HIGHLY SENSITIVE RAPID ISOTHERMAL METHOD FOR THE DETECTION OF POINT MUTATIONS AND SNPs, A SET OF PRIMERS AND A KIT THEREFOR

• US 20120171676A1
• Filed: 06/08/2010
• Published: 07/05/2012
• Est. Priority Date: 07/10/2009
• Status: Active Grant

• Current Assignee: DiaSorin Molecular LLC
• Original Assignee: Diasorin Ireland Ltd. (DiaSorin S.p.A.)
• Inventors: Adlerstein, Daniel, Amicarelli, Giulia, Minnucci, Giulia
• Granted Patent: US 9,404,149 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides

Abstract

The present invention refers to a method for detecting a point mutations of a nucleotide sequence by an improve- ment of the LAMP (loop amplification mediated polymerization) amplification method, as well as to a set of primers and kit there- for. As a non limitative embodiment, the invention refers to the G1849T mutation of the JAK2 gene.

Claims

1-28. -28. (canceled)

29. A method for detecting the presence of a point mutation in a target nucleic acid molecule, the method comprising the steps of:

1) providing a nucleic acid sample;

2) contacting said nucleic acid sample, under appropriate reaction conditions, with a solution comprising a mixture of oligonucleotides and a DNA polymerase having strand displacement activity under hybridization conditions, wherein said mixture of oligonucleotides comprises primers suitable for loop mediated isothermal amplification of the region of the target nucleic acid molecule including the nucleic acid position of the point mutation to be detected, said primers comprising ;

i. a first outer primer F3 and a second outer primer B3;

ii. a first inner primer FIP and a second inner primer BIP,

wherein FIP comprises a 3′

nucleic acid sequence F2 and a 5′

nucleic acid sequence F1c and BIP comprises a 3′

nucleic acid sequence B2 and a 5′

nucleic acid sequence B lc,

wherein F2 is able to recognize and hybridize to a region of the target nucleic acid molecule designated as F2c and B2 is able to recognize and hybridize to a region of the target nucleic acid molecule designated as B2c,

wherein F2c and B2c are different regions located on opposite strands of the target nucleic acid molecule,

wherein either B2c is downstream of the point mutation or F2c is upstream of the point mutation, and wherein

if B2c is downstream of the point mutation, then said point mutation is located in the F2c sequence or downstream of the F2c sequence and upstream of the F1c sequence, or

if F2c is upstream of the point mutation, then said point mutation is located in the B2c sequence or upstream of the B2c sequence and downstream of the B1c sequence;

iii. a stem-loop mutant extensible primer comprising;

a central loop sequence able to selectively recognize and hybridize to a region of the target nucleic acid molecule comprising the point mutation, such that the central loop sequence is capable of recognizing and hybridizing to the target nucleic acid molecule only if the point mutation is present, and

a 5′

end sequence and a 3′

end sequence which are complementary to each other such as to form a stem upon intramolecular hybridization,

the hybridization affinity of the central loop sequence to the region of the target nucleic acid molecule comprising the point mutation being higher than the intramolecular hybridization affinity of the 5′

sequence to the 3′

sequence such that, if the point mutation is present, the central loop sequence anneals to and amplifies the region of the target nucleic acid molecule comprising the point mutation; and

iv. a non extensible moiety which is capable of selectively recognizing and hybridizing to the wild type nucleic acid molecules;

3) incubating the resulting mixture at a constant temperature; and

4) detecting a signal indicative of amplification of the region of the target nucleic acid molecule comprising the point mutation.

30. The method of claim 29, wherein the point mutation is located in the region between the F2c sequence and the F1c sequence or between the B2c sequence and the B1c sequence.

31. The method of claim 29, wherein the point mutation is located in the F2c sequence or in the B2c sequence.

32. The method of claim 29, wherein each of the 5′

end sequence and the 3′

end sequence of the stem-loop mutant extensible primer is at least 3 nucleotides in length.

33. The method of claim 29, wherein the non extensible moiety is a Peptide Nucleic Acid (PNA).

34. The method of claim 33, wherein the PNA is at least 10 bases in length.

35. The method of claim 33, wherein the PNA base sequence is such that the melting temperature of the double stranded structure resulting from hybridization of the PNA with the target nucleic acid molecule in the absence of the point mutation (Tm=X) is higher than the incubation temperature and the melting temperature of the double stranded structure resulting from hybridization of the PNA with the target nucleic acid molecule in the presence of the point mutation (Tm=Y) is lower than the incubation temperature, and wherein X is at least 5°

C. higher than Y.

36. The method of claim 29, wherein the non-extensible moiety is a stem-loop wild type non extensible primer, comprising:

a central loop sequence able to selectively recognize and hybrizide to a region of the wild type nucleic acid molecule comprising the nucleic acid position of the point mutation to be detected;

a 5′

end sequence and a 3′

end sequence which are complementary to each other such as to form a stem upon intramolecular hybridization,

the hybridization affinity of the central loop sequence to the region of the wild type nucleic acid molecule comprising the nucleic acid position of the point mutation to be detected being higher than the intramolecular hybridization affinity of the 5′

end sequence to the 3′

end sequence, such that the central loop sequence anneals to the region of the wild type nucleic acid molecule comprising the nucleic acid position of the point mutation to be detected thereby blocking amplification.

