Method and reagents for analyzing the nucleotide sequence of nucleic acids

• US 7,399,844 B2
• Filed: 04/17/2001
• Issued: 07/15/2008
• Est. Priority Date: 07/09/1998
• Status: Expired due to Fees

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Sampson, Jeffrey R., Yakhini, Zohar H., Tsalenko, Anna M., Sampas, Nicholas M., Myerson, Joel, Webb, Peter G.
• Primary Examiner(s): Chunduru; Suryaprabha
• Application Number:
  US09/836,012
• Publication Number:
  US 20020182601A1
• Time in Patent Office: 2,646 Days
• Field of Search: 435/6, 435/7.1, 435/91.1, 435/91.2, 435/267.1, 536/22.1, 536/23.1, 536 243- 2433
• US Class Current: 536/22.1
• CPC Class Codes: B01J 2219/00452 Means for the recovery of reactants or products
• CPC Class Codes: B01J 2219/00454 by chemical cleavage from the solid support
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00581 Mass
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00599 Solution-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00641 the porous medium being continuous, e.g. porous oxide substrates
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00707 separated from the reactor apparatus
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2533/107 : Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2537/143 : Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2600/156 : Polymorphic or mutational markers
• CPC Class Codes: C40B 40/00 : Libraries per se, e.g. arrays, mixtures

Abstract

Methods and reagents are disclosed which provide for more sensitive, more accurate and higher through-put analyses of target nucleic acid sequences. The methods and reagents of the present invention may be generically applied to generally any target nucleic acid sequence and do not require a priori information about the presence, location or identity of mutations in the target nucleic acid sequence. The reagents of the invention are mixtures of oligonucleotide precursors having a high level of coverage and mass number complexity, and also having tags analyzable by mass spectrometry which are covalently linked to the precursors through cleavable bonds. A method is also disclosed for analyzing a target nucleic acid sequence employing the mixtures of oligonucleotide precursors having tags analyzable by mass spectrometry covalently linked to the oligonucleotide precursors through cleavable bonds, and chemical or enzymatic assays to alter the mass of the oligonucleotide precursors prior to mass spectral analysis. The enzymatic assay may be a polymerase extension assay or a ligation-based assay. The kits for carrying out the methods of the invention are also disclosed.

Claims

1. A mixture or set of sub-mixtures comprising X-mer precursors of different length,

wherein the X-mer precursors have a minimum length of 3 nucleotides;

wherein the mixture has a minimum mixture coverage complexity of at least 56/N or wherein the set of sub-mixtures has a composite mixture coverage complexity of at least 56/N, wherein N represents the number of distinct X-mer precursors in the mixture;

wherein each sub-mixture in said set has a reduced mixture coverage complexity as compared with the composite mixture coverage complexity;

wherein each sub-mixture comprises a plurality of X-mer precursors;

wherein said length is selected independently for each X-mer precursor; and

wherein the mixture or set of sub-mixtures further comprises a set of tags that are distinguishable by mass spectrometry, wherein each tag is covalently linked to at least one X-mer precursor through a cleavable linker such that any given oligonucleotide sequence in the mixture is attached to a single tag with a discrete molecular weight.

3. The mixture or set of sub-mixtures of claim 1 or 2 wherein said mixture has a mixture coverage complexity of at least about 1/2 when said mixture contains at least 128 discrete X-mers, or wherein said set of sub-mixtures has a composite mixture coverage complexity of at least about 1/2 when said set of sub-mixtures contains at least 128 discrete X-mers.

4. The mixture or set of sub-mixtures of claim 1 or 2, wherein said mixture has a mixture coverage complexity of at least about 1/4 when said mixture contains at least 256 discrete X-mers, or wherein said set of sub-mixtures has a composite mixture coverage complexity of at least about 1/4 when said set of sub-mixtures contains at least 256 discrete X-mers.

5. The mixture or set of sub-mixtures of claim 1 or 2, wherein said mixture has a mixture coverage complexity of at least about 1/8 when said mixture contains at least 512 discrete X-mers, or wherein said set of sub-mixtures has a composite mixture coverage complexity of at least about 1/8 when said set of sub-mixtures contains at least 512 discrete X-mers.

6. The mixture or set of sub-mixtures of claim 1 or 2, wherein nucleotide sequences of the precursors of said mixture or set of sub-mixtures are known.

2. A mixture or set of sub-mixtures comprising X-mer precursors of different length,

wherein said X-mer precursors have a minimum length of 3 nucleotides;

wherein said mixture has a minimum mixture coverage complexity of at least 56/N or wherein said set of sub-mixtures has a composite mixture coverage complexity of at least 56/N, wherein N represents the number of distinct X-mer precursors in the mixture;

wherein each sub-mixture in said set has a reduced mixture coverage complexity as compared with the composite mixture coverage complexity;

wherein each sub-mixture further comprises a plurality of X-mer precursors;

wherein said length is selected independently for each X-mer precursor;

wherein the mixture or set of sub-mixtures further comprises a set of tags, wherein each tag is covalently linked to at least one X-mer precursor through a cleavable linker such that any given oligonucleotide sequence in the mixture is attached to a single tag with a discrete molecular weight; and

wherein said X-mer precursors have a determined isotopic composition.