Compositions and methods for detecting single gene copies in-situ

Abstract

Compositions and methods are provided for detecting single copies of a gene in-situ using brightfield microscopy in detection of various target nucleic acid sequences, such as genes or nucleic acid of microorganisms in a cell. In particular, nucleic acid probes for various target nucleic acid sequences in a cell are utilized in conjunction with enzymes and their substrates to produce chromogenic composition associated with the target nucleic acid sequences in the cell.

1 . A composition comprising at least one cell containing target nucleic acid sequences associated with a product of an enzyme and a chromogen composition in the at least one cell, wherein the product is individually associated with each of at least one of the target nucleic acid sequences and each of the target nucleic acid sequences associated with the product is separate from each other such that the product is distinguishable from the product associated with other copies of the target nucleic acid sequences in the same cell such that one can determine the number of original copies of the target nucleic acid sequences in the at least one cell.  
   
   
       2 . The composition according to  claim 1 , wherein the product is located at the target nucleic acid sequence of a chromosomal DNA exclusively.  
   
   
       3 . The composition according to  claim 1 , wherein the enzyme is a phosphatase or a peroxidase.  
   
   
       4 . The composition according to  claim 1 , wherein the chromogen is NBT/BCIP, tetramethylbenzidine or diamino benzidine.  
   
   
       5 . The composition according to  claim 1  wherein the enzyme is alkaline phosphatase and the chromogen is NBT/BCIP.  
   
   
       6 . The composition according to  claim 1 , wherein the target nucleic acid sequence is Her-2/neu DNA.  
   
   
       7 . The composition according to  claim 1 , wherein the target nucleic acid sequence is a nucleic acid sequence selected from the group consisting of a nucleic acid sequence of human immunodeficiency virus (HIV), human papilloma virus (HPV), Epstein-Barr virus (EBV), cytomegalovirus (CMV),  T. palidium , and  M. tuberculosis.    
   
   
       8 . The composition according to  claim 1 , wherein the enzyme is associated with a nucleic acid probe specific for the target nucleic acid sequence and bound to the target nucleic acid sequence in the at least one cell.  
   
   
       9 . The composition according to  claim 8 , wherein the nucleic acid probe is bound to the target nucleic acid sequence via hybridization.  
   
   
       10 . The composition according to  claim 8 , wherein the nucleic acid probe is labeled with a detectable moiety.  
   
   
       11 . The composition according to  claim 10 , wherein the detectable moiety is selected from the group consisting of digoxygenin, biotin and fluorescein.  
   
   
       12 . The composition according to  claim 10 , wherein the enzyme is linked to an antibody specific to the detectable moiety on the labeled nucleic acid probe.  
   
   
       13 . The composition according to  claim 12 , wherein the enzyme is alkaline phosphatase; the detectable moiety is digoxygenin; and the antibody is an antibody specific to digoxygenin and linked to alkaline phosphatase.  
   
   
       14 . The composition according to  claim 1 , wherein the product is distinguishable from the product associated with other copies of the target nucleic acid sequences in the same cell under brightfield microscope conditions.  
   
   
       15 . A kit for detecting a single gene copy target in an intact cell, comprising: 
 a) a detectably-labeled nucleic acid probe specific for a Her-2/neu gene;    b) an anti-digoxigenin antibody having an enzyme covalently attached thereto; and    c) a chromogen for deposition in the immediate vicinity of the enzyme-antibody complex.    
   
   
       16 . The composition according to  claim 15 , wherein the enzyme is a phosphatase or a peroxidase.  
   
   
       17 . The composition according to  claim 15 , wherein the chromogen is NBT/BCIP, tetramethylbenzidine or diamino benzidine.  
   
   
       18 . The kit according to  claim 15 , wherein the enzyme is alkaline phosphatase and the chromogen is NBT/BCIP.  
   
   
       19 . The kit according to  claim 15 , further comprising: instruction for detecting a single copy of the Her-2/neu gene in a cell.  
   
   
       20 . The kit according to  claim 15 , further comprising: a specimen containing breast cells.