Cross-Coupled Peptide Nucleic Acids for Detection of Nucleic Acids of Pathogens

Abstract

The present invention concerns methods for detecting a nucleic acid comprising (i) contacting a solution comprising a first PNA having a first cross-reactive functional group with a substrate having a second PNA affixed thereto, the second PNA having a second first cross-reactive functional group, wherein the first PNA has a reporter molecule attached thereto and the first and second PNAs being complementary to different portions of a target DNA; (ii) contacting a sample suspected of containing the nucleic acid with the first and second PNAs; and (iii) determining the presence of the reporter molecule on the substrate.

1 . A method for detecting a nucleic acid comprising:
 contacting a solution comprising a first PNA having a first cross-reactive functional group with a substrate having a second PNA affixed thereto, said second PNA having a second first cross-reactive functional group, wherein the first PNA has a reporter molecule attached thereto and the first and second PNAs being complementary to different portions of a target DNA;   contacting a sample suspected of containing the nucleic acid with said first and second PNAs;   determining the presence of said reporter molecule on said substrate.   
     
     
         2 . The method of  claim 1 , wherein said substrate is visually observed to detect the appearance of color from the reporter molecule. 
     
     
         3 . The method of  claim 1 , wherein said substrate is washed prior to determining the presence of said reporter molecule. 
     
     
         4 . The method of  claim 1 , wherein said first and second PNAs are trans-cyclopentane-containing PNAs. 
     
     
         5 . The method of  claim 1 , where said detecting is performed visually by an observer. 
     
     
         6 . The method of  claim 1 , wherein said first cross-reactive group comprises a pyrrole-2,5-dione functionality. 
     
     
         7 . The method of  claim 1 , wherein said second cross-reactive group comprises a thiol functionality. 
     
     
         8 . The method of  claim 1 , wherein said reporter molecule is biotin. 
     
     
         9 . The method of  claim 8 , further comprising contacting said first PNA and second PNA with a biotin-labeled PNA detection probe. 
     
     
         10 . The method of  claim 9 , wherein said biotin-labeled PNA detection probe comprises an avidin-horseradish peroxidase conjugate 
     
     
         11 . The method of  claim 1 , wherein said sample comprises anthrax, avian flu, severe acute respiratory syndrome (SARS), tuberculosis (TB), human papilloma virus (HPV), or human immunodeficiency virus (HIV). 
     
     
         12 . The method of  claim 1 , wherein the detection is performed by a method comprising:
 contacting a solution comprising a first PNA with a substrate having a second PNA affixed thereto, wherein the PNA has a reporter molecule attached thereto and the first and second trans-cyclopentane PNAs being complementary to different portions of a target DNA;   contacting DNA with the first and second cyclopentane-containing PNAs;   visually observing the substrate to detect the appearance of color from the reporter molecule.   
     
     
         13 . A kit for detecting a nucleic acid comprising:
 solution comprising a first PNA having a first cross-reactive functional group; and   a substrate having a second PNA affixed thereto, said second PNA having a second first cross-reactive functional group,   wherein the first PNA has a reporter molecule attached thereto and the first and second PNAs being complementary to different portions of a target DNA.   
     
     
         14 . The kit of  claim 13 , wherein the kit is adapted for detecting an infectious agent, said infectious agent being anthrax, avian flu, severe acute respiratory syndrome (SARS), tuberculosis (TB), human papilloma virus (HPV), or human immunodeficiency virus (HIV). 
     
     
         15 . The kit of  claim 13 , further comprising a biotin-labeled PNA detection probe an avidin-horseradish peroxidase conjugate.