37. The method of claim 29, wherein the DNA polymerase is selected from the group consisting of:

Bst large fragment polymerase, Bca (exo-), Vent, Vent (exo-), Deep Vent, Deep Vent (exo-), φ

29 phage, MS-2 phage, Z-Taq, KOD, Klenow fragment and any combination thereof.

38. The method of claim 29, wherein the point mutation to be detected is a g→

t mutation at position 2343 of SEQ ID NO;

1.

39. The method of claim 38, wherein the first outer primer F3 comprises SEQ ID NO:

3, the second outer primer B3 comprises SEQ ID NO;

4, the first inner primer FIP comprises SEQ ID NO;

5, the second inner primer BIP comprises SEQ ID NO;

6 and the stem-loop mutant extensible primer comprises SEQ ID NO;

8.

40. The method of claim 39, further comprising a non-extensible moiety which is a PNA comprising the following base sequence:

41. The method of claim 29, further comprising the step of assessing whether the point mutation is in homozygotic or heterozygotic form, by quantitatively comparing the signal indicative of amplification of the region of the target nucleic acid molecule comprising the point mutation obtained from the sample with the signal obtained from at least one calibrator.

42. The method of claim 41, wherein the at least one calibrator comprises a predetermined percentage (%) of mutant target nucleic acid molecules in a background of wild-type nucleic acid molecules.

43. A set of primers for detecting, by loop mediated isothermal amplification, the presence of a point mutation in a target nucleic acid molecule in a background of wild type nucleic acid molecules, the set of primers comprising:

i. a first outer primer F3 and a second outer primer B3;

ii. a first inner primer FIP and a second inner primer BIP,

wherein FIP comprises a 3′

nucleic acid sequence F2 and a 5′

nucleic acid sequence Flc and BIP comprises a 3′

nucleic acid sequence B2 and a 5′

nucleic acid sequence B1c,

wherein F2 is complementary to a region of the target nucleic acid molecule designated as F2c and B2 is complementary to a region of the target nucleic acid molecule designated as B2c,

wherein F2c and B2c are non overlapping regions located on opposite strands of the target nucleic acid molecule,

wherein either B2c is downstream of the point mutation or F2c is upstream of the point mutation, and wherein

if B2c is downstream of the point mutation, then said point mutation is located in the F2c sequence or between the F2c sequence and the F1c sequence, or

if F2c is upstream of the point mutation, then said point mutation is located in the B2c sequence or between the B2c sequence and the B1c sequence; and

iii. a stem-loop mutant extensible primer comprising;

a central loop sequence complementary to a region of a target nucleic acid molecule comprising the point mutation, and

a 5′

end sequence and a 3′

end sequence which are complementary to each other such as to form a stem upon intramolecular hybridization,

the hybridization affinity of the central loop sequence to the region of the target nucleic acid molecule comprising the point mutation being higher than the intramolecular hybridization affinity of the 5′

sequence to the 3′

sequence.

44. The set of primers of claim 43, wherein each of the 5′

end sequence and the 3′

end sequence of the stem-loop mutant extensible primer is at least 3 nucleotides in length.

45. The set of primers of claim 43, further comprising a non extensible moiety capable of hybridizing to the wild type nucleic acid molecules.

46. The set of primers of claim 45, wherein the non extensible moiety is a Peptide Nucleic Acid (PNA).

47. The set of primers of claim 46, wherein the PNA is at least 10 bases in length.

48. The set of primers of claim 43, wherein the non extensible moiety is a stem-loop non extensible primer, comprising:

a central loop sequence complementary to a region of the wild type nucleic acid molecules comprising the nucleic acid position of the point mutation to be detected;

a 5′

end sequence and a 3′

end sequence which are complementary to each other such as to form a stem upon intramolecular hybridization,

the hybridization affinity of the central loop sequence to the region of the wild type nucleic acid molecules comprising the nucleic acid position of the point mutation to be detected being higher than the intramolecular hybridization affinity of the 5′

end sequence to the 3′

end sequence.

49. The set of primers of claim 43, wherein the first outer primer F3 comprises SEQ ID NO:

3, the second outer primer B3 comprises SEQ ID NO;

4, the first inner primer FIP comprises SEQ ID NO;

5, the second inner primer BIP comprises SEQ ID NO;

6 and the stem-loop mutant extensible primer comprises SEQ ID NO;

8.

50. The set of primers of claim 49, further comprising a non extensible moiety which is a PNA comprising the following base sequence: