.  
     
     
         37 . The method of  claim 35 , further comprising incubating said admixture with a deoxyribonuclease prior to the reverse transcription reaction.  
     
     
         38 . The method of  claim 35 , wherein the catabolic enzyme is proteinase K and the final concentration of the proteinase K between 0.0001 and 5 mg/ml in the admixture.  
     
     
         39 . The method of  claim 1 , wherein the catabolic enzyme is comprised in a buffer composition prior to admixing.  
     
     
         40 . The method of  claim 1 , wherein the admixture is incubated at between 0° C. and 100° C.  
     
     
         41 . A method for producing cDNA from one or more biological units comprising: 
 obtaining at least one biological unit;    obtaining at least one catabolic enzyme;    preparing an admixture of the biological unit and the catabolic enzyme; and    incubating the admixture at a temperature where the catabolic enzyme is active and with reverse transcriptase under conditions to allow reverse transcription.    
     
     
         42 . The method of  claim 41 , further comprising obtaining at least two catabolic enzymes.  
     
     
         43 . The method of  claim 42 , wherein the at least two catabolic enzymes are a protease and a nuclease.  
     
     
         44 . The method of  claim 43 , wherein the protease is Proteinase K and the nuclease is DNase I.  
     
     
         45 . The method of  claim 41 , wherein the reverse transcriptase is added to the admixture after a time sufficient to allow the catabolic enzyme to function.  
     
     
         46 . The method of  claim 41 , wherein the biological unit is a cell.  
     
     
         47 . The method of  claim 41 , wherein the biological unit is a virus.  
     
     
         48 . The method of  claim 41 , wherein the catabolic enzyme is a protease.  
     
     
         49 . The method of  claim 48 , wherein the protease is proteinase K.  
     
     
         50 . The method of  claim 41 , wherein the catabolic enzyme degrades carbohydrates.  
     
     
         51 . The method of  claim 41 , wherein the catabolic enzyme degrades lipids.  
     
     
         52 . The method of  claim 41 , wherein the catabolic enzyme degrades DNA.  
     
     
         53 . The method of  claim 41 , wherein preparing an admixture of the biological unit and the catabolic enzyme is further defined as comprising preparing an extract of the biological unit and preparing an admixture of the extract of the biological unit and the catabolic enzyme.  
     
     
         54 . The method of  claim 53 , wherein preparing an admixture of the extract of the biological unit and the catabolic enzyme comprises: 
 first preparing the extract; and    then mixing the extract with the catabolic enzyme.    
     
     
         55 . The method of  claim 53 , wherein preparing an admixture of the extract of the biological unit and the catabolic enzyme comprises: 
 first mixing the biological unit and the catabolic enzyme; and    then preparing the extract from the biological unit in the presence of the catabolic enzyme.    
     
     
         56 . The method of  claim 41 , further comprising amplifying the products of the reverse transcription.  
     
     
         57 . The method of  claim 41 , further comprising incubating said admixture with a deoxyribonuclease prior to the reverse transcription reaction.  
     
     
         58 . A kit for producing cDNA from a biological unit, comprising, in a suitable container: 
 a buffer; and    a catabolic enzyme.    
     
     
         59 . The kit of  claim 58 , wherein the buffer and the catabolic enzyme are comprised in the same container.  
     
     
         60 . The kit of  claim 58 , further comprising, in one or more container(s): 
 a reverse transcription buffer    a reverse transcriptase; and    a dNTP mix.    
     
     
         61 . The kit of  claim 58 , further comprising a deoxyribonuclease.  
     
     
         62 . The kit of  claim 58 , wherein said catabolic enzyme is proteinase K.  
     
     
         63 . The kit of  claim 58 , further comprising an RNase inhibitor.  
     
     
         64 . A kit for producing cDNA from a biological unit comprising, in one or more suitable container(s): 
 a biological unit lysis buffer;    a deoxyribonuclease;    an RNase inhibitor;    a reverse transcription buffer;    reverse transcriptase;    dNTPs; and    an Armored RNA® control.    
     
     
         65 . The kit of  claim 64 , further comprising a protease inhibitor.  
     
     
         66 . The kit of  claim 65 , wherein the protease inhibitor is PMSF.  
     
     
         67 . The kit of  claim 64 , further comprising a thermostable DNA polymerase.  
     
     
         68 . A Cell Lysis Buffer comprising a catabolic enzyme, 1 mM CaCl 2 , 3 mM MgCl 2 , 1 mM EDTA, 1% Triton X100, and 10 mM Tris pH 7.5.  
     
     
         69 . The Cell Lysis Buffer of  claim 68 , wherein the catabolic enzyme is Proteinase K.  
     
     
         70 . The Cell Lysis Buffer of  claim 69 , wherein Proteinase K is at a concentration of about 0.2 mg/ml.