Method for identifying one or more mutations in a hotspot mutation sequence
The present invention relates to an in vitro method for identifying and/or characterizing one or more mutations in a hotspot mutation sequence of at least one ESR1 target fragment from a DNA sample, with a drop-off digital polymerase chain reaction (PCR).
1 . An in vitro method for identifying, characterizing and discriminating individual mutations in a hotspot mutation sequence of a first and a second ESR1 target fragment from a DNA sample, said method comprising subjecting the DNA sample to a drop-off digital polymerase chain reaction (PCR) in the presence of a PCR solution comprising:
a pair of primers suitable for amplifying the first ESR1 target fragment;
an oligonucleotide reference (REF) hydrolysis probe, labeled with a fluorophore, wherein said REF oligonucleotide probe is complementary to a wild-type sequence of the first ESR1 target fragment located outside of the hotspot mutation sequence; and
an oligonucleotide hotspot (HOTSPOT) hydrolysis probe, labeled with another fluorophore, wherein said oligonucleotide HOTSPOT probe is complementary to a wild-type sequence of the hotspot mutation sequence of the first ESR1 target fragment, and, wherein the drop-off digital PCR is combined with a mutation-specific digital PCR for identifying a mutation in a hotspot mutation sequence of the second ESR1 target fragment from said DNA sample; and wherein the PCR solution further comprises:
a pair of primers suitable for amplifying the second ESR1 target fragment of the DNA sample;
an oligonucleotide hydrolysis probe (MS-MUT), labeled with a fluorophore, wherein said oligonucleotide MS-MUT probe is complementary to of the hotspot mutation sequence of the second ESR1 target fragment; and
an oligonucleotide reference (MS-REF) hydrolysis probe, labeled with another fluorophore, wherein the MS-REF oligonucleotide probe is complementary to a wild-type sequence of the second ESR1 target fragment located outside of the hotspot mutation sequence of the second ESR1 target fragment.
2 . The method according to claim 1 , wherein the first ESR1 target fragment is from the ligand binding domain of ESR1.
3 . The method according to claim 2 , wherein the first ESR1 target fragment of the DNA sample is exon 8.
4 . The method according to claim 3 , wherein the hotspot mutation sequence in the first ESR1 target fragment includes codons 536-538 of exon 8.
5 . The method according to claim 1 , wherein the second ESR1 target fragment is exon 5.
6 . The method according to claim 5 , wherein the hotspot mutation sequence of the second ESR1 target fragment includes codon 380.
7 . The method according to claim 6 , wherein the hotspot mutation sequence of the second ESR1 target fragment corresponds to E380Q.
8 . The method according to claim 1 , wherein the fluorophore associated with the REF probe is identical to the fluorophore associated with the MS-MUT probe and wherein the fluorophore associated with the HOTSPOT probe is identical to the fluorophore associated with the MS-REF probe.
9 . The method according to claim 1 , wherein the ESR1 target fragments are from genomic tumor DNA.
10 . The method according to claim 1 , wherein the DNA sample is selected from the group consisting of tumor tissue, disseminated cells, feces, blood cells, blood plasma, serum, lymph nodes, urine, saliva, semen, sputum, cerebrospinal fluid, tears, mucus, pancreatic juice, gastric juice, amniotic fluid, and serous fluids.
11 . The method according to claim 1 , wherein the DNA sample is from a subject suffering from advanced breast cancer.
12 . The method according to claim 1 , wherein the DNA sample is from a subject suffering from ER-positive breast cancer after the development of acquired secondary resistance to aromatase inhibitors.
13 . The method according to claim 1 , wherein the nucleotide sequences of the pair of primers suitable for amplifying the first ESR1 target fragment of the DNA sample consist of SEQ ID NO: 3 and SEQ ID NO: 4, the nucleotide sequence of the oligonucleotide reference (REF) hydrolysis probe consists of SEQ ID NO: 7; and the nucleotide sequence of the oligonucleotide hotspot (HOTSPOT) hydrolysis probe consists of SEQ ID NO: 8.
14 . The method according to claim 1 , wherein the nucleotide sequences of the pair of primers suitable for amplifying said second ESR1 target fragment of the DNA sample consist of SEQ ID NO: 1 and SEQ ID NO: 2, the nucleotide sequence of the oligonucleotide hydrolysis probe (MS-MUT) consists of SEQ ID NO: 6; and the nucleotide sequence of the oligonucleotide reference (MS-REF) hydrolysis probe consists of SEQ ID NO: 5.