Amplification and Sequencing of Transrenal Nucleic Acids

Abstract

The present invention provides highly sensitive methods used for diagnosing and monitoring various diseases and disorders by detecting and analyzing “ultra short” (20-50 base pair) nucleic acids obtained from bodily fluids.

1 . A method of analyzing a target nucleic acid sequence of 20-50 nucleotides in length, the method comprising:
 (a) obtaining a urine sample from a patient;   (b) assaying the urine sample for the presence of a target transrenal nucleic acid sequence of 20-50 nucleotides in length that has crossed the kidney barrier, wherein said assaying comprises the polymerase chain reaction (PCR) that amplifies the target transrenal nucleic acid sequence and the sequencing of the target transrenal nucleic acid sequence.   
     
     
         2 . The method of  claim 1 , wherein the target nucleic acid sequence is DNA. 
     
     
         3 . The method of  claim 1 , wherein the target nucleic acid sequence is RNA. 
     
     
         4 . The method of  claim 1 , wherein nucleic acid degradation in said urine sample is reduced. 
     
     
         5 . The method of  claim 4 , wherein reducing nucleic acid degradation comprises inhibiting nuclease activity by increased pH, increased salt concentration, heat inactivation, or by treating said urine sample with a compound selected from the group consisting of: ethylenediaminetetraacetic acid, guanidine-HCl guanidine isothiocyanate, N-lauroylsarcosine, and sodium dodecylsulphate. 
     
     
         6 . The method of  claim 1 , wherein said urine sample has been held in the bladder less than 12 hours. 
     
     
         7 . The method of  claim 1 , further comprising substantially isolating said nucleic acids in said urine sample. 
     
     
         8 . The method of  claim 7 , wherein said isolation is by precipitation or using a solid adsorbent material. 
     
     
         9 . The method of  claim 1 , wherein said assaying further comprises quantifying said nucleic acids. 
     
     
         10 . The method of  claim 1 , wherein the patient is a pregnant female and the target nucleic acid sequence is fetal in origin and indicative of a fetal genetic disorder. 
     
     
         11 . The method of  claim 1 , wherein the target nucleic acid sequence is indicative of cancer or a predisposition to cancer. 
     
     
         12 . The method of  claim 11 , wherein the cancer is a solid tumor or a hemtologic tumor or malignancy. 
     
     
         13 . The method of  claim 11 , wherein the cancer is selected from breast cancer, lung cancer, colorectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, renal carcinoma, hepatoma, HCC (hepatocelluar carcinoma), brain cancer, melanoma, multiple myeloma, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, childhood lymphomas, and lymphomas of lymphocytic and cutaneous origin, leukemia, childhood leukemia, hairy-cell leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, chronic myelogenous leukemia, and mast cell leukemia, myeloid neoplasms, mast cell neoplasms, hematologic tumor, and lymphoid tumor, including metastatic lesions in other tissues or organs distant from the primary tumor site. 
     
     
         14 . The method of  claim 11 , wherein the target transrenal nucleic acids are indicative of cancer and
 the method further comprises monitoring cancer treatment, anti-tumor therapy, or tumor re-growth in said patient.   
     
     
         15 . The method of  claim 1 , wherein the target nucleic acid sequence comprises a sequence variation comprising single base substitutions and/or deletions or insertions of one or more nucleotides.