METHODS AND KITS FOR THE DETECTION OF NUCLEOTIDE MUTATIONS USING PEPTIDE NUCLEIC ACID AS BOTH PCR CLAMP AND SENSOR PROBE

• US 20080176226A1
• Filed: 01/19/2007
• Published: 07/24/2008
• Est. Priority Date: 01/19/2007
• Status: Active Grant

• Current Assignee: Chang GUNG University
• Original Assignee: Chang GUNG University
• Inventors: LUO, Ji-Dung, CHIOU, Chiuan-Chian
• Granted Patent: US 7,803,543 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 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2525/107 incorporating a peptide nucleic acid
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2535/131 Allele specific probes
• CPC Class Codes: C12Q 2549/126 using oligonucleotides as clamps
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

Disclosed herein is a method for determining whether a target polynucleotide sequence contained in a nucleic acid sample has nucleotide variation(s) in a selected region thereof, the steps of which involve the use of a pair of primers that allows the formation of a PCR product having a sequence covering that of the selected region of the target polynucleotide sequence via a PCR process, and a peptide nucleic acid (PNA) that acts as a PCR clamp as well as a sensor probe. Also disclosed herein is a kit for use in determining the presence of nucleotide variation(s) in the target polynucleotide sequence, which includes the pair of primers and the PNA.

Claims

1. A method for determining whether a target polynucleotide sequence contained in a nucleic acid sample has nucleotide variation(s) in a selected region thereof, comprising the steps of:

providing a pair of a first primer and a second primer which allows the formation of a PCR product having a sequence covering that of the selected region of the target polynucleotide sequence via a PCR process, the first primer having a sequence identical to that of a first region located upstream of the selected region of the target polynucleotide sequence, the second primer having a sequence based on that of a second region located downstream of the selected region of the target polynucleotide sequence, wherein the 5′

-end of the first region is spaced apart from the 5′

end of the sequence of the selected region by 30 nucleotides or more;

providing a detectable peptide nucleic acid probe having a sequence that complements fully the sequence of the selected region of the target polynucleotide sequence having no nucleotide variation(s) therein, such that hybridization of the detectable peptide nucleic acid probe to the selected region of the target polynucleotide sequence having no nucleotide variation(s) results in the formation of a duplex having a melting temperature;

determining the melting temperature of the duplex;

admixing the detectable peptide nucleic acid probe and the pair of the first primer and the second primer with the nucleic acid sample to form a mixture;

subjecting the mixture to a PCR process including an extension reaction set to run at a temperature lower than the melting temperature of the duplex by 5 to 20°

C., such that a mixture of PCR products is obtained; and

subjecting the mixture of PCR products thus-obtained to a melting analysis to determine melting temperatures of the PCR products, wherein the presence of at least one melting temperature lower than the melting temperature of the duplex is indicative of the nucleotide variation(s) in the selected region of the target polynucleotide sequence contained in the nucleic acid sample.

2. The method according to claim 1, wherein the nucleic acid sample is obtained from an organism selected from viruses, bacteria, fungi, plants, and animals.

3. The method according to claim 2, wherein the organism is a mammal.

4. The method according to claim 3, wherein the mammal is human.

5. The method according to claim 1, wherein the target polynucleotide sequence comprises a gene selected from the group consisting of disorder-associated gene, drug-resistance gene, and virulence gene.

6. The method according to claim 5, wherein the disorder-associated gene is selected from the group consisting of cancer-associated genes, and genes associated with a hereditary disease.

7. The method according to claim 6, wherein the cancer-associated gene is selected from the group consisting of K-ras, H-ras, N-ras, p53 (TP53), CDKN2A (p16), PIC3K, PTEN, RB1, epidermal growth factor receptor gene, BRAF, BRCA1, BRCA2, STK11, and VHL.

8. The method according to claim 7, wherein the cancer-associated gene is K-ras.

9. The method according to claim 1, wherein the nucleotide variation(s) include one or more nucleotide deletion, insertion, substitution, or abnormal methylation.

10. The method according to claim 1, wherein the 5′

-end of the sequence of the first region is spaced apart from the 5′

-end of the sequence of the selected region by 30 to 2000 nucleotides.

11. The method according to claim 1, wherein the 5′

-end of the sequence of the first region is spaced apart from the 5′

-end of the sequence of the selected region by 50 to 1500 nucleotides.

12. The method according to claim 1, wherein the 5′

end of the sequence of the first region is spaced apart from the 5′

-end of the sequence of the selected region by 50 to 1000 nucleotides.

13. The method according to claim 1, wherein the extension reaction is set to run at a temperature lower than the melting temperature of the duplex by 5 to 18°

C.

14. The method according to claim 1, wherein the extension reaction is set to run at a temperature lower than the melting temperature of the duplex by 8 to 18°

C.

15. The method according to claim 1, wherein the detectable peptide nucleic acid probe ranges from 8 to 30 mer in length.

16. The method according to claim 1, wherein the detectable peptide nucleic acid probe is labeled with one of a fluorescent moiety, a photoluminescent moiety, a luminescent moiety, and a chemiluminescent moiety.

17. The method according to claim 16, wherein the detectable peptide nucleic acid probe is labeled with a fluorescent moiety.

18. The method according to claim 17, wherein the fluorescent moiety is one selected from the group consisting of fluorescein, rhodamine, FAM, TET, HEX, JOE, TAMA, NTB, TAMRA, ROX, VIC, NED, 4,7-dichloro-fluorescein, 4,7-dichloro-rhodamine, Cy5, Cy3, Texas Red, DABCYL, DABSYL, malachite green, cyanine, LC-Red610, LC-Red640, LC-Red670, LC-Red705, and derivatives thereof.

19. The method according to claim 18, wherein the fluorescent moiety is fluorescein.

20. The method according to claim 17, wherein an anchor probe is additionally added in the admixing step.

21. The method according to claim 20, wherein the anchor probe is labeled with a fluorescent dye.

22. The method according to claim 21, wherein the fluorescent dye is selected from the group consisting of fluorescein, rhodamine, FAM, TET, HEX, JOE, TAMA, NTB, TAMRA, ROX, VIC, NED, 4,7-dichloro-fluorescein, 4,7-dichloro-rhodamine, Cy5, Cy3, Texas Red, DABCYL, DABSYL, malachite green, cyanine, LC-Red610, LC-Red640, LC-Red670, LC-Red705, and derivatives thereof.

23. The method according to claim 22, wherein the fluorescent dye is LC-Red 640.

24. A kit for determining whether a target polynucleotide sequence contained in a nucleic acid sample has nucleotide variation(s) in a selected region thereof, comprising:

a detectable peptide nucleic acid probe having a sequence that complements fully the sequence of the selected region of the target polynucleotide sequence having no nucleotide variation(s) therein, such that hybridization of the detectable peptide nucleic acid probe to the selected region of the target polynucleotide sequence having no nucleotide variation(s) results in the formation of a duplex having a melting temperature;

a pair of a first primer and a second primer which allows the formation of a PCR product having a sequence covering that of the selected region of the target polynucleotide sequence via a PCR process, the first primer having a sequence identical to that of a first region located upstream of the selected region of the target polynucleotide sequence, the second primer having a sequence based on that of a second region located downstream of the selected region of the target polynucleotide sequence, wherein the 5′

-end of the sequence of the first region is spaced apart from the 5′

-end of the sequence of the sequence of the selected region by 30 nucleotides or more; and

an instruction sheet providing guidance for a user to use the detectable peptide nucleic acid probe and the pair of the first primer and the second primer in a method as defined in claim 1.