High density immobilization of nucleic acids

• US 6,818,394 B1
• Filed: 01/04/2000
• Issued: 11/16/2004
• Est. Priority Date: 11/06/1996
• Status: Expired due to Fees

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Cantor, Charles R., OʼDonnell-Maloney, Maryanne J., Köster, Hubert
• Primary Examiner(s): CELSA, BENNETT M
• Application Number:
  US09/297,575
• Time in Patent Office: 1,778 Days
• Field of Search: 435/89, 435/6, 435/91.1, 435/174, 435/177, 436/501
• US Class Current: 435/6.11
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00315 Microtiter plates
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00387 Applications using probes
• CPC Class Codes: B01J 2219/00468 by manipulation of individual beads
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00504 Pins
• CPC Class Codes: B01J 2219/00511 Walls of reactor vessels
• CPC Class Codes: B01J 2219/0052 in the shape of elongated tubes
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-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 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00659 : Two-dimensional arrays
• CPC Class Codes: B01J 2219/0072 : Organic compounds
• CPC Class Codes: B01J 2219/00722 : Nucleotides
• CPC Class Codes: B01J 2219/00725 : Peptides
• CPC Class Codes: B01L 2200/025 : Align devices or objects to ensure defined positions relative to each other
• CPC Class Codes: B01L 2300/0838 : Capillaries
• CPC Class Codes: B01L 2400/0439 : ultrasonic vibrations, vibrating piezo elements
• CPC Class Codes: B01L 2400/0487 : fluid pressure, pneumatics
• CPC Class Codes: B01L 3/0244 : using pins
• CPC Class Codes: B01L 3/0262 : using touch-off at substrate or container
• CPC Class Codes: B01L 3/0268 : using pulse dispensing or spraying, eg. inkjet type, piezo actuated ejection of droplets from capillaries
• CPC Class Codes: C07B 2200/11 : Compounds covalently bound to a solid support
• CPC Class Codes: C07F 9/2408 : of hydroxyalkyl compounds
• CPC Class Codes: C07F 9/2429 : of arylalkanols
• CPC Class Codes: C07H 21/00 : Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6816 : characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 : for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6834 : Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6858 : Allele-specific amplification
• CPC Class Codes: C12Q 1/6872 : involving mass spectrometry
• CPC Class Codes: C12Q 1/6883 : for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/6886 : for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2523/10 : Characterised by chemical treatment
• CPC Class Codes: C12Q 2525/197 : incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2531/113 : PCR
• CPC Class Codes: C12Q 2535/101 : Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2565/507 : characterised by the density of the capture oligonucleotide
• CPC Class Codes: C12Q 2565/518 : characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/537 : characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C12Q 2565/627 : being a mass spectrometer
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: G01N 2035/00237 : Handling microquantities of analyte, e.g. microvalves, capillary networks
• CPC Class Codes: G01N 2035/1037 : Using surface tension, e.g. pins or wires
• CPC Class Codes: G01N 2035/1069 : by adjusting the spacing between multiple probes of a single transferring head
• CPC Class Codes: G01N 35/1067 : for transfer to or from containers having different spacing
• CPC Class Codes: G01N 35/1074 : arranged in a two-dimensional array

Abstract

Processes and kits for immobilizing a high density of nucleic acids on an insoluble surface, which are particularly useful for mass spectrometric detection of nucleic acids, are disclosed. Arrays containing the immobilized nucleic acids and use of the immobilized nucleic acids in a variety of solid phase nucleic acid chemistry applications, including nucleic acid synthesis (chemical and enzymatic), hybridization and/or extension, and sequencing, are provided. Serial and parallel dispensing tools that can deliver defined volumes of fluid to generate multi-element arrays of sample material on a substrate surface are further provided. Tools provided herein can include an assembly of vesicle elements, or pins, wherein each of the pins can include a narrow interior chamber suitable for holding nanoliter volumes of fluid. Methods for dispensing tools that can be employed to generate multi-element arrays of sample material on a substrate surface are also provided. The tool can dispense a spot of fluid to a substrate surface by spraying the fluid from the pin, contacting the substrate surface or forming a drop that touches against the substrate surface. The tool can form an array of sample material by dispensing sample material in a series of steps, while moving the pin to different locations above the substrate surface to form the sample array, The prepared sample arrays may be passed to a plate assembly that disposes the sample arrays for analysis by mass spectrometry.

