Methods for detecting rare polymorphic variants in genomic DNA sequences
AssigneeMassachusetts Institute of Technology
InventorsXiao-Cheng Li-Sucholeiki, Elena Viktorovna Gostjeva, William G. Thilly
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Abstract
Methods for detecting low frequency nuclear mutations in a target sequence from a genomic DNA sequence are disclosed.
Claims25
A method for detecting low frequency mutations in a target sequence from a DNA sample comprising the steps of:
- a) enriching a DNA sample for one or more target sequences, wherein the enrichment step comprises sequence-specific hybridization to the target sequences with one or more labeled probes, wherein each labeled probe is complementary to a specific target sequence, resulting in about a 103-fold to about a 104-fold enrichment of target sequences from the DNA sample thereby obtaining a target-enriched sample;
- and
- b) detecting mutations in the target sequence or sequences from the target-enriched sample.
A method for detecting low frequency nuclear mutations in a target sequence from a genomic DNA sample comprising the steps of:
- a) enriching the DNA sample for molecules comprising one or more target sequences, thereby preparing a target-enriched sample comprising mutant and non-mutant sequences, wherein the enrichment step comprises sequence-specific hybridization with one or more labeled probes that hybridize to the target sequences resulting in about a 103-fold to about 104-fold enrichment of molecules comprising target sequences from the DNA sample;
- b) subjecting the target-enriched sample to constant denaturant capillary electrophoresis using a wide-bore capillary to separate mutant heteroduplexes from non-mutant homoduplexes;
- c) amplifying the heteroduplexes of step b) by high fidelity polymerase chain reaction to obtain amplified polymerase chain reaction products;
- d) subjecting the polymerase chain reaction products of step c) to constant denaturant capillary electrophoresis to further enrich the sample for mutants, thereby creating a mutant-enriched sample;
- e) subjecting the mutant-enriched sample of step d) to constant denaturant capillary electrophoresis to obtain a mutational spectra;
- and
- f) selecting one or more individual mutant fractions from the mutational spectra for sequence analysis to detect mutations.
A method of mutational analysis to detect nuclear gene mutations at mutant fractions at or above 10−6 in a target sequence comprising subjecting a DNA sample comprising one or more target sequences to constant denaturant capillary electrophoresis and high fidelity polymerase chain reaction to obtain a mutational spectrum to detect nuclear gene mutations, wherein, prior to constant denaturant capillary electrophoresis and high fidelity polymerase chain reaction, the DNA sample is enriched for a target sequence and wherein the enrichment comprises two steps wherein the first step comprises a sequence-specific hybridization coupled with a biotin-streptavidin capture system to enrich for DNA molecules comprising the target sequences, and wherein the second step comprises a mutant enrichment using constant denaturant capillary electrophoresis using a wide bore capillary.
Dependencies are inferred from the claim text.