MODIFIED HYBRIDIZATION PROBES

• US 20140057263A1
• Filed: 03/21/2012
• Published: 02/27/2014
• Est. Priority Date: 03/24/2011
• Status: Active Application

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Qiagen GmbH (Qiagen NV)
• Inventors: Engel, Holger, Azzawi, Alexander, Di Pasquale, Francesca
• Application Number:
  US14/006,794
• Publication Number:
  US 20140057263A1
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/119 incorporating abasic sites
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis

Abstract

The invention relates to a method for detecting a nucleic acid of an organism in a composition, comprising the steps of, (i) amplifying the nucleic acid to be detected, (ii) during or after amplification, hybridizing to said nucleic acid to be detected a first probe that comprises an abasic site additionally optionally carrying a detectable label, (iii) wherein the position of the abasic site corresponds to a position in said nucleic acid to be detected, known to have a polymorphism in said organism, and wherein said nucleic acid is detected if hybridization occurs.

Claims

1. Method for detecting a nucleic acid, comprising the steps of,

a. amplifying the nucleic acid to be detected,

b. during or after amplification, hybridizing to said nucleic acid to be detected a first probe that comprises an abasic site, additionally optionally carrying a detectable label, wherein the position of the abasic site corresponds to a position in said nucleic acid to be detected, known to have a polymorphism,

c. and wherein said nucleic acid is detected if hybridization occurs.

2. Method according to claim 1, wherein after hybridization the melting temperature and/or point of said first hybridization probe is determined and the probe carries a label which is detectable when the probe hybridizes to its target or dissociates from its target.

3. Method according to claims 1, wherein the method comprises at least one further probe which may optionally also comprise an abasic site but which carries the same label as said first hybridization probe and wherein the probes are distinguishable due to their difference in melting temperatures.

4. Method according to claims 1, wherein the abasic site is derived from a 1′

-2′

-dideoxyribose or a 1,3-propandiol.

5. Method according to claims 1, wherein

a. each of the probes is specific for a nucleic acid sequence,

b. at least two probes carry the same label;

and

c. each of the probes that carry the same label has a melting temperature (T_m) which differs by more than 1°

C. from the other probes.

6. Method according to claim 1, wherein the amplification method is selected from the group of polymerase chain reaction (PCR), real-time PCR (rtPCR), ligase chain reaction (LCR), transcription-based amplification system (TAS), nucleic acid sequence based amplification (NASBA), rolling circle amplification (RCA), transcription-mediated amplification (TMA), self-sustaining sequence replication (3SR) and Qβ

amplification.

7. Method according to claim 1, wherein the amplification method is selected from the group of polymerase chain reaction (PCR) and real-time PCR (rtPCR) and the melting temperature and/or point of said probes is determined during PCR or post PCR.

8. Method according to claim 1, wherein the probe is selected from the group of TaqMan probe, Scorpion probe, molecular beacon probe, light cycler probe, LUX probe and amplifluor probe.

9. Oligonucleotide probe selected from the group of TaqMan probe, Scorpion probe, molecular beacon probe, light cycler probe, LUX probe and amplifluor probe, wherein the probe comprises at least on abasic site, and wherein the abasic site corresponds to a position in a nucleic acid to be detected, known to have a polymorphism.

10. Kit for the detection of a nucleic acid, wherein the kit comprises at least one oligonucleotide probe with an abasic site, and wherein the abasic site corresponds to a position in said nucleic acids to be detected, known to have a polymorphism.

11. Kit according to claim 10 wherein, the probe is labelled.

12. Kit according to claim 10, wherein the probe is a TagMan probe, Scorpion probe, molecular beacon probe, light cycler probe, LUX probe or amplifluor probe.

13. Kit according to claim 10, wherein the kit comprises two oligonucleotide probes wherein these two carry the same label, wherein at least one probe carries an abasic site and both are specific for a given target.