Method for identifying one or more mutations in a hotspot mutation sequence

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.