Method for determining the presence of DNA variants using peptide nucleic acid probes

• US 20030203364A1
• Filed: 04/24/2002
• Published: 10/30/2003
• Est. Priority Date: 04/24/2002
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Spectrumedix LLC
• Inventors: Liu, Zhaowei
• Granted Patent: US 6,872,530 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2527/15 Gradients
• CPC Class Codes: C12Q 2565/125 Electrophoretic separation

Abstract

The present invention relates to a peptide nucleic acid probe-based method for generating data indicative of the presence of a nucleotide polymorphism, mutation, or methylated cytosine in a nucleotide containing compound. A peptide nucleic acid probe (PNAP) is subjected to temperature gradient electrophoresis in the presence of a nucleotide containing compound. The PNAP is irradiated to generate a spectroscopic signal. The spectroscopic signal is converted into data suitable for determining the presence of the nucleotide polymorphism or the mutation in the nucleotide-containing compound.

Claims

1. A peptide nucleic acid probe-based method for generating data indicative of the presence of a variant in a nucleotide containing compound, comprising:

subjecting a peptide nucleic acid probe (PNAP) having fewer than about 30 bases to temperature gradient electrophoresis in the presence of a nucleotide containing compound;

irradiating the PNAP to generate a spectroscopic signal; and

converting the spectroscopic signal into data suitable for determining the presence of the variant in the nucleotide-containing compound.

2. The method of claim 1, wherein the PNAP comprises fewer than about 20 bases.

3. The method of claim 1, wherein, at the beginning of the temperature gradient electrophoresis, the PNAP is bound to the nucleotide containing compound.

4. The method of claim 1, wherein the PNAP comprises a fluorescent tag and the spectroscopic signal results from the fluorescent tag.

5. The method of claim 1, wherein the data suitable for determining the presence of the variant are essentially free of data resulting from a intercalating dye fluorescence.

6. The method of claim 1, where the PNAP migrates during the temperature gradient electrophoresis.

7. The method of claim 1, wherein the temperature gradient electrophoresis is conducted using a sieving medium that is essentially free of entrapped PNAP.

8. A peptide nucleic acid probe-based method for determining the presence of a variant in a nucleotide containing compound, comprising:

obtaining a first parameter representative of a first spectroscopic signal resulting from a first peptide nucleic acid probe (PNAP), the first PNAP having been subjected to temperature gradient electrophoresis in the presence of the nucleotide containing compound;

obtaining a second parameter representative of a second spectroscopic signal resulting from a second PNAP, the second PNAP having been subjected, simultaneously with the first PNAP, to temperature gradient electrophoresis in the presence of the nucleotide containing compound;

comparing the first and second parameters to determine the presence of the variant in the nucleotide-containing compound.

9. The method of claim 8, wherein the first and second PNAP'"'"'s each comprise fewer than about 20 bases.

10. The method of claim 8, wherein the each PNAP comprises a respective fluorescent tag and the first and second spectroscopic signals result from the respective fluorescent tags.

11. The method of claim 8, wherein the respective fluorescent tags emit fluorescence at different wavelengths.

12. The method of claim 8, wherein the first and second spectroscopic signals used to obtain the first and second parameters are essentially free of fluorescence resulting from an intercalating dye.

13. A peptide nucleic acid probe-based method for determining the presence of variants in a plurality of respective nucleotide containing compounds, comprising:

subjecting a mixture comprising a plurality of peptide nucleic acid probes and a plurality of different nucleotide containing compounds to temperature gradient electrophoresis, different nucleotide containing compounds having a different length, peptide nucleic acid probes forming complexes with respective nucleotide containing compounds;

irradiating the peptide nucleic acid probes to generate spectroscopic signals; and

converting the spectroscopic signals into data suitable for determining the presence of variants in respective nucleotide-containing compounds.

14. The method of claim 13, wherein the different nucleotide containing compounds comprises at least 4 nucleotide containing compounds having lengths that differ by between about 2 and about 10 bases.

15. The method of claim 13, wherein the mixture is subjected to electrophoresis within a single separation lane.

16. The method of claim 13, wherein the mixture includes at least about 10 different peptide nucleic acid probes and at least about 10 different nucleotide containing compounds.

17. The method of claim 16, wherein the mixture includes at least about 25 different peptide nucleic acid probes and at least about 25 different nucleotide containing compounds.

18. The method of claim 13, wherein the peptide nucleic acid probes comprise a respective fluorescent tag.

19. The method of claim 13, wherein the spectroscopic signals are essentially free of fluorescence resulting from an intercalating dye.

20. A temperature gradient electrophoresis-based method for generating data indicative of the presence of one or more methylated cytosines in a sample comprising a first nucleotide containing compound (NCC) having non-methylated cytosines, comprising:

contacting the first NCC with a first compound to thereby provide a modified NCC wherein non-methylated cytosines of the first NCC are replaced with a different base;

amplifying the modified NCC to obtain first PCR products;

subjecting the first PCR products to temperature gradient electrophoresis in the presence of a peptide nucleic acid probe;

irradiating the first PCR products and peptide nucleic acid with light to thereby generate a spectroscopic signal; and

converting the spectroscopic signal into data indicative of the presence of the one or more methylated cytosines in the first NCC.

21. A temperature gradient electrophoresis-based method for generating data indicative of the presence of one or more methylated cytosines in a sample comprising a first nucleotide containing compound (NCC), comprising:

obtaining first PCR products, the PCR products having been prepared by;

(a1) contacting the first NCC with a first compound to thereby provide a modified NCC in which non-methylated cytosines are replaced with a different base, and (a2) amplifying the modified NCC to obtain said first PCR products;

subjecting the first PCR products and a peptide nucleic acid probe (PNAP) to temperature gradient electrophoresis,

irradiating the first PCR products and peptide nucleic acid (PNAP) with light to thereby generate a spectroscopic signal; and

converting the spectroscopic signal into data indicative of the presence of the one or more methylated cytosines in the first NCC.

22. A peptide nucleic acid probe-based method for generating data indicative of a sequence of at least a portion of the nucleotide-containing compound, comprising:

subjecting first and second peptide nucleic acid probes (PNAP'"'"'s) to temperature gradient electrophoresis in the presence of a nucleotide containing compound, the first and second PNAP'"'"'s having spectroscopically distinguishable tags;

irradiating the PNAP'"'"'s to generate a spectroscopic signal; and

converting the spectroscopic signal into data suitable for determining the sequence of at least a portion of the nucleotide-containing compound.

23. A peptide nucleic acid probe-based method for generating data indicative of the presence of a variant in a nucleotide containing compound, comprising:

subjecting a peptide nucleic acid probe (PNAP) to temperature gradient electrophoresis in the presence of a nucleotide containing compound, wherein the nucleotide-containing compound comprises at least about 10 times as many bases as the nucleotide containing compound;

irradiating the PNAP to generate a spectroscopic signal; and

converting the spectroscopic signal into data suitable for determining the presence of the variant in the nucleotide-containing compound.