Methods for determining sequences information in polynucleotides using mass spectrometry
First Claim
1. A method for identifying a putative point mutation in a polynucleotide using mass spectrometry, comprising the steps of:
- a. providing a primer having a sequence that hybridizes adjacent said putative point mutation of said single strand of said polynucleotide;
b. heating said polynucleotide and said primer to a temperature sufficient to melt said polynucleotide; and
c. cooling said melted polynucleotide and said primer to a temperature sufficient to hybridize said primer to said single strand of said polynucleotide, thereby forming a duplex wherein the 3'"'"'-end of said primer is hybridized adjacent the putative point mutation of said single strand of said polynucleotide;
d. extending the 3'"'"'-end of said primer by adding a single dideoxynucleotide base complementary to the putative point mutation of the polynucleotide, wherein said dideoxynucleotide base has a mass range of about 273 to about 313,using a polynucleotide thermostable polymerase having substantially the same relative rate of incorporation of dideoxynucleotide bases as deoxynucleotide bases, thereby forming an extended primer; and
e. analyzing the extended primer using mass spectrometry to determine the identity of the dideoxynucleotide base at the putative point mutationwherein steps a-d are repeated a sufficient number of times to permit detection of said extended primer by mass spectrometry.
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Abstract
The invention relates to methods for determining sequence information in polynucleotides by combining the recent disparate technologies of mass spectrometry and polynucleotide hybridization, amplification, extension and/or ligation techniques. Broadly, in a first step, the method for determining sequence information in a sample polynucleotide includes hybridizing with a sample nucleotide one or a mixture of oligonucleotide probes having a nucleotide sequence complementary to a portion of the sample polynucleotide, thereby forming a complex. Then, in a second step, the complex is contacted with at least a member selected from the group consisting of nucleosides, dideoxynucleosides, polymerases, nucleases, transcriptases, ligases and restriction enzymes to alter at least a subset of said oligonucleotide probes. In a third step, the method provides for determining the molecular weight of at least the subset of altered probes by mass spectrometry and infering the sequence information of the sample polynucleotide therefrom.
369 Citations
12 Claims
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1. A method for identifying a putative point mutation in a polynucleotide using mass spectrometry, comprising the steps of:
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a. providing a primer having a sequence that hybridizes adjacent said putative point mutation of said single strand of said polynucleotide; b. heating said polynucleotide and said primer to a temperature sufficient to melt said polynucleotide; and c. cooling said melted polynucleotide and said primer to a temperature sufficient to hybridize said primer to said single strand of said polynucleotide, thereby forming a duplex wherein the 3'"'"'-end of said primer is hybridized adjacent the putative point mutation of said single strand of said polynucleotide; d. extending the 3'"'"'-end of said primer by adding a single dideoxynucleotide base complementary to the putative point mutation of the polynucleotide, wherein said dideoxynucleotide base has a mass range of about 273 to about 313, using a polynucleotide thermostable polymerase having substantially the same relative rate of incorporation of dideoxynucleotide bases as deoxynucleotide bases, thereby forming an extended primer; and e. analyzing the extended primer using mass spectrometry to determine the identity of the dideoxynucleotide base at the putative point mutation wherein steps a-d are repeated a sufficient number of times to permit detection of said extended primer by mass spectrometry. - View Dependent Claims (4, 5, 6)
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2. A method for identifying a putative point mutation in a polynucleotide using mass spectrometry, comprising the steps of:
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a. hybridizing a primer comprising a mass tag to a single strand of said polynucleotide having a putative point mutation, the 3'"'"'-end of said primer being hybridized adjacent the putative point mutation; b. extending the 3'"'"'-end of said primer by adding a single dideoxynucleotide base complementary to the putative point mutation of the polynucleotide, wherein said dideoxynucleotide base has a mass range of about 273 to about 313, using a polynucleotide thermostable polymerase having substantially the same relative rate of incorporation of dideoxynucleotide bases as deoxynucleotide bases, thereby forming an extended primer; and c. analyzing the extended primer using mass spectrometry to determine the identity of the dideoxynucleotide base at the putative point mutation, wherein steps a and b are repeated a sufficient number of times to permit detection of said extended primer. - View Dependent Claims (3)
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7. A multiplex method for identifying a plurality of putative point mutations in at least one polynucleotide using mass spectrometry, comprising the steps of:
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a. providing a plurality of pairs each having a sequence that hybridizes adjacent a respective putative point mutation of said at least one polynucleotide; b. heating said at least one polynucleotide and said plurality of primers to a temperature sufficient to melt said at least one polynucleotide; and c. cooling said at least one melted polynucleotide and said plurality of primers to a temperature sufficient to hybridize the plurality of primers to said at least one single strand of said at least one polynucleotide, thereby forming a multiplex wherein the 3'"'"'-end of each one of said plurality of primers is hybridized adjacent a respective one of said plurality of putative point mutations; d. extending the 3'"'"'-end of said plurality of primers by adding one of a plurality of dideoxynucleotide bases to said plurality of hybridized primers, wherein said dideoxynucleotide base has a mass range of about 273 to about 313, using a polynucleotide thermostable polymerase having substantially the same relative rate of incorporation of dideoxynucleotide bases as deoxynucleotide bases, whereby one of said plurality of said dideoxynucleotide bases complementary to said respective one of said plurality of putative point mutations is added to the respective one of said plurality of hybridized primers, thereby forming a plurality of extended primers each having one added dideoxynucleotide base; and e. analyzing the plurality of extended primers using mass spectrometry to determine the identity of the dideoxycleotide base at each putative point mutation wherein steps a-d are repeated a sufficient number of times to permit detection of said extended primers by mass spectrometry. - View Dependent Claims (10, 11, 12)
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8. A multiplex method for identifying a plurality of putative point mutations in at least one polynucleotide using mass spectrometry, comprising the steps of:
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a. hybridizing a plurality of primers, each having a 3'"'"'-end and a 5'"'"'-end, to at least one single strand of said at least one polynucleotide having said plurality of putative point mutations, the 3'"'"'-end of each of said plurality of primers being hybridized adjacent a respective one of said plurality of putative point mutations; b. extending the 3'"'"'-end of said plurality of primers by adding one of a plurality of dideoxynucleotide bases to said plurality of hybridized primers, wherein said dideoxynucleotide base has a mass range of about 273 to about 313, using a polynucleotide thermostable polymerase having substantially the same relative rate of incorporation of dideoxynucleotide bases as deoxynucleotide bases, whereby one of said plurality of dideoxynucleotide bases complementary to said respective one of said plurality of putative point mutations is added to the respective one of said plurality of hybridized primers, thereby forming a plurality of extended primers each having one added dideoxynucleotide base; and c. analyzing the plurality of extended primers using mass spectrometry to determine the identity of the dideoxynucleotide base at each putative point mutation wherein steps a and b are repeated a sufficient number of times to permit detection of said extended primers by mass spectrometry and at least one primer comprises a mass tag. - View Dependent Claims (9)
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Specification