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Cross-coupled peptide nucleic acids for detection of nucleic acids of pathogens

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Title: 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. ...


Inventors: Daniel H. Appella, Christopher Micklitsch
USPTO Applicaton #: #20120107794 - Class: 435 5 (USPTO) - 05/03/12 - Class 435 
Chemistry: Molecular Biology And Microbiology > Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip >Involving Virus Or Bacteriophage

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The Patent Description & Claims data below is from USPTO Patent Application 20120107794, Cross-coupled peptide nucleic acids for detection of nucleic acids of pathogens.

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CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation in part of U.S. patent application Ser. No. 12/441,925 filed Mar. 19, 2009, which claims benefit to International Patent Application No. PCT/US2007/020466, filed Sep. 21, 2007, which claims benefit of U.S. Provisional Application Nos. 60/846,354, filed Sep. 22, 2006 and 60/896,667, filed Mar. 23, 2007, each of which is incorporated herein by reference in their entirety; and the disclosures of which are incorporated herein in their entirety.

TECHNICAL FIELD

The instant invention concerns methods for detecting nucleic acids.

BACKGROUND

Polymerase chain reaction (PCR) is a widely used technique for the detection of pathogens. The technique uses a DNA polymerase used to amplify a piece of DNA by in vitro enzymatic replication. The PCR process generates DNA that is used as a template for replication. This results in a chain reaction that exponentially amplifies the DNA template.

Technologies for genomic detection most commonly use DNA probes to hybridize to target sequences. To achieve required sensitivity, the use of PCR to amplify target sequences has remained standard practice in many labs. While PCR has been the principle method to identify genes associated with disease states, the method has remained confined to use within a laboratory environment. Most current diagnostic applications that can be used outside of the laboratory are based on antibody recognition of protein targets and use ELISA-based technologies to signal the presence of a disease. These methods are fast and fairly robust, but they can lack the specificity associated with nucleic acid detection

Recently, it was reported that incorporating trans-1,2-diaminocyclopentane into aminoethylglycine peptide nucleic acids (aegPNAs) significantly increases binding affinity and sequence specificity to complementary DNA. See, Pokorski, et al, J. Am. Chem. Soc. 2004, 126, 15067-15073 and Myers, et al, Org. Lett. 2003, 5, 2695-2698. Despite the promise of PNAs with 1,2-diaminocyclopentane residues in the backbone, commercially viable uses of such PNAs have not been realized.

There is a need for pathogen detection methods that are highly specific and robust for use outside of a laboratory environment.

SUMMARY

In some aspects, the invention concerns methods of 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. The substrate can be washed prior to determining the presence of said reporter molecule.

In some aspects, the substrate is visually observed to detect the appearance of color from the reporter molecule. The detecting can be performed visually by an observer.

Preferred PNAs for the first and second PNAs include trans-cyclopentane-containing PNAs.

Any suitable cross-reactive functional groups may be used. For example, pyrrole-2,5-dione and a thiol functionality can be used as the functional groups. In some embodiments, the first cross-reactive group comprises a pyrrole-2,5-dione functionality. In certain embodiments, the second cross-reactive group comprises a thiol functionality. In addition, in some preferred embodiments, the reporter molecule is biotin.

While the instant methods can be used to detect a wide variety of samples, particularly useful samples include anthrax, avian flu, severe acute respiratory syndrome (SARS), tuberculosis (TB), human papilloma virus (HPV), or human immunodeficiency virus (HIV).

In one aspect, the invention concerns methods where 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.

The invention also concerns kits 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;



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stats Patent Info
Application #
US 20120107794 A1
Publish Date
05/03/2012
Document #
12409159
File Date
03/23/2009
USPTO Class
435/5
Other USPTO Classes
435/611
International Class
/
Drawings
7



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