Method for detecting a mutation in a microsatellite sequence

Patent metadata

Field	Value
Publication number	US-12371741-B2
Application number	US-202418434179-A
Country	US
Kind code	B2
Filing date	Feb 6, 2024
Priority date	Jul 12, 2017
Publication date	Jul 29, 2025
Grant date	Jul 29, 2025

Abstract

The present disclosure relates to a method for detecting a mutation in a microsatellite sequence locus of a target fragment from a DNA sample, comprising a step of subjecting said DNA sample to a digital polymerase chain reaction (PCR) in the presence of a PCR solution comprising:
a pair of primers suitable for amplifying said target fragment of the DNA sample including said microsatellite sequence; a first MS oligonucleotide (MS) hydrolysis probe, labeled with a first fluorophore, wherein said first MS oligonucleotide probe is complementary to a wild-type sequence including the microsatellite sequence; a second oligonucleotide reference (REF) hydrolysis probe, labeled with a second fluorophore, wherein said second oligonucleotide REF probe is complementary to a wild-type sequence of said target DNA fragment which does not include said microsatellite sequence.

First claim

The invention claimed is:

1. A method for detecting a mutation in a microsatellite sequence locus of a target fragment from a DNA sample, comprising a step of subjecting said DNA sample to a digital polymerase chain reaction (dPCR) in the presence of a PCR solution comprising:
a pair of primers suitable for amplifying said target fragment of the DNA sample including said microsatellite sequence;
a first MS oligonucleotide (MS) hydrolysis probe, labeled with a first fluorophore, wherein said first MS hydrolysis probe is complementary to a wild-type sequence including the microsatellite sequence, wherein said MS hydrolysis probe is designed to confer its ability to bind properly to the wild-type microsatellite sequence while preventing hybridization in the presence of a mutation in the microsatellite sequence;
and wherein the MS hydrolysis probe covers the full wild-type microsatellite sequence and extends further between 1 to 10 nucleotides on each extremity; and
a second reference oligonucleotide hydrolysis probe, labeled with a second fluorophore, wherein said second reference oligonucleotide hydrolysis probe is complementary to a wild-type sequence of said target DNA fragment located outside of said microsatellite sequence and

wherein said hydrolysis probes have a fluorophore covalently attached to their 5′-end of the oligonucleotide probe and a quencher, and said method further comprises a step of measuring the fluorescence signals associated with the reference oligonucleotide hydrolysis and MS probes, wherein the maximal fluorescence intensity signal associated with both the reference oligonucleotide hydrolysis and MS probes indicates the presence of a wild-type microsatellite sequence in the target DNA fragment, while a shift in the fluorescence intensity signal associated with the MS probe indicates the presence of a mutation in the microsatellite sequence of the target DNA fragment.

2. The method according to claim 1 , wherein the target fragment of the DNA sample is constitutional genomic DNA.

3. The method according to claim 1 , wherein the target fragment of the DNA sample is genomic tumor DNA.

4. The method according to claim 1 , wherein the microsatellite sequence locus is selected from the group consisting of: BAT-25, BAT-26, BAT-34c4, BAT-40, NR21, NR24, MONO-27, D2S123, D5S346, D17S250, ACVR2A, DEFB105A, DEFB105B, RNF43, DOCK3, GTF2IP1, LOC100093631, PIP5K1A, MSH3, TRIM43B, PPFIA1 and TDRD1.

5. 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, stool, sputum, cerebrospinal fluid, tears, mucus, pancreatic juice, gastric juice, amniotic fluid, cerebrospinal fluid, and serous fluids.

6. The method according to claim 1 , wherein the target fragment is originating from a tumor in a patient with cancer, a disease associated with mismatch repair (MMR) genes or familial tumor predisposition.

7. A method for prognosis of cancers comprising the detection of a mutation in a microsatellite sequence locus of a target fragment from a DNA sample according to claim 1 , wherein the target fragment is originating from a tumor.

8. A method for predicting the efficacy of a treatment in a subject suffering from a cancer, comprising the detection of a mutation in a microsatellite sequence locus of a target fragment from a DNA sample according to claim 1 , wherein the target fragment is originating from a tumor and wherein the treatment is optionally immune therapy optionally immune checkpoint therapy.

9. The method according to claim 1 , wherein the target fragment of the DNA sample originates from a tumor in a patient diagnosed with a tumor associated with impaired DNA mismatch repair.

Assignees

• Inst Curie
• Inst Nat Sante Rech Med
• Univ Versailles Saint Quentin En Yvelines

Inventors

• Proudhon Charlotte
• Kasperek Amélie
• Bortolini Silveira Amanda
• Bidard François-Clément
• Stern Marc-Henri

Classifications

• G01N2021/6439

  with indicators, stains, dyes, tags, labels, marks · CPC title

• G01N21/6428

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q1/6886

  for cancer (immunoassay for cancer G01N33/575) · CPC title

• C12Q1/6858 Primary

  Allele-specific amplification · CPC title