Claims

1. A method for synthesizing DNA on the surface of a support, comprising:

reacting the surface of a silicon support or a support with a silicon surface with a solution of 3-aminopropyltriethoxysilane to produce a uniform layer of primary amines on the surface of the support;

derivatizing the surface of the support with iodoacetamido functionalities by reacting the uniform layer of primary amines with a solution of. N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB);

contacting the surface of the support with a thiol-containing strand of DNA, whereby the thiol-containing DNA is immobilized on the surface of the support by a covalent bond between the thiol group of the thiol-containing DNA and the iodoacetamrido functionality on the surface;

hybridizing a single-strand of DNA that is complementary to a portion of the immobilized thiol-containing DNA; and

adding at least one nucleotide to the 3′

-end of the hybridized single-strand of DNA by DNA synthesis, whereby DNA is synthesized on the surface of a support; and

determining a molecular weight by mass spectrometry of the single-strand of DNA to which at least one nucleotide has been added.

2. The method of claim 1, wherein the immobilized DNA is positioned on the support in the form of an array.

3. The method of claim 1, further comprising:

adding one or more dideoxynucleoside triphosphates during DNA synthesis.

4. The method of claim 1, wherein the mass spectrometry analysis is selected from the group consisting of Matrix Assisted Laser Desorption/lonization, Time-of-Flight (MALDI-TOF) analysis, Electronspray Electrospray (ES), Ion Cyclotron Resonance (ICR) and Fourier transform.

5. A method, comprising:

reacting thiol-containing DNA molecules with a solid support under conditions such that a covalent bond is formed, thereby immobilizing DNA molecules on the insoluble support, wherein;

the resulting covalent linkages and support are stable to laser desorption, and

the support is a silicon support or comprises a silicon surface or a silicon dioxide surface;

hybridizing a single-strand of DNA to a portion of the immobilized thiol-containing DNA molecule complementary thereto;

adding at least one deoxynucleotide or dideoxynucleotide to the 3′

-end of the hybridized single strand of DNA by enzymatic DNA synthesis; and

determining the molecular weight of the hybridized single-strand of DNA to which at least one deoxynucleotide or dideoxynucleotide has been added using mass spectrometry analysis, whereby the sequence of at least a portion of thiol-containing DNA molecule immobilized on the surface of a support is determined.

7. The method of claim 5 or 6, wherein the mass spectrometry analysis is selected from the group consisting of Matrix Assisted Laser Desorption/lonization, Time-of-Flight (MALDI-TOF) analysis, Electrospray (ES), Ion Cyclotron Resonance (ICR) and Fourier transform.

8. The method of claim 5, or 6 wherein the immobilized DNA is positioned on the support in the form of an array.

6. A method for sequencing DNA, comprising:

reacting the surface of a silicon support or support with a silicon surface with a solution of 3-aminopropyltriethoxysilane to produce a uniform layer of primary amines on the surface of the support,

derivatizing the surface of the support with iodoacetamido functionalities by reacting the uniform layer of primary amines with a solution of N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB),

reacting the surface of the support with a thiol-containing strand of DNA, whereby the thiol-containing DNA is immobilized on the surface of the support by a covalent bond between the thiol group of the thiol-containing DNA and the iodoacetamido functionality derivatized on the surface of the support,

hybridizing a single-strand of DNA to a portion of the immobilized thiol-containing DNA complementary thereto,

performing DNA synthesis in the presence of an appropriate mixture of deoxynucleotides containing one or more dideoxynucleotides, wherein at least one deoxynucleotide or dideoxynucleotide is added to the hybridized single-strand of DNA at its 3′

-end by enzymatic DNA synthesis; and

determining the molecular weight of the hybridized single-strand of DNA containing the enzymatically added deoxynucleotide(s) or dideoxynucleotide(s) using mass spectrometry analysis, whereby at least one base in the sequence is determined.