Diagnostic sequencing by a combination of specific cleavage and mass spectrometry
First Claim
1. A method for sequence analysis of one or more target nucleic acids for which a reference nucleic acid sequence is known, the method comprising the steps of:
- (a) deriving from one or more biological samples the one or more target nucleic acids;
(b) subjecting the one or more target nucleic acid to complementary cleavage reactions with one or more cleavage reagents thereby generating cleavage products;
(c) performing mass spectroscopical analysis on the cleavage products generated in step (b) to obtain one or more mass spectra; and
(d) comparing the one or more mass spectra of the cleavage products of the one or more target nucleic acids obtained in step c) with a known or predicted mass spectra for the reference nucleic acid sequence, and deducing therefrom, by systematic computational analysis, all or part of the nucleotide sequence of the one or more target nucleic acids, and comparing the deduced nucleic acid sequence with the reference nucleic acid to determine whether the one or more target nucleic acids have the same sequence or a different sequence from the reference nucleic acid.
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Accused Products
Abstract
The present invention is in the field of nucleic acid-based diagnostic assays. More particularly, it relates to methods useful for the “diagnostic sequencing” of regions of sample nucleic acids for which a prototypic or reference sequence is already available (also referred to as “re-sequencing”), or which may be determined using the methods described herein. This diagnostic technology is useful in areas that require such re-sequencing in a rapid and reliable way: (i) the identification of the various allelic sequences of a certain region/gene, (ii) the scoring of disease-associated mutations. (iii) the detection of somatic variations, (iv) studies in the field of molecular evolution, (v) the determination of the nucleic acid sequences of prokaryotic and eukaryotic genomes; (vi) identifying one or more nucleic acids in one or more biological samples; (vii) and determining the expression profile of genes in a biological sample and other areas.
55 Citations
56 Claims
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1. A method for sequence analysis of one or more target nucleic acids for which a reference nucleic acid sequence is known, the method comprising the steps of:
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(a) deriving from one or more biological samples the one or more target nucleic acids;
(b) subjecting the one or more target nucleic acid to complementary cleavage reactions with one or more cleavage reagents thereby generating cleavage products;
(c) performing mass spectroscopical analysis on the cleavage products generated in step (b) to obtain one or more mass spectra; and
(d) comparing the one or more mass spectra of the cleavage products of the one or more target nucleic acids obtained in step c) with a known or predicted mass spectra for the reference nucleic acid sequence, and deducing therefrom, by systematic computational analysis, all or part of the nucleotide sequence of the one or more target nucleic acids, and comparing the deduced nucleic acid sequence with the reference nucleic acid to determine whether the one or more target nucleic acids have the same sequence or a different sequence from the reference nucleic acid. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
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26. A method for scoring known nucleotide sequence variations of one or more target nucleic acids for which a known reference nucleic acid sequence is available, the method comprising the steps of:
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(a) deriving from one or more biological samples the one or more target nucleic acids;
(b) subjecting the one or more target nucleic acids to complementary cleavage reactions with one or more cleavage reagents thereby generating cleavage products;
(c) performing mass spectroscopical analysis on the cleavage products obtained in step (b) to obtain two or more mass spectra; and
(d) comparing the one or more mass spectra of the cleavage products of the one or more target nucleic acids obtained in step c) with the known or predicted mass spectra for the reference nucleic acid sequence, and scoring therefrom, by systematic computational analysis the presence or absence of the known nucleotide sequence variations of the one or more target nucleic acids. - View Dependent Claims (27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 50)
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49. The method of claim or claim wherein the one or more target nucleic acids are phosphorothioate- modified single-stranded DNA or RNA and the endonuclease is nuclease P1.
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51. A method for determining the sequence of one or more target nucleic acids, the method comprising the steps of:
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(a) deriving from a biological sample material the one or more target nucleic acids;
(b) subjecting the one or more target nucleic acids to complementary cleavage reactions with one or more cleavage reagents thereby generating cleavage products;
(c) performing mass spectroscopical analysis on the cleavage products obtained in step (b) to obtain a mass spectra of the nucleic acid fragments; and
(d) deducing therefrom the nucleotide sequence of the one or more target nucleic acids by systematic computational analysis on the mass spectra of the cleavage products obtained in step (c).
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52. A method for genome wide genotyping of one or more known or unknown target nucleic acids, the method comprising the steps of:
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(a) deriving from one or more biological samples the one or more target nucleic acids;
(b) subjecting the one or more target nucleic acids to one or more specific cleavage reactions with one or more cleavage reagents thereby generating cleavage products;
(c) performing mass spectroscopical analysis on the cleavage products obtained in step (b) to obtain one or more mass spectra; and
(d) comparing the mass spectra of the cleavage products obtained in step c) with reference mass spectra, and diagnosing therefrom genetically relevant nucleic acid sequence variations of the one or more known or unknown target nucleic acids.
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53. A method for identifying one or more target nucleic acids in one or more biological samples, the method comprising the steps of:
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(a) deriving from one or more biological samples the one or more target nucleic acids;
(b) subjecting the one or more target nucleic acids of step (a) to one or more specific cleavage reactions with one or more cleavage reagents thereby generating cleavage products;
(c) performing mass spectroscopical analysis on the cleavage products obtained in step (d) to obtain one or more mass spectra; and
(d) comparing the one or more mass spectra obtained in step c) with each other or with a plurality of mass spectra of reference nucleic acids, and deducing therefrom the identity of the one or more target nucleic acids. - View Dependent Claims (54, 55)
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56. A kit for sequence analysis of one or more target nucleic acids using mass spectroscopy, the kit comprising:
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(a) one or more sets of reference nucleic acids for which the nucleic acid sequence is known;
(b) one or more nucleotide triphosphates;
(c) one or more polymerases;
(d) one or more nucleic acid cleaving agents; and
(e) computer software for comparing the mass spectra of the one or more target nucleic acid with the mass spectra of the reference nucleic acid and deducing therefrom the nucleic acid sequence of the target nucleic acid.
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Specification