Target enrichment and amplification
OwnerShoulian Dong, Giulia Kennedy +2inventors
InventorsShoulian Dong, Giulia Kennedy, Linda McAllister, Xing Su
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Abstract
The presently claimed invention provides methods and kits for isolating and amplifying a specific target sequence from within a nucleic acid population. The presently claimed invention provides selection probes which are complementary to at least a portion of said target sequence and mechanisms for isolating the target-probe complexes from the rest of the nucleic acid population.
Claims28
A method of reproducibly obtaining a population of target fragments, wherein each of said target fragments comprises a region of interest, comprising:
- fragmenting a nucleic acid population to produce fragments;
- exposing said fragments to a plurality of selection probes to form a plurality of target-probe complexes, each of said selection probes comprising a separation element and a region which is complementary to said region of interest;
- separating said target-probe complexes from any non-hybridized fragments via said separation element;
- and
- denaturing said target-probe complexes to produce separated target fragments and selection probes.
A method of analyzing a nucleic acid sample comprising;
- fragmenting said nucleic acid population to produce fragments,
- exposing said fragments to selection probes to form a plurality of probe-target complexes, each of said selection probes comprising a separation element and a region which is complementary to said region of interest;
- separating said probe-target complexes from any non-hybridized fragments via said separation element;
- denaturing said probe-target complexes to produce target fragments and selection probes;
- amplifying said target fragments;
- and
- hybridizing said amplified target fragments to a nucleic acid array.
A kit for reproducibly obtaining a population of target fragments wherein each of said target fragments comprises a region of interest comprising;
- a population of cells comprising cloning vectors wherein said cloning vectors comprise nucleic acid insert sequences which are complementary to at least a part of said region of interest;
- a plurality of separation elements which are capable of being attached to fragments of said cloning vectors.
A method of genotyping an individual comprising:
- obtaining a sample of genomic DNA from said individual;
- fragmenting said sample to form genomic fragments;
- exposing said genomic fragments to selection probes, wherein said selection probes comprise a separation element and a region which is complementary to a region of DNA which is known or believed to be adjacent to a polymorphic site, under suitable conditions to allow for hybridization between said selection probes and said genomic fragments to form a plurality of probe-target complexes;
- separating said probe-target complexes from any non-hybridized fragments via said separation element;
- denaturing said probe-target complexes to produce target fragments and selection probes;
- amplifying said target fragments;
- hybridizing said amplified target fragments to an array of probes immobilized to a solid support at known locations, wherein said probes are able to interrogate said polymorphic site;
- and
- detecting the nucleic acid base at said polymorphic site thus genotyping said individual.
An isolated fragment comprising a region of interest obtained by:
- fragmenting a nucleic acid population of interest thereby producing fragments;
- exposing said fragments to a selection probe to form a probe-target complex, said selection probe comprising a separation element and a region which is complementary to said region of interest;
- separating said probe-target complex from any non-hybridized fragments via said separation element;
- denaturing said probe-target complex to produce a target fragment and selection probe;
- and
- isolating said target fragment from said selection probe.
A method for reproducibly obtaining target sequences for further analysis comprising:
- fragmenting a nucleic acid sample to produce nucleic acid fragments;
- exposing said fragments to a plurality of selection probes under suitable condition so as to produce hybridization between complementary sequences, each said plurality of selection probes being designed to be complementary to at least a portion of one of said target sequences, said plurality of selection probes being immobilized to a solid support;
- removing any non-hybridized fragments;
- and
- eluting said hybridized fragments to produce a pool of target sequences.
An apparatus for reproducibly obtaining a population of target fragments comprising an array of selection probes immobilized to a solid support, wherein each of said selection probes is designed to be complementary to at least a portion of one of said target fragments.
Dependencies are inferred from the claim text.