High-throughput screening method for identification of genetic mutations or disease-causing microorganisms using segmented primers
First Claim
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1. A method for screening genomic nucleic acid samples to identify a nucleic acid mutation comprising the steps of:
- (i) isolating a plurality of genomic nucleic acids;
(ii) exposing said nucleic acids to one or more segmented primers, each comprising a first primer having a length between 5 and 8 nucleotides, and a second primer having a length between 15 and 100 nucleotides and a 3'"'"' non-extendible nucleic acid, wherein said first and second primers hybridize to regions of a target nucleic acid that are separated by no more than one nucleotide on said target and wherein said second primer binds upstream of said first primer;
(iii) conducting a template-based nucleic acid extension reaction, thereby to add a single terminal nucleotide to one or more of said first probes;
(iv) isolating extended first probes; and
(v) determining the sequence of said extended first probes;
thereby to detect the presence or absence of a genetic alteration.
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Abstract
Methods are provided for high-throughput screening for the presence of genetic alterations and disease-causing microorganisms in a biological sample. These methods are particularly useful for identifying individuals with gene mutations indicative of early colorectal cancer.
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Citations
15 Claims
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1. A method for screening genomic nucleic acid samples to identify a nucleic acid mutation comprising the steps of:
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(i) isolating a plurality of genomic nucleic acids; (ii) exposing said nucleic acids to one or more segmented primers, each comprising a first primer having a length between 5 and 8 nucleotides, and a second primer having a length between 15 and 100 nucleotides and a 3'"'"' non-extendible nucleic acid, wherein said first and second primers hybridize to regions of a target nucleic acid that are separated by no more than one nucleotide on said target and wherein said second primer binds upstream of said first primer; (iii) conducting a template-based nucleic acid extension reaction, thereby to add a single terminal nucleotide to one or more of said first probes; (iv) isolating extended first probes; and (v) determining the sequence of said extended first probes;
thereby to detect the presence or absence of a genetic alteration. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A method for screening genomic nucleic acid samples to identify a mutation comprising the steps of:
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(i) immobilizing a plurality of genomic nucleic acids on a solid phase support; (ii) exposing said immobilized nucleic acids to one or more segmented primers, each comprising a first primer having a length between 5 and 8 nucleotides and a second primer having a length between 15 and 100 nucleotides and a 3'"'"' non-extendible nucleic acid, wherein said first and second primers hybridize to regions of a target nucleic acid that are separated by no more than one nucleotide on said target and wherein said second primer binds upstream of said first primer; (iii) conducting a template-based nucleic acid extension reaction, thereby to add a single terminal nucleotide to one or more of said first probes; (iv) isolating extended first probes; and (v) determining the sequence of said extended first probes;
thereby to detect the presence or absence of a genetic alteration. - View Dependent Claims (11, 12)
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13. A method for high-throughput screening of genomic nucleic acid samples to identify one or more genetic alterations in one or more target nucleic acid sequences present in said samples, comprising the steps of:
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(i) isolating a plurality of genomic nucleic acids; (ii) exposing said nucleic acids to an excess amount of one or more segmented primers, each comprising a first primer having a length between 5 and 8 nucleotides, and a second primer having a length between 15 and 100 nucleotides and a 3'"'"' non-extendible nucleic acid, wherein said first and second primers hybridize to regions of a target nucleic acid that are separated by no more than one nucleotide on said target and wherein said second primer binds upstream of said first primer; (iii) conducting a template-based nucleic acid extension reaction in the presence of a heat-stable polymerase and an excess amount of labeled non-wild-type nucleotides, thereby to add a single labeled terminal nucleotide to one or more of said first probes; (iv) heating the extension reaction mixture of step (iii), thereby to dissociate said first probes and said second probes from said target nucleic acids; (v) cooling the extension reaction mixture of step (iv), thereby to expose said target nucleic acids to said excess amount of segmented primers; (vi) repeating step (iii); (vii) isolating extended labeled first probes; and (viii) determining the sequence of said extended labeled first probes;
thereby to detect the presence or absence of a genetic alteration. - View Dependent Claims (14, 15)
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Specification