Diagnostic sequencing by a combination of specific cleavage and mass spectrometry

a mass spectrometry and specific cleavage technology, applied in the direction of microorganism testing/measurement, biochemistry apparatus and processes, etc., can solve the problems of inability to apply methods to diagnostic sequencing of nucleic acids, poor applicability of direct and indirect ms methodologies under current performance conditions, and inability to extend chain lengths beyond .about 30-50 nucleotides, etc., to achieve rapid and reliable results

Inactive Publication Date: 2006-11-09
AGENA BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0014] The present invention is directed to a mass spectroscopic method for detecting or analyzing a particular nucleic acid sequence. The present invention is useful for de novo sequencing or re-sequencing nucleic acid in a rapid and reliable way which permits, for example, the identification of the various allelic sequences of a certain region / gene, the identification and scoring of disease-associated mutations, the detection of somatic variations, determining genetic diversity in molecular evolution, and the determination of the genomic sequences e.g., of viral and bacterial isolates. The present invention is also useful for identification of all nucleic acid molecules in one or more biological samples including for expression profiling i.e., identification of all the mRNA species that are expressed in a given cell at a given time, by rapidly determining the sequence of the mRNA that is expressed.

Problems solved by technology

A parallel challenge is to characterize the type and extent of variation in the sequences of interest because it underlies the heritable differences among individuals and populations.
A severe limitation of both the direct and indirect MS methodologies under the current performance conditions is the poor applicability to chain lengths beyond .about.30-50 nucleotides.
The latter MS analyses are however considerably less informative in that they are essentially restricted to the detection of sequence variations.
The methods cannot be applied to diagnostic sequencing of nucleic acids, where the term diagnostic sequencing means the unequivocal determination of the presence, the nature and the position of sequence variations.
While most methods in the art do yield sequence-related information, they do not disclose that a combination of several different mass spectra, obtained after complementary digestion reactions, allows for the effective survey of a nucleic acid region and provides an unambiguous assignment of both known as well as previously unknown sequence variations that occur relative to a reference nucleic acid with a known nucleotide sequence.

Method used

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  • Diagnostic sequencing by a combination of specific cleavage and mass spectrometry
  • Diagnostic sequencing by a combination of specific cleavage and mass spectrometry
  • Diagnostic sequencing by a combination of specific cleavage and mass spectrometry

Examples

Experimental program
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example 4

[0106] Example 4 illustrates the analysis of a .about.1000 base-pair nucleic acid.

[0107] Example 5 illustrates the use of the present invention for genotyping, including multiplex genotyping.

[0108] Example 6 illustrates the use of the present invention for transcription profiling.

[0109] Example 7 illustrates the use of the present invention for whole genome resequencing.

example 1

Modeling the Diagnostic Sequence Analysis of a 1200 Base-Pair Region of HIV-1

[0110] The methods of the present invention have been utilized on a 1200 base-pair sequence derived from human immunodeficiency virus type 1 (HIV-1; HXB2 isolate; Genbank accession number K03455; position 2161 to 3360). This sequence was used as a model in computer simulations to examine the overall performance of the method, as well as the occurrence of ambiguities. The selected region encompasses the entire protease gene and the first .about.270 codons of reverse transcriptase [compare with Hertogs K. et al., Antimicrob. Agents Chemother. 42: 269-276 (1998)]. The genotyping / re-sequencing of this domain of clinical isolates of HIV is of special interest in order to monitor the emergence of drug resistance-associated mutations. Single as well as multiple changes have been implicated in the decreased sensitivity towards the antiviral activity of protease and RT inhibitors [Hertogs K. et al., Antimicrob. Agen...

example 2

Base-Specific Cleavage by Modification of the Template

[0116] The present example illustrates that the specificity of cleavage by a nucleolytic reagent may be further confined through the modification of the target template such that particular phosphodiester bonds resist cleavage. More particularly, it is demonstrated that RNase-A, which normally cleaves at the 3'-side of both C- and U-residues, becomes mononucleotide-specific when the target incorporates the 2'-deoxy analog of one of these nucleotides. A region of the plasmid vector pGEM3-Zf(+) (Promega, Madison, Wis.), encompassing the multi-cloning site as well as the phage T7 promoter sequences, was used as a model (see FIG. 5).

[0117] The first step towards the sequence analysis according to the present invention involved the amplification of the 158 base-pair test sequence. The reaction was carried out in a total volume of 50 1 using 12.5 pmol each of the forward and reverse primer, 200 M of each dNTP, 0.25 1 Taq DNA polymerase...

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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.

Description

[0001] 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.BACKGROUND OF INVENTION[0002] Comp...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68
CPCC12Q1/6872C12Q1/6855
Inventor ZABEAU, MARCSTANSSENS, PATRICK
Owner AGENA BIOSCI
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