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AssigneeCancer Research Technology Limited
InventorsJohn Burn, Mohammed Ghanim Mehdi Alhilal, Franscisco Mauro Santibanez-Koref, Lisa Redford, Michael Stewart Jackson
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The present invention relates to methods and kits for identifying microsatellite instability (MSI) in a sample. In particular it relates to identifying microsatellite instability in a tumor sample, which may be from a subject suspected of having colorectal cancer or Lynch syndrome. The methods and kits can be used to identify mismatch repair defects. More particularly the invention relates to a panel of markers for a sequencing based MSI test, that can differentiate between MSI-H and MSS CRCs. The invention also allows for determination of biological significance, differentiating between PCR and sequencing errors and MSI induced indels/mutations.
A method for evaluating levels of microsatellite instability in a sample comprising:
A method as in any of the previous claims wherein, in step (a) primers are also provided for amplifying the microsatellite loci DEPDC2 and AP003532_2.
A method as in any of the previous claims wherein, in step (a) primers are provided for amplifying at least 10 of the selected group of microsatellite loci of human genomic DNA of Table A and/or Table B or at least 17 of the selected group of microsatellite loci of human genomic DNA of Table A and/or Table B or at least 18 of the selected group of microsatellite loci of human genomic DNA of Table A and/or Table B.
A method as in any of the previous claims wherein step (a) is providing primers for amplifying at least 10 of the selected group of microsatellite loci of human genomic DNA comprising;
A method as in any of the previous claims wherein step (a) is providing primers for amplifying at least 10 of the selected group of microsatellite loci of human genomic DNA comprising;
A method as in any of the previous claims wherein step (a) is providing primers for amplifying the selected group of microsatellite loci of human genomic DNA comprising;
A method as in any of the previous claims wherein step (a) is providing primers for amplifying at least 10 of the selected group of microsatellite loci of human genomic DNA comprising;
A method as in any of the previous claims wherein step (a) is providing primers for amplifying the selected group of microsatellite loci of human genomic DNA comprising;
A method as in any of the previous claims wherein the primers are for co-amplifying the selected microsatellite loci.
A method as in any of the previous claims wherein the step of (b) amplifying from the sample the plurality of selected microsatellite mono-nucleotide repeat loci to give microsatellite amplicons, comprises co-amplifying the set of selected loci in a multiplex amplification reaction.
A method as in any of the previous claims wherein the primer pairs are selected to allow for multiplexing.
A method as in any of the previous claims wherein a number of separate microsatellite mono-nucleotide repeat loci are amplified and sequenced as separate reactions.
A method as in any of the previous claims wherein the step of (c) uses high throughput or next generation sequencing.
A method as in any of the previous claims wherein step (c) uses sequencing-by-synthesis.
A method as in any of the previous claims wherein step (c) uses ion semiconductor sequencing or ion torrent sequencing.
A method as in any of the previous claims wherein step (c) uses pyrosequencing.
A method as in any of the previous claims wherein amplification is by polymerase chain reaction and uses using primer pairs, each primer pair comprising a forward primer which is complimentary to a portion upstream from a selected microsatellite mono-nucleotide repeat loci, and a reverse primer which is complimentary to a portion downstream from said selected microsatellite mono-nucleotide repeat loci.
A method as in any of the previous claims wherein the selected group of microsatellite loci also include a single nucleotide polymorphism (SNP) within a short distance of the microsatellite loci.
A method as in any of the previous claims wherein the selected group of microsatellite loci also include a single nucleotide polymorphism (SNP) within 100 base pairs of the microsatellite loci.
A method as in any of the previous claims wherein the selected group of microsatellite loci also include a single nucleotide polymorphism (SNP) within 80 base pairs of the microsatellite loci.
A method as in any of the previous claims wherein the selected group of microsatellite loci also include a single nucleotide polymorphism (SNP) within 50 base pairs of the microsatellite loci.
A method as in any of the previous claims wherein the selected group of microsatellite loci also include a single nucleotide polymorphism (SNP) within 30 base pairs of the microsatellite loci.
A method as in any of the previous claims wherein the single nucleotide polymorphism (SNP) has a minor allele frequency between 0.05-0.95.
A method as in any of the previous claims wherein the single nucleotide polymorphism (SNP) has a high frequency.
A method as in any of the previous claims wherein the primers are selected to amplify both the microsatellite loci and the SNP.
A method as in any of the previous claims wherein amplification step (b) gives microsatellite amplicons including both microsatellite and SNP.
A method as in any of the previous claims wherein the method includes the step of determining allelic imbalance.
A method as in any of the previous claims wherein the method includes the step of diagnosing MSI induced CRC or Lynch syndrome.
A method as in any of the previous claims wherein deviation from the predetermined sequences for two or more microsatellite mono-nucleotide repeat loci is indicative of a mismatch repair defect.
A method as in any of the previous claims wherein deviation from the predetermined sequences for two or more microsatellite mono-nucleotide repeat loci is indicative of the sample having high levels of microsatellite instability (MSI-H).
A method as in any of the previous claims wherein the sample is a tumor sample or a body tissue or fluid suitable for detecting tumor cells.
A method as in any of the previous claims wherein the sample is a human tissue or fluid sample.
A method as in any of the previous claims wherein the sample is a nucleic acid sample.
A kit for evaluating levels of microsatellite instability in a sample, comprising:
A kit as in any of 35 to 42 wherein the primers are for amplifying the selected group of microsatellite loci of human genomic DNA comprising;
A method for evaluating the biological significance of mutations identified during sequencing:
Dependencies are inferred from the claim text.