Detection of short homopolymeric repeats
AssigneeBiocartis NV
InventorsBart Claes, Rudi Rossau
Life of this patent
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Abstract
The present application relates to detection of changes in the number of nucleotides in short homopolymeric nucleic acid repeats, in particular in short homopolymeric microsatellites, for example for the purpose of diagnosing microsatellite instability (MSI) and/or mismatch repair (MMR-) deficiency in tumors. Accordingly, methods are provided for detecting changes in the number of nucleotides present in short homopolymeric nucleotide repeat sequences as well as kits and cartridges for automated detection of said changes.
Claims13
A method for deducing the number of nucleotides present in a target homopolymeric repeat sequence equal to or shorter than 15bp in length, the method comprising the steps of:
- a) generating amplicons by amplifying a nucleic acid sequence comprising the target homopolymeric repeat sequence;
- b) heating the generated amplicons in the presence of at least one signal-generating oligonucleotide probe comprising a sequence capable of hybridizing to the target homopolymeric repeat sequence, and detecting the changes in the strength of the signal generated by said probe in the function of temperature to obtain at least one melting curve;
- and
- c) deducing the number of nucleotides present in the target homopolymeric repeat sequence from the at least one melting curve;
- wherein the sequence capable of hybridizing to the target homopolymeric repeat sequence comprises a sequence identical to or complementary to a mutant sequence comprising a deletion of at least one homonucleotide in said target homopolymeric repeat sequence, and
- wherein the step of generating amplicons is performed in a PCR comprising a polymerase having 3′-5′ exonuclease activity.
Dependencies are inferred from the claim text.